基于NLRP3炎症小体探讨半夏泻心汤对炎症性结直肠癌小鼠的影响

Exploration of the Effect of Banxia Xiexin Decoction on Inflammatory Colorectal Cancer Mice Based on NLRP3 Inflammatory Body

目的 基于NLRP(NOD-like receptor thermal protein domain associated protein,NLRP)3炎症小体探讨半夏泻心汤对炎症性结直肠癌(colorectal cancer,CRC)小鼠的改善作用。方法 小鼠CRC模型采用腹腔注射氧化偶氮甲烷联合口服葡聚糖硫酸钠法制备。100只小鼠按照随机数字表法分为正常对照组、模型组、半夏泻心汤低剂量、半夏泻心汤高剂量组及美沙拉嗪组,每组各20只,连续给药14 d。记录各组小鼠一般情况;光镜观察小鼠结肠和脾脏病理改变;TUNEL染色检测结肠组织肿瘤细胞凋亡情况、荧光定量PCR检测结肠组织NLRP3、芳香烃受体(aryl hydrocarbon receptor,AhR)、三叶因子(trefoil factor,TFF)3和p53肿瘤蛋白(tumor protein 53,P53)m RNA表达水平;免疫组化和Western Blot分别检测各组结肠组织中核增殖抗原Ki-67(Ki-67 antigen,Ki67)、AhR及NLRP3、AhR、TFF3和基质金属蛋白酶(matrix metalloproteinase,MMP)-9蛋白表达。结果 与正常对照组比较,模型组小鼠体质量明显减轻,结肠瘤体数目增多,脾脏和胸腺指数升高,血清中肿瘤坏死因子(tumor necrosis factor,TNF)-α,白介素(interleukin,IL)-6和干扰素(interferon,IFN)γ含量显著升高(P<0.01),IL-10含量下降;结肠组织中Ki67、AhR、NLRP3、AhR、MMP-9蛋白以及NLRP3、AhR和P53mRNA表达明显增高(P<0.05);而TFF3 m RNA和蛋白显著降低(P<0.01)。与模型组比较,半夏泻心汤和美沙拉嗪组上述指标的变化均发生逆转,均具有统计学差异(P<0.05),且以半夏泻心汤高剂量组变化更为显著。结论 半夏泻心汤可减轻CRC小鼠炎症反应,诱导肿瘤细胞凋亡从而改善肠黏膜屏障功能,其机制与阻断AhR通路活化抑制NLRP3炎症小体生成有关。

Objective Exploration of the effect of Banxia Xiexin decoction(BXD) on inflammatory colorectal cancer mice based on NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammatory Body.Methods The mouse CRC model was prepared by intraperitoneal injection of azomethane oxide combined with oral administration of sodium dextran sulfate.100 mice were randomly divided into a normal control group,model group,Banxia Xiexin decoction group,and mesalazine groups.The body weight,colon length,tumor formation rate,and spleen and thymus index of mice in each group were recorded.HE staining,ELISA,and TUNEL staining were applied to examine the colonic and spleen tissue morphology,the content of inflammatory cytokines in serum,and the apoptosis of tumor cells in each group,respectively.The expressions of Ki-67 antigen(Ki67) and AhR proteins in the colon were evaluated by immunohistochemistry.RT-qPCR was performed to determine the levels of NLRP3,aryl hydrocarbon receptor(AHR),trefoil factor(TFF)3,and tumor protein 53(p53) m RNA expression in colon tissue.Western blot was used to detect the regulatory effect of BXD on the expression of NLRP3,AHR,TFF3,and matrix metalloproteinase(MMP)-9 proteins in the colon of CRC mice.Results Compared with the normal control group,the model group had significantly reduced body weight and colon tumors,while increased spleen index and thymus index.The levels of tumor necrosis factor(TNF)-α,interleukin(IL)-6,and interferon(IFN)-γ in serum were increased remarkably( P< 0.01).However,IL-10 was decreased significantly(P<0.05).Besides,the expression of Ki67 and AhR protein in immunohistochemistry increased noticeably.The expression of NLRP3,AhR,and p53 m RNA in the colon of CRC mice increased significantly(P<0.01),however,the expression of TFF3 m RNA decreased evidently(P<0.01).The expression of NLRP3,AhR,and MMP-9 proteins in the colon was markedly increased(P<0.01),while TFF3 protein was significantly decreased(P<0.01).Compared with the model group,the changes in the above indexes of CRC mice in BXD group and mesalazine groups were reversed,and the differences had remarkable statistical significance( P< 0.05).Conclusion Banxia Xiexin decoction can reduce the inflammatory response of CRC mice,induce apoptosis of tumor cells,and improve intestinal mucosal barrier function,and its mechanism may be related to blocking AhR pathway activation and inhibiting NLRP3 inflammasome formation.