To Explore The Effect And Mechanism Of Modified Shuyu Pills On Reducing Oxidative Stress Injury Of Neurons In APP/PS1 Mice Based On Mitochondrial Mass Regulation

ObjectiveThis study explores the pathogenesis of Alzheimer's disease(AD)from the perspective of insufficient Medullary sea and phlegm and blood stasis,and clarifies the theoretical basis for the treatment of AD with Modified Shuyu Pills;Combined with behavioral detection,immunofluorescence staining,Western Blot,real-time quantitative PCR,transmission electron microscope scanning and other methods,from the perspectives of mitochondrial dynamics,mitophagy,and mitochondrial biosynthesis,exploring the effect of Modified Shuyu Pills on the oxidative stress level of neurons in APP/PS1 double transgenic mice,clarifying the mechanism of Modified Shuyu Pills in improving learning and memory ability of APP/PS1 double transgenic mice.Methods1.Theoretical exploration:Discussing the understanding of AD in TCM from the aspects of disease location,pathogenesis and pathogenic factors;The theoretical basis for the treatment of AD with Modified Shuyu Pills is clarified from three parts:the analysis of the composition of the Modified Shuyu Pills,the preliminary research,and the modern pharmacological effects of a single drug.2.Experimental Study:Thirty APP/PS1 double transgenic mice were randomly divided into model group,donepezil group,and Modified Shuyu Pills group,10 mice in each group,and 10 C57BL/6J mice were set as normal group.The donepezil group was administered with donepezil hydrochloride solution at a dose of 0.45 mg·kg^-1·d^-1,and the Modified Shuyu Pills group was given the Modified Shuyu Pills decoction 10 g·kg^-1·d^-1by gavage,the normal group and the model group were given normal saline 10 m L·kg^-1·d^-1by gavage once a day for 35 days.After the drug intervention,Morris water maze test and novel object recognition test were used to test the learning and memory ability of mice.After the behavioral test,brain slices were taken for in situ end labeling test(TUNEL)to observe the apoptosis of hippocampal neurons;Fresh hippocampal tissue was used for biochemical detection,JC-1staining,transmission electron microscopy observation after glutaraldehyde fixation,Western Blot and real-time fluorescence quantitative PCR detection.Biochemical detection was used to detect the levels of ROS,SOD and ATP in hippocampal neurons;JC-1 staining to detect the level of mitochondrial membrane potential in hippocampus;The ultrastructure of mitochondria in hippocampal CA1 region was observed by transmission electron microscope;Western Blot detection of hippocampal mitochondrial PINK1,Parkin,p62,LC3II protein expression levels;The expression levels of SIRT1,PGC-1?,MFN-1,MFN-2,OPA-1,FIS-1,DRP-1,FOXO1,PINK1 and Parkin m RNA in hippocampal mitochondria were detected by real-time fluorescence quantitative PCR.Results1.Morris Water Maze Test:In the positioning navigation experiment,the escape latency of each group of mice decreased with the number of training days.Compared with the normal group,the escape latency of mice in the model group increased significantly(P<0.05);After the intervention of Modified Shuyu Pills and donepezil,the escape latency of APP/PS1 mice was significantly shortened compared with the model group(P<0.05).In the space exploration experiment,compared with the normal group,the number of times of crossing the platform and the proportion of time spent in the target quadrant were significantly reduced in the model group(P<0.05);Compared with the model group,the times of crossing the platform and the residence time in the target quadrant of mice in the Modified Shuyu Pills group and Donepezil group increased significantly(P<0.05),At the same time,the results showed that the improvement effect of the Modified Shuyu Pills group was more than that of the Donepezil group,but the difference was not statistically significant.2.New Object Recognition Test:In the recognition stage,compared with the normal group,the relative discrimination index of the mice in the model group was significantly reduced,and the ability to recognize new objects was weakened(P<0.05);Compared with the model group,the Modified Shuyu Pills group and the Donepezil group could significantly increase the relative resolution index and the ability to recognize new objects in mice(P<0.05).3.TUNEL staining:The apoptotic cells of the hippocampal neurons in each group showed red fluorescence,and the nuclei showed blue fluorescence.Compared with the normal group,the apoptotic cells of neurons in the hippocampal CA1 area of the mice in the model group were significantly increased(P<0.05);Compared with the model group,after the intervention of Modified Shuyu Pills,the apoptotic cells in the Modified Shuyu Pills group were significantly decreased(P<0.05).4.Biochemical detection:Compared with the normal group,the levels of ROS in the hippocampal neurons of the model group and each intervention group were significantly increased(P<0.01),and the levels of SOD and ATP were significantly decreased(P<0.01);Compared with the model group,the levels of ROS in each intervention group were significantly decreased(P<0.01),while the levels of SOD and ATP were significantly increased(P<0.05);Compared with the donepezil group,the SOD and ATP levels of the mice in the Modified Shuyu Pills group were significantly increased(P<0.05).5.JC-1 staining:The ratio of H1-UR/H1-LR showed that compared with the normal group,the mitochondrial membrane potential of the mice in the model group,donepezil group and Modified Shuyu Pills group was significantly decreased(P<0.01);Compared with the model group,the mitochondrial membrane potential of mice in the Modified Shuyu Pills group was significantly increased(P<0.01).6.Transmission Electron Microscope scan:Compared with the normal group,the hippocampal mitochondria of the mice in the model group were severely changed,with twisted strips or swollen shape,the cristae were arranged in disorder,twisted,sparse,or even disappeared,and the matrix was cloudy and uneven,after the intervention of Modified Shuyu Pills and donepezil,the mitochondrial morphology and various structures were significantly improved,and the improvement of mitochondria in the Modified Shuyu Pills group was the best.7.Western Blot detection:Compared with the normal group,the expression of PINK1 protein in the hippocampus of mice in the model group and each intervention group increased(P<0.05),while the expression of Parkin protein in the hippocampus of mice in the model group and donepezil group decreased(P<0.05),and the expression of p62,LC3II proteins increased(P<0.01);Compared with the model group,after the intervention of donepezil and Modified Shuyu Pills,the expressions of PINK1 and Parkin proteins in mice were significantly increased(P<0.01),and the expressions of p62 and LC3II proteins were significantly decreased(P<0.05,P<0.01);There was no significant difference between the donepezil group and the Modified Shuyu Pills group.8.Real-time PCR detection:Compared with the normal group,the m RNA expressions of FIS-1,DRP-1,OPA-1,MFN-1,MFN-2,SIRT1,PGC-1?,FOXO1,PINK1 and Parkin in the hippocampus of mice in the model group were all decreased(P<0.05);Compared with the model group,after intervention of donepezil and Modified Shuyu Pills,the m RNA expressions of FIS-1,DRP-1,OPA-1,MFN-1,MFN-2,SIRT1,PGC-1?,FOXO1,PINK1,and Parkin in mice were all improved.(P<0.05,P<0.01);Compared with donepezil group,the FIS-1,MFN-2,SIRT1,FOXO1 m RNA expressions in the Modified Shuyu Pills group were significantly increased(P<0.05),while the expression of other genes was not significantly different.Conclusion1.Insufficient marrow sea and brain Losing nourish are the root of AD pathogenesis,and phlegm and blood stasis are important pathological factors in AD pathogenesis,Modified Shuyu Pills with invigorating kidney and marrow,resolving phlegm and removing blood stasis have a solid theoretical basis in the treatment of AD;2.Modified Shuyu Pills can effectively improve learning and memory ability and oxidative stress injury of hippocampal neurons in APP/PS1 mice;3.Modified Shuyu Pills can up-regulate the key factor of mitochondrial biosynthesis SIRT1/PGC-1?,increase the level of mitochondrial dynamics-related genes,and play a positive role in the regulation of mitochondrial mass in the hippocampus of APP/PS1 mice;4.Modified Shuyu Pills can maintain the steady state of mitochondrial mass in the hippocampus of APP/PS1 mice and exert antioxidant protection,the mechanism may be through up-regulating the autophagy transcription factor FOXO1 and promoting the mitophagy pathway mediated by the PINK1/Parkin pathway.