Effect of Anwei Decoction-Containing Serum on Autophagy and Apoptosis of Gastric Intestinal Metaplasia Model Cells
Objective To investigate the effects of Anwei Decoction-containing serum(Pinelliae Rhizoma,Coptidis Rhizoma,Zingiberis Rhizoma Recens,Linderae Radix,Lilii Bulbus,etc.)on autophagy and apoptosis of gastric intestinal metaplasia(GIM)model cells.Methods GIM cell model was prepared by inducing human gastric mucosal epithelial cell GES-1 with chenodeoxycholic acid(CDCA).The experiment was divided into normal group(normal gastric mucosal epithelial GES-1 cells were cultured routinely),model group(GIM model was replicated by CDCA and cultured routinely),blank control group(GIM model cells were cultured with the same amount of blank serum),and Anwei Decoction group(GIM model cells+7%Anwei Decoction-containing serum).The cells in each group were detected after 24 hours of intervention.Apoptosis was detected by flow cytometry(Annexin V-PE/7-AAD double staining).The expressions of intestinal metaplasia-related proteins SOX2 and CDX2 were detected by immunofluorescence double labeling method.The ultrastructure and autophagy of cells were observed by transmission electron microscopy.The mRNA expressions of LC3 and Bax were detected by qRT-PCR.The protein expressions of MCU2,KLF4,LC3 and Bax were detected by Western Blot.Results Compared with the normal group,the protein expressions of MUC2 and KLF4 in the model group were significantly up-regulated(P＜0.01),and the apoptosis rate of the cells was significantly decreased(P＜0.05).The fluorescence intensity of SOX2 was significantly decreased(P＜0.05),and the fluorescence intensity of CDX2 was significantly increased(P＜0.01).The cell structure was disordered,the nucleus was atrophic and deformed,the organelles such as endoplasmic reticulum and mitochondria were swollen,ruptured and vacuolated,and the number of autophagosomes and autolysosomes were significantly increased.The mRNA and protein expressions of Bax were significantly down-regulated(P＜0.05),the mRNA expression of LC3 was significantly up-regulated(P＜0.01),and the protein expression ratio of LC3 II/LC3 I was significantly increased(P＜0.01).Compared with the blank control group,the apoptosis rate of Anwei Decoction group was significantly increased(P＜0.01).The fluorescence intensity of SOX2 was significantly increased(P＜0.01),and the fluorescence intensity of CDX2 was significantly decreased(P＜0.05).The cell structure was relatively complete,and the autophagosomes and autolysosomes were slightly reduced under the microscope.The mRNA and protein expressions of Bax were significantly up-regulated(P＜0.01),the mRNA expression of LC3 was significantly down-regulated(P＜0.01),and the protein expression ratio of LC3 II/LC3 I was significantly decreased(P＜0.01).Conclusion Anwei Decoction may improve the intestinal metaplasia of gastric mucosal epithelial cells by regulating autophagy and promoting apoptosis in GIM model cells.
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