CN108398549A - The method for obtaining the drug with mitochondria uncoupling - Google Patents

Abstract

The invention belongs to biology and field of medicaments, disclose a kind of method for leading to mitochondria uncoupling and activating Adenylate cyclase：I.e. sulfur-containing compound causes the inversion electron of mitochondrial respiratory chain composite I level to transmit by the oxidation of SQR, reduces across mitochondrial membrane potential, so as to cause mitochondria uncoupling；And mitochondria uncoupling can cause intracellular ATP synthesis level to decline, and then activate AMPK.The invention also discloses a kind of methods obtaining mitochondrial uncoupler and AMPK activator based on SQR.The present invention passes through experimental verification：1) SQR is the new method for causing mitochondria uncoupling or AMPK to activate to the oxidation of sulfur-containing compound；2) novel mitochondrial uncoupler or AMPK activator can be obtained by being based on SQR；3) and obtain compound have cell-specific mitochondria uncoupling or AMPK activations, toxic side effect is small, can be used for treating correlative diseases.

Description

The method for obtaining the drug with mitochondria uncoupling

Technical field

The invention belongs to biology and field of medicaments, disclose a kind of new method for causing mitochondria uncoupling or AMPK to activate, And it discloses based on sulfide-quinone oxidoreductase (SQR, Sulfide:Quinone oxidoreductase) it obtains with line The method of the substance of plastochondria uncoupling or AMPK activations, and the substance obtained in this way are preparing treatment Purposes in the drug of relevant disease.

Background technology

Mitochondria is the key cells device of cell metabolism effect, is intracellular high-energy synthetic substance Adenosine triphosphate thuja acid (ATP) main place.ATP provides the main energetic of vital movement.Under normal circumstances, the substrate oxidations such as glucose in cell The process to release energy utilizes the energy phosphorylation adenosine diphosphatase (ADP) of substrate oxidation release with mitochondrial ATP synthesis The process for generating ATP is coupling, i.e. the energy of the substrate oxidations such as glucose release is used to synthesize by mitochondrial ATP synthesis ATP.It is this coupling formed mechanism be：The substrates such as glucose are during glycolysis and tricarboxylic acid cycle by oxidation (de- electricity Son), the electronics taken off is transferred to molecular oxygen through mitochondrial respiratory chain composite I-IV；The energy that electronics discharges in transmittance process Proton is then driven to enter intermembrane space inside and outside mitochondria from mitochondrial matrix cross-line mitochondrial membrane, the cross-line mitochondrial membrane being consequently formed Proton gradient (film potential) drives ATP synzyme phosphorylations ADP to generate ATP.

Mitochondria uncoupling refers to that the energy of the substrate oxidations such as glucose release cannot be by mitochondrial ATP synthesis for closing At ATP, i.e. the oxidation process of substrate synthesizes the process uncoupling of ATP with mitochondria.It is now recognized that uncoupling mechanism is：With solution The allogenic material of coupled action or endogenous uncoupling protein make proton outer membrane out of mitochondria as proton carrier or proton channel Gap comes back to matrix, to reduce cross-line mitochondrial membrane current potential, under the proton motive force for leading to ATP synthesis enzymatic synthesis ATP Drop.In the case of uncoupling, the oxidation of the substrates such as glucose still provides for, but energy is not used to synthesize ATP by mitochondria, but It is dissipated with form of thermal energy.Mitochondria uncoupling can lead to following effect：1) substrate oxidation release energy not by with synthesis in ATP causes intracellular ATP levels to decline；2) increase the oxidation for promoting the substrates such as lipid and glucose；3) intracellular ATP levels decline Lead to the activation of Adenylate cyclase (AMPK), therefore mitochondria uncoupling has the function of significantly activating AMPK.

It is existing research shows that mitochondrial uncoupler can treat a variety of diseases, including but not limited to metabolic syndrome, obesity, Neurodegenerative disease, aging, cerebral apoplexy, diabetes and cancer etc..

Uncoupler is divided into two types：Exogenous chemical uncoupler and endogenous uncoupling protein.It has now found that Chemical uncoupler is divided into two classes, wherein main is proton carrier, additionally including some other ions (such as potassium ion) Carrier.Their mechanism of action is that transhipment proton (hydrogen ion) or other ions cross mitochondrial inner membrane.For example, including proton carrier Intermembrane space is combined rear cross-film with proton, to which proton is taken back matrix again, mitochondrial membrane potential is caused to reduce.For example, weak acid Proton type uncoupler can dissociate or combine proton in different pH environment.Ring of the weak acid proton type uncoupler in pH7.0 Exist in the form of dissociating in border, is then protonated in the acidic environment of intermembrane space inside and outside the mitochondria, becomes fat-soluble non-solution From form (UH).UH can penetrate mitochondrial inner membrane phospholipid bilayer.Under alkaline condition in mitochondrial matrix, non-dissociation shape The UH dissociation release H+ of formula, so that a proton is brought on the outside of inner membrance the base value of inner side, to reduce cross-line grain The electrochemical proton gradient of internal film, and energy is discharged in the form of thermal energy, inhibit the formation of ATP.2,4- dinitrophenol (2,4-dinitrophenol, DNP) is most common weak acid proton uncoupler, and as first mitochondrial uncoupler Once be used to treat fat.But DNP toxicity is big, has been prohibited Clinical practice.Other ionophore chemical uncouplers can be with spy Ions binding is determined, to improve mitochondrial inner membrane to certain ion permeabilities.Ionophore type chemical uncoupler is represented as A kind of valinomycins, the annular small peptide antibiotic being made of 12 amino acid residues generated by streptomycete, belongs to potassium ion (K+) carrier.After valinomycins is inserted into liposome, increase permeability of the mitochondrial inner membrane to K+, to reduce the current potential ladder of cross-film Degree inhibits ATP synthesis.

Endogenous uncoupling protein (uncoupling protein, UCP) is a kind of mitochondrial inner membrane albumen.It is now recognized that UCP is substantially to transport proton as proton channel and enter mitochondrial matrix, to eliminate the cross-film of mitochondrial inner membrane both sides Proton concentration is poor, reduces the generation of ATP.The uncoupling protein having now been found that is UCP1, UCP2, UCP3, UCP4 and UCP5.

To sum up, it has now been found that chemical uncoupler be all proton or ionophore, their uncoupling machine in itself Primarily as carrier, intermembrane space is combined rear cross-line mitochondrial membrane with proton to system inside and outside mitochondria, to take back proton again Mitochondrial matrix causes mitochondrial transmembrane potentials to reduce.The effect of proton or ionophore type mitochondrial uncoupler lacks special The opposite sex, toxic side effect are big.For example, once in the effective dose and toxic amount of the proton carrier type uncoupler DNP of clinical application Cause side effect very close to, easy overdose.The mankind, which disposably take 20-50mg/kg DNP, can directly result in death.Therefore Year ends 1938, DNP are forced from American market undercarriage and are prohibited to be used for clinical treatment.So far safe mitochondria solution is not yet found Coupling agent is applied to clinic.Thus, it is found that the mitochondrial uncoupler that mechanism of action is innovated, safe dose is high has important clinical Meaning.

Invention content

Sulfide-quinone oxidoreductase (SQR) is a kind of oxidoreducing enzyme, and it is over cure that function, which is by Oxidation of Hydrogen Sulfide, Object.SQR bacterium, protist until vertebrate mammals in be widely present, and in SQR albumen with it is catalysed curing The closely related amino acid sites of hydroxide are conservative.The oxidative function of SQR is acted in vertebrate mammals at present Understanding be：1) vertebrate SQR plays the detoxication to hydrogen sulfide by oxidative metabolism hydrogen sulfide；2) in certain detail , may be similar with bacterium SQR in born of the same parents, mammal SQR provides approach (i.e. to the energy that the oxidation of hydrogen sulfide may be also cell ATP synthesis is caused to rise).

And inventor then has found by further investigation, (or these sulfide are in cell by aoxidizing sulfur-containing compound by SQR Or the product after conversion in vivo or metabolism) inversion electron of mitochondrial respiratory chain composite I level can be caused to transmit (RET), into And mitochondria uncoupling or AMPK is caused to activate.Under normal circumstances, the electron transmission that Respiratory Chain Complex I mediates is positive 's：I.e. it is auxiliary from the nicotinamide adenine dinucleotide (NADH) of reduction-state to take off electron transmission oxidizing type for Respiratory Chain Complex I Enzyme Q (CoQ)；To generate the ubiquinone (CoQH2) of reduced form, and NADH is then oxidized to the nicotinamide adenine two of oxidation state Nucleotide (NAD+).The inversion electron of mitochondrial complex I levels transmits exactly the opposite：Composite I is from reduced coenzyme Q (CoQH2) NAD+ for taking off electron transmission oxidizing type, to generate reduced-NAD H.Therefore mitochondrial respiratory chain composite I water Flat inversion electron transmission can reduce mitochondria NAD+/NADH ratios.Theoretically, SQR leads the oxidation of hydrogen sulfide or sulfide It causes the CoQ of oxidized form to be reduced to the CoQH2 of reduced form, CoQH2/CoQ ratios is made to increase；And research shows that CoQH2/CoQ ratios It is one of the key condition for driving the horizontal reversed electron transmissions of mitochondrial complex I that value, which rises,.But now all of research is Show：The inversion electron transmission of Respiratory Chain Complex I level always needs high across mitochondrial membrane potential.But exceed to anticipate Material, inventor have found that sulfide aoxidizes the horizontal reversed electron transmission of caused composite I by SQR and but leads to cross-line plastochondria Inner membrance film potential is remarkably decreased, and to make ATP synzyme lose the proton motive force of synthesis ATP, that is, leads to mitochondria uncoupling. Applicant further found that mitochondria uncoupling caused by sulfide is aoxidized by SQR reduces ATP and synthesizes, this also with have now been found that SQR Oxidation of sulfureted hydrogen provides energy (ATP synthesis is caused to rise) difference for cell.

Existing mitochondria uncoupling method be using proton (or ion) carrier cross-line mitochondrial membrane transhipment proton or Ion, so as to cause mitochondria uncoupling.And mitochondria uncoupling method disclosed by the invention is totally different from existing method： Sulfide be not be directly as proton or ionophore and lead to mitochondria uncoupling.Sulfide (or sulfide is in cell Or the product after conversion in vivo or metabolism) it is to cause the inversion electron of Respiratory Chain Complex I level to transmit by SQR oxidations, into And lead to mitochondria uncoupling.

Therefore, the sulfide obtained using the method for the present invention causes mitochondria uncoupling multiple dependent on mitochondria solution respiratory chain Close the function of object I；And the effect for the mitochondrial uncoupler having now found that is not required to the function of mitochondrial respiratory chain composite I.

It is existing research shows that：The oral half lethal doses of proton type carrier uncoupler DNP in rats be 30 milligrams/ Kilogram；And ADT (mitochondrial uncoupler based on SQR effects obtained using the method for the present invention) is oral in mouse Half lethal dose is up to 3850 mgs/kg.Inventor has found that the expression of SQR has cell-specific.And not all cell SQR is expressed, as nerve cell does not express SQR.Therefore, cause the effect of the substance of mitochondria uncoupling that there is cell by SQR Specificity, without leading to uncoupling in all cells.Therefore, the method security of mitochondria uncoupling is caused to be wanted by SQR Far above the existing method for leading to mitochondria uncoupling using nonspecific proton or ionophore.Therefore, with existing side Method is compared, and the present invention causes the method for mitochondria uncoupling also to have cell-specific and higher safety.The present invention discloses Acquisition sulfide have cell-specific, toxic side effect is small, safe.

Intracellular ATP levels decline caused by mitochondria uncoupling can activate AMPK.Therefore, disclosed by the invention to lead to line grain The method of body uncoupling can be declined by intracellular ATP levels, and then activate AMPK.Compared with the conventional method, the present invention discloses The method of activation AMPK be also mechanism innovation, and there is cell-specific and high security.

On this basis, being obtained the invention discloses a kind of cell based on SQR or animal model has mitochondria solution even The method of the substance of connection effect.It the described method comprises the following steps：

(a) mitochondria for handling the cell of expression SQR with candidate substances or being extracted from the cell of expression SQR, in candidate In the presence of substance, detect mitochondria uncoupling characteristics index, with solvent handle cell/mitochondria or untreated cell/ Mitochondria compares, and compares the mitochondria uncoupling characteristics index of candidate substances processing group and control group, if statistically Variant, candidate substances are potential mitochondrial uncouplers；

(b) it is not expressed with candidate substances processing or low expression SQR, SQR activity reduces or the cell of inactivation, or with candidate Matter processing detects mitochondria uncoupling characteristics index from the mitochondria of above-mentioned cell extraction, the cell/line grain handled with solvent Body or untreated cell/mitochondria compare, and compare candidate substances processing group and the mitochondria uncoupling characteristic of control group Index, if statistically indifference, and candidate substances are potential mitochondrial uncouplers in (a), then and candidate substances are The substance with mitochondria uncoupling by SQR is mediated.

Wherein, the mitochondria uncoupling characteristics index is：1) mitochondria uncoupling can be at Adenosine triphosphate thuja acid (ATP) Cause the oxygen demand of cell or mitochondria to rise when synzyme is suppressed, 2) reduces cross-line Mitochondria Membrane film potential and 3) reduce cell Interior ATP is horizontal.

In some embodiments, Adenosine triphosphate thuja acid (ATP) synthetase inhibitors can be Oligomycin.

In some embodiments, the method for obtaining the substance with mitochondria uncoupling is before step (a) It further include the steps that preliminary screening substance with mitochondria uncoupling by SQR is mediated.

In some embodiments, preliminary screening substance with mitochondria uncoupling by SQR is mediated Step is specially：With the mitochondria that candidate substances handle the cell of expression SQR or extracted from the cell of expression SQR, in candidate Under conditions of substance exists and inhibits mitochondrial respiratory chain composite I, mitochondria uncoupling characteristics index is detected, with only with time It selects cell/mitochondria of the expression SQR of substance processing to compare, compare candidate substances and inhibits mitochondrial respiratory chain composite I The mitochondria uncoupling characteristics index of coprocessing group and control group, if such as variant in statistics, candidate substances are potential By SQR is mediated with mitochondria uncoupling substance.In some embodiments, mitochondrial respiratory chain may be used Composite I inhibitor rotenone.

Further, the method for obtaining the substance with mitochondria uncoupling further includes cell experiment and/or moves Object test procedure has medicative substance to obtain for relevant disease.

On the other hand, the acquisition of the present invention provides a kind of cell or animal model based on SQR has cell-specific The method of substance act on, that there is activation to AMPK.It the described method comprises the following steps：

(A) cell that expression SQR is handled with candidate substances, in the presence of candidate substances, the activated water of detection cell AMPK Flat, the cell or untreated cell handled with solvent compares, and compares candidate substances processing group and control group A MPK activated waters Flat, as statistically variant, candidate substances are potential AMPK activator；

(B) it is not expressed with candidate substances processing or low expression SQR, SQR activity reduces or the cell of inactivation, in candidate substances In the presence of, the activation levels of detection cell AMPK, the cell or untreated cell handled with solvent compares, and compares candidate Matter processing group and control group A MPK activation levels, such as statistically indifference, and candidate substances are potential in (A) AMPK activator, then candidate substances are to lead to the substance that AMPK is activated by SQR.

In some embodiments, described obtain to the method for substances of the AMPK with activation further includes that experiment is candidate The step of whether the AMPK activations of substance depend on mitochondrial respiratory chain composite I.

In some embodiments, whether the AMPK activations of the experiment candidate substances are multiple dependent on mitochondrial respiratory chain Close object I the step of be specially：The cell that expression SQR is handled with candidate substances is existed in candidate substances and inhibits mitochondrial respiratory Under conditions of chain cpd I, AMPK activation levels are detected, the cell only to handle expression SQR with candidate substances compares, and compares Candidate substances and inhibition mitochondrial respiratory chain composite I coprocessing group and control group A MPK activation levels, such as have difference in statistics Different, then candidate substances are potentially to lead to the substance that AMPK is activated by SQR..In some embodiments, it can be exhaled with mitochondria Inhale chain cpd I inhibitor rotenone.

Wherein, the cell of the expression SQR includes but not limited to following cell：The primary cell of endogenous expression SQR or Cell line；Lead to the primary cell for expressing or being overexpressed SQR or cell line, from expression with genetic manipulation means or pharmacological techniques The primary or passage cell that the genetic engineering animal of SQR obtains.

In some embodiments, the primary small colloid that the cell of expression SQR of the present invention is endogenous expression SQR is thin Born of the same parents.In some embodiments, the BV2 microglias system that the cell of expression SQR of the present invention is endogenous expression SQR. In other embodiments, the cell of heretofore described expression SQR is to be overexpressed the primary nerve of SQR carefully by slow virus technology Born of the same parents.

In the above method, it is described do not express or the cell of low expression SQR, SQR activity reduction/inactivation include but not limited to Lower cell：Endogenous low expression or the primary cell or cell line for not expressing SQR；Or endogenous SQR protein inactivations or activity drop Low primary cell or cell line；Or it utilizes genetic manipulation means or pharmacological techniques reduction/knockout/to strike and subtracts endogenous SQR tables The primary cell or cell line for reaching or making endogenous SQR activity to reduce or inactivate；And it knocks out or strikes from SQR and subtract what animal obtained Primary or passage cell.

And the genetic manipulation means include but not limited to, as small interfering RNA technology (siRNA) reduces endogenous SQR tables It reaches, knocks out/strike with CRISP-CAS9 technologies, TALEN technologies etc. and subtract endogenous SQR.

In some embodiments, the cell of the present invention for not expressing SQR is the mouse primary god that endogenous does not express SQR Through cell.In some embodiments, the present invention selects RNA perturbation techniques (short hairpin RNA, shRNA) technology of lentivirus mediated It is struck in primary microglia and subtracts the cell that SQR obtains SQR expression/activity reduction/inactivation.In further embodiments, originally The technology of invention selection transfection siRNA is struck in BV2 microglias subtracts the thin of SQR acquisition SQR expression/activity reduction/inactivation Born of the same parents.

Further, the acquisition with cell-specific effect, to the method for substances of the AMPK with activation also Including cell experiment and/or animal experiment step, has medicative substance for relevant disease to obtain.

The present invention is by the above method, mitochondria solution of the screening with cell-specific effect from 100 multiple compounds Coupling agent/AMPK activator finally obtains three kinds of sulfur-containing compounds.Three kinds of sulfur compounds that the screening obtains are respectively 5- pairs Hydroxy phenyl -3H-1,2- dithiacyclopentene -3- thioketones (ADT-OH, as shown in Equation 1), 5- p-methoxyphenyls -3H-1,2- bis- Sulphur cyclopentene -3- thioketones (ADT, as shown in Equation 1) and N- (benzoyl sulfenyl) benzamide (abbreviation 5a, as shown in Equation 2).

Wherein, R is-H or-CH in formula 1₃。

It is 5- p-hydroxybenzenes -3H-1,2- dithiacyclopentene -3- thioketones (ADT-OH) when R is-H in formula 1.In formula 1 Middle R is-CH₃When, it is 5- p-methoxyphenyls -3H-1,2- dithiacyclopentene -3- thioketones (ADT).

Further, the present invention by specific embodiment confirm above-mentioned three kinds of sulfide be mediated by SQR, effect machine System innovation and mitochondrial uncoupler/AMPK activator with cell-specific effect.Therefore the present invention provides above-mentioned three kinds Purposes of the sulfide (ADT-OH, ADT, 5a) as mitochondrial uncoupler, AMPK activator.

On the other hand, the method that inventor also uses cell and/or animal experiment, is identified through the object of above method acquisition Matter, such as therapeutical uses of the above-mentioned sulfide to relevant disease.Therefore the present invention also provides obtained using the method for the invention Mitochondrial uncoupler/AMPK activator prepare treatment cerebral hemorrhage, metabolic syndrome, obesity, neurodegenerative disease, decline Always, the purposes in the drug of cerebral ischemia, diabetes or cancer.

Wherein, inventor selects cerebral hemorrhage cell and animal model to carry out experiment, especially selects and passes through genetic manipulation hand Section (the shRNA technologies of lentivirus mediated) causes endogenous SQR expression to strike the mouse subtracted.

Further, the present invention also provides a kind of method leading to mitochondria uncoupling, sulfur-containing compound (or these vulcanizations Object cell or in vivo conversion or metabolism after product) by the oxidation of SQR cause mitochondrial respiratory chain composite I level it is anti- To electron transmission, reduce across mitochondrial membrane potential, so as to cause mitochondria uncoupling.

The present invention also provides a kind of method of activation AMPK, sulfur-containing compound (or these sulfur-containing compounds in cell or Product after conversion in vivo or metabolism) cause the inversion electron of mitochondrial respiratory chain composite I level to pass by the oxidation of SQR It passs, reduces across mitochondrial membrane potential, decline so as to cause mitochondria uncoupling and intracellular ATP levels, and then activate AMPK。

The present invention also provides the substances obtained using the above method as mitochondrial uncoupler or AMPK activator Purposes, the especially purposes in the drug for preparing treatment relevant disease.

As shown from the above technical solution, the invention discloses a kind of method for causing mitochondria uncoupling and AMPK to activate, Sulfur-containing compound (or the product of sulfur-containing compound after cell or conversion in vivo or metabolism) leads to mitochondria by the oxidation of SQR The inversion electron of Respiratory Chain Complex I level transmits, and to reduce across mitochondrial membrane potential, leads to mitochondria uncoupling； Mitochondria uncoupling makes intracellular ATP levels decline, and then activates AMPK.The invention also discloses obtain mitochondria based on SQR The method of uncoupler and AMPK activator.By this method, inventor, which obtains, has mitochondria uncoupling or AMPK The compound of activation, and by experimental verification using the method for the present invention obtain substance mitochondria uncoupling and AMPK activations are dependent on SQR to the oxygen of sulfide (or the product of these sulfide after cell or conversion in vivo or metabolism) The inversion electron of mitochondrial respiratory chain composite I level caused by changing transmits.By further experiment, the present invention also shows to apply The mitochondria uncoupling and AMPK activations for the substance that the method for the invention obtains have cell-specific；In particular, phase Than with the mitochondrial uncoupler that has now found that, the substance toxic side effect obtained using the method for the present invention is small, can be used for treating more Kind disease such as cerebral hemorrhage, metabolic syndrome, obesity, neurodegenerative disease, cerebral ischemia, cancer and diabetes etc..

Description of the drawings

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technology description to be briefly described.

Fig. 1 shows the result of mitochondrial uncoupler of 1 preliminary screening of embodiment based on SQR；At candidate substances or solvent The BV2 microglias of endogenous expression SQR are managed, then cell is handled to indicate mitochondrial membrane potential with TMRM fluorescence probes；With Solvent (Vehicle) is compared, in 100 multiple compounds, three kinds of sulfur-containing compounds：Two sulphur rings of 5- p-hydroxybenzenes -3H-1,2- Amylene -3- thioketones (abbreviation ADT-OH) 5- p-methoxyphenyl -3H-1,2- dithiacyclopentene -3- thioketones (abbreviation ADT) and N- (benzoyl sulfenyl) benzamide (abbreviation 5a) can be substantially reduced mitochondrial membrane potential in anoxic at 50 μM and (it is red show as TMRM Color fluorescence decline) and these substances reduce mitochondrial membrane potential effect can be by mitochondrial complex I inhibitor rotenone (Rot) (Figure 1A, B, C) is blocked；Proton type mitochondrial uncoupler FCCP can also reduce cell TMRM red fluorescences, but FCCP drops The effect of low mitochondrial membrane potential is not blocked by rotenone；* or $ $：The p compared with solvent<0.01；##：With candidate is only added Matter (ADT-OH, ADT or 5a) compares p<0.01, p<0.01 indicates statistically there is pole significant difference；

Fig. 2 shows that embodiment 2 shows that candidate substances reduce work(of the horizontal effects of intracellular ATP dependent on mitochondrial complex I Energy；Compared with solvent (Vehicle), ADT-OH (50 μM), ADT (50 μM) and 5a (50 μM) are substantially reduced intracellular ATP levels And increase ADP/ATP ratios and these effects can be blocked by rotenone (Rot), the mitochondrial uncoupler FCCP of proton type Also cellular ATP levels are reduced and increase ADP/ATP ratios, but the effect of FCCP is not blocked by rotenone (Rot)；Vehicle:Add Enter the control cell of solvent, * * or $ $：The p compared with Vehicle<0.01；##：With only be added candidate substances (ADT-OH, ADT or 5a) compare p<0.01, p<0.01 indicates statistically there is pole significant difference；

Fig. 3 shows that 2 candidate substances of embodiment increase cell oxygen demand in the presence of ATP synthetase inhibitors oligomycin (OCR) function of the effect dependent on mitochondrial complex I：Endogenous expression SQR primary microglia (Fig. 3 A) or In BV2 microglias (Fig. 3 B), candidate substances ADT-OH (50 μM) increased in the presence of oligomycin cell OCR and The effect that ADT-OH increases OCR can be blocked by rotenone (Rot)；As shown, in A points addition oligomycin, about 30 points ADT-OH and/or Rot, * is added in B points after clock：Oligomycin+ADT-OH groups and oligomycin+ADT-OH+Rot groups and Solvent group (oligomycin) compares p<0.05, p<0.05 statistically there were significant differences.

Fig. 4 shows that embodiment 3 shows that the expression of endogenous SQR has cell-specific；Figure A-B show Western blot and QPCR results：SQR albumen (Fig. 4 A) and SQR mRNA (Fig. 4 B) are expressed in primary microglia and star spongiocyte, and It is hardly expressed (using liver liver as positive control) in primary neuron.* * and liver (liver), microglia (Microglia) p is compared with star spongiocyte (Astrocyte)<0.0001, difference is extremely notable；Figure C-E shows to be examined with qPCR Survey microglia specific marker CD-11B, star spongiocyte specific marker GFAP and neuron specific markers NeuN's as a result, to show primary microglia (figure C), star spongiocyte (figure D) and the neuron (figure of separated culture E purity)；* * indicate the p compared with another specific cell marks<0.0001, difference is extremely notable；

Fig. 5 shows embodiment 4 in the primary neuron that no endogenous SQR is expressed, and it is even that candidate substances do not have mitochondria solution The result figure of connection effect；In the presence of figure A shows ATP synthetase inhibitors oligomycin, candidate ADT-OH is in primary neuron In cannot increase cell OCR values, and proton carrier type uncoupler FCCP increases primary neuron in the presence of oligomycin Oligomycin and ADT-OH/FCCP is added at corresponding time point as shown in the figure in OCR；Scheme B-C to show compared with solvent, candidate ADT-OH (50 μM) cannot reduce primary neuron intracellular ATP horizontal (Fig. 5 B) and increase ADP/ATP ratios (Fig. 5 C), and FCCP There is above-mentioned effect in primary neuron；Figure D shows that TMRM coloration results are shown:Candidate ADT-OH (50 μM) cannot reduce primary Neuron linear mitochondrial membrane potential, and FCCP has above-mentioned effect in primary neuron；Figure E-F shows that candidate 5a (50 μM) cannot drop Low primary neuron intracellular ATP levels and raising ADP/ATP ratios；Figure G shows that TMRM coloration results, candidate 5a cannot reduce original For neuron linear mitochondrial membrane potential；**：The p compared with solvent or ADT-OH<0.01；

Fig. 6 shows embodiment 4 in the microglia of endogenous expression SQR, and candidate substances are made with mitochondria uncoupling With；Figure A-B shows that in the presence of ATP synthetase inhibitors oligomycin, candidate ADT-OH is in primary microglia (figure OCR values 6A) or in BV2 microglias (Fig. 6 B, 50 μM) are increased, as shown, it is added oligomycin in A points, about 30 points ADT-OH is added in B points after clock；Figure C-D shows in primary microglia, and compared with solvent, candidate ADT-OH reduces intracellular ATP level (Fig. 6 C) and increase ADP/ATP ratios (Fig. 6 D), FCCP also have similar effect；Figure E shows that TMRM coloration results are aobvious Show, candidate ADT-OH reduces primary small colloid mitochondrial membrane potential；Figure F shows to be deposited in ATP synthetase inhibitors oligomycin When, candidate 5a increases OCR values in primary microglia, as shown in the figure corresponding time point be added oligomycin and 5a；Figure G-H shows in primary microglia that (50 μM) of candidate 5a reduces intracellular ATP horizontal (Fig. 6 G) and increases ADP/ATP ratios It is worth (Fig. 6 H)；Fig. 6 A and 6B：* indicate to be added ADT-OH processing group and solvent processing group after oligomycin is added (oligomycin) p is compared<0.05；In Fig. 6 C, 6D, 6E, 6G and 6H, * * indicate the p compared with solvent (Vehicle)<0.01；

Fig. 7 shows that embodiment 5 is struck using slow virus technology in primary microglia and subtracts endogenous SQR expression (Fig. 7 A), SiRNA rotaring dyeing technologies strike in BV2 microglias subtracts endogenous SQR expression (Fig. 7 B) and slow virus technology in primary nerve SQR (Fig. 7 C) is overexpressed in cell；Show that SQR albumen strikes with Western blot and subtracts or be overexpressed efficiency；shSQR：Express needle To the slow virus of the stem ring RNA interfering (shRNA) of SQR；siSQR:For the siRNA of SQR；NC：Negative control virus or Negative control siRNA；Con is uninfected by the control cell of slow virus or untransfected siRNA；OE SQR：It is overexpressed the slow disease of SQR Poison；* p compared with Con or NC<0.01；

Fig. 8 shows embodiment 6 in the primary neuron for being overexpressed SQR, shows that candidate substances are made with mitochondria uncoupling Result figure；Figure A shows that in the primary neuron (OE SQR) that infection is overexpressed SQR slow virus, candidate ADT-OH is in ATP Cell OCR values are increased in the presence of synthetase inhibitors oligomycin, and cannot increase the god of infection negative control virus (NC) OCR values through member；Oligomycin is added in A points as shown in the figure in each processing group cell, and ADT-OH is added in B points；Figure B-C shows It is being overexpressed in the primary neuron (OE SQR) of SQR using slow virus technology, it is horizontal that candidate ADT-OH reduces intracellular ATP (Fig. 8 B) and increase ADP/ATP ratios (Fig. 8 C), and infection negative control virus neuron (NC) in ADT-OH do not have Above-mentioned effect；Figure D shows TMRM coloration results, shows in the primary neuron (OE SQR) for being overexpressed SQR, candidate ADT-OH drops Low mitochondrial membrane potential (showing as the decline of TMRM red fluorescences), and the ADT- in the neuron (NC) of infection negative control virus OH does not have above-mentioned effect, slow virus while enhanced green fluorescent protein, therefore slow virus has been infected in green positive cell expression Cell；Scheme A:* Oligomycin+ADT-OH+OE SQR and solvent processing group after oligomycin is added are indicated (oligomycin) p is compared<0.05；Scheme * * or $ $ in B-D：With Con (control cell of uninfecting virus) or NC+ADT-OH (50-10 μM) is compared p<0.01；

Fig. 9 shows that embodiment 6 shows that in striking the primary microglia for subtracting endogenous SQR expression, candidate substances do not have The result of mitochondria uncoupling:Figure A, which shows, is striking the primary small glue for subtracting endogenous SQR expression using slow virus shRNA technologies In cell plastid (shSQR), candidate ADT-OH no longer increases cell OCR in the presence of ATP synthetase inhibitors oligomycin Value, and OCR values can be increased infecting negative control virus (NC) or being uninfected by ADT-OH in the primary microglia of slow virus, Each processing group cell is added oligomycin in A points, ADT-OH is added in B points after about 30 minutes；For infection comparison virus and ADT-OH (oligomycin+ADT-OH+NC) is added or is uninfected by slow virus but ADT-OH (oligomycin+ADT-OH) is added Primary microglia, * or # indicate after oligomycin is added (oligomycin) OCR values compared with solvent processing group Compared to p<0.05；Scheme B-C to show in striking the primary microglia (shSQR) for subtracting endogenous SQR expression, candidate ADT-OH (50 μM) cannot reduce intracellular ATP horizontal (Fig. 9 B) and increase ADP/ATP ratios (Fig. 9 C), and in infection negative control virus (NC) ADT-OH still has above-mentioned effect in microglia；Figure D-E shows TMRM coloration results, shows to subtract endogenous striking The primary microglia (shSQR) of SQR expression, candidate ADT-OH (Fig. 9 D) and 5a (Fig. 9 E) no longer reduce line at 50 μM Mitochondrial membrane potential (is indicated) with red fluorescence, and still has above-mentioned work in the microglia of infection negative control virus (NC) With, slow virus while enhanced green fluorescent protein, therefore green positive cell indicates to have infected the cell of slow virus；Figure F-G shows The primary microglia (shSQR) for subtracting endogenous SQR expression is being struck, it is horizontal that candidate 5a (50 μM) cannot reduce intracellular ATP (Fig. 9 F) and ADP/ATP ratios (Fig. 9 G) are increased, and still had in the microglia of infection negative control virus (NC) above-mentioned Effect；*, with the small glue for being uninfected by the primary microglia (Con) of slow virus, the infection negative control virus that solvent is added Cell plastid (NC) or the primary microglia (shSQR) for infecting shSQR that solvent is added compare p<0.01；## is indicated and is added The primary microglia (NC+ADT-OH or NC+5a) of the infection NC of ADT-OH or 5a compares p<0.01；

Figure 10 shows embodiment 7BV2 microglias with intracellular CoQH after (50 μM) processing of ADT-OH (50 μM) or 5a₂/ CoQ ratios comparison diagram (10A) and BV2 microglias are with ADT-OH and/or mitochondrial respiratory chain composite I inhibitor trifoliate jewelvine Mitochondria NADH and NAD+/NADH ratio comparison diagram (10B-C) after ketone (Rot) processing；Vehicle：Solvent；* indicated with * * with Solvent (Vehicle) compares p<0.05 or p<0.01；## indicates the p compared with ADT-OH or the single processing group of rotenone (Rot)< 0.01。

Figure 11 show embodiment 8 expressed in endogenous expression or not the effect of candidate substances activation AMPK in the cell of SQR according to Rely in SQR；Figure A-B shows western blot as a result, showing in the primary microglia of endogenous expression SQR, candidate (50 μM, Figure 11 B) of ADT-OH (50-100 μM, Figure 11 A) and 5a increase AMPK phosphorylations (p-AMPK), i.e. AMPK activates water It is flat, * * p compared with the ADT-OH (25 μM) or 5a (25 μM) of control cell or low concentration of solvent is added<0.01；Figure C-D shows Endogenous is not expressed in the primary neuron of SQR, and candidate ADT-OH (Figure 11 C) and 5a (Figure 11 D) cannot increase AMPK phosphoric acid Change (p-AMPK), i.e. AMPK activation levels, and proton carrier type uncoupler FCCP can still increase AMPK phosphorus in nerve cell It is acidified (activation), * * p compared with the cell of control cell or addition ADT-OH (50-100 μM) that solvent is added<0.01；

Figure 12 shows that the effect of candidate substances activation AMPK in striking the cell for subtracting or being overexpressed SQR of embodiment 9 depends on SQR；Scheme A to show in striking the primary microglia (shSQR) for subtracting endogenous SQR expression using the shRNA of lentivirus mediated, ADT-OH (Figure 12 A, 50 μM) no longer increases AMPK phosphorylations (p-AMPK), i.e. AMPK activation levels, and in infection negative control AMPK phosphorylations (activation) are still increased in the primary microglia (NC) of virus；Figure B-C shows in transfection SQR siRNA strike and subtract In the BV2 microglias (siSQR) of source property SQR expression, ADT-OH (Figure 12 B, 50 μM) and 5a (Figure 12 C, 50 μM) no longer rise High AMPK phosphorylations (p-AMPK), i.e. AMPK activation levels, and in the BV2 cells of transfection negative control siRNA (NC), ADT- OH and 5a still increases AMPK phosphorylations (activation) level；Figure D is shown in the primary neuron for being overexpressed SQR using slow virus technology In (OE SQR), candidate ADT-OH can increase AMPK phosphorylations (p-AMPK), i.e. AMPK activation levels, and negative right in infection AMPK phosphorylations (activation) are not increased according to ADT-OH in the neuron (NC) of virus；Con：Uninfecting virus transfects siRNA's Control cell；Scheme ## in A-C and indicates the p compared with NC or shSQR (or siSQR) cell of Con and addition solvent<0.01, $ $ Indicate the p compared with NC+ADT-OH (or NC+5a)<0.01；Scheme NC or OE that ## or $ $ in D indicated with Con and be added solvent (50-100 μM) of SQR or NC+ADT-OH compare p<0.01；

Figure 13 shows that embodiment 10 is handled with ADT-OH and/or mitochondrial respiratory chain composite I inhibitor rotenone (Rot) AMPK activates (AMPK phosphorylations, p-AMPK) horizontal comparing result after BV2 microglias；Vehicle：Solvent, * * indicate with Vehicle compares p<0.01；## indicates the p compared with the mono- processing groups of ADT-OH or Rot<0.01；

Figure 14 shows therapeutic effects of the 11 candidate substances ADT-OH of embodiment to cerebral hemorrhage；After figure A shows cerebral hemorrhage 3 days, injection The mouse of candidate ADT-OH and solvent (Vehicle) injection mouse striaturn (Striatum) brain edema (brain water content, Water content) comparison diagram；Ipsil：Bleeding side, Contra：Bleeding offside, Sham：Sham-operation mouse, * * indicate p< 0.01；After figure B-D shows cerebral hemorrhage 3 days, injection candidate ADT-OH is tied with the detection of solvent injection control group mice three behaviors Fruit comparison diagram：Garcia nervous functions comprehensive detection (Figure 14 B, Garcia neuroscores), right limb touching experiment (Figure 14 C, Right paw placement) and corner experiment (Figure 14 D, Right turns), * * p compared with Sham group mouse< 0.01, # with solvent (Vehicle) inject mouse compared with p<0.05；

Figure 15 shows that the therapeutic effect of the candidate substances ADT-OH in intracerebral hemorrhage models of embodiment 12 depends on SQR；Scheme A Show that striatum injection expressed SQR shRNA slow virus (shSQR) or negative control virus (NC) after 14 days, mouse receives cerebral hemorrhage Receive ADT-OH or solvent after operation daily

(Vehicle) it injects, after cerebral hemorrhage 3 days, ADT-OH and solvent injection group mouse (Vehicle) corpus straitum (Striatum) brain edema (brain water content, water content) comparison diagram, Ipsil：Bleeding side, Contra：Bleeding offside； Figure B-D showed striatum injection expression SQR shRNA slow virus or comparison virus (NC) after 14 days, after mouse receives cerebral hemorrhage operation Receive ADT-OH or solvent (Vehicle) injection daily, injection candidate ADT-OH and solvent injection control group after cerebral hemorrhage 3 days Mouse three behaviors testing result comparison diagram：Garcia nervous functions comprehensive detection (Figure 15 B, Garcia Neuroscores), right limb touching experiment (Figure 15 C, Right paw placement) and corner test (Figure 15 D, Right Turns), * or * * indicate sham group mouse p compared with other group of mouse<0.05 or p<0.01, # indicates in injection NC shRNA In the control mice (NC) of slow virus, NC+ADT-OH injections group p compared with NC+Vehicle injection group mouse<0.05；

Figure 16 shows 12 mouse striaturn of embodiment injection expression SQR shRNA slow virus (shSQR) or comparison virus (NC) After 14 days, then receive ADT-OH or solvent injection daily after cerebral hemorrhage operation, after cerebral hemorrhage 3 days, injects candidate ADT-OH With the proinflammatory inflammation factor mRNA expression comparison diagrams of control mice corpus straitum of solvent injection；Wherein Ipsil：Bleeding side, Contra：Bleeding offside, IL-1 β (Figure 16 A)：Interleukin-11 β, IL-6 (Figure 16 B)：Interleukin 6, iNOS (Figure 16 C)：Induction type Nitricoxide synthase, TNF-α (Figure 16 D)：Tumor necrosis factor α；

Figure 17 shows that therapeutic effect of 12 candidate substances of embodiment in cerebral hemorrhage cell model depends on the result of SQR；With Erythrocyte cracked liquid (LY) is handled microglia and is activated with the inflammation for simulating microglia after cerebral hemorrhage, and the small colloids of BV2 are thin After dysuria with lower abdominal colic dye is for the siRNA (siSQR) or negative control siRNA (NC) of SQR, then with erythrocyte cracked liquid (LY) and ADT-OH Or solvent handles BV2 microglias, and after 6 hours, the proinflammatory inflammation factor mRNA tables of microglia of ADT-OH and solvent processing Up to horizontal comparing result；Wherein IL-1 β (Figure 17 A)：Interleukin-11 β, IL-6 (Figure 17 B)：Interleukin 6, iNOS (Figure 17 C)：It lures Conductivity type nitricoxide synthase, TNF-α (Figure 17 D)：Tumor necrosis factor α；* indicates that solvent and erythrocyte cracked liquid processing is added (LY) microglia of transfection NC p compared with untreated control cell (Con)<0.01；# or ## is indicated in transfection NC In the microglia (NC) of siRNA, solvent is added and erythrocyte cracked liquid (LY) processing is split with addition ADT-OH and red blood cell It solves liquid processing (LY+ADT-OH) and compares p<0.05 or p<0.01；＆＆ or $ $ ($) are indicated and ADT-OH and erythrocyte cracked liquid are added The NC microglias that activation handles (LY+ADT-OH) compare p<0.01(p<0.05)；

Figure 18 shows that embodiment 13 shows that ADT-OH is to the activation of microglia AMPK in cerebral hemorrhage cell model Result dependent on SQR：It is small after cerebral hemorrhage to simulate that microglia handled with erythrocyte cracked liquid (LY) in cell model The inflammation of spongiocyte activates；After the transfection of BV2 microglias is for the siRNA (siSQR) or control siRNA (NC) of SQR, with Erythrocyte cracked liquid (LY) and ADT-OH or solvent handle BV2 microglias, after activating 2 hours, with untransfected siRNA and not It is compared by the LY control BV2 microglias (Con) activated, erythrocyte cracked liquid (LY) reduces microglia AMPK and activates water Flat (p-AMPK is horizontal), in the BV2 cells (NC) of transfection NC siRNA slow virus, ADT-OH is deposited in erythrocyte cracked liquid (LY) When raise AMPK activation levels, but transfection SQR siRNA BV2 microglias (siSQR) in, ADT-OH is to AMPK's Activation is suppressed；* with untransfected siRNA and without LY activation control cell (Con) compared with p<0.01；＆＆＆ is indicated and is added The microglia (NC) for entering solvent and the transfection NCsiRNA of erythrocyte cracked liquid (LY) processing compares p<0.001；$ $ indicate with The microglia (NC) that ADT-OH and the transfection NC siRNA of erythrocyte cracked liquid activation processing (LY+ADT-OH) is added is compared p<0.01；

Figure 19 shows that the AMPK in cerebral hemorrhage cell model of embodiment 13 mediates the therapeutic effect of ADT-OH：With erythrocyte splitting Liquid (LY) is handled microglia and is activated with the inflammation for simulating microglia after cerebral hemorrhage, and BV2 microglias transfect AMPK SiRNA (siAMPK) compareed siRNA (NC) after 48 hours, then small with erythrocyte cracked liquid and ADT-OH or solvent processing BV2 Spongiocyte, after activating 6 hours, ADT-OH and the proinflammatory inflammation factor mRNA expressions comparison of microglia of solvent processing are tied Fruit；IL-1 β (Figure 19 A)：Interleukin-11 β；IL-6 (Figure 19 B)：Interleukin 6；INOS (Figure 19 C)：Nitric oxide synthase type； TNF-α (Figure 19 D)：Tumor necrosis factor α；* indicates the small glue that solvent and the transfection NC of erythrocyte cracked liquid processing (LY) is added Cell plastid p compared with untreated control cell (Con)<0.01；# or ## indicates the microglia in transfection NC siRNA (NC) in, solvent and erythrocyte cracked liquid processing (LY) is added and the small colloid of addition ADT-OH and erythrocyte cracked liquid processing are thin Born of the same parents (LY+ADT-OH) compare p<0.05 or p<0.01；＆＆ or $ $ ($) are indicated and ADT-OH and erythrocyte cracked liquid processing are added (LY+ADT-OH) NC siRNA microglias (NC) compare p<0.01(p<0.05)；

Specific implementation mode

The invention discloses a kind of method obtaining the substance with mitochondria uncoupling and in this way Purposes of the substance of acquisition in treating relevant disease.Those skilled in the art can use for reference present disclosure, be suitably modified technique Parameter is realized.In particular, it should be pointed out that all similar substitutions and modifications are apparent for a person skilled in the art , they are considered as being included in the present invention.The method and product of the present invention is described by preferred embodiment, phase Pass personnel can obviously not depart from the content of present invention, method described herein is being modified or suitably changed in spirit and scope With combine, to realize and apply the technology of the present invention.

The embodiment of the invention discloses a kind of new method leading to mitochondria uncoupling and AMPK activations and acquisitions The method of substance with mitochondria uncoupling or AMPK activations, and disclose the object obtained using the method for the present invention Purposes of the matter in treating relevant disease.The method of existing mitochondria uncoupling is to utilize proton (or ion) carrier cross-line grain Body membrane transport proton or ion decline (i.e. uncoupling) so as to cause mitochondrial membrane potential.Method provided by the invention is then： Sulfide (or the product of these sulfide after cell or internal conversion/metabolism) leads to mitochondrial complex 1 by SQR oxidations Horizontal RET, and then cross-line mitochondrial membrane current potential is caused to decline.Sulfide with these effects can the side through the invention Method obtains, or carries out structure of modification design as primer using the compound of acquisition and obtain.These sulfide are not intended as directly Proton (or ion) carrier lead to mitochondria uncoupling.Therefore, this method is totally different from existing mitochondria uncoupling Method, and there is cell-specific.In addition, it is also the important of AMPK activation that mitochondria uncoupling, which causes intracellular ATP levels to decline, Mechanism.Therefore, it is based on method provided by the invention, also can get mechanism of action innovation, the line grain with cell-specific effect Body uncoupler or AMPK activator.The present invention also by preferred embodiment, provides the substance of acquisition in treatment relevant disease In purposes.

It should be understood that these embodiments are merely to illustrate the present invention rather than limit the scope of the invention.Such as the present invention carries The embodiment of confession shows：Therapeutic effect needs of the mitochondrial uncoupler that the present invention obtains to specified disease-cerebral hemorrhage The activation of downstream AMPK.AMPK activation is a downstream effects caused by mitochondria uncoupling.Although AMPK activation itself also has There are important biology and therapeutic potential, but not all effects caused by mitochondria uncoupling all rely on AMPK activation. Therefore, the embodiment of the present invention is merely to illustrate this that the mitochondria uncoupling substance that obtains of the present invention can be activated by AMPK The mediation of one downstream effects and play therapeutic effect, rather than by the protection domain of patent of the present invention --- mitochondria uncoupling object The purposes of matter is only restricted in AMPK activations caused by mitochondria uncoupling.

Unless otherwise defined, all professions used herein and scientific terminology and meaning known to one skilled in the art It is identical.In addition, the change of any method and material and experimental procedure order similar or impartial to described content with again Combination all can be used for and the present invention.Those skilled in the art can use for reference present disclosure, be suitably modified technique or technical parameter, Or application as described herein is modified or is suitably changed and combined not departing from spirit and scope of the invention, realizing and Using the technology of the present invention.For example, mitochondria uncoupling and AMPK activation can lead to other biological effect and pharmacological action, such as increase The oxidation etc. of the substrates such as stuffing class and glucose.It therefore, can be it should be evident that those skilled in the art utilizing institute of the present invention When the cell or animal model stated, line described in the invention can be substituted by detecting these biological effects or pharmacological action Plastochondria uncoupling characteristics index or AMPK activate index, to obtain line grain based on SQR, with cell-specific effect Body uncoupler or AMPK activator.For another example, the present invention is determined with cerebral hemorrhage cell and animal model in preference by upper State application of the substance of method acquisition in treating relevant disease.It is existing to grind as inventor is described in technical background Study carefully and have shown that mitochondrial uncoupler or AMPK activator can treat a variety of diseases, such as metabolic syndrome, obesity, neurological Property disease, aging, cerebral apoplexy, diabetes and cancer etc..It should be evident that those skilled in the art can utilize it is corresponding other The cell or animal model of disease are related in treatment with the substance clearly obtained using method as described herein or the like Application in disease.For another example, in another preferred example, inventor also using by genetic manipulation means (lentivirus mediated ShRNA technologies) cause endogenous SQR expression to strike the mouse subtracted, to show treatment of the substance of the invention obtained to relevant disease Mediation of the effect dependent on SQR.It should be evident that in conjunction with cell model provided by the present invention, those skilled in the art can It is struck using this animal model or using the SQR that is obtained by other genetic manipulation means and subtracts/knockout/animal being overexpressed/carefully Born of the same parents' model obtains the mitochondrial uncoupler or AMPK activator based on SQR.

To sum up, related personnel obviously can not depart from the content of present invention, in spirit and scope to method described herein into Row change is suitably changed and is combined, to realize and apply the technology of the present invention.In particular, these all similar are replaced Change, change or these methods reconfigure using apparent to those skilled in the art, they all by It is considered as and is included in the present invention.Preferable implementation described herein and material only do demonstration and are used, and application of the invention is It is described by preferred embodiment.With reference to specific embodiment, the present invention is further illustrated.

Unless otherwise specified, reagent involved in the embodiment of the present invention is commercial product, can pass through business canal Road purchase obtains.

Wherein, the experiment material and method that each embodiment is related to are as follows：

1. cell：Microglia system (BV-2) cell, mouse primary nerve cell (Neuron), primary small colloid are thin Born of the same parents, primary astroglial cells.

2. animal：The pregnant mouse of C57BL/6J (pregnant 16-18d, Shanghai Si Laike Experimental Animal Centers)；C57BL/6J newborn mices (birth 1-2d, Shanghai Si Laike Experimental Animal Centers)；(weight 25-30g, Shanghai Si Laike are tested CD-1 adult male mices Animal center)

3. drug and reagent：

4. antibody and instrument：

5. prepared by reagent：

5.1 cell culture related reagents are prepared

1 × PBS (0.01M, pH7.35)：NaH₂PO₄.2H₂O 0.296g

Na₂HPO₄.12H₂O 2.90g

NaCl 8.50g

PH 7.35, ultra-pure water (ddH are adjusted with HCl₂O) it is settled to 1L.

BV-2 cell culture fluids：DMEM

Fetal calf serum (FBS) 1:10

Twin antibiotic 1:100

Primary microglia culture solution：DMEM/F12

Fetal calf serum (FBS) 1:15

Twin antibiotic 1:100

Neuronal cultured solution：Neuron basal+B27 (1:50)

Glutamine 0.5mmol/L

Dual anti-1:200

Hank ' s equilibrium liquids：Hank ' s balance reagent

NaHCO₃ 350mg

HEPES(1M) 10ml

Glucose 6g

Ultra-pure water (ddH₂O it) is settled to 1L, adjusts pH7.3, is filtered, 4 DEG C of storages are spare.

5.2 Western Blot protein immunoblot related reagents are prepared：

30% polyacrylamide mixed liquor：Methylene bisacrylamide 1g

Acrylamide 29g

Ultra-pure water (ddH₂O it) is settled to 100ml, is filtered, stored protected from light.

1.5M Tris-HCl(pH8.8)：Tris 18.15g, ultra-pure water (ddH₂O it) is settled to 100ml and adjusts pH 8.8, Room temperature storage.

0.5M Tris-HCl(pH6.8)：Tris 6.0g, ultra-pure water (ddH₂O it) is settled to 100ml and adjusts pH 6.8, room Temperature storage.

10%SDS：SDS 10g, ultra-pure water (ddH₂O 100ml, room temperature storage) are settled to.

10%APS：APS 1g, ultra-pure water (ddH₂O 10ml, 4 DEG C of preservations) are settled to.

1 × electrophoretic buffer (pH8.3)：Tris 2.72g

Glycine 18.8g

SDS 1g

Ultra-pure water (ddH₂O 1L, room temperature storage) are settled to

1 × transferring film buffer solution：Tris 3.03g

Glycine 14.4g

Absolute methanol 200ml

Ultra-pure water (ddH₂O 1L, room temperature storage) are settled to

10 × TBS buffer solutions (pH7.5)：Tris 24.2g,

NaCl 80g,

Ultra-pure water (ddH₂O 1L, room temperature storage) are settled to

PH7.5,1 × TBST buffer solution：Ultra-pure water (ddH₂O):10×TBS:Tween-20=900:100:1, room temperature storage It deposits.

Confining liquid：Milk 1g

1×TBST 20ml

It dissolves and is uniformly mixed, it is now with the current.

6. experimental method

6.1 BV2 microglias are cultivated

6.1.1 cell recovery:3ml cultures are taken to be based on 15ml centrifuge tubes；The BV2 cells frozen are taken out rapidly from liquid nitrogen And cells frozen storing liquid is made to be dissolved in 37 DEG C of water-baths in 1 minute；A little culture is added to be based on cell in cell cryopreservation tube then Mixed liquor is soft to be moved in 15ml centrifuge tubes；1000 × g centrifuges 3min and removes supernatant, and gently bullet, which beats centrifugation bottom of the tube, makes cell It scatter, then 3ml culture mediums is added slowly to blow and beat mixing；Appropriate culture medium is first added in culture dish, culture base lid is made completely to cultivate Ware bottom.Cell pipette is equably moved in culture dish along ware in a manner of drawing a circle again, moves into cell incubator.

6.1.2 the passage of BV2 cells and bed board：Culture medium is sucked, 2-3ml PBS are added and wash 1-2 times；Pancreatin is added 3ml, digestion to after about 30% cell levitating plus 3ml culture mediums terminate digestion, gently blown and beaten with pipette do not float it is thin Born of the same parents cause its whole to hike up, and shift whole culture mediums and cell to 15ml centrifuge tubes, centrifuge 1000 × g 3min, remove supernatant, use Hand gently bullet beat centrifugation bottom of the tube to precipitation be suspended in remaining minimal amount of culture medium, then be added 3ml culture mediums slowly blow Beat mixing.Cell count counts if there are many cell quantity after needing dilution.Per ware 100-150 ten thousand if cell passes on, if paving Plate is then taped against 96 orifice plates according to requirement of experiment.

6.1.3 BV2 microglias transfect siRNA

BV2 cell inoculations when 24 orifice plates, transfection cell density 30% or so.The full culture containing serum is replaced before transfection Base (contributes to this reagent serum to promote transfection efficiency).The SQR or siRNA for taking 0.67 μ g, are added a certain amount of serum-free Dilution mixes well, and RNA dilutions are made, and final volume is 25 μ L；The Entranster of 2 μ L is taken simultaneously^TM23 μ L are added in-R Serum-free diluent liquid, mixes well, and Entranster is made^TM- R dilutions, final volume are 25 μ L, are stored at room temperature 5 minutes； By Entranster^TM- R dilutions and RNA dilutions are sufficiently mixed, and are stored at room temperature 15-30min；It is finally that 50 μ L transfections are compound Object is added drop-wise in the full culture mediums of 0.45mL, is moved forward and backward culture dish, is uniformly mixed；Fresh complete culture solution is replaced after transfection 6h.

6.2 primary microglia cultures:

Materials：1) after newborn rat (is born 24 hours) alcohol disinfecting twice, the eyes and ear of newborn rat are pinned with ophthalmic tweezers Piece brain tissue is taken out with fixed, is placed in Hank ' the s liquid placed on ice；2) meninx, olfactory bulb and rete vasculosum are divested under body formula mirror Film, while cerebellum is removed, whole cerebral cortexes are taken out, move to super-clean bench operation by brain stem and whole Basal ganglias；3) eye scissors are used Tissue block is shredded as possible, 0.25% pancreatin containing ETDA of 2.5ml (37 DEG C of preheatings) is added and is moved into together with 2.5ml Hank ' s liquid Be placed in incubator in the big ware of 10cm base diameters and digest 10min, rock within every three minutes it is primary, taken out after 10min big ware to Isometric culture medium is wherein added and terminates digestion, then all moving into the centrifuge tube of 50ml gently to be blown and beaten with pipette makes it Dissipate into single cell suspension；4) single cell suspension is filtered, filtered liquid is transferred to 1000 × g of centrifugation in 15ml centrifuge tubes Supernatant is discarded after 5min, appropriate culture solution piping and druming is added, single cell suspension is made again；Be inoculated in poly-D-L be coated in advance to It is cultivated in few 2 hours and the 75ml Tissue Culture Flasks that cleaned.

Purifying：1) it all replaces a culture solution within every 3 days, and observes cell state；2) about 10 days or so the first generation are small At this moment culture bottle is placed in 180r/min in 37 DEG C of constant-temperature tables and vibrates 120min by spongiocyte from levitating on star cellula adhesiae；It receives Supernatant is abandoned in collection 1000 × g of culture solution 5min centrifugations, is counted after adding culture solution；It is inoculated in 6 orifice plates or 24 orifice plates according to experiment demand Or 96 orifice plate culture.

Subsequent experimental：If 1) directly add candidate substances processing, cell spreads at least 12 hours in plate and carries out lower step behaviour again Make.It is such as used to ATP/TMRM detect, candidate substances detect after handling 30 minutes；Western Blot detections AMPK is such as used to live Change, is detected after adding candidate substances to handle 2 hours.2) if cell, which is added slow virus and strikes, subtracts endogenous SQR：Then cell is spread at least 2 Slow virus (MOI=4) is added in hour, changes liquid entirely within 10-12 hours.Observation transfection efficiency (express simultaneously by slow virus after transfection three days Green fluorescent protein GFP), the GFP positives show cell infection slow virus, infection cell ratio>95%, with Western Blot into Row Testing and appraisal strikes decreasing effect rate.

The culture of 6.3 primary neurons：

1) pregnant mouse is dislocated and is put to death, abdomen takes out embryo cutting embryo with 70% alcohol disinfecting abdominal cut and connects with mesenterium It connects；2) stripping takes out embryo, is fixed at tire rathole eyeball and ear with curved tweezer, and small scissors is cut off the full brain of skull taking-up and is put into as ice On Hank ' s plates in；3) cerebellum, olfactory bulb and blood nethike embrane, remaining akrencephalon are removed under the microscope and take cortex, extremely by cortex In being placed in Hank ' s liquid on ice；4) cortex is caught broken as possible with sharp tweezer, is then moved in 50ml centrifuge tubes；5) Hank ' s liquid It washes and (does not blow and beat) three times；With 0.25% pancreatin 1:1 is added, and tissue is made to be totally immersed into liquid, and lid upper tube cap rocks centrifuge tube, Tissue will be embraced into agglomerate at this time, if being formed without agglomerate, be needed to add a small amount of 0.25% pancreatin, be subsequently placed in 37 DEG C of water-baths 10min is digested, is rocked within every three minutes primary；6) supernatant is removed after 10min rapidly, Hank ' s liquid cleans twice, and NB washes (note twice Meaning not be drawn onto cell).Then being added under 5ml culture mediums, 5 μ lDNase (1000 ×) piping and druming 30 makes tissue digest completely, then All liq is transferred in 15ml centrifuge tubes；7) 1000 × g 5min are centrifuged, and are abandoned and are reset and added 3ml culture mediums piping and druming mixing, mistake Sieve, then 2ml culture mediums is added to rinse sieving；8) cell is counted with trypan blue, and 2.5 × 10 are added per hole for 24 orifice plates⁵A cell, 6 holes 1.6-2 × 10 are added per hole for plate⁶2-3 × 10 are added per hole for a cell, 96 orifice plates⁴A cell replaces a culture solution every three days, Half changes liquid.

2) subsequent experimental：Subsequent experimental can be just carried out when cell culture was to the 10th day, is such as detected for ATP/TMRM, it is candidate Substance detects after handling 30 minutes；It is such as used for Western Blot detection AMPK activation, is added after candidate substances are handled 2 hours and examines It surveys.

3) slow-virus infection primary neuron

To be overexpressed the slow-virus infection of SQR：Carry out virus transfection within three days after primary neuron bed board, transfection 10-12 is small Shi Jinhang changes liquid entirely, continues culture to the 10th day, observes transfection efficiency (slow virus while enhanced green fluorescent protein GFP), GFP The positive shows cell infection slow virus, infection cell ratio>95%, and identification SQR is detected with Western Blot and crosses table Up to efficiency；Or as measured ATP contents, mitochondrial membrane potential, AMPK activated water equalitys after above-mentioned addition candidate substances.

6.4 measure mitochondria NAD+/NADH ratios (PROMEGA cat:G9071USA)

The BV2 cells for being grown on the big wares of diameter 10cm are taken, with being centrifuged after pancreatin TE all digestion, make cell aggregation.Centrifugation After removing pancreatin, reagent A (50mM Tris-HCl, pH 7.4,225mM mannitol, 75mM sucrose, the 1mM benzyl sulphurs of 500 μ l Acyl fluorides, 10 μ l protease inhibitor cocktails) cell is resuspended, then smash cell to pieces with grinding rod.4 DEG C of 600 × g are centrifuged 7.5 minutes. Supernatant is collected, 4 DEG C of 7000 × g is centrifuged 10 minutes.Supernatant is removed, precipitation is received.This precipitation is mitochondria, with 50 μ L PBS weights Outstanding precipitation.

Prepare NAD+ fluorescein detection reagents working solution (recombination fluorescein detection reagent, PROMEGA):Make reassembly buffer liquid It thaws, the two is all mixed, overturned repeatedly with after freeze-dried powder Luciferin detection reagents to room temperature by reassembly buffer lyolysis jelly Several times to be thoroughly mixed.Then packing is stored in -20 DEG C of refrigerators, in case using.Note：It needs to equilibrate to room temperature before each use.

The 50 μ L PBS mitochondria solution being resuspended is transferred in 96 orifice plates, then 50 μ L 1% are added wherein and are dissolved in 0.2N The DTAB of NaOH.96 orifice plates are jiggled, wherein reagent is made to be uniformly mixed.Total volume is 100 μ L in every hole at this time, is denoted as the holes A； It draws per in the 50 μ l liquid to corresponding one new hole of hole, is denoted as the holes B；25 holes μ L 0.4N HCL, A are added in the holes B to be not added with. Plate lid is covered, is incubated 15 minutes in 60 DEG C of insulating boxs.

Equilibrium at room temperature 10 minutes.25 μ l 0.5M Trizma base are added in the holes A, 50 μ l HCL/ are added in the holes B Trizma solution.The holes A are still 100 μ l with liquor capacity in B at this time.Prepare NAD/NADH Glo by following table lattice^TMDetection Reagent (NAD/NADH Glo^TMKit purchases Promega):

Component	Dosage (1Well)
		Recombinate fluorescein detection reagent	1ml
Reductase	5μl
		Restore zymolyte	5μl
NAD cyclophorases	5μl
		NAD recycles zymolyte	25μl

By 100 μ l NAD/NADH Glo^TMDetection reagent sequentially adds in all holes.Jiggling 96 orifice plates makes wherein liquid Body mixes well.It is placed at room temperature for 30-60 minutes.Microplate reader is noted in luminous reading numerical values：The holes A are NAD+, and the holes B are NADH。

Preparation, brain edema and the behaviouristics detection of 6.5 mouse cerebral hemorrhage molds

6.5.1 the self blood of striatum injection leads to the production method of cerebral hemorrhage mold

It chooses weight and is placed on stereotaxic instrument with chloral hydrate anesthesia in 25g-30g or so ICR (CD-1) male mice, It is fixed, start to perform the operation, Iodophor is applied to the crown, cuts off the wound of a length 1cm overhead, is starched with hydrogen peroxide and find bregma Position, after determining bregma position, open 0.5mm in the past, 2.0mm is opened on side, left brain find inserting needle position, then use dentistry Brill gently drills.Tail portion blood sampling is selected in blood sampling, and an openning is cut open on tail, and 15 μ l are drawn with the micro syringe of 50 μ l Arterial blood, syringe is moved to above stereotaxic instrument rapidly, slow inserting needle, depth of needle 3.5mm, then starts to inject The 15 self blood of μ l, speed are 2 μ l/min, and constant temperature is kept in injection process, after the completion of injection, is pulled out after stopping needle 15 minutes Then needle is sewed up a wound, mouse is put into above electric blanket, until revival.The striatum injection of sham-operation group mouse is isometric Physiological saline.

Animal packet:With reference to Garcia Neuroscore point systems, the mouse that do not perform the operation is grouped at random：It does evil through another person Art group (sham), cerebral hemorrhage+solvent group (Veh groups), cerebral hemorrhage+ADT-OH groups.Cerebral hemorrhage animal：Striatum revealed injection is self After arteria caudalis blood 3 hours, solvent or ADT-OH (50mg/kg/day) is injected intraperitoneally.The preparation of ADT-OH injections：15mg's ADT-OH is diluted to whole solubility as the solution of 15mg/mL first with 250 μ L DMSO dissolvings, then with 750 μ L corn oils.Self It is injected intraperitoneally by the dosage of 50mg/kg when blood injects 3h.Solvent injection control group mice gives jade of the same volume containing DMSO Rice bran oil (Veh).

6.5.2 neurological deficits score (Neurologic deficit score):After modelling, with reference to Garcia Neuroscore point systems, to postoperative mouse after anaesthetic is awake, into row stochastic grouping, score assigns to 20 16/ Between be model successfully group.It is randomly divided into solvent group and ADT-OH groups.

6.5.3 right limb touching percentage (Percent of right paw placement)：Measure the right side of mouse recklessly The when of falling desktop must be being touched, right side limbs touch the probability of desktop simultaneously.10 experiments are carried out, are write down in 10 experiments, right limb Touch the number of desktop.

6.5.4 corner percentage (Percent of left turns)：Mouse is forced into the angle that an angle is 30 degree It falls, normal mouse freely walks out corner to from left to right, and the selection in direction is random；After cerebral hemorrhage, mouse, which turns to, to be partial to Blood side (left side).It is repeated 10 times, the number on record selection right side.

6.5.5 brain water content (Water Content)：72 hours after animal cerebral hemorrhage, mouse is put to death, is broken immediately Head opens cranium and takes out brain tissue, after removing cerebellum and brain stem, brain tissue is divided into along center line after two hemisphere in left and right be divided into cortex and Bleeding side brain tissue is placed in after electronic analytical balance weighs weight in wet base in closed can rapidly by corpus straitum, and 95 DEG C of constant temperature are dry Its dry weight is weighed again after dry 3 days.By Blliot formula：Brain water content (%)=(weight in wet base-dry weight)/weight in wet base × 100%.

6.6 erythrocyte cracked liquids (Rbc Lysis) activate microglia model

Mouse anesthesia extracts mouse left ventricle blood 1ml, so with one milliliter of syringe of heparin (100u/ml) rinse Afterwards at 4 DEG C, centrifuge centrifuges 5min with the rotating speed of 1000RPM/min.Supernatant is abandoned, solution of red blood cells is frozen 10min in liquid nitrogen, so Afterwards again in 37 DEG C of water-bath water-bath 5min, multigelation 4 times.Centrifugation, obtains supernatant, i.e. erythrocyte cracked liquid (RBC Lysis).It is small Spongiocyte the previous day spreads 24 orifice plates (100,000 cells/every hole),.Amount processing by+10 μ l Lysis of 500 μ l culture mediums per hole is small Spongiocyte, candidate substances or solvent (DMSO) are added simultaneously.LYSIS and/or candidate substances detect proinflammatory disease after handling 6 hours Factor mRNA level in-site.

6.7 Western Blot

6.7.1 cell protein extracts：It after cell abandons supernatant, is cleaned one time with PBS, appropriate RIPA Protein Extractions is added Liquid (50 μ lRIPA cracking 10⁶A cell) after, cell scraper is removed with cell scraper, 4 DEG C of standings 30min, 4 DEG C of 12000rpm from Heart 30min, it is total protein of cell to take supernatant.

6.7.2 animal tissue protein extracts：Appropriate RIPA lysates are added into tissue sample (according to materials size to be estimated Amount), it is ground into homogenate with grinding rod, stands 4 DEG C of centrifugation 30min of 12000rpm after 30min on ice, it is tissue to collect supernatant It is spare to be stored in -80 DEG C of refrigerators for total protein

6.7.3 BCA methods detect albumen concentration：Prepare working solution:It is formulated by A liquid and B liquid；A liquid:B=50:1, point The two volume, mixing are not calculated.Use ddH₂O and RIPA respectively dilutes standard items BSA and sample, then each addition working solution 400ul；Absorbance value is measured at 37 DEG C of incubations 30min, 562nm, counter sample concentration is calculated according to mark song absorbance.

6.7.4 electrophoresis:Take protein sample, 30 μ g albumen and albumen sample-loading buffer mixing, 95 DEG C of water-bath 5min, through 12% SDS-PAGE is separated by electrophoresis, concentrate glue voltage 60V, separation gel voltage 100V.

6.7.5 transferring film:It is permanent with electricity of wet process robin by protein delivery to polyvinylidene fluoride (PVDF) film after electrophoresis Flow 350mA time 115min.

6.7.6 closing：After transferring film, pvdf membrane is placed in 5% skim milk and is closed, is slowly shaken on shaking table, room Temperature closing 2h.Wash film：1 × TBST is washed three times, each 5min.

6.7.7 primary antibody is incubated：The calf serum dilution AMPK/p-AMPK antibody (l with 5% is needed according to experiment:1000)、 SQR antibody (1:500), β-actin antibody (1:2000 internal references), 4 DEG C of refrigerators are incubated overnight.

6.7.8 secondary antibody is incubated：It dilutes and is slowly shaken on corresponding secondary antibody shaking table, be incubated at room temperature 2h.

6.7.9 film is washed：L × TBST washes film 3 times, each 5min.

6.7.10 gel imaging：After secondary antibody is incubated, developed with gel imager；Image J softwares analysis knot is used in combination Fruit.

6.8 real-time fluorescence quantitative PCRs (Real-time Quantitative PCR, Q-PCR)

6.8.1 the extraction of RNA

The RNA extractions of cell sample are (by kit specification operation TIANGEN cat:DP420CHINA)

Cell culture medium is sucked out completely, 350 μ l or 75 μ l are added, and (cell number is less than 10⁴When a) lysate RL (uses 1% beta -mercaptoethanol of preceding addition), piping and druming repeatedly makes cell cracking.It collects cell pyrolysis liquid and transfers them to 1.5ml In RNase-Free centrifuge tubes.1 times of 70% ethyl alcohol of volume is added, with pipettor mixing, carries out immediately in next step.By all solution And precipitation is fully transferred on adsorption column CR1 (adsorption column CR1 is placed in the collecting pipe of 2mlRNase-Free), on soft lid Pipe lid, 12,000rpm (≈ 13,4000 × g) centrifuge 30-60sec, abandon waste liquid, adsorption column CR1 is put back in collecting pipe.To suction 350 μ l protein liquid removals RW1,12,000rpm (≈ 13,4000 × g) centrifugation 30-60sec are added in attached column CR1, outwell collecting pipe In waste liquid, adsorption column CR1 is put back in collecting pipe.The preparation of DNase I working solutions:10 μ l DNase I storing liquids are taken to be put into In new RNase-Free pipes, 70 μ lRDD solution, soft mixing is added.The DNase I of 80 μ l are added to the centers adsorption column CR1 Working solution is placed at room temperature for 15min.It is added 350 μ l protein liquid removals RW1 into adsorption column CR1,12,000rpm (≈ 13,4000 × G) 30-60sec is centrifuged, the waste liquid in collecting pipe is outwelled, adsorption column CR1 is put back in collecting pipe.It is added into adsorption column CR1 500 μ l rinsing liquids RW (ethyl alcohol has been added using preceding), are stored at room temperature 2min, and 12,000rpm (13,4000 × g of ≈) centrifuges 30- 60sec outwells the waste liquid in collecting pipe, and adsorption column CR1 is put back in collecting pipe.After repeating the above steps, 12,000rpm (≈ 13,4000 × g) centrifugation 2min, outwells waste liquid.Adsorption column CR1 is placed in and is placed at room temperature for a few minutes, thoroughly to dry sorbing material The rinsing liquid of middle remnants.

Adsorption column CR1 is put into a new RNase-Free pipe, 14 μ lRNase-Free H are added to film center₂O, Soft lid upper tube cap is placed at room temperature for 2min, and 12,000rpm (≈ 13,4000 × g) centrifuge 1min, and acquired solution is RNA molten Liquid.

The RNA extractions of animal sample are (by kit specification operation TIANGEN cat:DP431CHINA)

Tissue plus 300 μ l lysates RL (1% beta -mercaptoethanol has been added using preceding) per 10-20mg, with grinding rod by group Knit thorough grinding；Then to 590 μ lRNase-Free H are added in homogenate₂O and 10 μ lProteinase K, turns upside down several 56 DEG C of processing 10-20min after secondary mixing.12,000rpm (≈ 13,4000 × g) centrifuge 2-5min, take supernatant into following operation. It is slowly added to 0.5 times of supernatant volume absolute ethyl alcohol, mixing, obtained solution and precipitation are transferred to (adsorption column in adsorption column CR3 together It is placed in collecting pipe), 12,000rpm (≈ 13,4000 × g) centrifuge 30-60sec, abandon waste liquid, adsorption column CR3 is put back to collection Guan Zhong.350 μ l protein liquid removals RW1,12,000rpm (≈ 13,4000 × g) are added into adsorption column CR3 and centrifuge 30-60sec, Fall the waste liquid in collecting pipe, adsorption column CR3 is put back in collecting pipe.The preparation of DNase I working solutions:Take 10 μ l DNase I storages Liquid storage is put into new RNase-Free pipes, and 70 μ lRDD solution, soft mixing is added.It is added 80 μ l's to the centers adsorption column CR3 DNase I working solutions, are placed at room temperature for 15min.Be added into adsorption column CR3 350 μ l protein liquid removals RW1,12,000rpm (≈ 13, 4000 × g) centrifugation 30-60sec, outwells the waste liquid in collecting pipe, adsorption column CR3 is put back in collecting pipe.Into adsorption column CR3 500 μ l rinsing liquids RW (ethyl alcohol has been added using preceding) are added, are stored at room temperature 2min, 12,000rpm (≈ 13,4000 × g) centrifugations 30-60sec outwells the waste liquid in collecting pipe, and adsorption column CR3 is put back in collecting pipe.After repeating the above steps, 12,000rpm (≈ 13,4000 × g) centrifuges 2min, outwells waste liquid.Adsorption column CR3 is placed in and is placed at room temperature for a few minutes, thoroughly to dry absorption Remaining rinsing liquid in material.

Adsorption column CR3 is put into a new RNase-Free pipe, 30-100 μ lRNase-Free are added to film center H₂O, soft lid upper tube cap are placed at room temperature for 2min, and 12,000rpm (≈ 13,4000 × g) centrifuge 2min, and acquired solution is RNA solution.

RNA concentration mensurations and quantitative：RNA concentration (optium concentrations are measured using enzyme-linked immunosorbent assay instrument NaNoDrop softwares For 300-500ng/ μ l), with RNase-Free H₂O is quantitative at 10 μ l of 1000ng total volumes (1 μ g/10 μ l).

The synthesis (20 μ l reaction systems) of cDNA：

Be gently mixed uniformly, in PCR instrument reverse transcription synthesis cDNA (25 DEG C of 10min, 37 DEG C of 120min, 85 DEG C of 5min, 4 ℃)。

QPCR (20 μ l reaction systems)：MRNA expression is carried out on ABI 7500PCR instrument：Per hole in 96 hole Q-PCR reaction plates It is added:

Non-specific amplification and primer dimer are analyzed after PCR amplification by observing solubility curve whether there is, Ct values are with 2 ∧-△ △ Ct methods calculate gene expression amount difference, using 18S as internal reference gene.

1 primer sequence table of table

6.9 intracellular ATP levels and the detection of ADP/ATP ratios (operate SIGMA-ALDRICH according to kit specification cat:MAK-135-1KT USA)

It tests on the previous day paving cell and 96 hole blanks, per 10,000, hole cell.Before detection, equilibrium at room temperature detection reagent is surveyed Determine buffer solution, substrate, cosubstrate, and ATP/ADP enzymes are placed on ice.After test agent to be checked equilibrates to room temperature, as following table is matched ATP reaction mixtures processed.

Reagent	Volume (1well)
		Measure buffer solution	95μl
Substrate	1μl
		Cosubstrate	1μl
ATP enzyme	1μl

Before detection, after candidate processing cell 30 minutes, culture medium is sucked, ATP reactions are sequentially added by 90 μ l of every hole Mixture.Incubation at room temperature 1 minute, microplate reader is denoted as RLU in luminous reading numerical values_A.Continue incubation at room temperature 10 minutes. During this, as following table prepares ADP reaction mixtures.

Reagent	Volume (1well)
		Water	5μl
ATP enzyme	1μl

It reads a pass value again after ten minutes, is denoted as RLU_B。Then it sequentially adds, mixes by 5 μ lADP reaction mixtures of every hole It is even, it is incubated at room temperature 1 minute, reading numerical values are denoted as RLU_C.Calculate the ratio of ADP/ATP.ADP/ATP=(RLU_C-RLU_B)/RLU_A

6.10 mitochondrial membrane potential detects

Cell quantity need to be in 100,000/hole by taking 24 orifice plates as an example.Candidate handles cell 30 minutes, sucks pastille culture medium, PBS cleanings are primary.With TMRM, (Tetramethylrhodamine methyl ester, tetramethylrhodamine methyl esters, sigma are public Department, Ref:T668)):Culture medium=1:1000 dilution proportion dyestuff (final concentration of 100nM), is added in culture medium, 37 DEG C Cell incubator culture 10 minutes.Dyestuff is sucked, PBS cleans a cell, adds appropriate culture medium.Under Laser Scanning Confocal Microscope Feux rouges cy3/555nm wavelength periods shoot cell fluorescence intensity.Using Image J software statistics fluorescence intensities.

6.10 cell oxygen demand (OCR) measure

1. cell plates used and reagent are all from Seahorse Bioscience.(24 porocyte plates PartNo:100777- 004；Cell cage plate：102328-000；XF correcting fluids：100840-000；XF detects liquid：102365-100)；2. primary small glue and The cell number of primary neuron need to control 100,000/hole；3. primary small glue virus transfection：Corresponding disease is added in cell after spreading 2 hours Poison (shRNA slow virus or comparison virus of the expression for SQR), progress in 10-12 hours changes liquid entirely, later more per 24-36 hours A culture solution is changed, four days observation transfection efficiencies of virus transfection carry out seahorse detections.4. primary neuron virus treated： Corresponding virus (slow virus or the comparison virus that are overexpressed SQR) is added in cell seeding after 3 days, carry out cell within 10-12 hours and change entirely Liquid replaces a culture solution, can carry out seahorse detections within 7 days after transfection every three days later.

Cell is laid on 24 hole detection plates, per hole 100,000 cell, and is stayed on cell plates and does not spread cell as benchmark there are three hole, (hole A1, C3, D6).Experiment the previous day XF correcting fluid is added to every hole 1ml correcting fluids in 24 orifice plates in registering, correct placement Registering makes it at 37 DEG C without CO₂Overnight incubation is protected from light in incubator.

The ratio of liquid+0.5ml Sodium Pyruvate+0.5ml glutamine+0.5ml glucose is detected needed for empirically with 48.5ml Example prepares detection liquid.Cell is cleaned with detection liquid, it is each per hole 1ml.After cleaning twice.525 μ l are added per hole and detect liquid, again 37 DEG C are put into without CO₂In incubator.

A points (oligomycin), B points (candidate substances), C (mitochondrial complex I inhibitor rotenone ROT) are prepared respectively The drug of point.Drug has been prepared, and registering is taken out, and is covered label grouping in registering plate, is added to the drug of preparation is corresponding The corresponding position of each detection hole, per 75 μ l of hole, drug ROT is added in all the points of blank well.Cell plates are put into seahorse Machine detects.Experiment terminates export excle data and is analyzed.

6.11 efficient liquid phases-tandem mass spectrum (HPLC-MS/MS) measures intracellular CoQ10H2 (reduced form)/CoQ10 (oxidations Type) ratio：

Cell handled with candidate substances after with normal propyl alcohol (the internal standard CoQ containing 0.02 μ g/ml₉) cracking, then with oscillation Device vibrates 1min, then 4500rmp centrifuges 3min, takes supernatant to be washed three times with n-hexane, it includes n-hexane to take organic layer, organic layer And normal propyl alcohol.Liquid is evaporated with nitrogen evaporator, is eventually adding with 95:Methanol-n-hexane mixed liquor of 5 ratio mixing is allowed to again Dissolving.HPLC-MS/MS detections use Symmetry C18 analytical columns (2.1 × 100mm, Waters), and mass spectrograph is Thermo TSQ Vantage.Mobile phase is methanol/normal propyl alcohol and increases ammonium formate, gradient elution.Whole process has 10min altogether, and flow velocity is 200μL/min.Mass Spectrometer Method is positive ion mode, and ion source injection electric is+3500V, and temperature is 350 DEG C, and sheath air pressure is 40Arb.LC Quan softwares are used for data collection and analysis.Mass-to-charge ratio (m/ is passed through using selective reaction monitoring (SRM) pattern Z) transformation for detecting following parent ions and daughter ion, to oxidized form and reduced coenzyme CoQ₁₀It is quantified.CoQ₁₀H₂：M/z from 882.5 are changed into 150.8 and 196.8；CoQ₁₀：M/z is changed into 148.8 and 196.8 from 863.5；CoQ₁₀：M/z is from 795.500 It is changed into 150.8 and 196.8.

6.12 mouse striaturn injecting lentivirus：

ICR (CD-1) male mice weight 20g-25g is fixed on mouse brain with 4% chloral hydrate anesthesias of 0.1ml/10g On stereotaxic instrument, it is applied to crown disinfection with Iodophor, dissecting scissors cuts off the wound of a length 1cm, uses hydrogen peroxide overhead Wiping skull surface searches out the position of bregma, after determining position, opens 0.5mm in the past respectively, 2.0mm, depth of needle are opened in side 3.5 and before open 1mm, the position that 1.5mm depth of needle 3.2mm finds inserting needle coordinate in left brain is opened on side, then gently with dental burr The hole of drill straight diameter 1mm.1.5 μ L expression SQR shRNA slow virus or negative control virus are drawn with the micro syringe of 5 μ l, it is fast Syringe is fixed on above stereotaxic apparatus by speed, slow inserting needle.Start to inject after reaching above-mentioned position, speed is 0.3 μ L/ Min keeps mouse temperature constant in injection process, after the completion of injection, stops needle and pulls out needle after ten minutes and then sew up a wound, will be old Mouse is put into above electric blanket, until revival.Virus injection mouse is followed by by cerebral hemorrhage operation for 14 days.

Embodiment 1：Preliminary screening

Inventor it has been investigated that, (or these sulfide are in vivo or after cellular transformation/metabolism for specific sulfur-containing compound Product) through SQR oxidations can lead to the electronics back transfer (RET) of Respiratory Chain Complex I level, so as to cause mitochondria solution idol Connection.Therefore, the mitochondria uncoupling for inhibiting Respiratory Chain Complex I that SQR can specifically be inhibited to mediate:Including inhibiting cross-line The decline of mitochondrial membrane film potential (referred to as mitochondrial membrane potential), the decline for inhibiting intracellular ATP levels and The rising of OCR values in the presence of oligomycin.And the mitochondrial uncoupler having now been found that is proton or ionophore, they Uncoupling independent of Respiratory Chain Complex I.Using this feature, can using preliminary screening based on SQR as the line of target spot Plastochondria uncoupler or AMPK activator.

For example, do primary screening with 20 kinds of compounds, will expression BV2 microglias (this kind of cell expresses SQR) kind in 24 orifice plates, with candidate substances, Respiratory Chain Complex I inhibitor rotenone (rotenone, Rot), candidate substances+trifoliate jewelvine after staying overnight Ketone handles cell, is control with the cell that solvent (vehicle) is handled.After cell is handled 30 minutes, TMRM index line grains are utilized Body film potential：Mitochondrial membrane potential declines, then intracellular TMRM red fluorescence intensities decline, and assay method is for example aforementioned.Experiment knot Fruit such as Fig. 1 shows：Compared with the control cell of solvent (vehicle) processing, simple rotenone (rotenone, Rot) processing can Cell TMRM red fluorescence intensities are caused to decline (mitochondrial membrane potential declines, Figure 1A).And generally acknowledged proton type mitochondria solution is even Join agent FCCP (p- this hydrazone of trifluoromethoxy of carbonyl-cyanogen-, Trifluoromethoxy carbonylcyanide Phenylhydrazone) also mitochondrial membrane potential is caused to decline, but rotenone cannot reverse caused by FCCP with FCCP coprocessing Mitochondrial membrane potential declines, and shows function of the effect independent of composite I of proton type mitochondrial uncoupler.

Inventor has found that following three kinds of sulfur-containing compounds not only reduce cell mitochondrial film electricity from 100 multiple compounds Position, and the effect of these three compounds reduction mitochondrial membrane potential can be reversed by rotenone (Rot)：5- p-hydroxybenzenes -3H- 1,2- dithiacyclopentene -3- thioketones (ADT-OH, as shown in Equation 1) 5- p-methoxyphenyls -3H-1,2- dithiacyclopentene -3- sulphur Ketone (ADT, as shown in Equation 1) and N- (benzoyl sulfenyl) benzamide (abbreviation 5a, as shown in Equation 2).The results are shown in Figure 1： ADT-OH (Figure 1A, 50 μ Μ), ADT (Figure 1B, 50 μ Μ) and 5a (Fig. 1 C, 50 μ Μ).As a result prompt these three compounds that may lead to Crossing SQR oxidations leads to mitochondria uncoupling.

It is 5- p-hydroxybenzenes -3H-1,2- dithiacyclopentene -3- thioketones (ADT-OH) when R is-H in formula 1.

R is-CH in formula 1₃When, it is 5- p-methoxyphenyls -3H-1,2- dithiacyclopentene -3- thioketones (ADT).

Embodiment 2：Confirm that the mitochondria uncoupling of candidate substances depends on the function of mitochondrial respiratory chain composite I

Can inventor further has detected mitochondrial respiratory chain composite I inhibitor rotenone on microglia hinder Disconnected candidate substances lead to the another two important feature of mitochondria uncoupling：1) reduce that intracellular ATP is horizontal (and to increase ADP/ATP Ratio)；2) cell oxygen demand (OCR) is increased in the presence of oligomycin.

With after the BV2 microglias of substance to be screened or solvent processing endogenous expression SQR, detection intracellular ATP is horizontal And ADP/ATP ratios.The result shows that simple rotenone (Rot) processing can cause ATP levels in BV2 microglias to decline (figure 2A, 2C, 2E) and ADP/ATP ratios rising (Fig. 2 B, 2D, 2F).Proton type mitochondrial uncoupler FCCP also leads to intracellular ATP Level declines, but rotenone (Rot) coprocessing cannot reverse intracellular ATP levels caused by FCCP to decline (Fig. 2A) and ADP/ATP The rising (Fig. 2 B) of ratio, shows function of the effect independent of composite I of proton type mitochondrial uncoupler.But rotenone (Rot) the small colloid intracellular ATP levels of BV2 caused by ADT-OH (50 μ Μ), ADT (50 μ Μ) and 5a (50 μ Μ) is reversed to decline (figure 2A, 2C, 2E) and ADP/ATP ratios rising (Fig. 2 B, 2D, 2F).Correspondingly rotenone (Rot) also inhibits ADT-OH to exist The effect of primary microglia (Fig. 3 A) and BV2 microglia OCR values (Fig. 3 B) is increased in the presence of oligomycin.It is above-mentioned The result shows that the mitochondria uncoupling of these three sulfide depends on the function of mitochondrial respiratory chain composite I.

Embodiment 3：Confirm that the expression of endogenous SQR has cell-specific

Inventor has detected three kinds of cells using Western blot and Quantitative Reverse Transcription PCR (qPCR) SQR albumen and mRNA expressions, specific method see aforementioned.

Inventors be surprised to learn that:1) SQR albumen (Fig. 4 A, liver liver are positive control) and SQR mRNA (Fig. 4 B, liver Dirty is positive control) it is a large amount of in primary microglia (Microglia) and primary astroglial cells (Astrocyte) Expression, and almost without expression in primary neural cell (Neuron).In order to show separated culture primary microglia, The purity of astroglia and nerve cell, inventor utilize qPCR detection microglia specific markers CD-11B, star The mRNA expressions of shape spongiocyte specific marker GFAP and Neuron-specific label NeuN.Microglia (MIC) high expression CD-11B mRNA, and hardly express GFAP and NeuN mRNA (Fig. 4 C)；And neuron height expresses NeuN, Without expressing GFAP and CD-11B mRNA (Fig. 4 E), show that cell purity is high.

Embodiment 4：The cell for not expressing SQR using endogenous expression SQR or endogenous confirms the mitochondria of candidate substances Uncoupling depends on SQR

To show that the oxidation of SQR is necessary to candidate leads to mitochondria uncoupling, in inventor's utilization The microglia of source property expression SQR and endogenous do not express the primary neural cell of SQR.Cell with candidate substances and solvent at After reason, three features of mitochondrial uncoupler are detected：1) intracellular ATP horizontal (increasing ADP/ATP ratios) is reduced；2) in ATP Cell oxygen consumption (OCR) value is increased in the presence of synzyme oligomycin；3) mitochondrial membrane potential is reduced.

The results show that in the presence of oligomycin, candidate substances ADT-OH (50 μ Μ) cannot increase primary god Expressed without endogenous SQR through member) OCR (Fig. 5 A), and ADT-OH cannot reduce the horizontal (figures of ATP of primary neuron intracellular 5B) and increases ADP/ATP ratios (Fig. 5 C), reduces mitochondrial membrane potential (Fig. 5 D).And proton type uncoupler FCCP (0.5 μ Μ) then have above-mentioned effect in primary neuron, show different from ADT-OH, FCCP is in the primary neuron for not expressing SQR In also have mitochondria uncoupling.Equally, candidate 5a (50 μ Μ) can not reduce the horizontal (figures of ATP of primary neuron 5E), ADP/ATP ratios (Fig. 5 F) are increased and reduce mitochondrial membrane potential (Fig. 5 G).

But candidate substances ADT-OH (50-100 μ Μ) increases primary microglia (figure in the presence of oligomycin 6A) with BV2 microglias (Fig. 6 B) oxygen consumption (OCR), the level (Fig. 6 C) of primary microglia ATP is reduced, is increased ADP/ATP ratios (Fig. 6 D) simultaneously reduce mitochondrial membrane potential (Fig. 6 E).Equally, candidate 5a (50 μ Μ) is deposited in oligomycin When can result in primary microglia OCR and increase (Fig. 6 F), reduce the level (Fig. 6 G) of ATP in primary microglia, Increase ADP/ATP ratios (Fig. 6 H).

To sum up as a result, the mitochondria uncoupling of candidate substances depends on SQR.

Embodiment 5：The cell for being overexpressed SQR is built using genetic manipulation means or the cell subtracted is struck in endogenous SQR expression

Inventor also utilizes the short stem loop RNA perturbation technique (shRNA) of lentivirus mediated, in the original of endogenous expression SQR Subtract SQR for being struck in microglia.To express the slow virus of SQR shRNA (interfering shRNA for the short stem ring of SQR) (shSQR) infector is compared for microglia with infecting negative control virus (NC) or uninfecting virus cell (Con).Western blot the result shows that：Compared with the control, microglia endogenous SQR protein expression levels are significantly struck Subtract (Fig. 7 A).

Inventor, which is also struck using siRNA rotaring dyeing technologies in the BV2 microglias of endogenous expression SQR, subtracts SQR.With SQR interferes tiny RNA (siSQR) to transfect BV2 microglias, to transfect negative control siRNA's (NC) or untransfected siRNA Cell (Con) compares.Western blot the result shows that：Compared with the control, microglia endogenous SQR protein expression water Flat significantly struck subtracts (Fig. 7 B).

Inventor also crosses table with the overexpression technology of lentivirus mediated in the primary neural cell for not expressing endogenous SQR Up to SQR：Slow virus (OE SQR) to be overexpressed SQR infects primary neuron, to infect the nerve of negative control virus (NC) The neuron of member or uninfecting virus compares (Con).Western blot the result shows that：Compared with the control, SQR expressions Significantly (7C) is increased in the neuron of infection OE SQR.

Embodiment 6:Using the cell for being overexpressed SQR or the cell for subtracting endogenous SQR and expressing is struck, confirms the line of candidate substances Plastochondria uncoupling depends on SQR：

ADT-OH (50-100 μ Μ) is being overexpressed raising in the primary neuron (OE SQR) of SQR using slow virus technology Cell oxygen consumption (OCR) in the presence of oligomycin；And in the primary neuron of infection negative control virus (NC), ADT- OH is without this effect (Fig. 8 A).Equally, ADT-OH (50-100 μ Μ) is reduced in the primary neuron (OE SQR) for being overexpressed SQR Intracellular ATP horizontal (Fig. 8 B), ADP/ATP ratios (Fig. 8 C) are increased and reduce mitochondrial membrane potential (Fig. 8 D)；And it is infecting In the primary neuron of negative control virus (NC), ADT-OH is acted on without this.Con is the control cell of uninfecting virus.

Correspondingly, the primary microglia subtracted is struck by the shRNA perturbation techniques of lentivirus mediated in endogenous SQR expression In, the uncoupling of ADT-OH is blocked, and is shown as：ADT-OH (50 μ Μ) is primary infection negative control virus (NC) The cell oxygen consumption (OCR) in the presence of oligomycin is improved in microglia；And in the slow disease of infection expression SQR shRNA In the primary microglia of poison (shSQR), ADT-OH is without this effect (Fig. 9 A).Equally, compared with solvent, ADT-OH (50 μ Intracellular ATP horizontal (Fig. 9 B) Μ) is reduced in the primary microglia of infection negative control virus (NC), is increased ADP/ATP ratios (Fig. 9 C) and reduction mitochondrial membrane potential (Fig. 9 D)；And in the primary of the slow virus of infection expression SQR shRNA In microglia (shSQR), ADT-OH is acted on without these.Equally, compared with solvent processing, candidate 5a (50 μ Μ) is feeling Mitochondrial membrane potential (Fig. 9 E) is reduced in the primary microglia of dye negative control virus (NC), reduces intracellular ATP Horizontal (Fig. 9 F) and high ADP/ATP ratios (Fig. 9 G)；And the primary microglia of the slow virus in infection expression SQR shRNA In (shSQR), 5a without these act on.Con：It is uninfected by the control cell of slow virus.

To sum up as a result, the mitochondria uncoupling of candidate substances depends on SQR.

Embodiment 7：Confirm that candidate substances transmit (RET) by the inversion electron of Mitochondria complex I levels and lead to line grain Body uncoupling

Above example shows that the mitochondria uncoupling of candidate substances is multiple dependent on SQR and mitochondrial respiratory chain Close the function of object I.Theoretically, SQR causes the CoQ of oxidized form to be reduced to reduced form the oxidation of hydrogen sulfide and sulfide CoQH₂, lead to CoQH₂/ CoQ ratios rise；And CoQH₂The rising of/CoQ ratios is the driving horizontal inversion electrons of mitochondrial complex I One of key factor of transmission.Therefore, inventor inquired into candidate sulfide in embodiment 7 can be endogenous expression SQR's CoQH is increased in cell₂/ CoQ ratios simultaneously cause the inversion electron of Respiratory Chain Complex I level to transmit.First, the present invention utilizes Efficient liquid phase-tandem mass spectrum has detected in the BV2 microglias of endogenous expression SQR that can candidate ADT-OH and 5aIt rises It is highCoQH₂/CoQ.The result shows that：ADT-OH (50 μM) and 5a (50 μM) is significantly increased under the dosage for leading to mitochondria uncoupling Cell CoQH₂/ CoQ ratios (Figure 10 A).

There has been no the inversion electron transmission that experiment can directly detect composite I level at present.But composite I level is reversed Electron transmission theoretically can cause the NAD+ of mitochondrial oxidative type to be reduced to NADH, to increase mitochondria NAD+/NADH Ratio.Therefore, the ratio of mitochondria NAD+/NADH can be used as the inversion electron transmission of characterization suction chain chain cpd I levels Index.

The experimental results showed that:1) compared with solvent group, candidate ADT-OH increases NADH in BV2 microglia mitochondrias Content (Figure 10 B) and reduce NAD⁺The ratio (Figure 10 C) of/NADH.The above result shows that candidate substances are in endogenous expression The microglia of SQR leads to the RET of complex I levels.In particular, inventor has found that rotenone (Rot) inhibits ADT-OH liters The effect (Figure 10 A-10B) of NADH contents and reduction NAD+/NADH ratios in high microglia mitochondria；Show rotenone (rotenone, Rot) specificity inhibits the inversion electron of Respiratory Chain Complex I level to transmit.And in embodiment 2, inventor is Confirm that the mitochondria uncoupling of ADT-OH can be blocked by rotenone (rotenone).These results synthesis shows candidate Matter leads to mitochondria uncoupling by the RET of Mitochondria complex I levels.

Embodiment 8：Confirm that candidate substances pass through dependent on SQR's using endogenous expression SQR or the cell for not expressing SQR Mechanism activation AMPK

Inventor first with Western blot have detected embodiment 1 screening obtain candidate substances can activate it is endogenous Property expression SQR BV2 microglias AMPK.ADT-OH (50-100 μ Μ, Figure 11 A) and 5a (50 μ Μ, Figure 11 B) can be with The activation levels for increasing AMPK show as the rising of the AMPK horizontal (p-AMPK) of phosphorylation.But ADT-OH (50-100 μ Μ, Figure 11 C) and 5a (50 μ Μ, Figure 11 D) cannot activate primary neuron (no endogenic SQR expression)

AMPK, and proton type uncoupler FCCP can activate primary neuron AMPK (Figure 11 C).

Embodiment 9：Using genetic manipulation means in being overexpressed the cell of SQR or striking the cell for subtracting endogenous SQR expression, Confirm that candidate substances can activate AMPK by the mediation of SQR

Inventor is in primary microglia using slow-virus transfection shRNA (shSQR), in BV-2 microglias system It is middle to be transfected using siRNA (SiSQR) to knock out endogenous SQR, using NC shRNA or NC siRNA as negative control (NC).As a result Show:It is struck in primary (Figure 12 A) and BV2 microglias (Figure 12 B) and subtracts SQR ADT-OH (50 μ Μ) can be blocked to AMPK's Activate (p-AMPK) effect.After knockout SQR (siSQR) being transfected in BV-2 microglias system with siRNA, activation of the 5a to AMPK (p-AMPK) effect is also blocked (12C).

Compared with solvent, ADT-OH (50-100 μ Μ) improves AMPK in the primary neuron (OE SQR) for being overexpressed SQR Activation levels (p-AMPK is horizontal), but cannot promote to infect the AMPK activation (Figure 12 D) of the neuron of negative control virus (NC).

To sum up as a result, candidate substances can specifically activate AMPK by the mediation of SQR.

Embodiment 10：Confirm that candidate substances depend on the activation of AMPK the function of mitochondrial complex I

Can inventor further has detected mitochondrial complex I inhibitor rotenone (Rot) on microglia hinder The effect of disconnected candidate substances activation AMPK.The result shows that compared with solvent (Vehicle), simple rotenone (Rot) and ADT-OH Processing can cause BV2 microglias AMPK activation (p-AMPK) is horizontal to rise (Figure 13).And work as rotenone (Rot) and ADT-OH When coprocessing, Rot reverses ADT-OH (50-100 μ Μ) to lead to the horizontal work risen of the small colloid born of the same parents AMPK activation (p-AMPK) of BV2 With (Figure 13).The above results show function of the candidate ADT-OH activation AMPK effects dependent on mitochondrial complex I.

Embodiment 11：Confirm therapeutic effect of the candidate substances to relevant disease in animal model

Cerebral hemorrhage (intracerebral hemorrhage, ICH) is because one kind that rupture of blood vessel in brain causes is in brain reality Cerebral apoplexy (stroke) hypotype with the characteristics of hemotoncus is formed in matter.Cerebral hemorrhage is case fatality rate and the highest cerebral apoplexy class of disability rate The case fatality rate of type, acute stage is up to 30%~40%, and survivor usually has different degrees of movement, cognition and linguistic function barrier Hinder.Cerebral hemorrhage also lacks effective medicine so far.After cerebral hemorrhage, self-blood can cause a system in the siltation of focal zone Arrange the inflammatory reaction occurred due to hemotoncus content and hemotoncus form crack after bleeding.For example, red blood cell be hemotoncus it is main at Point, body complement system activity after cerebral hemorrhage causes erythrocyte splitting, pyrolysis product (such as fibrin ferment, ferrous ion, oxidation blood Red pigment etc.) excessive activation that intracerebral microglia and blood-borne macrophages can be caused, excessive response is generated, is largely released The factor for putting proinflammatory disease, causes vasogenic edema.Therefore, inhibit cerebral hemorrhage after neuroinflamation have have to cerebral hemorrhage treatment Significance.It is there is no so far research shows that mitochondrial uncoupler has therapeutic effect to cerebral hemorrhage.

Inventor shows the candidate substances of the invention filtered out to correlation so that ADT-OH is to the therapeutic effect of cerebral hemorrhage as an example The therapeutical uses of disease.The present inventor has found through further investigation, compared with injection solvent (vehicle), mouse cerebral hemorrhage 3 hours Pneumoretroperitoneum injects the uncoupler ADT-OH (50mg/kg/ days) that the screening of embodiment 1 obtains, and continuous three days, significantly reduces bleeding side (Ipsil) brain edema (Figure 14 A) of corpus straitum (striatum)；Three behaviors detection (Garcia nervous functions synthesis inspection Survey, right limb touching experiment and corner experiment) result also indicates that (Figure 14 C-D)：It is god after 3 days that ADT-OH, which injects mouse in bleeding, It significantly improves through behavioral function obstacle.Sham-operated control group (Sham) animal does not show apparent brain edema and neural row Dysfunction.The above results show that candidate ADT-OH has therapeutic effect to relevant disease.

Embodiment 12：Confirm that candidate substances depend on SQR to the therapeutic effect of relevant disease in animal and cell model

In order to show that the therapeutic effect of ADT-OH depends on SQR, inventor also to strike reduction using the shRNA of lentivirus mediated The endogenic SQR expression in mouse cerebral hemorrhage position (i.e. corpus straitum).With Naoliqing capsule technology expression SQR is injected to mouse striaturn The slow virus (shSQR) of shRNA, the mouse to inject the slow virus (NC) of negative control shRNA are control mouse.Mouse injection disease Poison receives cerebral hemorrhage operation after 14 days.The uncoupler that the mouse cerebral hemorrhage pneumoretroperitoneum injection screening of embodiment 1 in 3 hours obtains ADT-OH (50mg/kg/ days), continuous three days, control-animal received solvent injection.In the control of the slow virus of injection NC shRNA In mouse (NC), ADT-OH reduces corpus straitum (Striatum) brain edema (Figure 15 A) compared with solvent injection (Vehicle)；And Three behaviors detection (Garcia nervous functions comprehensive detection, corner experiment and right limb touching experiment) result also indicates that (figure 15C-D)：ADT-OH injects mouse neurological dysfunction after bleeding 3 days and significantly improves.But express SQR in injection In the mouse (shSQR) of the slow virus of shRNA, compared with solvent injection (Vehicle), ADT-OH, which is no longer showed, reduces brain edema (Figure 15 A) and the therapeutic effect (Figure 15 C-D) for improving neurological dysfunction.

In addition, compared with sham-operation group animal (Sham) and bleeding offside (Contra), bleeding side (Ipsil) corpus straitum portion Proinflammatory inflammation factor (IL-1 β, IL-6, iNOS, TNF-α) mRNA expressions of position obviously rise.In the slow of injection NC shRNA In the control mice (NC) of virus, compared with solvent injection (Vehicle), ADT-OH reduces by 3 days bleeding sides after cerebral hemorrhage (Ipsil) induced expression level of the proinflammatory inflammation factor of corpus straitum (IL-1 β, IL-6, iNOS, TNF-α) mRNA；But it is expressed in injection In the mouse (shSQR) of the slow virus of SQR shRNA, ADT-OH inhibits these proinflammatory inflammation factors in bleeding part induced expression Effect is apparent to weaken (Figure 16).This shows inhibition of the candidate substances to proinflammatory inflammation factor induced expression in cerebral hemorrhage mouse model Effect depends on SQR.

Utilize with erythrocyte cracked liquid (LY) cause microglia inflammation activate cerebral hemorrhage cell model, inventor with For ADT-OH, further demonstrate that the therapeutic effect of candidate substances depends on SQR.BV2 microglias transfect negative control NC SiRNA (NC) or SQR siRNA (siSQR) for SQR.After transfection 48 hours, cause small colloid thin with erythrocyte cracked liquid While born of the same parents activate inflammation activation, handled with ADT-OH or solvent.After processing 6 hours, qPCR results are shown：Erythrocyte cracked liquid (LY) proinflammatory inflammation factor (IL-1 β, IL-6, iNOS, TNF-α) mRNA expressions are raised.In transfection NC siRNA slow virus It compares in BV2 cells (NC), ADT-OH can significantly reduce induced expressions of the LY to proinflammatory inflammation factor；But in transfection SQR siRNA BV2 microglias in (siSQR), ADT-OH in the presence of LY to the inhibition of these proinflammatory inflammation factor mRNA induced expressions make With being obviously reduced (Figure 17).

The above results show the candidate substances obtained with the method for the invention screening to the therapeutic effect of relevant disease according to Rely in SQR.

Embodiment 13：Confirm that AMPK activation mediates therapeutic effect of the candidate substances to relevant disease in cell model

Utilize with erythrocyte cracked liquid (LY) cause microglia inflammation activate cerebral hemorrhage cell model, inventor with For ADT-OH, show that AMPK mediates therapeutic effect of the candidate substances to cerebral hemorrhage.With not by LY activation compare the small colloids of BV2 Cell (Con) is compared, and erythrocyte cracked liquid (LY) reduces microglia AMPK activation levels (p-AMPK is horizontal).In transfection NC In the control BV2 cells (NC) of siRNA, ADT-OH raises AMPK activation level (phosphoric acid in the presence of erythrocyte cracked liquid (LY) Change horizontal p-AMPK)；But in the BV2 microglias of transfection SQR siRNA, ADT-OH is blocked AMPK activations (Figure 18).Result shown in 11 (SQR siRNA can block therapeutic effects of the ADT-OH to cerebral hemorrhage) in conjunction with the embodiments, these results Prompt ADT-OH may be by what AMPK activations caused by mitochondria uncoupling mediated to the therapeutic effect of cerebral hemorrhage.

In order to further illustrate inventor transfects BV2 microglias with NC siRNA or AMPK siRNA (siAMPK) After 48 hours, microglia inflammation is caused to activate with erythrocyte cracked liquid (LY), while being handled with ADT-OH or solvent.Processing 6 hours, qPCR results were shown：Erythrocyte cracked liquid (LY) raises proinflammatory inflammation factor (IL-1 β, IL-6, iNOS, TNF-α) mRNA Expression.In the control BV2 cells (NC) of transfection NC siRNA, ADT-OH significantly reduces inductions of the LY to proinflammatory inflammation factor Expression；But in the BV2 microglias (siSQR) of transfection SQR siRNA, ADT-OH is proinflammatory to these in the presence of LY The inhibiting effect of inflammation factor mRNA induced expressions is obviously reduced (Figure 19).The above results show that AMPK mediates the screening of embodiment 1 to obtain Therapeutic effects of the ADT-OH obtained to cerebral hemorrhage.

Claims (10)

1. a kind of method obtaining the substance with mitochondria uncoupling, which is characterized in that include the following steps：

(a) mitochondria for handling the cell of expression SQR with candidate substances or being extracted from the cell of expression SQR, in candidate substances In the presence of, mitochondria uncoupling characteristics index is detected, and with cell/mitochondria of solvent processing or untreated cell/line Plastochondria compares, and compares the mitochondria uncoupling characteristics index of candidate substances processing group and control group, if statistically having Then candidate substances are potential mitochondrial uncouplers to difference；

(b) with candidate substances handle do not express or low expression SQR, SQR activity reduce or inactivation cell, or with candidate substances at Manage from the mitochondria of above-mentioned cell extraction, detect mitochondria uncoupling characteristics index, cell/mitochondria for being handled with solvent or Untreated cell/mitochondria compares, and compares the mitochondria uncoupling characteristics index of candidate substances processing group and control group, If statistically indifference, and candidate substances are potential mitochondrial uncouplers in (a), then and candidate substances are to pass through SQR is mediated and the substance with mitochondria uncoupling.

2. according to the method described in claim 1, it is characterized in that, the mitochondria uncoupling characteristics index is：1) line grain Body uncoupling can cause the oxygen demand of cell or mitochondria to rise when atriphos acid enzyme is suppressed, 2) reduce cross-line Mitochondrial membrane film potential and 3) reduction intracellular ATP levels.

3. a kind of method obtained to the AMPK substances with activation, which is characterized in that include the following steps：

(A) cell that expression SQR is handled with candidate substances, in the presence of candidate substances, the activation levels of detection cell AMPK, with The cell or untreated cell of solvent processing compare, and compare candidate substances processing group and control group A MPK activation levels, such as Statistically variant, candidate substances are potential AMPK activator；

(B) it is not expressed with candidate substances processing or low expression SQR, SQR activity reduces or the cell of inactivation, existed in candidate substances Under, the activation levels of detection cell AMPK, the cell or untreated cell handled with solvent compares, and compares at candidate substances Reason group and control group A MPK activation levels, such as statistically indifference, and candidate substances are that potential AMPK swashs in (A) Agent living, then candidate substances are to lead to the substance that AMPK is activated by SQR.

4. method according to claim 1 or 3, which is characterized in that the cell of the expression SQR includes endogenous expression The primary cell or cell line of SQR lead to the primary cell for expressing or being overexpressed SQR with genetic manipulation means or pharmacological techniques Or cell line, from the primary or passage cell that obtains of genetic engineering animal of expression SQR.

5. method according to claim 1 or 3, which is characterized in that it is described do not express or low expression SQR, SQR activity drop The cell of low/inactivation includes endogenous low expression or does not express the primary cell or cell line, endogenous SQR protein inactivations of SQR Activity reduce primary cell or cell line, utilize the technologies such as genetic manipulation means or pharmacology reduction/knockout/strike subtract it is endogenous Property SQR expression or endogenous SQR activity is made to reduce or the primary cell of inactivation or cell line, knock out or strike from SQR and subtract animal and obtain The primary or passage cell obtained.

6. purposes of the compound as mitochondrial uncoupler and AMPK activator shown in formula 1 or formula 2,

Wherein, R is-H or-CH in formula 1₃。

7. compound shown in formula 1 or formula 2 prepare treatment cerebral hemorrhage, metabolic syndrome, obesity, neurodegenerative disease, aging, Purposes in the drug of cerebral ischemia, diabetes or cancer,

Wherein, R is-H or-CH in formula 1₃。

8. a kind of method leading to mitochondria uncoupling, which is characterized in that sulfur-containing compound leads to mitochondria by the oxidation of SQR The inversion electron of Respiratory Chain Complex I level transmits, and reduces across mitochondrial membrane potential, so as to cause mitochondria uncoupling.

9. a kind of method of activation AMPK, which is characterized in that sulfur-containing compound causes mitochondrial respiratory chain multiple by the oxidation of SQR The inversion electron for closing object I levels transmits, and reduces across mitochondrial membrane potential, so as to cause cell mitochondrial uncoupling and ATP Synthesis is horizontal to be declined, and then activates AMPK.

10. the substance obtained using the method as described in claim 1 and 3 is as mitochondrial uncoupler or AMPK activator Purposes and the purposes in the drug for preparing treatment relevant disease.