蛇床子素介导Toll样受体4/核因子-κB通路调控肾癌小鼠免疫系统抑制肿瘤细胞增殖和迁移的作用机制

Mechanism of osthole mediated Toll-like receptor 4/nuclear factor-κB pathway in regulating immune system inhibition of tumor cell proliferation and migration in renal carcinoma mice

目的探究蛇床子素介导Toll样受体4/核因子-κB(TLR4/NF-κB)通路调控肾癌小鼠免疫系统抑制肿瘤细胞增殖和迁移的机制。方法通过皮下注射人肾癌细胞系786-0的方法构建肾癌小鼠模型。将45只模型小鼠随机分为模型组、蛇床子素组和蛇床子素+AXO-102组,每组15只。蛇床子素组小鼠灌胃蛇床子素(100 mg/kg),蛇床子素+AXO-102组小鼠灌胃蛇床子素(100 mg/kg)+AXO-102(50 ng/kg),模型组小鼠给予相同体积的生理盐水灌胃,均连续20天。比较3组小鼠肿瘤体积及质量。采用免疫组化染色检测3组小鼠Ki-67蛋白表达水平、Western blot检测蛋白相对表达水平、流式细胞术检测T细胞亚群水平、RT-qPCR检测肿瘤组织中TLR4和NF-κB mRNA相对表达水平并进行比较。结果蛇床子素组和蛇床子素+AXO-102组的肿瘤体积、质量、Ki-67、基质金属蛋白酶(MMP)2、MMP9、TLR4和NF-κB mRNA和蛋白表达水平均显著低于模型组,且蛇床子素+AXO-102组显著低于蛇床子素组(P<0.05)。蛇床子素组和蛇床子素+AXO-102组的CD4^(+)T淋巴细胞表达水平、CD4^(+)/CD8^(+)T淋巴细胞表达水平比值均显著高于模型组,且蛇床子素+AXO-102组显著高于蛇床子素组(P<0.05)。结论蛇床子素可能通过调控TLR4/NF-κB抑制肾癌细胞的生长和迁移,并提高免疫力。

Objective To investigate the mechanism of osthole mediated Toll-like receptor 4/nuclear factor-κB(TLR4/NF-κB)pathway to regulate immune system and inhibit tumor cell proliferation and migration in renal carcinoma mice.Methods The mouse model of renal carcinoma was established by subcutaneous injection of human renal carcinoma cell line 786-0.Forty-five model mice were randomly divided into model group,osthole group and osthole+AXO-102 group,with 15 mice in each group.Osthole group was intragastrically administered with osthole(100 mg/kg),osthole+AXO-102 group was intragastrically administered with osthole(100 mg/kg)+AXO-102(50 ng/kg),and model group was intragastrically administered with the same volume of normal saline for consecutive 20 days.The tumor volume and quality of the three groups were compared.Immunohistochemical staining was used to detect Ki-67 protein expression level,Western blotting was used to detect relative protein expression level,flow cytometry was used to detect T cell subsets,RT-qPCR was used to detect relative mRNA expression level of TLR4 and NF-κB in tumor tissues,and comparison was performed.Results Tumor volume,mass,mRNA and protein expression levels of Ki-67,MMP2,MMP9,TLR4 and NF-κB in osthole group and osthole+AXO-102 group were significantly lower than those in model group,and osthole+AXO-102 group was significantly lower than that in osthole+AXO-102 group(P<0.05).The expression level of CD4^(+)T lymphocyte and the ratio of CD4^(+)/CD8^(+)T lymphocyte expression level in osthole group and OSthole+AXO-102 group were significantly higher than that in model group,and osthole+AXO-102 group was significantly higher than that in osthole group(P<0.05).Conclusion Osthole may inhibit the growth and migration of renal cancer cells by regulating TLR4/NF-κB,and alleviate and improve immunity.