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    化瘀消痞汤调控NLRP3/ASC/Caspase-1通路抑制巨噬细胞焦亡改善胃癌前病变的机制研究

    Study on the Mechanism of Huayu Xiaopi Decoction Regulating NLRP3/ASC/Caspase-1 Pathway to Inhibit Macrophage Pyroptosis and Improve Gastric Precancerous Lesions

      摘要
    • 摘要:
      目的 基于NLRP3/ASC/Caspase-1信号通路研究化瘀消痞汤对胃癌前病变(precancerous lesions of gastric cancer，PLGC)模型大鼠的干预作用及机制。
      方法 通过N-甲基-N’-硝基-N-亚硝基胍联合氨水自由饮用、饥饱失常、乙醇及雷尼替丁灌胃的复合造模法构建PLGC模型大鼠，并随机分为模型组、对照组及化瘀消痞汤高、中、低剂量实验组，每组12只；另取12只健康大鼠设为空白组。化瘀消痞汤高、中、低剂量组每天分别予24.8、12.4、6.2 g·kg−1化瘀消痞汤药液灌胃，对照组每天予等量0.002 g·kg−1叶酸溶液灌胃，空白组和模型组每天均予等量0.9% NaCl灌胃，每日1次，共90 d。观察并记录各组大鼠一般生存状况及体质量，H&E染色及AB-PAS染色观察各组大鼠胃组织病理学变化，实时荧光定量聚合酶链式反应法(reverse transcription quantitative polymerase chain reaction，RT-qPCR)检测各组大鼠胃组织中IL-1β、IL-18 mRNA表达水平，western blotting检测各组大鼠胃组织NLRP3、Caspase1、ASC、GSDMD-N蛋白表达水平，多重免疫荧光(multiplex immunohistochemistry，mIHC)检测各组大鼠胃组织巨噬细胞中NLRP3、GSDMD-N蛋白表达水平。
      结果 与空白组相比，模型组大鼠一般生存状况差，体质量显著减轻，胃组织有明显的病理损伤，胃组织IL-1β、IL-18 mRNA表达水平，NLRP3、ASC、 Caspase-1及 GSDMD-N蛋白表达水平明显升高，胃组织巨噬细胞内NLRP3、GSDMD-N表达水平明显升高；相较于模型组，各给药组大鼠生存状况明显提升，体质量明显增加，病理损伤显著改善，胃组织IL-1β、IL-18 mRNA表达水平，NLRP3、ASC、 Caspase-1及 GSDMD-N蛋白表达水平明显降低(P<0.05)，胃组织巨噬细胞内NLRP3、GSDMD-N表达水平明显减少。
      结论 化瘀消痞汤能够显著减轻胃黏膜炎症损伤，阻断“炎-癌”转化，有效防治PLGC，其具体机制与抑制NLRP3/ASC/Caspase-1通路介导的巨噬细胞焦亡有关。

       

      Abstract:
      OBJECTIVE To investigate the intervention effect and mechanism of Huayu Xiaopi decoction(HYXPD) on precancerous lesions of gastric cancer(PLGC) model rats based on the NLRP3/ASC/Caspase-1 signaling pathway.
      METHODS PLGC model rats were established using a composite modeling method combining free drinking of N-methyl-N’-nitro-N-nitrosoguanidine solution, irregular feeding/fasting, ethanol gavage, and ranitidine gavage. A total of 60 model rats were randomly divided into model group, control group, and high-, medium-, low-dose HYXPD experimental groups(12 rats per group), with an additional 12 healthy rats as the blank group. The high-, medium-, and low-dose HYXPD groups were gavaged with HYXPD at doses of 24.8, 12.4, and 6.2 g·kg−1, respectively The control group received 0.002 g·kg−1 folic acid solution. Both blank and model groups were gavaged with an equal volume of 0.9% NaCl, once daily for 90 d. General survival status and body weight of rats in each group were observed and recorded. Pathological changes of gastric tissue were observed by HE staining and AB-PAS staining. IL-1β, IL-18 mRNA expression levels were detected by RT-qPCR. Western blotting was used to measure the protein expression levels of NLRP3, Caspase-1, ASC, and GSDMD-N. Multiplex immunohistochemistry was used to detect the expression of NLRP3 and GSDMD-N protein in macrophages of gastric tissue of rats in each group.
      RESULTS Compared with the blank group, the rats in the model group exhibited a poor general survival status, a significantly reduction in body weight, and evident pathological damage in gastric tissue. Moreover, the expression of IL-1β, IL-18 mRNA, as well as the protein of NLRP3, ASC, Caspase-1 and GSDMD-N, were significantly increased in gastric tissue. Correspondingly, the expression of NLRP3 and GSDMD-N in gastric tissue macrophages was markedly increased. In contrast, all treatment groups showed significant improvements compared to the model group, including better overall health status, increased body weight, and ameliorated pathological damage in the gastric tissue. Furthermore, the expression of IL-1β, IL-18 mRNA, along with NLRP3, ASC, Caspase-1, and GSDMD-N protein in gastric tissue were significantly reduced. The expression of NLRP3 and GSDMD-N in macrophages of gastric tissue decreased significantly.
      CONCLUSION HYXPD can significantly reduce gastric mucosal inflammation, block the “inflammation-cancer” transformation, effectively prevent PLGC, and its specific mechanism is related to inhibition of NLRP3/ASC/Caspase-1 pathway mediated macrophage pyroptosis.

       

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