| Objective:Alzheimer’s disease（AD）is a chronic progressive central nervous system degenerative disease characterized by progressive memory disorder,cognitive dysfunction,personality change,language disorder and other neuropsychiatric symptoms.It is the most common senile dementia.The pathogenesis of the disease is complex,difficult to summarize the single mechanism,and there is no effective treatment.At present,the widely accepted hypotheses about the pathogenesis include:-amyloid cascade hypothesis,tau protein hypothesis,oxidative stress hypothesis,inflammatory mechanism hypothesis,gene mutation and mitochondrial dysfunction hypothesis,etc.Recent studies have confirmed that mitochondria injury induced cell apoptosis is involved in the pathogenesis of alzheimer’s disease process,in the early AD can be caused by abnormal mitochondria autophagy mitochondrial dysfunction,reduce ATP synthesis,AMPK downstream pathways are activated and continue to further curb energy metabolism,a vicious cycle,eventually lead to neuronal loss.Therefore,timely clearance of damaged mitochondria is very important.Mitochondrial autophagy is a process of selectively identifying and degrading damaged and dysfunctional mitochondria to prevent further cell damage and activate apoptosis.Among them,mitochondrial autophagy regulated by PINK1/Parkin is the focus of current studies.Butylphthalide（Dl-3-n-butylphthalide,NBP）is a commonly used drug in clinical neurology,which has a variety of neuroprotective effects such as improving mitochondrial function,resisting oxidative stress,inhibiting apoptosis,and promoting neuron regeneration.Some studies have confirmed that NBP can improve cognitive impairment symptoms in patients with AD,but the specific mechanism is unknown.This study take AD cell and animal models as the research object,given different concentration of NBP through the AD pathology classic indicators,PINK1/Parkin channel related proteins,autophagy level and mitochondrial morphological structure detection and observation,validated for the following research contents:1)NBP can promote mitophagy by PINK1/Parkin pathways play a role of resistance to the AD;2)Parkin is a key effecting factor of NBP’s anti-AD effect through regulating mitophagy mechanism;3)NBP enhances the ubiquitination activity of Parkin and promotes autophagy by interfering with the expression of the deubiquitination enzyme USP30.Methods:1.APP/PS1 double-transgenic mice aged 6 months and 12 months were used as the animal model of AD,respectively representing the middle stage and the late stage of AD disease.AD model mice representing different disease courses were divided into four groups:AD group（6 months old）,AD group（12 months old）,NBP group（6 months old）and NBP group（12 months old）.C57BL/6J mice aged 6 months were used as blank control group.NBP group mice were intraperitoneally injected 0.25mg/ml sodium butylphthalide injection 0.2ml/10g twice a day,and AD group and blank control group were intraperitoneally injected 0.2ml/10g sodium chloride injection twice a day for a total of 8 weeks.2.After 8 weeks,Morris experiment system and video analysis system were used to evaluate the changes in learning and memory ability of mice in each group:Detection process,a total of 7 days,1 day no adaptation of platform for mice,2-6 days for mice learning period,water maze in the platform,platform hidden in a 0.5 1 cm below the surface,the position of SW（southwest）,mice to study 4 times a day,each entry point respectively,E（east）,N（north）,NW（northwest）,SE（southeast）,random sequence,each time a total of 60 s,if find the platform within the given time in mice,the 30 s to remember platform in platform.If the mice did not find the platform within the prescribed 60s,the mice were guided to take the stage and kept on the stage for 30s.The evasive incubation period was recorded for 5 days.The 7th day was the exploratory period for mice.After the learning experiment was over 24 hours,the platform was removed and the mice were immersed into the water in the direction of NE（northeast）.The recording time was 60s,and the time the mice stayed in the quadrant of the original platform and the number of times they crossed the original platform were recorded.The learning and memory abilities of mice were evaluated by incubation period,time of target quadrant and times of crossing platform.The obtained data were analyzed by anova and LSD.3.Hippocampal tissue samples were obtained from each group of mice after water maze experiment:（1）The expressions of PINK1/Parkin channel related proteins PINK1,Parkin,PHB2,USP30,and autophagy related proteins LC3B and P62 were detected by western blot.The regulation of NBP on the levels of PINK1/Parkin channel and mitochondrial autophagy in AD with different disease course was determined.(2Oobserve the morphological and structural changes of hippocampal neuron mitochondria and autophagosome by transmission electron microscopy--detect the gold standard of mitochondrial autophagy level;（3）Immunohistochemistry was used to detect the expression of senile plaque deposition and autophagy indexes in brain tissue,and to evaluate the improvement of butylphthalide on different disease courses of AD.4.SH-SY5Y cells were cultured in vitro and treated with Aβ25-35（10,20,40,80 M）at different concentrations.The survival rate of the cells was determined by CCK8 method,and the AD cell model was established.Similarly,the optimal concentration（5,10,20,40,80,160 M）of NBP was determined by CCK8 assay.The cells were divided into three groups:blank control group,AD model group and NBP group.5.Detection and comparison of cells between groups:（1）Western blot analysis:PINK1/Parkin channel related proteins PINK1,Parkin,PHB2,USP30,and autophagy related proteins LC3B and P62 were expressed to determine the regulation effect of butylphthalide on mitochondrial autophagy in this channel.（2）Western blot analysis:AD pathological indexes:changes in the expression levels of app-c,A and p-tau,to determine the repair effect of butylphthalide on AD pathological injury;（3）Detection of apoptosis by flow cytometry;（4）Transmission electron microscopy observation:mitochondrial morphological and structural changes and the formation of autophagosome,the gold standard of mitochondrial autophagy level was evaluated;（5）JC-1 detection:mitochondrial membrane potential changes,to clarify the mitochondrial damageResults:1.NBP group improved the learning and memory ability of mice compared with AD group:In terms of learning ability,the escape latency of mice in the AD group was longer than that in the normal group,and the difference was greater with the increase of the age of months,while the escape latency of mice in the NBP group was shorter than that in the AD group.In terms of memory ability,mice in the AD group spent significantly less time in the target quadrant and crossed the platform than mice in the normal group,and the change became more obvious with the increase of the age of months.Mice in the NBP group spent more time in the target quadrant and crossed the platform than mice in the AD group.It is suggested that NBP can improve the learning and memory of AD mice.2.Compared with the AD group,the PINK1/Parkin pathway protein and autophagy markers in the hippocampus of the NBP group were increased:Western blot results showed that,compared with the corresponding AD group,the expression of autophagocyte-related protein LC3-II/LC3-I in the hippocampal tissues of the NBP group at 6 months and 12 months was increased,P62 expression was decreased,PINK1 content was not significantly changed,Parkin content was increased,and USP30was decreased,and the above changes were significantly changed with the age of months.It is suggested that in NBP animal model,mitochondrial autophagy mediated by PINKl/Parkin pathway is enhanced,and Parkin may be the key effection factor of NBP.3.Compared with the AD group,the NBP group had complete mitochondrial morphological structure and increased autophagosome formation in the hippocampal tissue of mice:The ultrastructure of neurons in each group was observed under electron microscopy.It was found that the normal group had complete mitochondrial bilayer membrane structure,clear mitochondrial ridge,complete and smooth cell nuclear membrane structure,clear bilayer membrane structure,and uniform nuclear chromatin.The mitochondria of neurons in NBP group were mostly complete in structure,and some of them were slightly swollen,indicating that the arrangement of mitochondrial crest was disordered,the cell nuclear membrane structure was more regular,the chromatin in the nucleus was less uniform,and multiple autophagosomes were seen in the field of vision.In the AD group,the mitochondria with abnormal neuron structure were further increased,which were manifested as the dissolution of the double-layer membrane structure and the destruction of the inner ridge.At the age of 12 months,AD mice had a large number of degenerated mitochondrial pyknosis in the hippocampus,and the matrix became deeper and accumulated in the axons to form medullary bodies.It suggested that in AD animal models,mitochondria and nuclear morphological structures were destroyed and autophagy was reduced,and NBP could play a role in maintaining and protecting mitochondria and nuclear morphological structures and promoting autophagy.4.Compared with the AD group,the NBP group showed reduced deposition of A in the hippocampus,up-regulated expression of autophagy marker LC3B,and intact morphology of neurons:Changes in the hippocampal CAl area of each group with HE staining:neurons in the normal group were arranged in a neat,round and regular shape with a number of neuronsThe amount was normal,the cytoplasm was uniformly red stained,the nucleus was large and round with pale blue color,and the boundary of nuclear membrane was clear.The neurons in AD group were disordered,the number of them was significantly reduced,the cytoplasm was wrinkled and vacuoles were formed,and the cytoplasmic pyknosis was blue-purple.In the butylphthalide group,a large number of neurons were found to be alive,with clear boundaries and regular shapes.The nuclei were more clearly defined than the regular boundaries.A few cells showed denaturation such as deepened nuclear staining and partial disappearance of cytoplasm.Changes of the hippocampal CAl in each group with immunohistochemical staining:compared with the normal group,the AD group mice 12 The percentage of A positive staining cells was significantly increased and that of LC3B positive staining cells was decreased at the age of 6 months.The NBP group had fewer positive staining cells and more positive staining cells than the AD group.5.CCK8 assay showed no significant change in cell activity for 24 h when the concentration of A 25-35 was 20umol/L.When NBP concentration was 40umol/L,there was no significant change in cell activity and no cytotoxicity.6.Compared with the AD group,the protein and autophagy markers of the PINK1/Parkin pathway in the NBP group were increased,and the pathological indicators of AD were decreased:Western blot results indicated that compared with the AD group,the expression of autophagocyte-related protein lc3-11/lc3-1 in sh-sy5y cells in the NBP group increased,while the expression of P62 decreased.There was no significant change in PINK1content,Parkin content increased,and the content of USP30,app-c,A,tau and p-tau increased.It is suggested that in the NBP cell model,mitochondrial autophagy mediated by PINKl/Parkin pathway is enhanced and has a certain repair effect on the pathological damage of AD.7.Compared with the AD group,the NBP group had more complete mitochondrial morphological structure and increased autophagosome formation in sh-sy5y neurons:The ultrastructure of neurons in each group was observed under electron microscopy.It was found that the normal group had complete mitochondrial bilayer membrane structure,clear mitochondrial ridge,complete and smooth cell nuclear membrane structure,clear bilayer membrane structure,and uniform nuclear chromatin.In the AD group,the number of mitochondria with abnormal structure of neurons was significantly increased,which was manifested by the dissolution of the double-layer membrane structure and the destruction of the inner crista.A large number of degenerated mitochondria were observed in the cells,and the matrix became deeper and accumulated to form medullary bodies.The mitochondria of neurons in NBP group were mostly complete in structure,and some of them were slightly swollen,indicating that the arrangement of mitochondrial crest was disordered,the cell nuclear membrane structure was more regular,the chromatin in the nucleus was less uniform,and multiple autophagosomes were seen in the field of vision.It suggested that in AD cell model,mitochondria and nuclear morphological structure were destroyed and autophagy was reduced,and NBP could play a role in maintaining and protecting mitochondria and nuclear morphological structure and promoting autophagy.8.The apoptosis rate in the NBP group was lower than that in the AD group:The apoptosis rate in the AD group was significantly higher than that in the normal group after the treatment of sh-sy5y cells by A 25-35,while that in the NBP group was lower than that in the AD group.9.The detection NBP JC-1 can improve AD group cell mitochondria damage:Comparison between groups of cellsΔbits of the change of m,Δbits of m high,gathered to form polymers,JC-1 red fluorescence,otherwise produce green fluorescent,red cell fluorescence AD group was obviously lower,obviously increase the green fluorescence and red fluorescence to green fluorescent,after pretreatment with NBP red fluorescence trends are suppressed the shift towards green fluorescence.Conclusion:Butylphthalide by promoting Parkin and interference to the expression of ubiquitin digesting enzyme USP30 improve Parkin ubiquitin activity promoting mitochondria autophagy,to improve the symptoms of AD animal model of cognitive impairment,reducing A beta deposition and abnormal tau protein phosphorylation,inhibition of mitochondrial damage,reduce the apoptosis,maintain normal ultrastructure of nerve protective effect.Parkin is a key effection factor of butylphthalide regulating mitochondrial autophagy,providing theoretical and laboratory basis for the search of new therapeutic targets for the early prevention of AD and the slowing down of disease progression. |
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