Study On The Effects And Mechanism Of DTCA And ETCA Isolated From Trillium Tschonoskii Maxim.in Improving AD Via Autophagy Induction

Objective: Study on the effects and mechanism of DTCA and ETCA isolated from Trillium tschonoskii Maxim.in improving AD via autophagy induction.Methods: In this experiment:(1)MTT and flow cytometry method were used to detect the effect of DTCA/ETCA on the cell viability of HT-22 and PC-12;(2)Thioflavin method to detect the inhibitory effect of DTCA/ETCA on the formation of Aβ(25-35)and Aβ(1-42)oligomers;(3)Bio-Layer interferometry method to detect the interaction between DTCA/ETCA and Aβ(1-42);(4)MTT,flow cytometry,Hochest/PI staining and fluorescence microscopy imaging methods to detect the effects of DTCA/ETCA on the corresponding protein expression and cell viability in HT-22 and PC-12 cells induced or overexpressed by A β,APP or Tau;(5)Western blot and fluorescence microscopy imaging methods to detect the effect of DTCA/ETCA on the expression of autophagy marker proteins and signal pathway proteins that regulate autophagy;(6)Using Atg7 knockout MEF cells and autophagy inhibitors to investigate whether DTCA/ETCA clears AD-related proteins by activating autophagy;(7)Using nematode models expressing autophagy genes and AD-related protein genes to study the effects of DTCA/ETCA in worms that activate autophagy,inhibit the expression of AD-related proteins and improve behavioral ability;(8)APP/PS1 transgenic mice to study the effect and mechanism of ETCA on improving the cognitive ability of AD mice.Results: We have achieved the following main results:(1)DTCA/ETCA has no significant effect on the cell viability of HT-22 and PC-12 in the range of 0.3-2μM;(2)DTCA/ETCA inhibits the formation of Aβ(25-35)/Aβ(1-42)oligomers;DTCA/ETCA and Aβ(1-42)have a mutually binding effect;(3)DTCA/ETCA effectively removes AD-related proteins in HT-22 and PC-12 cells and inhibits apoptosis induced by them;(4)DTCA/ETCA concentration-dependently increased the expression of LC3-II protein in HT-22 cells;increased the formation of GFP-LC3 spots in HT-22 cells;increased the mitochondrial dye Mito-tracker and GFP-LC3 in HT-22 cells fluorescence co-localization indicates the occurrence of autophagy/mitochondrial autophagy;autophagy inhibitor3-methyladenine(3-MA)significantly inhibits DTCA/ETCA-induced LC3-II protein expression and GFP-LC3 spot formation in HT-22 cells;Bafilomycin A1(Baf)further promote the expression of LC3-II protein and the formation of GFP-LC3 spots in HT-22 cells induced by DTCA and ETCA;(5)3-MA and Baf significantly inhibit the clearance of DTCA/ETCA on AD-related proteins in HT-22 or PC-12 cells;(6)DTCA/ETCA activates autophagy and clears AD-related proteins through Atg7;(7)DTCA/ETCA significantly inhibited the protein expression of p-PI3 K,p-AKT,p-m TOR,p-P70S6 K and p-ULK1(Ser757)in HT-22 cells;enhanced p-AMPK,p-ULK1(Ser555),PINK1 and Parkin protein expression;AMPK inhibitor Compound C significantly inhibited the expression of LC3-II protein and the formation of GFP-LC3 spots in HT-22 cells treated with DTCA/ETCA;(8)DTCA/ETCA induces the occurrence of cell autophagy;(9)DTCA/ETCA significantly reduced the expression of GFP-P62 in C.elegans BC12921 and increased the formation of GFP-LGG-1 fluorescent spots in C.elegans DA2123;DTCA/ETCA significantly reduced the paralysis rate of AD transgenic nematode model CL4176,and improved the food perception behavior of BR5270 transgenic nematode;DTCA/ETCA can activate autophagy through unc-51,pdr-1,beca-1,and ps-34 genes to inhibit the paralysis rate of AD transgenic nematode model CL4176;(10)ETCA can significantly shorten the escape time and total run length of APP/SP1 mice in the water maze experiment;(11)WB results show that: ETCA inhibits the protein expression of Aβ and p-Tau in the APP/SP1 mouse brain,ETCA inhibits p-m TOR,p-ULK1(Ser757),p-P70S6 K and Bax/Bcl-2 ratio,ETCA increases the protein expression of p-AMPK,p-ULK1(Ser555),PINK1,Parkin and LC3-II;(12)The results of immunohistochemistry show that: ETCA reduces the expression of Aβ,p-Tau,GFAP and Iba-1 in brain tissue,and ETCA increases the expression of LC3 B,PINK1,Parkin and Neu N.Conclusion: The trillium saponin component DTCA/ETCA isolated from trillium can eliminate AD-related pathological proteins and improve the behavior and cognitive function of AD models in vivo and in vitro.Its main molecular mechanism is related to m TOR,AMPK/ULK1 and the autophagy/mitochondrial autophagy regulated by the PINK1/Parkin signal pathway are closely related.