Protective Effects Of Autophagy Induced By Tenuifolin PINK1-parkin Pathway On Brain Mitochondrial Damage In APP/PS1 Mice

Objective: In this study,APP/PS1 double transgenic mice were usedas the animal model of Alzheimer’s disease(AD)to study the effects of Tenuifolin(TEN)on the biosynthesis,fission,fusion and autophagy of brain mitochondriain model mice,and to explore the protective effect of TEN on brain mitochondria through mitochondrial dynamic balance and mitochondrial autophagy and its related mechanism.Methods: 50 APP/PS1 double transgenic mice and 10 wild type mice were selected as subjects.50 APP/PS1 mice were randomly divided into model group(Model),low dose group(TEN-L),medium dose group(TEN-M),high dose group(TEN-H)and inhibitor group(TEN-M+3-MA),with 10 mice in each group.10 wild type mice were used as normal control group(Control).Mice in TEN-L group,TEN-M group,TEN-H group and TEN-M+3-MA group were given intragastric administration of TEN 20 mg/kg/d,TEN 40 mg/kg/d,TEN 80 mg/kg/d and TEN-M 40 mg/kg/d+3-MA 30 mg/kg/d for 3 months,once a day.Mice in control group and model group were fed with the same volume of0.3%CMC-Na solution for 3 months,once a day.After administration,the mice were killed by cervical vertebra removal method,and the brain tissues were collected and brain mitochondria were extracted.The morphological structure of mouse brain mitochondria was observed by transmission electron microscope.The level of mitochondrial membrane potential in mouse brain was detected by JC-1 method.The mRNA expression of mitochondrial production gene(PGC-1α),mitochondrial mitosis gene(Drp1)and mitochondrial fusion gene(Mfn2)in mouse brain was detected by RT-q PCR technique.The mRNA expression of LC3 and p62 genes related to autophagy level in mouse brain was detected.The mRNA expression of lysosome function related genes Cathepsin D and Rab7 was detected.The mRNA expression of PINK1 and Parkin related genes in mouse brain mitochondrial autophagy was detected.The expression of mitochondrial production related protein(PGC-1α),mitochondrial division associated protein(Drp1)and mitochondrial fusion related protein(Mfn2)in mouse brain was detected by Western Blot technique,the protein expression of LC3 and p62 related proteins in mouse brain mitochondrial autophagy level was detected,and the protein expression of PINK1 and Parkin related proteins in mouse brain mitochondrial autophagy initiation was detected.In addition,the positive distribution of autophagy marker protein LC3 in mouse brain was observed by immunohistochemical staining.Results:1.The results of transmission electron microscope showed that the membrane structure of brain mitochondria was intact,the ridge of mitochondria was clearly visible,the swelling of mitochondria decreased and the vacuolation of mitochondria decreased in TEN group.The results of mitochondrial membrane potential showed that the level of brain mitochondrial membrane potential increased in TEN group.RT-q PCR results showed that TEN could increase the expression level of PGC-1 αand Mfn2 mRNA,and decrease the expression level of Drp1 mRNA.Western Blot results showed that TEN could increase the expression of PGC-1α and Mfn2 protein and decrease the expression level of Drp1 protein.The results showed that TEN could maintain the dynamic balance of mitochondria and protect the brain mitochondria of APP/PS1 mice by improving the morphology and structure of mitochondria,increasing the level of mitochondrial membrane potential and regulating the factors related to mitochondrial production,division and fusion.2.The results of immunohistochemical staining showed that the average optical density of LC3 protein increased in TEN group.RT-q PCR results showed that TEN could increase the expression level of LC3,Cathepsin D and Rab7 mRNA,and decrease the expression level of P62 mRNA.Western Blot results showed that TEN could increase the expression level of LC3 protein and decrease the expression level of p62 protein.However,autophagy inhibitor 3-MA antagonized the effect of TEN on LC3 and p62 to some extent.The results showed that TEN could accelerate the clearance of damaged mitochondria in APP/PS1 mice by increasing the level of mitochondrial autophagy and improving the function of lysosome.3.RT-q PCR results showed that TEN could increase the expression level of PINK1 and Parkin mRNA.Western Blot results showed that TEN could increase the expression of PINK1 and Parkin protein.Autophagy inhibitor 3-MA antagonized the effect of TEN on PINK1 and Parkin to some extent.The results showed that TEN could activate the brain mitochondrial autophagy of APP/PS1 mice by inducing the initiation of PINK1-Parkin pathway.Conclusion: TEN can enhance the generation and fusion of mitochondria,weaken the division of mitochondria,improve the morphology and structure of mitochondria,increase the level of membrane potential of mitochondria,regulate the dynamic balance of mitochondria and protect the brain mitochondria of APP/PS1 mice.In addition,TEN can induce the initiation of PINK1-Parkin signal pathway,improve the ability of PINK1 on mitochondrial membrane to recruit Parkin,up-regulate the expression of LC3,down-regulate the expression of p62,increase the level of mitochondrial autophagy,up-regulate the expression of Cathepsin D and Rab7,improve the degradation function of lysosome,accelerate the clearance of damaged abnormal mitochondria gathered in APP/PS1 mouse brain neurons,and then protect APP/PS1 mouse brain neurons.