安胃汤抑制CAG大鼠PD-1/PD-L1信号轴免疫逃逸机制研究

Mechanism for Anwei decoction to inhibit immune escape induced by programmed cell death protein 1/programmed cell death ligand 1 axis in rats with chronic atrophic gastritis

背景众多证据表明免疫逃逸在肿瘤形成过程中扮演重要角色,慢性萎缩性胃炎(chronic atrophic gastritis,CAG)是胃癌的癌前疾病.安胃汤被发现可改善CAG临床症状及病理表现,实现CAG的逆转,该作用是否与免疫逃逸机制相关有待进一步研究.目的从细胞免疫逃逸角度,探讨程序性死亡受体-1(programmed cell death protein 1,PD-1)/程序性死亡受体配体-1(programmed cell death ligand 1,PD-L1)信号轴与安胃汤对CAG模型大鼠疗效之间的关系.方法采用1-甲基-3-硝基-1-亚硝基胍(1-Methyl-3-nitro-1-nitrosoguanidine,MNNG)慢性萎缩性胃炎大鼠模型,应用不同剂量安胃汤及维酶素片进行干预;HE染色观察安胃汤对CAG模型大鼠胃黏膜炎症细胞浸润及组织形态改变的影响;免疫组化检测CAG模型大鼠胃黏膜组织PD-1、PD-L1蛋白表达;ELISA检测血清CD4^(+)、CD8^(+)水平变化;qPCR检测CAG模型大鼠胃黏膜PD-1mRNA、PD-L1mRNA表达;Western-blot检测CAG模型大鼠胃黏膜组织PD-1、PD-L1蛋白表达.结果免疫组化结果示:与模型组和维酶素组比较,安胃汤高、低剂量组PD-L1表达均较低(P<0.01,P<0.05).ELISA实验结果示:与模型组比较,安胃汤高剂量组CD4^(+)表达及CD4^(+)/CD8^(+)比值升高(P<0.01,P<0.05),安胃汤各组和维酶素组CD8^(+)表达降低(P<0.01);与维酶素组比较,安胃汤高剂量组CD8^(+)表达降低(P<0.05).qPCR实验结果显示:与模型组比较,安胃汤高剂量组和维酶素组PD-1mRNA表达下降(P<0.01),安胃汤高、中剂量组PD-L1mRNA表达下降(P<0.01,P<0.05).Western-blot实验结果显示:与模型组比较,安胃汤高、中剂量组PD-1/Actin,PD-L1/Actin表达下降(P<0.01,P<0.05).结论安胃汤抗CAG作用可能与抑制PD-1/PD-L1信号通路诱导的细胞免疫逃逸有关.

BACKGROUND Numerous studies suggest that immune escape plays an important role in tumor formation.Chronic atrophic gastritis(CAG)is a precancerous lesion of gastric cancer.Anwei decoction has been found to improve the clinical symptoms and pathological manifestations of CAG and reverse it,but whether this effect is related to the immune escape mechanism needs to be further investigated.AIM To explore the relationship between the programmed cell death protein 1(PD-1)/programmed cell death ligand 1(PD-L1)axis and the curative effect of Anwei decoction in CAG rats from the perspective of cellular immune escape.METHODS Rats with CAG induced by 1-methyl-3-nitro-1-nitrosog-uanidine(MNNG)were treated with different doses of Anwei decoction and Vitacoenzyme tablets.Hematoxylin-eosin staining was used to observe the effect of Anwei decoction on inflammatory cell infiltration and histomorp-hological changes in the gastric mucosa of CAG rats.Immunohistochemistry assay was used to detect the expression of PD-1 and PD-L1 in the gastric mucosa of CAG rats.The changes of serum CD4^(+)and CD8^(+)T-cell levels were detected by ELISA.The expression of PD-1 and PD-L1 mRNA and protein in the gastric mucosa of CAG rats was detected by qPCR and Western blot,respectively.RESULTS Immunohistochemistry analysis showed that the expression level of PD-L1 in the high-and low-dose Anwei decoction groups was lower than that in the model group and the Vitacoenzyme group(P<0.01 and P<0.05,respectively).ELISA showed that compared with the model group,the level of CD4^(+)T-cells and the CD4^(+)/CD8^(+)ratio in the high-dose Anwei decoction group increased(P<0.01 and P<0.05,respectively),and the level of CD8^(+)T-cells in all the Anwei decoction groups and Vitacoenzyme group decreased(P<0.01);compared with the Vitacoenzyme group,the expression of CD8^(+)T-cells in the high-dose Anwei decoction group was decreased(P<0.05).qPCR results showed that compared with the model group,the expression level of PD-1 mRNA in high-dose Anwei decoction group and Vitacoenzyme group decreased(P<0.01),and the expression level of PD-L1 mRNA decreased in the high-and middle-dose Anwei decoction groups(P<0.01 and P<0.05,respectively).Western blot analysis showed that compared with the model group,the relative expression level of PD-1 and PD-L1 to actin in the high-and middle-dose Anwei decoction groups decreased(P<0.01 and P<0.05,respectively).CONCLUSION The apparent anti-CAG effect of Anwei decoction may be related to the inhibition of immune escape induced by the PD-1/PD-L1 axis.