五子衍宗方对D-半乳糖诱导的GC-1细胞凋亡的保护作用OA

Objective To explore the protective effect of Wuzi Yanzong recipe(WZ)against D-galactose(D-gal)-induced apoptosis in GC-1 cells and its mechanism.Methods GC-1 cells were divided into control group,model group,and low-,middle-,and high-dose WZ groups(0.1,0.2,0.4 mg/mL).GC-1 cells were stimulated with 250 mmol/L D-gal for 48 h to establish the spermatogonial apoptosis model.GC-1 cells in low-,middle-,and high-dose WZ groups were pretreated with 0.1,0.2,0.4 mg/mL Wuzi Yanzong recipe for 12 h before modeling,respectively.The cell viability was detected by MTT assay,the cell apoptosis was assessed by flow cytometry,the changes in intracellular ROS and calcium ion levels were measured using fluorescent probes,the expression and locali-zation of Nrf2 signaling pathway-related proteins Nrf2,HO-1,and NQO1 were examined by immunofluorescence,the protein expres-sion levels of Nrf2,the key protein of UPRER(GRP78),and its related pathway proteins(p-PERK,p-IRE1,p-eIF2α,XBP1,ATF4 and ATF6),and endoplasmic reticulum stress(ERS)-mediated apoptosis-related proteins(Caspase-12,Caspase-9,and Caspase-3)were determined by Western blot.Results Compared with control group,the cell viability significantly decreased in model group(P<0.01),the cell apoptosis rate increased(P<0.01);the levels of intracellular ROS,calcium ions,Caspase-12,Caspase-9 and Caspase-3 significantly increased(P<0.01);and the expression levels of Nrf2,HO-1,NQO1,GRP78,p-PERK,p-IRE1,p-eIF2α,XBP1,ATF4 and ATF6 decreased(P<0.01).Compared with model group,the cell viability was increased in low-,middle-,and high-dose WZ groups(P<0.01),the cell apoptosis rate was dose-dependently decreased(P<0.01);the levels of intracellular ROS,calcium ions,Caspase-9 and Caspase-3 were decreased in a dose dependent manner(P<0.01),and Caspase-12 expression also decreased,but there was no significant difference between three WZ groups;the expression levels of Nrf2,HO-1,NQO1,p-PERK,p-IRE1,p-eIF2α,XBP1,ATF4 and ATF6 increased in a dose dependent manner(P<0.05),and GRP78 also significantly increased(P<0.05),but there was no significant difference between three WZ groups.Conclusion WZ can alleviate D-gal-induced apoptosis in GC-1 cells via the activation of Nrf2 signaling pathway,which enhances the antioxidant capacity of GC-1 cells,thereby strengthening the UPRER and reducing ERS-mediated apoptotic pathways.