CN103948585B - Application in preparation preventing and treating nervous system disease medicine for the arteannuin - Google Patents
Application in preparation preventing and treating nervous system disease medicine for the arteannuin Download PDFInfo
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Abstract
The invention discloses the application in preparation preventing and treating nervous system disease medicine of arteannuin and its derivant.The present invention is with Clonal Rat Pheochromocytoma tumor PC12 neurocyte strain, retinal neuronal cell strain RGC 5 and Primary cortical neurons are representative as experimental subject, with sodium nitroprusside, hydrogen peroxide simulates the cell injury such as oxidative stress and the apoptosis of inducing nerve cell, research finds arteannuin in sodium nitroprusside, in the cell injury of hydrogen peroxide induction, there is protective effect, and the Primary Study Protective effects of arteannuin and its derivant, result explanation arteannuin and its derivant can be used for preventing and treating various nervous system diseasies as neuroprotective drug, as various neurodegenerative diseases, acute and chronic neurodegenerative disease, nerves oculopathy etc., these main and oxidativestress damage, apoptosis-related nervous system disease.The present invention is that the treatment and prevention of nervous system disease bring new direction.
Description
Technical field
The present invention relates to application in preparation preventing and treating nervous system disease medicine for the arteannuin.
Background technology
Arteannuin (artemisinin) is a kind of compound extracting from feverfew artemisia Herba Artemisiae annuae, wide
The general treatment for various malaria, arteannuin mainly acts on erythrocyte stage to plasmodial killing action, for erythrocyte
Outer phase and pre-erythrocytic stage, then effect was very weak.In addition to having prominent Antimalarial, arteannuin also has significant antibacterial, resists
Virus function, its antimicrobial spectrum includes staphylococcus epidermidiss, micrococcus catarrhalises, anthrax bacillus, diphtheria corynebacterium, simultaneously to golden yellow Fructus Vitis viniferae
Coccus, bacillus pyocyaneus, dysentery bacterium, tuberculosis stalk bacterium etc. also have certain inhibitory action.Extract in Herba Artemisiae Annuae the sitosterol obtaining and
Stigmasterol also has antivirus action.Separately, arteannuin also has immunoregulation effect, has obvious inhibitory action to humoral immunization,
Then there is certain facilitation to cellular immunization., in clinical practice, toxicity is relatively low for artemisine goods and materials, using safety, typically
No obvious adverse reaction.With regard to arteannuin central nervous system disease effect, current research is also little, has research display
The cell proliferation of the mouse neuroblastoma N2a to In vitro culture for the dihydroartemisinine has certain inhibitory action, points out Herba Artemisiae Annuae
The tumor of plain Central nervous system may have certain inhibitory action.About arteannuin to the protective effect of neurocyte and its
Treat neurodegenerative diseases as neuroprotective and optic neuropathy yet there are no report.
NO is a kind of less bioactive molecule, under low concentration, can participate in the regulation of intracerebral many physiological functions,
Although its chemical constitution is simple, function or the physiological function of neuron can be controlled in the way of relatively special；And more highly concentrated
Under degree, NO has toxic action to people's cortical neuron and neurocyte.As exogenous NO donor, sodium nitroprusside（SNP）Preparation
Neuronal damage model be Related Experimental Study preferable in vitro models.
The progress of contemporary medical science extends the life of the mankind, and thus leads to the rising of old acute and chronic nervous system disease,
Such as Alzheimer disease, apoplexy, Parkinson's disease etc..These nervous system diseasies are with neuronal degeneration specific in disease process
Constantly development is characterized.Because mature neuron cell can not regenerate after its death, neuron in above-mentioned nervous system disease
Death may lead to brain basic function (include identification, sensation and action function) incurable damage and thus produce
Raw economy crosses HD with society.
Oxidative stress (oxidativestress) and apoptosis (apoptosis) are considered as the master of neuronal death
Want approach, oxidative stress (oxidativestress) and multiple nervous system disease（As various neurodegenerative diseases and nerve
Property oculopathy）Relevant.
At present, to the effect of neurocyte, seldom meanwhile, arteannuin is in the oxygen of neurocyte is understood to artemisinin-based drug
Change stress with apoptosis in whether to have protective effect still unclear.
Content of the invention
In order to solve the above problems, the present invention is with Clonal Rat Pheochromocytoma tumor PC12 neurocyte strain, retina god
It is representative through cell strain RGC-5 and Primary cortical neurons, as experimental subject, with sodium nitroprusside (SNP), hydrogen peroxide (H2O2) come
Simulate oxidative stress and the apoptosis of inducing nerve cell, find that arteannuin and its derivant induce in sodium nitroprusside, hydrogen peroxide
Corresponding cell injury in there is protective effect, and the Primary Study mechanism of arteannuin and its derivant protective effect, explanation
Arteannuin and its derivant can be used for preventing and treating various nervous system diseasies as neuroprotective drug, such as various nervus retrogressions
Disease, acute and chronic neurodegenerative disease, nerves oculopathy etc., these main and oxidativestress damage, apoptosis-related god
Through property disease.
It is an object of the invention to provide the application in preparation preventing and treating nervous system disease medicine of arteannuin and its derivant.
The technical solution used in the present invention is：
The application in preparation preventing and treating nervous system disease medicine of arteannuin and its derivant.
Further, above-mentioned nervous system disease includes acute and chronic neurodegenerative disease, nerves ophthalmic diseasess.
Further, above-mentioned nervous system disease is the nervous system disease related to oxidativestress damage.
Further, above-mentioned artemisinin derivative is compound with arteannuin as parent nucleus or it is pharmaceutically acceptable
Salt.
Further, the above-mentioned compound with arteannuin as parent nucleus is selected from Artemtherin, artesunate, dihydro Herba Artemisiae Annuae
Element, deoxyartemisin.
The invention has the beneficial effects as follows：
1）Present invention demonstrates that arteannuin and its derivant are in SNP, H2O2Have certain in the neural cell injury causing
Protective effect, and the desk study mechanism of action of arteannuin and its derivant, it may be by activation or part activates
MAPK/Erk signal path realizing the protective effect in SNP damaged nerve cells, because artemisinin-based drug has been used to
Clinic, studies the neuroprotective of these medicines, for finding its possible target spot, new pharmacological action, to such medicine
Safe handling has great importance.
2）Present invention illustrates arteannuin and its derivant can be used for preventing and treating various nerves as neuroprotective drug
Property disease, such as various neurodegenerative diseases, acute and chronic neurodegenerative disease, nerves oculopathy etc., be controlling of nervous system disease
Treat and prevention brings new direction.
Brief description
Fig. 1 is the cell viability situation that PC12 cell is processed in the SNP of variable concentrations, and # represents P<0.05, ## P<
0.01；
Fig. 2 is the arteannuin protective effect that damages in PC12 cell in SNP of variable concentrations, and # representsP<0.05, ##
RepresentP<0.01；
Fig. 3 dyes detection arteannuin for Hochest and induces the protective effect in PC12 apoptosis in SNP；
Fig. 4 is the statistical analysiss to apoptosis number in Fig. 3, * *P<0.01, ## representsP<0.01；
Fig. 5 is the protection situation that arteannuin damages in PC12 cell in SNP in the presence of inhibitor PD98059, and A is Herba Artemisiae Annuae
Element, LY is LY294002, and PD is PD98059, * *P<0.01, ## representsP<0.01；
Fig. 6 may damage shielding in PC12 cell by MAPK/Erk signal path in SNP for arteannuin；
Fig. 7 is the damaging action to RGC-5 cell for the SNP, *P<0.01；
Fig. 8 is that the arteannuin of variable concentrations damages the protective effect in RGC-5 cell in SNP（* represents P<0.01）；
The apoptotic protective effect of RGC-5 that Fig. 9 induces to SNP for arteannuin, A is Hoechst Coloration experiment, and B is
Statistical analysiss to apoptosis ratio in each group cell in A, ART：Arteannuin（Artemisnin）,## P<0.01, * *P<0.01；
Figure 10 is the damaging action to cortical neurons for the SNP, *P<0.01；
Figure 11 is that the arteannuin of variable concentrations damages the protective effect in cortical neurons in SNP（*P<0.05, * *P
<0.01）；
Figure 12 is H2O2Damaging action to RGC-5 cell, *P<0.01；
Figure 13 is the arteannuin of variable concentrations in H2O2Damage the protective effect in RGC-5 cell（# represents P<0.01）.
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
Embodiment 1 arteannuin damages the protective effect in PC12 neurocyte in SNP
First, materials and methods
（1）Material and main agents
Arteannuin is purchased from Dalian U.S. logical sequence Science and Technology Ltd.；Sodium nitroprusside（SNP）It is purchased from green skies biotechnology research
Institute；DMEM culture medium, hyclone and horse serum are purchased from Gibco company of the U.S.；Tetrazolium bromide (MTT), dimethyl sulfoxide (
DMSO), it is purchased from Sigma Co., USA；LY294002, PD98059 are purchased from Sigma-Aldrich；Western
Blot related reagent is purchased from the green skies in Guangzhou biotechnology research institute；Antibody A nti-phospho-Akt used by Western
(Ser473) antibody and Anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody
Purchased from Cell Signaling Technology (Woburn, USA)；Horseradish peroxidase（HRP）The two of labelling resist
（IgG）Purchased from Santa Cruz company of the U.S.；Chemical luminous substrate test kit is purchased from Thermo company of the U.S..
（2）The double one side of key instrument purifies super-clean bench（Purifying Equipment Co., Ltd., Suzhou）；Model 2323 dioxy
Change carbon incubator（SHELLA company of the U.S.）；LX71 fluorescence inverted phase contrast microscope, S8F-3 photomicrographic system（Japan
Olympus company）；Western blot vertical electrophoresis and transferring system（Bio-Rad company）.
（3）Method
1）Cell culture
Differentiated PC12 cell strain is by Canadian Mcgill university Douglas research center Remi
Quirion presents, and the method according to foundation such as Zheng Wenhua is cultivated：It is respectively 5% hyclone and 5% using volume fraction
Horse serum, penicillin 100U/ml, the DMEM culture medium of streptomycin 100U/ml, are placed in 37 DEG C, and volume fraction is 5% CO2
Cultivate in the incubator of gas.Standby with 0.25% pancreatin -0.02%EDTA had digestive transfer culture every two days.
2）Primary neuronal culture
A. prepare before testing：Operating theater instruments used for experiment are immersed in the ethanol of 75 %, need to soak 30 min with
On, be prepared in advance the separately equipment such as the little culture dish needed for cortex, centrifuge tube, prepares 1 × HBSS, digestion terminate liquid and culture
Base.
B. Poly-D-Lysine is coated：The every hole of 6 orifice plates adds 1.5 mL Poly-D-Lysine, and other orifice plates are pressed such
Push away, washed twice with 1 × PBS after 10-30 min.
C. the pregnant Mus used by primary neuronal culture are derived from Zhongshan University's Experimental Animal Center, choose the Kunming of pregnant 18 days
Pregnant mice, the ethanol of Mus abdominal part spray 75%, dislocation is put to death, along ventrimeson hara kiri, is found uterus, quickly remove, be placed in advance
In the culture dish preparing, it is transferred quickly to super-clean bench.
D. cut off uterus, take out pregnant Mus, broken end, separate skull, take out full brain, be placed in the culture dish filling 1 × HBSS
Interior.
E., in superclean bench, with ophthalmic forceps, operating scissorss, careful separation Hippocampus and cortex, it is placed in 1 × HBSS
Rinsing.
F. carefully remove meninges and the blood streak on cortex and hippocampal tissue surface with ophthalmic forceps, use tweezer in culture dish
Son tears up cerebral tissue as far as possible, and the tissue tearing up is transferred in 10 mL EP pipes.
G. plus 1 × HBSS rinses for several times, and find to have the blood streak of suspension, remove immediately, plus pancreatin, put 37 DEG C of CO2Culture
Case digests 8 min.
H. the culture medium plus containing 10 % FBS terminates digestion, flicks centrifuge tube, continues 1.5-2 min.
I. suck FBS, plus 1 × HBSS 1 mL, pasteur pipet is blown and beaten until in unicellular and be uniformly dispersed repeatedly, such as
See block or connective tissue in a organized way, then pick up and discard.
J. after blowing and beating uniformly, 10 μ L cell suspension are taken to be counted, 800 rpm × 10 min centrifugations.
K. suck supernatant, plus preprepared cell culture medium, make single cell suspension, according to cytometer quantity kind
Plate, is placed in CO2Incubator is cultivated.When planting plate, used medium is：DMEM+10% FBS+0.2 % PSA, adherent after 6 h after
Change liquid, be changed to Neurobasal+2 % B27+0.2 % PSA, routine observation cell.
3）Mtt assay detects cell viability
By PC12 cell with 1 × 104Individual/hole density is inoculated in 96 orifice plates, after cell attachment, adds arteannuin（3~
100μM）, SNP (500 μM), in administration after 37 DEG C, volume fraction be 5% CO2Cultivate in the incubator of gas
24h, adds MTT liquid（0.5mg/ml）, supernatant discarded after 37 DEG C of incubation 4h, every hole adds 100 μ L DMSO vibrations, waits to tie
Crystalline substance detects absorbance (A at 490 nm wavelength after being completely dissolved in microplate reader490), detect cell viability.
4）Immunoblotting detects downstream signaling pathway protein phosphorylation level
Carry out cell counting by after the PC12 cell dissociation of above-mentioned culture, with the DMEM culture medium inoculated containing 1%FBS to use
In coated 12 orifice plates of poly-D-lysine.After cell attachment, after being processed with LY294002, PD98059, wash away cell culture fluid,
Gently rinsed with ice-cold PSB, obtain total protein of cell after harvesting, after BCA method protein quantification, protein concentration is adjusted to
Concentration.With the protein sample of 30 μ g/well through 8% polyacrylamide gel（SDS-PAGE）Electrophoretic separation, then it is transferred to PVDF
On film.With the TBST room temperature closing transfer film 1.5h containing 5% defatted milk powder, add various antibody（Anti-phospho-Akt
(Ser473)antibody, Anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody）
Incubated at room is overnight.After washing film, then two anti-incubated at room 1h with horseradish peroxidase-labeled.ECL chemiluminescence system is examined
Survey protein signal.
5）Statistical method
Statistical analysis are carried out using SPSS 16.0, multigroup of dosage information compares and adopts one factor analysis of variance（One-
way ANOVN）, the multiple comparisons of each group mean are using minimum notable interpolation method（LSD）, P<0.05 has statistics for difference
Meaning.
2nd, result
（1）SNP can induce PC12 cell injury
After PC12 cell deprives serum, using the SNP of variable concentrations（200～1000 μM）Process PC12 cell, another setting
Containing 1% FBS（v/v）And do not contain serum（CTL）DMEM medium treatment PC12 cell as comparison, pass through after cultivating 24h
MTT method detects the change of cell viability, and testing result is as shown in figure 1, it can be seen that increase with SNP concentration, PC12
The vigor of cell is gradually lowered, when SNP concentration reach 800 μM and above when, the damage of PC12 cell viability is had extremely notable
Difference（P<0.01）.
The above results explanation SNP has dose dependent to the damage of PC12 cell, and 800 μM of SNP can be used for inducing
The experiment of PC12 cell injury.
（2 ）Arteannuin damages the protective effect in PC12 cell viability in SNP
After PC12 cell deprives serum, add the arteannuin of variable concentrations（0、3.125、6.25、12.5、25、50、100μ
M, is prepared with DMEM culture medium）Pretreatment 1h, adds 800 μM of SNP immediately（Cultivate basigamy with DMEM）, after processing 24h,
Cell viability is detected by MTT method, in addition sets up matched group CTL（Add the DMEM culture medium of same volume）, testing result
As shown in Fig. 2 it can be seen that under the process of 800 μM of SNP, with arteannuin（Artemisnin）The increase of concentration,
The degree that PC12 cell viability is damaged is gradually lowered, when arteannuin concentration reach 25 μM and above when, reduce SNP to PC12
The damaging action of cell viability has significant difference P<0.01.
The above results illustrate, arteannuin has protective effect in the PC12 cellular oxidation stress damage that SNP induces, and this
Kind of protective effect has dose dependent, when arteannuin concentration reach 25 μM and above when almost can block the poison to PC12 for the SNP
Property damaging action.
（3）Arteannuin induces the protective effect in PC12 apoptosis in SNP
Above-mentioned MTT result shows that arteannuin can protect the cell injury that SNP is induced, and next adopts further
Hoechst staining suppresses the effect of SNP inducing cell apoptosis to observe arteannuin.
After PC12 cell deprives serum, add 25 μM of arteannuin pretreatment 1h, add 800 μM of SNP process immediately
After 24h, the setting of related control group as shown in Figure 3 and Figure 4, carries out Hochest33258 dyeing after process, detect each group cell
Apoptosis situation.
Hoechst coloration result as shown in figure 3, cellular control unit karyon is mellow and full full, uniform coloring；Give SNP process
Afterwards, it is dispersed in the nucleus being distributed some and apoptosis feature occurring between normal PC12 nucleus, such as nucleus volume becomes rugula
Contracting, it is seen that karyorrhexiss become little lobate or assume the fine and close granule bulk of dense dye, assumes the blue-fluorescence of high intensity（As in figure arrow
Shown in head）；But giving 25 μm of ol/L arteannuin（Artemisinin）Under intervention, nucleus shrinkage number reduces and karyorrhexiss
Phenomenon alleviated.This shows that arteannuin has certain protective effect in SNP induced PC 12 cells apoptosis.
Apoptosis count results are as shown in Figure 4：Compared with the PC12 cell of normal culture, after SNP is processed, PC12 cell
Apoptosis quantity is significantly higher than normal group（P<0.01）, and after giving drug artemisinin intervention, can reverse SNP's to a certain extent
Toxic action, the cell number of its apoptosis necrosis substantially reduces, and its protective effect has significant difference（P<0.01）.
The above results illustrate the cell injury such as oxidative stress and the apoptosis of the thin PC12 of nerve that arteannuin induces in SNP
In (the SNP damage model of PC12 cell), there is protective effect.
（5）Arteannuin may damage shielding in PC12 cell by MAPK/Erk signal path in SNP
Studies have reported that artemisinin-based drug may play drug action in the cell by MAPK signal path, in order to
Exploring arteannuin further is by way of the effect playing protection PC12 cell by what kind of signal, and the present invention chooses PI3K/Ak
Explored with MAPK/Erk signal path.
After PC12 cell deprives serum, respectively using the specific inhibitor LY294002 of PI3K/Akt signal path（25μ
M）, the specific inhibitor PD98059 of MAPK/Erk signal path（50μM）After pretreatment PC12 cell 30min, add 25 μM
Arteannuin processes 1h, after adding SNP to process 24h immediately, is lived by the cell of MTT method detection process group and corresponding matched group
Power, as shown in Figure 5.From figure 5 it can be seen that inhibitor LY294002 and PD98059 itself can't substantially to reduce PC12 thin
Born of the same parents' vigor, and arteannuin and SNP simultaneously in the presence of PC12 cell viability almost also unaffected, but when arteannuin, SNP and
Inhibitor PD98059 simultaneously in the presence of, PC12 cell viability substantially reduces, and this explanation is blue or green in the presence of inhibitor PD98059 when have
Protective effect forfeiture in SNP damage PC12 cell for the artemisin is thus it is speculated that arteannuin may be existed by MAPK/Erk signal path
SNP damages shielding in PC12 cell.
Whether correct in order to explore above-mentioned supposition further, detect MAPK/ further followed by western blot
Phosphorylation status under artemisinin action for the related protein kinase in Erk signal path, cellular processes ibid, experimental design
As shown in Figure 6 with testing result.
As can be seen from Figure 6, after SNP processes PC12 cell with arteannuin simultaneously, p-Erk level significantly increases；Add
The specific inhibitor LY294002 of PI3K/Akt can not reduce the phosphorylation level of Erk, and adds the specificity of MAPK/Erk
After inhibitor PD98059, Erk phosphorylation level drops to matched group level.Illustrate that arteannuin damages the protective effect of PC12 to SNP
May be by MAPK/Erk signal path to play a role.
Mitogen activated protein kinase (Mitogen-activated protein kinases, MAPK) signal transduction
Path is one of study hotspot in recent years, and it can be transduceed cell-surface signal to nucleus.It is dynamic that this family passes through impact
The transcription of thing genes within cells and regulation and control, thus affect the propagation of cell, differentiation, conversion, apoptosis etc..Thin in mammal at present
It is found that altogether 4 parallel MAPK signal paths in born of the same parents：The signal path of MAPK family mainly includes extracellular signal and adjusts
The protein kinase of control（ERK）, c-Jun N-terminal kinases（JNK）/ the protein kinase that stress activate（SAPK）, P38MAPK and
Tetra- approach of ERK5/BMK1.Wherein, Ras-Raf-ERK approach is studied so far and must be compared a clearly path, and this path is joined
Multiple physiology, pathological process with cell growth, growth, propagation, differentiation etc..Somatomedin activates tyrosine-kinase with receptor binding
Enzyme, by adaptin by signal transmission to Ras albumen, Ras-GTP is directly combined with Raf, forms an of short duration film
Anchor signal.The kinases of the protein kinase that the Raf of activation is activated by phosphorylation mitogen（MEK）Serine on ring is residual
Base and activated.The protein kinase that mitogen is activated by MEK again（ERK）Activation, and then phosphorylation many and kytoplasm and born of the same parents
Film be connected substrate and promote cell proliferation.Find in our study, PD98059 can suppress arteannuin that SNP is damaged
The protective effect of PC12 cell, you can illustrate that arteannuin protects the PC12 cell of SNP damage it may be possible to pass through activation or part
Activate MAPK/Erk signal path and promote the propagation of cell, thus reaching neuroprotective.And activate the concrete of this path
Mechanism, then need further to be studied.
In sum, this research proves that in PC12 cell the neurocyte that arteannuin damages to SNP has protection and makees
With, its effect may be by activation or part activation MAPK/Erk signal path, but its specific mechanism need deeper
The research entering.Because artemisinin-based drug has been used to clinic, study the neuroprotective of these medicines, can for finding it
Energy target spot, new pharmacological action, the safe handling to such medicine has great importance.
Embodiment 2 arteannuin damages the protective effect in retinal neuronal cell RGC-5 in SNP
First, materials and methods
With embodiment 1.
2nd, result
（1）SNP can induce RGC-5 cell injury
After RGC-5 cell deprives serum, using the SNP of variable concentrations（62.5～1000 μM）Process RGC-5 cell and
DMEM culture medium culturing RGC-5 cell is as comparison（CTL）, the change of cell viability is detected after culture 24h by MTT method,
Testing result is as shown in fig. 7, it can be seen that the SNP of low concentration promotes cell proliferation（1～250 μM), when reaching 500 μM
Toxicity in SNP, is reduced to 59% about in 750 μM of cell survival rates, and this concentration is as SNP cytotoxicity model.
（2）Arteannuin damages the protective effect in RGC-5 cell viability in SNP
Foundation SNP induction cell injury model basis on, using 750 μm of ol/L SNP to RGC-5
Cell is processed, and MTT method detects that the RGC-5 cell injury whether arteannuin induces to SNP has protective effect, arteannuin
Solubility select 6.25,12.5,25,50 and 100 μm of ol/L respectively, testing result is as shown in figure 8, increasing with arteannuin concentration
Plus, SNP induces the effect of RGC-5 cell injury gradually to weaken, and the survival rate of cell is gradually increased, when the concentration of arteannuin reaches
During 50 μm of ol/L, its protective effect has had significant difference（P<0.01）.This shows, the arteannuin of low concentration lures to SNP
The RGC-5 cell injury led has protective effect, and is in dose dependent（0～50 μm of ol/L）.
（3）Arteannuin induces the protective effect in RGC-5 apoptosis in SNP
Above-mentioned MTT result shows that arteannuin can protect the cell injury that SNP is induced, and next adopts further
Hoechst staining suppresses the effect of SNP inducing cell apoptosis to observe arteannuin.
, as shown in Fig. 9 A, cellular control unit karyon is mellow and full full, uniform coloring for Hoechst coloration result；Give at SNP
After reason, it is dispersed in the nucleus being distributed some and apoptosis feature occurring between normal RGC-5 nucleus, such as nucleus volume diminishes
Shrinkage, it is seen that karyorrhexiss become little lobate or assume the fine and close granule bulk of dense dye, assumes the blue-fluorescence of high intensity（As in figure
Shown in arrow）；But under giving 25 μm of ol/L arteannuin interventions, the phenomenon of the minimizing of nucleus shrinkage number and karyorrhexiss is
Alleviate.This shows that arteannuin has certain protective effect in SNP induction RGC-5 apoptosis.
Apoptosis count results are as shown in Fig. 9 B：Compared with the RGC-5 cell of normal culture, after SNP is processed, RGC-5
Apoptosis quantity is significantly higher than normal group（P<0.01）, and after giving drug artemisinin intervention, can reverse to a certain extent
The toxic action of SNP, the cell number of its apoptosis necrosis substantially reduces, and its protective effect has significant difference（P<0.01）.
The above results explanation arteannuin has in the oxidativestress damage of the RGC-5 retinal neuronal cell that SNP induces
Protective effect.
Embodiment 3 arteannuin damages the protective effect in cortical neuron in SNP
First, materials and methods
With embodiment 1.
2nd, result
（1）SNP can induce cortical neurons to damage
After cortical neurons deprive serum, using the SNP of variable concentrations（12.5～1000 μM）Process Cortical neurons
First cell and DMEM culture medium culturing cortical neurons are as comparison（CTL）, detected by MTT method after culture 24h
The change of cell viability, testing result is as shown in Figure 10, it can be seen that from 12.5 μm of ol/L to 100 μm of ol/L
SNP can obviously reduce the survival rate of neuron, and the value of MTT assumes dose dependent and declines, and the SNP of 50 μm of ol/L can be notable
The survival of inhibitory neuron, the dosage choice of the SNP of follow-up neuronal damage is 50 μm of ol/L.
（2）Arteannuin damages the protective effect in cortical neurons vigor in SNP
Foundation SNP induction cell injury model basis on, using 50 μm of ol/L SNP to Cortical neurons
First cell is processed, and MTT method detects that the cortical neurons damage whether arteannuin induces to SNP has protective effect,
The solubility of arteannuin selects 6.25,12.5,25,50 and 100 μm of ol/L respectively, and testing result is as shown in figure 11, with arteannuin
The increase of concentration, SNP induces the effect that cortical neurons damage gradually to weaken, and the survival rate of cell is gradually increased, and works as green grass or young crops
When the concentration of artemisin reaches 25 μm of ol/L, its protective effect has had significant difference（P<0.01）.This shows, 25-
In 100 μm of ol, arteannuin has significant inhibitory action to the cortical neurons apoptosis that SNP induces, and is in obvious dosage
Effect.
The above results explanation arteannuin has protective effect in the damage of the cortical neurons that SNP induces.
Embodiment 4 arteannuin is in H2O2Damage the protective effect in retinal neuronal cell RGC-5
First, materials and methods
With embodiment 1.
2nd, result
（1）H2O2RGC-5 cell injury can be induced
After RGC-5 cell deprives serum, using the H of variable concentrations2O2（10～100 μM）Process RGC-5 cell and DMEM
Culture medium culturing RGC-5 cell is as comparison（CTL）, the change of cell viability, detection knot after culture 24h, is detected by MTT method
Fruit is as shown in figure 12, it can be seen that the H of high concentration2O2Concentration (60～100 μM) causes RGC-5 cell dead dependency
Die, 100 μM of H2O2About 40～60% cell deaths can be caused.
（2）Arteannuin is in H2O2Damage the protective effect in RGC-5 cell viability
H in foundation2O2On the basis of cell injury model of induction, using the H of 100 μm of ol/L2O2To RGC-
5 cells are processed, and whether MTT method detection arteannuin is to H2O2The RGC-5 cell injury of induction has protective effect, arteannuin
Solubility respectively select 6.25,12.5,25,50 and 100 μm of ol/L.Testing result is as shown in figure 8, increasing with arteannuin concentration
Plus, H2O2The effect of induction RGC-5 cell injury gradually weakens, and the survival rate of cell is gradually increased, when the concentration of arteannuin reaches
During 25 μm of ol/L, its protective effect has had significant difference（P<0.05）.This shows, the arteannuin of low concentration is to H2O2Lure
The RGC-5 cell injury led has protective effect, and is in dose dependent（0～100 μm of ol/L）.
The above results explanation arteannuin is in H2O2The neurocyte of induction（PC12）Oxidative stress and the cell such as apoptosis
There is in damage protective effect.
Equally, to artemisinin derivative（As blue or green in Artemtherin Artemether, artesunate Artesunate, dihydro
Artemisin Dihydroartemisinin and deoxyartemisin Deoxyartemisinin etc.）Or on its pharmaceutically acceptable salt carries out
State in embodiment similar experimentation, find its derivant all have with artemisine as protection neurocyte, strengthen nerve
The effect of cell resistance lesion capability, thus can also illustrate that the analog with arteannuin as parent nucleus also has similar effect.
Above-mentioned correlation arteannuin and its derivant structure formula are as follows：
Arteannuin Artemisinin
Artemtherin (Artemether)
Artesunate (Artesunate)
Dihydroartemisinine (Dihydroartemisinin)
Deoxyartemisin (Deoxyartemisinin)
Artemisia ether woods acid (Artelinic acid)
Arteether (Artemotil)
According to above-mentioned experimental result, arteannuin and its derivant have and protection neurocyte, enhancing neurocyte opposing
The effect of lesion capability, can be used for preventing and treating various nervous system diseasies, including various mainly by oxidative stress, apoptosis
The neurodegenerative diseases that cause, acute and chronic neurodegenerative disease, nerves ophthalmic diseasess etc..
Claims (2)
1. application in preparation preventing and treating nervous system disease medicine for the arteannuin；Described nervous system disease is thin with retina neural
The relevant nerves ophthalmic diseasess of born of the same parents' oxidativestress damage.
2. application according to claim 1 is it is characterised in that described oxidativestress damage is H2O2Or/and NO causes
Oxidativestress damage.
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| CN104523679A (en) * | 2015-01-09 | 2015-04-22 | 中国人民解放军第三军医大学第一附属医院 | Application of artesunate in preparing medicine for treating and preventing central nerve injury |
| CN105147666A (en) * | 2015-09-09 | 2015-12-16 | 云南大学 | Compound for treating and relieving neurodegenerative disease |
| CN105732654B (en) * | 2016-01-29 | 2017-12-12 | 暨南大学 | Dihydroartemisinine Memantine dyad compound and its preparation method and use |
| CN106309433B (en) * | 2016-09-13 | 2019-04-12 | 南昌大学 | Qinghaosu is preparing the application in neuropathic pain drug |
| CN107802621A (en) * | 2017-12-06 | 2018-03-16 | 中国中医科学院中药研究所 | The purposes of the anti-neuroinflamation of artemisinin B and treatment nerve degenerative diseases |
| CN109364061A (en) * | 2018-10-30 | 2019-02-22 | 澳门大学 | Application of the Artemether in prevention and treatment cerebral apoplexy |
| CN109364064A (en) * | 2018-10-30 | 2019-02-22 | 澳门大学 | Application of the qinghaosu in prevention and treatment cerebral apoplexy |
| CN114668758A (en) * | 2021-05-17 | 2022-06-28 | 澳门大学 | Application of artemisinin and derivatives thereof in preparation of ChAT activity enhancer |
| CN113262301B (en) * | 2021-05-18 | 2022-06-24 | 南京邮电大学 | Multifunctional anti-tumor nano-drug and preparation method and application thereof |
| CN116334109B (en) * | 2022-07-29 | 2024-04-05 | 西南大学 | Application of overexpression of AaMAPK6 gene in sweet wormwood herb in improving artemisinin content and method |
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