芪贞汤对结肠炎相关癌症小鼠肠道细菌诱导的Toll样受体4/核因子κB信号通路的影响

Effect of Qizhen decoction on intestinal bacteria induced Toll-like receptor-4/nuclear factor-κB signaling pathway in colitis-associated cancer mice

目的探讨芪贞汤对结肠炎相关癌症(CAC)小鼠肠道细菌诱导的Toll样受体4(TLR4)/核因子κB信号通路的影响,为其抗炎抗肿瘤作用提供实验依据。方法将24只C57BL/6J雄性小鼠按照随机数字表法分为空白组、模型对照组、氟尿嘧啶组和芪贞汤组,每组6只。除空白组外,余小鼠采用氧化偶氮甲烷联合葡聚糖硫酸钠周期性诱导建立CAC小鼠模型。空白组与模型对照组以0.9%氯化钠注射液灌胃,余2组以对应药物灌胃,连续给药14 d。采用16S rDNA测序分析各组小鼠肠道菌属特征;运用酶联免疫吸附试验法测定各组小鼠血清中内毒素含量;运用免疫组织化学法检测并比较各组小鼠结肠组织中TLR4和核因子KB p65蛋白表达。结果测序结果显示革兰阴性菌拟杆菌属在模型对照组和氟尿嘧啶组中较多,芪贞汤组多为革兰阳性菌乳球菌属和芽孢杆菌属。模型对照组小鼠内毒素水平、TLR4和核因子κB p65蛋白表达水平明显高于空白组;氟尿嘧啶组和芪贞汤组较模型对照组明显降低[(14.2±3.1)、(8.6±1.8)U/L比(19.0±2.9)U/L,(2.0±1.1)、(1.2±0.7)分比(12.8±2.7)分,(2.3±1.4)、(1.7±0.5)分比(9. 3±2. 3)分],差异均有统计学意义(均P <0.01)。其中芪贞汤组内毒素水平明显低于氟尿嘧啶组,差异有统计学意义(P<0.01)。结论芪贞汤可通过抑制革兰阴性致病菌,减少抗原物质内毒素释放,抑制TLR4/核因子κB信号通路,而发挥抗CAC的作用。

Objective To explore the effect of Qizhen decoction on intestinal bacteria induced Toll-like receptor-4/nuclear factor-κB (TLR4/NF-κB)signaling pathway in colitis-associated cancer (CAC)mice. Methods Twenty-four C57BL/6J male mice were randomly divided into blank group,model control group, 5-fluorouracil group and Qizhen decoction group,with 6mice in each group.The latter 4groups were made CAC model by azoxymethane and sodium dextran sulfate.The blank group and the model control group were given 0.9%sodium chloride injection by gavage,and the remaining two groups were given corresponding drugs by gavage for 14days.Characteristics of intestinal flora were analyzed by 16S rDNA sequencing.Endotoxin content in serum was determined by enzyme linked immunosorbent assay.Expressions of TLR4and NF-κB p65protein in colon tissue were detected by immunohistochemistry.Results Sequencing results showed that Gram-negative bacteroides were commonly detected in the model control group and 5-fluorouracil group;intestinal flora in the Qizhen decoction group were mostly Gram-positive lactococcus and bacillus.Expression levels of endotoxin,TLR4 and NF-κB p65protein in the model control group were significantly higher than those in the blank group;the levels in the 5-fluorouracil group and Qizhen decoction group were significantly lower than those in the model control group[(14.2±3.1),(8.6±1.8)U/L vs (19.0±2.9)U/L;(2.0±1.1),(1.2±0.7)scores vs (12.8±2.7)scores,(2.3±1.4),(1.7±0.5)scores vs (9.3±2.3)scores ](all P <0.01);endotoxin level in the Qizhen decoction group was significantly lower than that in the 5-fluorouraeil group (P <0.01).Conclusion Qizhen decoction prevents CAC by inhibiting Gram-negative pathogens,reducing the release of endotoxin and inhibiting TLR4/NF-κB signaling pathway.