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 Pyrolysis and Improve Gastric Precancerous Lesions

关键词:  化瘀消痞汤  胃癌前病变  巨噬细胞  细胞焦亡  炎-癌转化

Key words:  Huayu Xiaopi Decoction  precancerous lesions of gastric cancer (PLGC)  macrophages  pyroptosis  inflammation-cancer transformation

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(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。中药高、中、低剂量组每天分别予24.8、12.4、6.2 g/kg化瘀消痞汤药液灌胃,对照组每天予等量0.002 g/kg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天。观察并记录各组大鼠一般生存状况及体质量,HE染色及AB-PAS染色观察各组大鼠胃组织病理学变化,用实时荧光定量聚合酶链式反应法(RT-qPCR)检测各组大鼠胃组织中IL-1β、IL-18 mRNA表达水平,用蛋白质印迹法(Western blot)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(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,体重显著减轻,胃组织有明显的病理损伤,胃组织IL-1β、IL-18 mRNA表达、NLRP3、ASC、 Caspase-1及 GSDMD-N蛋白表达水平明显升高(P<0.05),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,体重明显增加、病理损伤显著改善,胃组织IL-1β、IL-18 mRNA表达、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 (MNNG) solution, irregular feeding/fasting, ethanol gavage, and ranitidine gavage. A total of 72 rats were randomly divided into model group, control group, and high-, medium-, low-dose experimental groups (12 rats per group), with an additional 12 healthy rats as the blank group. The experimental groups were gavaged with HYXPD at doses of 24.8, 12.4, and 6.2 g/kg, respectively; the control group received 2.00×10?3 g/kg folic acid solution; the blank and model groups were gavaged with an equal volume of 0.9% NaCl, once daily for 90 days. General survival status and body weight of rats in each group were observed and recorded. Pathological changes of stomach tissue were observed by HE staining and AB-PAS staining. IL-1β, IL-18mRNA expression levels were detected by RT-qPCR. Western blot was used to measure the protein expression levels of NLRP3, Caspase-1, ASC, and GSDMD-N. Multiplex immunofluorescence 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 had poor general survival status, significantly reduced body weight, obvious pathological damage in stomach tissue, significantly increased expression of IL-1β, IL-18mRNA, NLRP3, ASC, Caspase-1 and GSDMD-N protein in stomach tissue (P<0.05), and significantly increased expression of NLRP3 and GSDMD-N in macrophages in stomach tissue. Compared with the model group, the survival status of rats in each treatment group was significantly improved, the body weight was significantly increased, the pathological damage was significantly improved, and the expression levels of IL-1β, IL-18mRNA and NLRP3,ASC,Caspase-1 and GSDMD-N protein in gastric tissue were significantly reduced (P<0.05). The expression of NLRP3 and GSDMD-N in macrophages of gastric tissue decreased significantly (P<0.05). CONCLUSION Huayu Xiaopi Decoction 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 charring death.