Study On The Protective Effect And Mechanism Of Shanzhiside On The Cell Model Of Parkinson’s Disease Based On Molecular Docking

Background and Objective: As an iridoid compound,shanzhiside is found in traditional Chinese medicine Gardenia jasminoides Ellis,and is often used as one of the markers for the study of the quality of traditional Chinese Herbal Slices and Chinese patent medicine.However,in the common anti-oxidation and anti-inflammatory tests,it is not as good as other iridoid glycosides such as geniposide.In this study,molecular docking was used to test the docking of the three proteins in the main endoplasmic reticulum stress pathway.And then,the SH-SY5 Y cells induced by 6-OHDA were used to construct an in vitro model of Parkinson’s disease to detect the protective effect of shanzhiside and the expression of related pathway proteins,and provide basis and ideas for the activity study of shanzhiside.Methods:(1)Using the Autodock software to perform the virtual molecular docking of the PERK,Bi P,and IRE1 proteins related to ER stress,and analyze the docking results.(2)Using SH-SY5 Y cells treated by 6-OHDA to construct an in vitro model of Parkinson’s disease,using CCK-8 and LDH assay to detect the protective effect of shanzhiside,and the tunicamycin pretreatment was used to activate endoplasmic reticulum stress to observe the changes in the protective effect of shanzhiside.Apoptosis was observed by optical image,Hoechst33258 fluorescence and Annexin VFITC/PI.Endoplasmic reticulum was observed by DIO-C6(3)staining,autophagy was observed by MDC staining,and the protein expressions of PERK-e IF2α-ATF4-CHOP,autophagy-related protein LC3,apoptosis-related proteins Bcl-2 and Bax were detected by Western blot.Results:(1)The docking results showed that the optimal docking conformation binding energy of shanzhiside and PERK protein is-5.06 kcal/mol,the inhibition constant(Ki)was 196.74 μM.The binding ability was better than the shanzhiside and Bi P protein(-3.43 kcal/mol),shanzhiside and IRE1 protein(-2.79 kcal/mol).(2)CCK-8 showed that 50 μM shanzhiside pretreatment could increase the survival rate of SH-SY5 Y cells from 64% to 93% under the condition of 300 mΜ 6-OHDA injuried(P<0.05).Shanzhiside also reduces 6-OHDA-induced LDH release.While a safe concentration of tunicamycin pretreatment inhibits this protection.(3)Cellular optical images,Hoechst33258 staining and Annexin V-FITC/PI double staining showed that 50 μM of shanzhiside could reduce the apoptosis of SH-SY5 Y induced by 6-OHDA.(4)DIO-C6(3)staining and MDC staining showed that shanzhiside pretreatment inhibited 6-OHDA-induced endoplasmic reticulum morphological changes and autophagy in SH-SY5 Y cells.(5)Western blot analysis showed that the pre-treated shanzhiside inhibited the phosphorylation of PERK and e IF2α protein and the expression of ATF and CHOP protein in SH-SY5 Y cells induced by 6-OHDA(P<0.05).The expression of autophagy protein LC3-II was decreased(P<0.05),and the ratio of anti-apoptotic protein Bcl-2 to pro-apoptotic protein Bax was increased(P<0.05).Conclusion: Shanzhiside can inhibit 6-OHDA-induced apoptosis of SH-SY5 Y cells,which may be achieved by reducing the phosphorylation level of PERK protein,inhibiting the PERK-e IF2α-ATF4-CHOP pathway,and regulating autophagy.