大补脾汤联合奥沙利铂对胃癌荷瘤小鼠瘤组织TLR4/MyD88/NF-κB信号通路及下游相关分子的影响

Effects of Dabupi Decoction combined with oxaliplatin on TLR4/MyD88/NF-κB signaling pathway related molecules in tumor tissues of mice bearing gastric cancer

目的:研究大补脾汤联合奥沙利铂对胃癌荷瘤小鼠瘤组织TLR4/MyD88/NF-κB信号通路及下游相关分子的影响,探讨其可能的作用机制。方法:采用昆明小鼠建立胃癌荷瘤模型,造模成功后将小鼠随机分为5组:模型组、奥沙利铂组(10 mg/kg)、大补脾汤高、中、低剂量(21.58、10.79、5.40 g/kg)联合奥沙利铂组,每组10只。造模成功后开始给药,连续给药14 d;末次给药后次日眼球取血,处死小鼠,摘取肿瘤组织称重,计算抑瘤率;采用苏木素-伊红(HE)染色后观察瘤组织形态,实时荧光定量聚合酶链式反应(RT-qPCR)、免疫组化法(IHC)和Western blot分别检测小鼠肿瘤组织中Toll样受体4(TLR4)、髓样分化因子88(MyD88)、肿瘤坏死因子受体相关因子6(TRAF6)和核因子κB(NF-κB)mRNA和蛋白相对表达,采用RT-qPCR检测小鼠肿瘤组织中TNF-α和IL-6 mRNA相对表达。结果:奥沙利铂组、大补脾汤高、中、低剂量联合奥沙利铂组瘤质量均较模型组显著降低(P<0.01),抑瘤率分别为37.12%、49.48%、42.10%、40.65%,大补脾汤高剂量联合奥沙利铂组与奥沙利铂组相比差异具有统计学意义(P<0.05);HE结果显示:与模型组相比,各给药组能明显降低肿瘤细胞密度,引起肿瘤细胞坏死;与模型组相比,各给药组肿瘤组织中TLR4、MyD88、TRAF6和NF-κB p65 mRNA和蛋白相对表达均显著降低(P<0.01,P<0.05);与奥沙利铂组相比,大补脾汤高、中剂量联合奥沙利铂组肿瘤组织中TLR4、MyD88、TRAF6和NF-κB p65 mRNA和蛋白相对表达均显著降低(P<0.05);与模型组相比,各给药组肿瘤组织中TNF-α和IL-6 mRNA相对表达均显著降低(P<0.01);与奥沙利铂组相比,大补脾汤高剂量联合奥沙利铂组肿瘤组织中TNF-α和IL-6 mRNA相对表达均显著降低(P<0.05)。结论:大补脾汤可通过抑制TLR4/MyD88/NF-κB信号通路下调下游炎症因子TNF-α和IL-6的表达,调节免疫微环境,发挥抗肿瘤作用,抑制胃癌发生发展。

Objective:To study the effects of Dabupi Decoction combined with oxaliplatin on TLR4/MyD88/NF-κB signaling pathway and downstream related molecules in tumor tissues of gastric cancer-bearing mice,and to explore its related mechanism.Methods:Kunming mice were used to establish tumor-bearing models of gastric cancer.After successful modeling,mice were randomly divided into five groups:model group,oxaliplation group(10 mg/kg),high,middle and low doses of Dabupi Decoction(21.58,10.79,5.40 g/kg)combined with oxaliplatin,with 10 mice in each group.After successful modeling,administration was started for 14 d;the next day after the last administration,the eyeball blood was taken,the mice were killed,the tumor tissues were taken and weighed,and the tumor inhibition rate was calculated.The tumor tissue morphology was observed after hematoxylin-eosin(HE)staining,relative expression levels of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),tumor necrosis factor receptor-related factor 6(TRAF6)and nuclear factorκB(NF-κB)mRNA and protein in mice tumor tissues were detected by real-time quantitative polymerase chain reaction(RT-qPCR),immunohistochemistry(IHC)and Western blot,respectively.RT-qPCR was used to detect the relative expression levels of TNF-αand IL-6 mRNA in mice tumor tissues.Results:Compared with the model group,the tumor quality of the oxaliplatin group,the high-dose,middle-dose and low-dose groups of Dabupi Decoction combined with oxaliplatin was significantly lower(P<0.01),and the tumor inhibition rates were 37.12%,49.48%,42.10%and 40.65%,respectively.The difference between the high-dose group of Dabupi Decoction combined with oxaliplatin and the oxaliplatin group were significant(P<0.01).The results of HE showed that compared with the model group,each administration group could significantly reduce the density of tumor cells and cause tumor cell necrosis.Compared with the model group,the relative expression levels of TLR4,MyD88,TRAF6 and NF-κB p65 mRNA and protein in the tumor tissues of each treatment group were significantly reduced(P<0.01,P<0.05).Compared with the oxaliplatin group,the relative expression levels of TLR4,MyD88,TRAF6 and NF-κB p65 mRNA and protein in tumor tissues in the high and middle doses of Dabupi decoction combined with oxaliplatin group were significantly reduced(P<0.05).Compared with the model group,relative expression levels of TNF-αand IL-6 mRNA in tumor tissues of each drug administration group were significantly reduced(P<0.01).Compared with the oxaliplatin group,relative expressions levels of TNF-αand IL-6 mRNA in tumor tissues of the high-dose Dabupi decoction combined with oxaliplatin group were significantly lower(P<0.05).Conclusion:Dabupi Decoction can down-regulate the expression of downstream inflammatory factors TNF-αand IL-6 by inhibiting TLR4/MyD88/NF-κB signaling pathway,regulate immune microenvironment,play an anti-tumor role and inhibit the occurrence and development of gastric cancer.