CN111939163A

CN111939163A - Application of kuduoning in preparing medicine for preventing and treating Huntington disease

Info

Publication number: CN111939163A
Application number: CN201910407227.0A
Authority: CN
Inventors: 马丽香; 赛音贺西格; 杨炜圻
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2020-11-17

Abstract

The invention belongs to the technical field of medicines, relates to a new application of a medicine kudzu vine root bark nin, and particularly relates to an application of an Indian traditional medicine kudzu vine root bark nin (Gedunin) in preparation of a medicine for preventing and treating Huntington diseases. The invention uses cellular immunofluorescence chemistry, immunoblotting and qRT-PCR, detect cell line Neuro-2a of transfection mutation Huntington protein from protein level and mRNA level separately, and Huntington patient fibroblast and Huntington patient can induce the multipotential stem cell to differentiate the neuron directionally, the result shows, Kudnin can degrade the cell line Neuro2a transfection mutation Huntington protein, degrade Huntington patient source fibroblast and can induce multipotential stem cell differentiation neuron endogenous mutation Huntington protein aggregate and enter into the nucleus mutation Huntington protein, its effect has dose dependence and time dependence. Further can be used for preparing medicines for preventing and treating Huntington's disease.

Description

Application of kuduoning in preparing medicine for preventing and treating Huntington disease

Technical Field

The invention belongs to the technical field of medicines, relates to a new application of a medicine kudzu vine root bark nin, and particularly relates to an application of an Indian traditional medicine kudzu vine root bark nin (Gedunin) in preparation of a medicine for preventing and treating Huntington diseases. The Indian traditional medicine kudzu vine root bark tannin can achieve the effect of treating Huntington disease by degrading abnormal mutant Huntington protein aggregates in cells of Huntington patients and mutant Huntington protein entering cell nuclei.

Background

The prior art discloses that Huntington's Disease (HD) is a monogenic dominant-inherited neurodegenerative disease in which IT15 gene on chromosome 4 is mutated, and the abnormal amplification of CAG repeat polyglutamine (PolyQ) results in the encoding of abnormal mutant huntingtin protein aggregates; there is currently no effective treatment (Kieburtz et al, 2018; MacDonald et al, 1993; Squitieri et al, 2016). Practice shows that the HD patients often suffer uncontrolled decline of choreoid movements and cognitive functions, as well as mental disorders such as depression, anxiety, dementia, and eventually life cannot take care of oneself, swallowing and breathing difficulties occur, until death (Walker, 2007); statistically, the disease course from morbidity to mortality lasts for 10-25 years. Studies have shown that the number of CAG repeats in huntington patients is above 37, that abnormally amplified polyglutamine in the mutant protein is structurally converted to β -sheet, forming entangled fibrillar and non-fibrillar aggregates, and that this incorrect folding can cause the mutant huntingtin protein to enter the nucleus, sequestering important cellular components, causing intracellular transcriptional disturbances, leading to loss of cellular normal function and increased cytotoxicity (Aylward et al, 2011; difigia et al, 1997; Saudou and Humbert, 2016); abnormal mutant huntingtin aggregates and mutant huntingtin entry into the nucleus selectively cause gamma aminobutyric acid (GABA) capable neurons in the striatum to die, resulting in corresponding neurological dysfunction with severe uncontrolled movements (Fisher and Hayden, 2014). The research of the inventor finds that: small molecule drugs can degrade mutant huntingtin proteins, reduce abnormal protein accumulation and cytotoxicity, and may be a therapeutic approach for effectively treating huntington's disease.

Heat shock protein 90 (Hsp 90) is the most abundant chaperone in the cell, and is associated with other chaperone molecules to form chaperone complexes, which are involved in protein assembly, folding, transport and degradation (Massey, 2010). Hsp90 forms a complex with accessory chaperones that stabilizes downstream proteins to which it binds, inhibiting downstream protein degradation, while inhibitors of Hsp90 promote downstream proteins to form proteasome-targeted chaperone complexes with Hsp90, enhancing degradation of Hsp90 downstream proteins through the ubiquitin-proteasome system pathway (Bagatell et al, 2001). Both mutant huntingtin aggregates and wild-type huntingtin are typical Hsp90 downstream proteins, and the inhibitor of Hsp90, NVP-AUY922, degrades mutant huntingtin in a dose-dependent manner (Baldo et al, 2012). Reports disclose that gedunin is a natural inhibitor of Hsp90 isolated from the neem tree and has been used primarily in indian traditional medicine for the treatment of malaria (patward han et al, 2013).

Based on the current situation and the foundation of the prior art, the inventor of the present application intends to provide a new application of the traditional medicine Gedunin, in particular to the application of the indian traditional medicine Gedunin (gelonin) in the preparation of the medicine for preventing and treating huntington diseases.

References relevant to the present invention:

Aylward,E.H.,Nopoulos,P.C.,Ross,C.A.,Langbehn,D.R.,Pierson,R.K.,Mills,J.A.,Johnson,H.J.,Magnotta,V.A.,Juhl,A.R.,and Paulsen,J.S.(2011).Longitudinal change in regional brain volumes in prodromal Huntington disease.J Neurol Neurosurg Psychiatry 82,405-410.

Bagatell,R.,Khan,O.,Paine-Murrieta,G.,Taylor,C.W.,Akinaga,S.,and Whitesell,L.(2001).Destabilization of steroid receptors by heat shock protein 90-binding drugs:a ligand-independent approach to hormonal therapy of breast cancer.CLIN CANCER RES 7,2076-2084.

Baldo,B.,Weiss,A.,Parker,C.N.,Bibel,M.,Paganetti,P.,and Kaupmann,K.(2012).A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90(Hsp90)client protein.J BIOL CHEM 287,1406-1414.

DiFiglia,M.,Sapp,E.,Chase,K.O.,Davies,S.W.,Bates,G.P.,Vonsattel,J.P.,and Aronin,N.(1997).Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.SCIENCE 277,1990-1993.

Fisher,E.R.,and Hayden,M.R.(2014).Multisource ascertainment of Huntington disease in Canada:Prevalence and population at risk.MOVEMENT DISORD 29,105-114.Kieburtz,K.,Reilmann,R.,and Olanow,C.W.(2018).Huntington's disease:Current and future therapeutic prospects.MOVEMENT DISORD.

MacDonald,M.E.,Ambrose,C.M.,Duyao,M.P.,Myers,R.H.,Lin,C.,Srinidhi,L.,Barnes,G.,Taylor,S.A.,James,M.,and Groot,N.,et al.(1993).A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.CELL 72,971-983.

Massey,A.J.(2010).ATPases as drug targets:insights from heat shock proteins 70 and 90.J MED CHEM 53,7280-7286.

Patwardhan,C.A.,Fauq,A.,Peterson,L.B.,Miller,C.,Blagg,B.S.,and Chadli,A.(2013).Gedunin inactivates the co-chaperone p23 protein causing cancer cell death by apoptosis.J BIOL CHEM 288,7313-7325.

Saudou,F.,and Humbert,S.(2016).The Biology of Huntingtin.NEURON 89,910-926.Squitieri,F.,Griguoli,A.,Capelli,G.,Porcellini,A.,and D'Alessio,B.(2016).Epidemiology of Huntington disease:first post-HTT gene analysis of prevalence in Italy.CLIN GENET 89,367-370.

Walker,F.O.(2007).Huntington's disease.LANCET 369,218-228.。

disclosure of Invention

The invention aims to provide a new application of a medicine kudzu disease medicine based on the current situation and the foundation of the prior art, and particularly relates to an application of an Indian traditional medicine kudzu disease medicine (Gedunin) in preparing a medicine for preventing and treating Huntington diseases.

The present invention has been experimentally confirmed to confirm that the conventional medicine gedunin can be used for preparing a novel medicine for preventing and treating huntington's disease by using the medicine dose and time for gedunin to degrade abnormal mutant huntingtin aggregates in cells of huntington patients and mutant huntingtin entering into cell nuclei, and the way for gedunin to degrade mutant huntingtin.

Experiments of the invention show that the gedunin can degrade mutant huntingtin protein which is a pathological protein of huntington disease in neurodegenerative diseases;

in the invention, the kudzu degradation abnormal mutant huntingtin protein aggregate in the fibroblasts of the Huntington patients is acted by 5-15 mu M for 12-24 h;

in the invention, the kudzu degradation huntingtin mutant huntingtin which enters into cell nucleus from fibroblast of huntington patient is acted by 10-20 mu M in dosage and 12-24h in action time;

in the invention, the nodulin degrades abnormal mutant huntingtin protein aggregates in neurons of the huntington patient and the mutant huntingtin protein entering the cell nucleus, the dosage is 5-15 mu M, the time is 12-24h, the effect appears after 8-10 days, and the nodulin degrades the mutant huntingtin protein in the neurons and has long-acting effect;

in the present invention, gedunin degrades mutant huntingtin in huntington patients via the ubiquitin-proteasome system pathway rather than the autophagy-lysosomal system pathway.

In the present invention, the effect of gedunin on degrading mutant huntingtin was detected separately from three different cell types using the following method:

degradation of EGFP-HDQ74 transfected by mouse neuroblastoma (Neuro2a) cells by gedunin

Culturing mouse neuroblastoma (Neuro2a) cells in DMEM and 10% FBS culture solution, transfecting enhanced green fluorescent protein and Huntington protein first exon fusion gene EGFP-HDQ74 containing 74 CAG trinucleotide repeats to Neuro-like Neuro2a cells by lipofectamine2000 when the growth reaches about 80%, and allowing gedunin with different doses of 5 muM, 10 muM, 15 muM and 20 muM for 24 hours and allowing gedunin with the same concentration of 20 muM for 8 hours, 16 hours and 24 hours respectively; after the drug treatment, carrying out experiments such as immunoblotting, cell immunofluorescence staining and qRT-PCR;

effect of Kudzuvine on protein degradation pathways

MG132 and Bafilomycin a1 are lysosomal and proteasome inhibitors, respectively, to observe which pathway gedunin-degrading mutant huntingtin protein is degraded through: after transfection, the mutant huntingtin expression levels were observed after 16 hours of action with 20 μ M gedunin per group, 6 hours of action with low concentration, high concentration of MG132(5 μ M,10 μ M) or Bafilomycin a1(0.5 μ M,1 μ M), DMSO and gedunin (20 μ M) as blank and positive controls, respectively, for immunoblotting and cytoimmunofluorescence staining;

(III) Gedu Ning degradation of mutant Huntington protein in Huntington's lineage-derived fibroblasts

Selecting two groups of normal human and three groups of Huntington patients with different CAG repetitive sequences, culturing human-derived fibroblasts by DMEM and 10% FBS culture solution, adding 5 mu M and 10 mu M of gedunin respectively for 24 hours, performing cell immunofluorescence staining, scanning by laser confocal method, and detecting and counting the area size of mutant Huntington protein and the number of cells entering the mutant Huntington protein in the cell nucleus;

(IV) the proteasome system inhibitor MG132 prevents the effect of gedunin on the degradation of huntingtin-derived fibroblast mutant huntingtin;

in the invention, a Huntington patient with 55 CAG repetitive sequences is selected, the fibroblast of the Huntington patient is cultured by DMEM and 10% FBS culture solution, 10 mu M kudzu is added for 12 hours, then 10 mu M MG-132 is added for further incubation for 12 hours, and a control group is arranged; after cell immunofluorescence staining is carried out, scanning is carried out through laser confocal, and the area size of the mutant huntingtin protein and the number of cells entering the mutant huntingtin protein in a cell nucleus are detected and counted;

(V) degradation of mutant huntingtin proteins in huntingtin patient-derived neurons differentiated from Induced Pluripotent Stem Cells (iPSCs)

Adopting a Matrigel-supported feeder layer-free induced pluripotent stem cell culture system and culturing by using E8 culture solution; when the clone growth density reaches 85% (about 5 days after passage), digesting by Dispase, centrifuging, culturing by an induced differentiation culture medium (DMEM/F12, 100xN2, 100xNEAA, 100x-Glutmax) until the culture reaches 40 days, digesting neurospheres and adherent neurons; adding 5-15 μ M of puerarin on 47 days of neuron adherence, and acting for 24 hr; continuously culturing for 62 days; neurons were fixed at 50 days, 57 days and 62 days, respectively; after cellular immunofluorescent staining, scanning was performed by a laser confocal microscope to detect and count the size of the area of the neuronal mutant huntingtin protein into which huntington patient-derived iPSCs were differentiated and the number of cells entering the mutant huntingtin protein within the nucleus.

The results of the experiments of the invention show that:

the method comprises the following steps that (I) 20 mu M of gedunin is added after transfected mutant Huntington protein in a cell line Neuro2a is detected by adopting an immunoblotting experiment, the mutant Huntington protein can be degraded, the gedunin has dose dependence on the degradation of the mutant Huntington protein, the expression level of the mutant Huntington protein is gradually reduced along with the prolonging of the action time of the gedunin after the action of the gedunin is carried out for 8 hours, and the group in which the gedunin acts for 24 hours has statistical significance compared with a control group and is consistent with a cell fluorescence result; the mRNA content of the mutant Huntington protein in the group with 20 μ M Kudurin acting for 24 hours and the control group is not statistically different by using qRT-PCR, which indicates that the degradation of the mutant Huntington protein by Kudurin is not at the transcription level, but at the protein level; the results confirmed that 20. mu.M gedunin was able to degrade the mutant Huntington protein exogenously transferred into the cells for 24 hours (as shown in FIG. 1).

(II) immunoblotting tests detect that in transfected Neuro2a cells, the proteasome system inhibitor MG132 can save the degradation effect of gedunin on mutant Huntington protein, but the lysosomal inhibitor BafilomycinA1 can not save the degradation effect of gedunin on mutant Huntington protein, which is consistent with the cell fluorescence result; the results demonstrate that the proteasome inhibitor MG132 antagonizes gedunin, i.e., gedunin exerts activity by promoting proteasome hydrolysis, degrading the mutant huntingtin protein (shown in figure 2).

(III) after cellular immunofluorescence staining, scanning through laser confocal scanning, detecting and counting the size of the area of mutant Huntington protein aggregates in normal human fibroblasts and mutant Huntington protein aggregates in Huntington patient fibroblasts, and the number of cells entering the mutant Huntington protein in cell nucleus, adding 5 μ M and 10 μ M of gedunin respectively for 24 hours, wherein the area of Huntington protein in normal human fibroblasts is not obviously changed statistically, the area of Huntington protein aggregates added in 5 μ M of mutant Huntington protein aggregates in Huntington patient fibroblasts is reduced, but the number of cells entering the mutant Huntington protein in cell nucleus is not obviously changed, the area of mutant Huntington protein aggregates added in 10 μ M of kudingoin protein aggregates is reduced, and the number of cells entering the mutant Huntington protein in cell nucleus is reduced; the results demonstrated that 5 μ M and 10 μ M gedunin were able to degrade mutant huntingtin aggregates and were dose dependent, and that gedunin was targeted to degrade endogenous mutant huntingtin in huntingtin patients without affecting the normal function of normal human huntingtin (as shown in figure 3).

(IV) after cellular immunofluorescent staining, scanning by laser confocal microscopy to detect and count the size of the area of mutant huntingtin protein aggregates in the fibroblasts of the Huntington patients and the number of cells entering the mutant Huntington proteins in the cell nucleus, wherein 10 mu MMG-132 reduces the degradation of Kudurin not only to the endogenous mutant Huntington protein aggregates but also to the mutant Huntington proteins in the cell nucleus; the results demonstrate that MG132 can rescue the effect of geduninfluenced mutant huntingtin degradation by the proteasome pathway (as shown in figure 4).

(V) after cell immunofluorescence staining, scanning through laser confocal, detecting and counting the area size of mutant huntingtin protein aggregates in neurons differentiated from the Huntington patient-derived iPS cultured to different days after adding drugs and the number of cells entering the mutant Huntington protein in cell nuclei; the results showed that the addition of 5 μ M gedunin did not affect the morphology of the neurons differentiated from the huntington-derived iPSCs; in the drug-free control group, the area size of mutant huntingtin aggregates and the number of cells entering mutant huntingtin in the nucleus gradually increased from 50 days to 62 days; added to the 5 μ M gedunin group, the area size of mutant huntingtin aggregates and the number of cells in the nucleus into mutant huntingtin were reduced on day 57, with a slight rebound occurring on day 62, but still less than the non-dosed control; the results further indicate that gedunin is effective in degrading endogenous mutant huntingtin aggregates and mutant huntingtin that enters the nucleus (as shown in figure 5).

The invention provides application of Indian traditional medicine kudzu vine root bark nin (Gedunin) in preparing a novel medicine for preventing and treating Huntington disease. The present invention has been experimentally confirmed to confirm that the conventional medicine gedunin can be used for preparing a novel medicine for preventing and treating huntington's disease by using the medicine dose and time for gedunin to degrade abnormal mutant huntingtin aggregates in cells of huntington patients and mutant huntingtin entering into cell nuclei, and the way for gedunin to degrade mutant huntingtin.

Drawings

FIG. 1 shows the degradation of EGFP-HDQ74 transfected by Neuro2a cells by gedunin.

Figure 2 shows the effect of gedunin on the proteolytic pathway.

FIG. 3 shows the degradation of mutant huntingtin aggregates in huntingtin-derived fibroblasts and huntingtin-derived fibroblasts by gedunin.

FIG. 4 shows the degradation of huntingtin patient-derived fibroblast mutant huntingtin by the proteasome system inhibitor MG132 rescue gedunin.

Figure 5 shows the degradation of mutant huntingtin in neural cells differentiated from huntington patient-derived Induced Pluripotent Stem Cells (iPSCs) by gedunin.

Detailed Description

Example 1

The degradation effect of gedunin on EGFP-HDQ-74 transfected by mouse neuroblastoma (Neuro2a) cells is as follows:

1. cell culture: each passage at 1:5

(1) Removing the culture solution by suction, washing with 3-10ml 0.01M PBS for 1 time;

(2) adding 3-5ml of 0.25% pancreatin-EDTA for digestion for 2-3 minutes;

(3) adding 3-5ml DMEM culture solution containing 10% FBS to stop digestion, and gently blowing and beating the separated cells;

(4) sucking the cell suspension in the dish into a 15ml centrifuge tube;

(5) centrifuging (1200rpm 2min), and removing the supernatant by aspiration;

(6) add 1ml of DMEM medium containing 10% FBS to resuspend, aspirate 200. mu.l into a 10cm dish, seed both dishes, aspirate 67. mu.l into a 6cm dish, and incubate at 37 ℃ with 5% CO 2.

2. Cell transfection: one day before transfection, two 6-well plates and one 24-well plate were plated

(1) Taking a 10cm culture dish with cells growing to 90%, digesting and centrifuging by using 0.25% pancreatin-EDTA, then re-suspending the cells by using 1ml culture solution, taking a 6-hole plate, culturing 150 mu l of each cell in each hole for 24 hours, and then transfecting 74Q; the other 6-well plate operates the same; taking a cleaned round glass slide with the diameter of 13mm from absolute ethyl alcohol, putting the round glass slide into a 24-hole plate, and drying the round glass slide in an ultra-clean bench; taking 6cm dish, resuspending the cells with 1ml of culture solution, wherein 50 mu l of each slide cell is planted, and adding 500 mu l of culture solution into each hole after 2 hours;

(2) transfection was performed in 6-well plates according to the Lipofectamine2000 kit: adding 4ug X6 wells of plasmid DNA into 250ul of OPTI-MEM culture medium, and mixing; adding 10ul of Lipofectamine2000 with 6 holes into another 250ul of OPTI-MEM, gently mixing, and standing for 5 minutes at room temperature; mixing the DNA suspension and the Lipo2000 suspension, gently mixing the mixture in a total volume of 500ul multiplied by 6 holes, and standing the mixture for 20 minutes at room temperature; fourthly, abandoning the old culture medium, adding 500ul of mixed solution of DNA and Lipo2000 into each hole, shaking the hole plates back and forth and left and right to mix uniformly, placing the mixed solution into a 5% CO2 incubator at 37 ℃ for culturing for 4 to 6 hours, replacing 10% FBS DMEM culture solution, and culturing for 24 hours; the other 6-well plate operates the same;

(3) transfection was performed in 24-well plates according to the Lipofectamine2000 kit: adding 0.8ug of plasmid DNA with 24 holes into 50ul of OPTI-MEM culture medium, and mixing; 2ul of Lipo2000 with 24 holes is added into the other 50ul of OPTI-MEM with 12 holes, and the mixture is gently mixed and stands for 5 minutes at room temperature; mixing the DNA suspension and the Lipo2000 suspension, gently mixing the mixture with the total volume of 100ul, and standing the mixture for 20 minutes at room temperature; and fourthly, abandoning the old culture medium, adding 100ul of the mixed solution of the DNA and the Lipo2000 into each hole, shaking the hole plates back and forth and left and right to mix uniformly, placing the mixture into an incubator at 37 ℃ and 5% CO2 for culturing for 4 to 6 hours, replacing 10% FBS DMEM culture solution, and culturing for 24 hours.

3. Adding medicine: drug treatment was started 3 hours after transfection

(1) Diluting 10mM Kuduling with culture medium to different concentrations, such as 5 μ M,10 μ M, 15 μ M, and 20 μ M, and allowing to act for 24 hr;

(2) acting at the same concentration (20 μ M) for different time periods, namely 8 hours, 16 hours and 24 hours respectively;

4.6 well plate medicine-added cell extraction of total protein

(1) Add 500. mu.l of 0.01M PBS per well of cells; the cells were gently shaken for 1 minute for washing, and then the washing solution was discarded; repeating the operation twice, washing the cells three times, and washing off serum in the culture solution;

(2) adding 500 μ l of 0.01M PBS, collecting cells with a clean scraper in a 1.5ml centrifuge tube, centrifuging at 4 deg.C and 2500g for 10 min;

(3) absorbing supernatant, estimating the volume of the sample, adding 30-60ul of cell lysate containing PMSF, and uniformly mixing by using an oscillator; standing for 30 minutes at 4 ℃;

(4) centrifuging at 12000g at 4 deg.C for 10 min;

(5) taking the supernatant, wherein one part of the supernatant is diluted by 1uL of sample and 4 uL of PBS, and then 2uL of the supernatant is taken for measuring the protein concentration; preparing a BCA working solution A: b, diluting BSA standard substances with different concentrations, and diluting the sample with PBS; adding 20ul of sample into each well, adding 20ul of standard substance, adding 200ul of BCA working solution, mixing, incubating at 37 ℃ for 30min or at room temperature for 60min, and reading OD value by a 560nm wavelength filter of an enzyme-labeling instrument; concentration values were analyzed with Excel 2007;

(6) the other part was boiled with 5 Xloadingbuffer for 5 minutes and stored.

5. Immunoblotting experiment for detecting the clearance effect of the gedunin with different concentrations on the mutant huntingtin and the clearance effect of the gedunin with different time on the mutant huntingtin

(1) Preparing separation gel and concentrated gel;

(2) protein: taking out from-20 deg.C, adding 5 × loading buffer solution to dilute, and decocting in boiling water for 5min to obtain denatured protein;

(3) sample adding: adding 10-20 mul (the general protein loading amount is 10-20ug) samples into each hole;

(4) electrophoresis: electrophoresis is carried out for 40-50 minutes at a constant voltage of 200V, and the electrophoresis is stopped when bromophenol blue reaches the bottom of the lower layer gel;

(5) film transfer: the following materials were placed in order: black face → sponge → filter paper → glue → PVDF membrane → filter paper → sponge, under constant voltage 100V, rotate membrane for 90 min;

(6) and (3) sealing: adding 5% skimmed milk powder in PBS, sealing, and shaking for 1 hr at normal temperature;

(7) adding a primary antibody: removing the sealing solution by suction, adding primary antibody, and incubating overnight in a side-swinging shaking table at 4 ℃;

(8) adding a secondary antibody: recovering primary antibody, washing membrane with TBST for 5min for 3 times, adding secondary antibody, and incubating for 1 hr by rocking bed at normal temperature; absorbing and removing the secondary antibody, washing the membrane for 10min by TBST, and totally 3 times;

(9) putting the PVDF film on a preservative film with the front surface facing upwards, dripping ECL luminescent agent on the film, and removing the luminescent agent on the film after 1min (note that the film cannot be dried);

6. cellular immunofluorescence staining

(1) Fixing: cells were fixed with ice 4% PFA (paraformaldehyde) for 15-20 min, washed 3 times with 0.01M PBS for 5min each;

(2) membrane breaking: rupture the membrane for 20 minutes with 0.2% Triton-100, wash 1 time with 0.01M PBS;

(3) and (3) sealing: blocking with donkey serum (10%) for 1 hour;

(4) adding DAPI: adding DAPI according to the concentration of the working solution (1: 1000), and placing for 15-20 minutes at room temperature in a dark place;

(5)0.01M PBS 3 times, each time for 5 minutes;

(6) and (3) sealing: mounting the wafer with a water-soluble anti-quenching mounting agent.

7. Scanning by laser confocal microscope and counting fluorescence expression quantity

(1) 3D scanning is adopted;

(2) the Z-axis was scanned by Image J software to obtain a Z-stabilized Image, and then the expression level of green fluorescence was counted.

RT-PCR method to detect the effect of Kudnin on the mRNA expression level of mutant Huntington protein

(1) RNA extraction: in order to prevent RNA pollution, the whole process is carried out in a super clean bench, and a gun head and a centrifuge tube used in the experiment are all RNA-free enzyme products;

firstly, culturing cells in a monolayer way, namely washing 0.01MPBS once, adding TRIZOL lysed cells into a culture plate, adding 1 ml/hole into a 6-hole plate, adding 500 ul/hole into a 12-hole plate, adding 250 ul/hole into a 24-hole plate, and sucking and beating by using a pipette for several times. The amount of TRIZOL to be used depends on the area of the culture plate and does not depend on the number of cells;

adding 1/5 volumes of chloroform, fully whirling for 15 seconds, and standing for 3 minutes at room temperature;

③ centrifuging at 12000rpm at 4 ℃ for 15 minutes; the sample was divided into three layers: the bottom layer is a yellow organic phase, and the upper layer is a colorless aqueous phase and a middle layer;

fourthly, transferring the water phase to a new tube. Precipitating the RNA in the aqueous phase with isopropanol of equal volume to the supernatant; turning upside down, mixing gently, and standing at room temperature for 20 min;

centrifuging at 12000rpm at 4 deg.c for 15 min; removing the supernatant;

sixthly, washing the RNA sediment with 1ml 75% ethanol, centrifuging the RNA sediment at 8000rpm and 4 ℃ for 5 minutes, and removing the supernatant;

seventhly, the concentration and the purity of the RNA are measured by a micro-spectrophotometer, and OD260/280 is in the range of 1.8-2.0. The RNA pellet was vacuum-dried.

(2) Reverse transcription, RNA was taken out from a freezer at-80 ℃ and thawed, and a reaction solution was prepared in a 0.2. mu.l PCR tube

The reverse transcription reaction conditions were 37 ℃ for 15min (reverse transcription reaction), 85 ℃ for 5sec (reverse transcriptase inactivation reaction), 4 ℃ temperature reduction or short-term storage.

(3) RT-PCR is carried out to prepare PCR reaction solution (the reaction solution is prepared on ice),

wherein the sequences of the primers used in the experiment are shown in the table,

。

(4) two-step PCR amplification standard procedure: pre-denaturation at 95 ℃ for 90 seconds, PCR reaction for 40 cycles at 95 ℃ for 5 seconds; 60 ℃ for 30 seconds.

(II) the influence of the gedunin on the protein degradation pathway comprises the following specific processes:

1. cell culture: passage at 1:5 each time;

2. cell transfection: paving plates one day before transfection, and paving two 6-hole plates and one 24-hole plate;

3. adding medicine: drug treatment was started 3 hours after transfection;

the expression level of mutant huntingtin protein DMSO and gedunin (20 μ M) were observed for each group after 16 hours of action with 20 μ M addition of gedunin and 6 hours of action with low concentration, high concentration of MG132(5 μ M,10 μ M) or Bafilomycin A1(0.5 μ M,1 μ M) as a blank control and a positive control, respectively;

4.6 adding medicine into the pore plate to extract total protein;

5. the immunoblotting experiment detects the influence of gedunin on protein degradation pathway MG132 and Bafilomycin A1(Baf A1);

6. performing cellular immunofluorescence staining;

7. scanning by a laser confocal microscope and counting the fluorescence expression quantity.

(III) the degradation effect of gedunin on mutant huntingtin in fibroblasts derived from normal human and mutant huntingtin aggregates in fibroblasts derived from huntington patient is specifically as follows:

1.24 well plate is laid with slide, cultured fibroblast from Huntington patient with moderate density is planted on the slide, put into incubator to culture overnight;

2. diluting 10mM Kudu-ning mother liquor into 5 μ M and 10 μ M with culture solution, respectively adding fibroblast from different sources, and reacting for 24 hr, wherein the rest materials are used as control;

3. cellular immunofluorescence staining

(3) and (3) sealing: blocking with 10% Donkey serum (Donkey-serum) for 60 min;

(4) adding a primary antibody: antibody 3B5H10 was expressed as 1: adding 0.2% triton-100, 5% Donkey-serum and PBS to 5000 concentration, 100 μ l each, 4 ° overnight;

(5) removing primary antibody, washing with 0.01M PBS for 3 times, and standing for 5-10 min each time;

(6) adding a secondary antibody: fluorescent secondary antibody and DAPI were added as 1: at a concentration of 1000, 100. mu.l of incubation solution of 5% donkey serum and PBS was added simultaneously (1: 1000). Placing the mixture for 60 minutes at room temperature in a dark place;

(7) removing II antibody, washing with 0.01M PBS for 3 times, and standing for 5-10 min each time;

(8) and (3) sealing: and sealing the water-soluble fluorescent anti-quenching sealing agent, and standing overnight at room temperature in a dark place.

4. Scanning by laser confocal microscope and counting fluorescence expression quantity

(1) The uniform field of view of the cells was looked for under a Leica SP 840-fold dry mirror and the thickness of the cells was determined on the Z-axis using a Z-stack scan. Scanning parameters of the Leica SP8 laser confocal microscope are as follows: pixel: 1024 × 1024(pixel), scanning speed: 100 Hz; linear (liner): 2; the Z-stack spacing is 1 mu m; a certain dyeing background difference exists, so other parameters can be adjusted according to requirements;

(2) z-axis scanning pictures of Z-stage (maximum intensity projection) are processed by Image J software, and then the area of the mutant Huntington protein aggregate and the number of cells entering the mutant Huntington protein in the cell nucleus are counted;

cell counting of the mutant huntingtin protein entering the nucleus was performed using a cell counting (cell counter) menu counting function in the software, as follows:

firstly, initializing an image (initial) and entering a counting interface;

selecting a count type button (counter type);

thirdly, counting the cells entering the mutant huntingtin protein in the nucleus by using the right button of the mouse;

fourthly, storing the data into an excel data table after counting;

(3) statistical analysis was performed using SPSS20.0 software.

(IV) the proteasome system inhibitor MG132 can rescue the degradation effect of Kudzuvine on the Huntington patient-derived fibroblast mutant Huntington protein, and the concrete process is as follows:

2. adding 10 μ M Kudnin into fibroblast derived from Huntington patient for 12 hr, changing culture medium for control group after 12 hr, adding 13210 μ M MG into another group, and culturing for 12 hr;

3. cellular immunofluorescence staining, primary antibody using 3B5H 10;

4. scanning by a laser confocal microscope and counting the fluorescence expression quantity.

(five) Kudurin degradation of mutant Huntington protein in neural cells differentiated from induced pluripotent stem cells (iPS) derived from Huntington patients (since neuron differentiation method is very complicated and briefly described here)

1. Culturing adherent neurons on slide by using differentiation induction culture medium (DMEM/F12, 0.5x N2, 1xNEAA, 1xL-G), and half-changing the liquid every other day;

2. adding 5 μ M of gedunin on day 47, and acting for 24 hr; continuously culturing for 62 days; neurons were fixed at 50 days, 57 days and 62 days, respectively;

3. cellular immunofluorescence staining, primary antibody using 3B5H10 and Tuj 1;

The results show that:

1. exogenous mutant huntingtin for degrading kudingvine root

The cell line Neuro2a transfected mutant Huntington protein 3 hours after adding kudingtin 20u M for 24 hours can degrade the mutant Huntington protein, kudingtin exogenous into the cell of mutant Huntington protein degradation dose dependent and time dependent, and kudingtin on mutant Huntington protein degradation is not in gene level, but in protein level; the enzyme inhibitor MG132 antagonizes gedunin, i.e., gedunin exerts its activity by promoting proteasome hydrolysis, degrading the mutant huntingtin protein.

2. Endogenous mutant huntingtin protein degraded by gedunin

The mutant huntingtin in normal human fibroblasts and the mutant huntingtin in huntington patient fibroblasts are added with 5 mu M and 10 mu M of gedunin respectively to act for 24 hours, and the area of the mutant huntingtin in the normal human fibroblasts is not statistically obviously changed; the area of the huntingtin patient fibroblasts added to the 5 μ M mutant huntingtin aggregates was reduced, but there was no significant change in the number of cells entering the mutant huntingtin in the nucleus; the area of the huntingtin patient fibroblasts added to the 10 μ M mutant huntingtin aggregates decreases and the number of cells in the nucleus that enter the mutant huntingtin decreases; gedunin is a targeted degradation of endogenous mutant huntingtin in huntington patients and has the effect that dose dependence does not affect the normal function of huntingtin in normal humans; MG132 can save the degradation effect of kudingvine on endogenous mutant Huntington protein through a proteasome pathway; adding medicines to the neurons differentiated from the iPS derived from the Huntington patient, and culturing the neurons for different days, wherein the form of the neurons differentiated from the iPS derived from the Huntington patient cannot be influenced by adding 5 mu M of gedunin; the drug free control group and the 5 μ M added gedunin group further demonstrated that gedunin was effective in degrading endogenous mutant huntingtin aggregates and mutant huntingtin that entered the nucleus.

Claims

1. Use of gedunin in the preparation of a medicament for the prevention and treatment of huntingtin by degrading intracellular and neuronal abnormal mutant huntingtin (mHTT) aggregates and mutant huntingtin entering the nucleus, with dose-and time-dependent effects.

2. The use of claim 1, wherein said gedunin-degrading mutant huntingtin protein passes through the ubiquitin-proteasome system pathway.

3. Use according to claim 1, wherein the gedunin degrades abnormal mutant huntingtin (mHTT) aggregates in cells at a dose of 5-15 μ M for a time of 12-24 h.

4. Use according to claim 1, wherein the mutant huntingtin (mHTT) degraded by gedunin into the nucleus is administered in an amount of 10 to 20 μ M for a period of 12 to 24 hours.

5. The use according to claim 1, wherein the nodulin-degrading abnormal mutant huntingtin aggregates in neurons and mutant huntingtin entering nuclei are administered at a dose of 5 to 15 μ M for a period of 12 to 24 hours.