苦参碱经TLR4/NF-κB通路对膀胱癌小鼠免疫机制的调控作用-东南大学学报医学版

苦参碱经TLR4/NF-κB通路对膀胱癌小鼠免疫机制的调控作用

单位：咸宁市中心医院/湖北科技学院附属第一医院泌尿外科, 湖北咸宁 437100

关键词：膀胱癌苦参碱 Toll样受体4/核因子-κB途径免疫抑制 T淋巴细胞小鼠

分类号：R737.14

出版年·卷·期（页码）：2021·40·第六期（813-819）

摘要：

目的: 探究苦参碱通过Toll样受体4(TLR4)/核因子(NF)-κB通路对膀胱癌小鼠免疫机制的调控作用。方法: 通过皮下注射膀胱癌细胞系T24的方法构建膀胱癌小鼠模型。将36只模型小鼠随机分为对照组、苦参碱低剂量组和苦参碱高剂量组(n=12)。苦参碱腹腔注射干预, 剂量分别为5、10 mg·kg^-1。测量肿瘤生长情况, 采用蛋白质印迹法检测增殖相关蛋白细胞周期蛋白D1(CyclinD1)和增殖细胞核抗原(PCNA)表达, TUNEL染色检测细胞凋亡。比较各组TLR4/NF-κB通路的转录和翻译水平。采用流式细胞术检测各组CD4⁺、CD8⁺细胞百分比, 免疫组化染色检测肿瘤组织中CD4⁺细胞浸润情况。结果: 3组小鼠各项指标比较差异均有统计学意义(P < 0.05)。苦参碱低剂量组和苦参碱高剂量组的肿瘤体积、质量, Cyclin D1和PCNA蛋白表达水平, TLR4、NF-κB mRNA及蛋白表达水平均显著低于对照组, 凋亡指数均显著高于对照组(P < 0.05);苦参碱高剂量组的肿瘤体积、质量, Cyclin D1和PCNA蛋白表达水平, TLR4、NF-κB mRNA及蛋白表达水平均显著低于苦参碱低剂量组, 凋亡指数显著高于苦参碱低剂量组(P < 0.05)。3组小鼠的CD4⁺细胞百分比、CD4⁺/CD8⁺和CD4⁺细胞浸润情况比较差异有统计学意义(P < 0.05)。苦参碱低剂量组和苦参碱高剂量组的CD4⁺细胞百分比、CD4⁺/CD8⁺、CD4⁺细胞浸润水平均显著高于对照组(P < 0.05);苦参碱高剂量组的CD4⁺细胞百分比、CD4⁺/CD8⁺、CD4⁺细胞浸润水平均显著高于苦参碱低剂量组(P < 0.05)。结论: 苦参碱可能通过调控TLR4/NF-κB途径抑制膀胱癌小鼠模型的肿瘤生长和诱导凋亡, 并提高CD4⁺/CD8⁺水平促进CD4⁺细胞的浸润。

Objective: To explore the effects of matrine on bladder cancer mice through Toll like receptor 4 (TLR4)/nuclear factor (NF)-κB pathway and its regulation of immunity.Methods: A mouse model of bladder cancer was constructed by subcutaneous injection of bladder cancer cell line T24. 36 model mice were randomly divided into control group, low-dose matrine group and high-dose matrine group (n=12). Matrine was injected intraperitoneally for intervention, the doses were 5 and 10 mg·kg^-1. The tumor growth was measured. The proliferation-related proteins CyclinD1 and PCNA were detected by Western blot. TUNEL staining was applied to detect cell apoptosis. The transcription and translation levels of TLR4/NF-κB pathway in each group were compared. The percentage of CD4⁺ and CD8⁺ in each group were detected by flow cytometry. The infiltration of CD4⁺ cells in tumor tissue was detected by immunohistochemical staining.Results: The indicators of the mice in three groups had significant differences (P < 0.05). The tumor volume, mass, Cyclin D1 and PCNA protein, TLR4 and NF-κB mRNA and protein levels of the low-dose matrine group and the high-dose matrine group were significantly lower than those of the control group, and the apoptosis index was significantly higher than that of the control group (P < 0.05). The tumor volume, mass, Cyclin D1 and PCNA protein, TLR4 and NF-κB mRNA and protein levels of the high-dose matrine group were significantly lower than those of the low-dose matrine group, and the apoptosis index was significantly higher than that of the low-dose matrine group ( P < 0.05). The CD4⁺ percentage, CD4⁺/CD8⁺ and CD4⁺ infiltration of the three groups were significantly different (P < 0.05). The CD4⁺ percentage, CD4⁺/CD8⁺ and CD4⁺ infiltration levels of low-dose matrine and high-dose matrine groups were significantly higher than those of the control group (P < 0.05). The CD4⁺ percentage, CD4⁺/CD8⁺ and CD4⁺ infiltration levels of the high-dose matrine group were significantly higher than those of the low-dose matrine group (P < 0.05).Conclusion: Matrine may inhibit tumor growth and induce apoptosis in mouse models of bladder cancer by regulating the TLR4/NF-κB pathway, and increase the level of CD4⁺/CD8⁺ to promote CD4⁺ infiltration.

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