目的 观察逍遥散对胃癌荷瘤共病抑郁小鼠程序性死亡受体1(programmed cell death protein 1,PD-1)抑制剂治疗的增敏作用,并探讨其作用机制.方法 采用皮下移植胃癌细胞系——MCF细胞构建荷瘤小鼠共60只,随机分为荷瘤对照组、荷瘤共病抑郁组、PD-1抑制剂组、逍遥散联合PD-1抑制剂组,每组15只.荷瘤对照组不做任何干预,共饲养56天;荷瘤共病抑郁组是在荷瘤对照组的基础上每天以慢性不可预知温和应激干预,每天行适量生理盐水灌胃,分别在第1天、15天、29天、43天小鼠尾静脉注射适量生理盐水;PD-1抑制剂组是在荷瘤共病抑郁组的基础上,将小鼠尾静脉注射生理盐水改为PD-1抑制剂;逍遥散联合PD-1抑制剂组是在PD-1抑制剂组的基础上,将生理盐水灌胃改为逍遥散水提物灌胃.分组后第57天,荷瘤对照组、荷瘤共病抑郁组进行糖水偏好测试、陌生环境摄食实验,分析两组小鼠的行为学变化.计算荷瘤共病抑郁组、PD-1抑制剂组、逍遥散联合PD-1抑制剂组小鼠的生存率并绘制生存曲线;获取小鼠瘤体,并测量瘤体体积及重量,计算抑瘤率;采用ELISA法检测小鼠肿瘤组织吲哚胺-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)、犬尿氨酸(kynurenine,Kyn)、芳香烃受体(aryl hydrocarbon receptor,AhR)的数值;对肿瘤组织通过免疫组化法检测小鼠肿瘤组织叉头样转录因子3(forkhead box P3,Foxp3)表达情况.结果 (1)行为学改变:与荷瘤对照组比较,荷瘤共病抑郁组的糖水偏好率显著降低(P＜0.01),摄食潜伏时间显著延长(P＜0.01).(2)生存率差异:荷瘤共病抑郁组小鼠生存率为20％,PD-1抑制剂组小鼠生存率为26.7％,逍遥散联合PD-1抑制剂组小鼠生存率为40％.(3)肿瘤增值差异:逍遥散联合PD-1抑制剂组瘤体体积小于PD-1抑制剂组(P＜0.05),PD-1抑制剂组小于荷瘤共病抑郁组(P＜0.05);逍遥散联合PD-1抑制剂组瘤体重量显著小于PD-1抑制剂组(P＜0.01),PD-1抑制剂组显著小于荷瘤共病抑郁组(P＜0.01);逍遥散联合PD-1抑制剂组的抑瘤率为35.4％优于PD-1抑制剂组的22.1％.(4)相关蛋白表达差异:逍遥散联合PD-1抑制剂组肿瘤组织中IDO的表达显著低于PD-1抑制剂组及荷瘤共病抑郁组(P＜0.01);逍遥散联合PD-1抑制剂组肿瘤组织中Kyn的表达显著低于PD-1抑制剂组(P＜0.01),PD-1抑制剂组小于荷瘤共病抑郁组(P＜0.05);逍遥散联合PD-1抑制剂组肿瘤组织中AhR的表达显著低于PD-1抑制剂组及荷瘤共病抑郁组(P＜0.01);(5)各组小鼠肿瘤组织Foxp3表达情况:与荷瘤共病抑郁组比较,PD-1抑制剂组、逍遥散联合PD-1抑制剂组肿瘤组织中Foxp3蛋白表达均显著降低(P＜0.01);与PD-1抑制剂组比较,逍遥散联合PD-1抑制剂组肿瘤组织中Foxp3蛋白表达显著降低(P＜0.01).结论 逍遥散对PD-1抑制剂的增敏作用可能与通过下调IDO、Kyn、AhR水平,进而有效降低调节性T淋巴细胞的增值与分化而实现的.
Study on sensitizing effect and mechanism of Xiaoyaosan on PD-1 inhibitor treatment in mice with gastric cancer comorbidity depression
Objective To observe the sensitizing effect of Xiaoyaosan on PD-1 inhibitor therapy in depressed mice with gastric cancer and explore its mechanism.Methods A total of 60 tumor-bearing mice were constructed by subcutaneous transplantation of gastric cancer cell lines-MCF cells.They were randomly divided into tumor-bearing control group,tumor-bearing comorbidity depression group,PD-1 inhibitor group,Xiaoyaosan combined PD-1 inhibitor group,15 mice in each group.The control group with tumor was fed for 56 days without any intervention.The tumor-bearing depression group was treated with chronic unpredictable mild stress every day on the basis of the control group.Normal saline was injected into the caudal vein on the first day,the 15th day,the 29th day and the 43th day,respectively.The PD-1 inhibitor group was changed into PD-1 inhibitor by injecting Radix Saline into the tail of mice on the basis of comorbidity depression group.On the basis of the PD-1 inhibitor group,the Xiaoyaosan combined with PD-1 inhibitor group changed saline into water extract of Xiaoyaosan.On the 57th day after grouping,sugar water preference test and unfamiliar environment feeding test were performed in the tumor-bearing control group and the tumor-bearing comorbidity depression group to analyze the behavioral changes of mice in the two groups.The survival rate of mice in the tumor-bearing comorbidity depression group,the PD-1 inhibitor group and the Xiaoyaosan combined PD-1 inhibitor group was calculated and the survival curve was drawn.The tumor volume and weight of mice were measured and the tumor inhibition rate was calculated.The values of IDO,Kyn,AhR were detected by ELISA.The expression of Foxp3 in tumor tissues of mice was detected by the method of virus-free organization.Results(1)Behavioral changes:compared with the tumor-bearing control group,the sugar water preference rate was significantly decreased(P＜0.01)and the latent time of ingestion was significantly increased(P＜0.01)in the tumor-bearing depression group.(2)The survival rate difference:the survival rate of mice in the tumor-bearing depression group was 20％,the survival rate of mice in the PD-1 inhibitor group was 26.7％,and the survival rate of mice in the Xiaoyaosan combined PD-1 inhibitor group was 40％.(3)Tumor increment difference:the tumor volume of Xiaoyaosan combined with PD-1 inhibitor group was smaller than that of PD-1 inhibitor group(P＜0.05),and that of PD-1 inhibitor group was smaller than that of depression group(P＜0.05).The tumor weight of Xiaoyaosan combined with PD-1 inhibitor group was significantly lower than that of PD-1 inhibitor group(P＜0.01),and that of PD-1 inhibitor group was significantly lower than that of comorbidity depression group(P＜0.01).The anti-tumor rate of Xiaoyaosan combined with PD-1 inhibitor group was 35.4％better than that of the PD-1 inhibitor group(22.1％).(4)The expression of IDO was significantly decreased in the group of Xiaoyaosan combined with PD-1 inhibitor than that in the group of PD-1 inhibitor and depression(P＜0.01);the expression of Kyn in tumor tissues in the Xiaoyaosan combined with PD-1 inhibitor group was significantly decreased than that in the PD-1 inhibitor group(P＜0.01),and that in the PD-1 inhibitor group was lower than that in the comorbidity depression group(P＜0.05).The expression of AhR in the group of Xiaoyaosan combined with PD-1 inhibitor was significantly lower than that in the group of PD-1 inhibitor and depression with comorbidity(P＜0.01).(5)The expression of Foxp3 in tumor tissues of mice in each group:compared with the tumor comorbidity depression group,the expression of Foxp3 protein in tumor tissues of PD-1 inhibitor group and Xiaoyaosan combined with PD-1 inhibitor group was decreased significantly(P＜0.01);compared with the PD-1 inhibitor group,the expression of Foxp3 protein in the tumor tissues of the group was significantly decreased(P＜0.01).Conclusion The sensitization effect of Xiaoyaosan on PD-1 inhibitor may be related to the decrease of proliferation and differentiation of Treg by down-regulating the levels of IDO,Kyn,AhR.
万方数据
维普
