基于mTOR/Beclin1通路探讨血竭散对克罗恩病大鼠结肠组织自噬和炎症小体的影响

Effect of Xuejie Powder on Autophagy and Inflammasomes in Colon Tissue of Rats with Crohn's Disease Based on the mTOR/Beclin1 Pathway

目的研究血竭散对克罗恩病大鼠结肠组织自噬和炎症小体的影响,探讨其治疗克罗恩病的作用机制。方法采用2,4,6-三硝基苯磺酸诱导复制克罗恩病大鼠模型。模型复制后,将36只大鼠随机分为空白组,模型组,血竭散低、中、高剂量组和柳氮磺砒啶(sulfasalazine,SASP)组,每组6只。苏木精-伊红染色法观察大鼠结肠组织病理学改变,酶联免疫吸附法检测结肠组织中炎症细胞因子表达水平,实时荧光定量PCR法检测Beclin1、NOD样受体家族含pyrin结构域蛋白3(NOD-like receptor family pyrin domain containing 3,NLRP3)和黑素瘤缺乏因子2(absent in melanoma 2,AIM2)mRNA表达水平,蛋白免疫印迹法检测哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、p-mTOR、Beclin1、NLRP3、AIM2、含Caspase募集结构域的凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a Caspase recruitment domain,ASC)和Caspase-1蛋白表达水平,免疫荧光法检测微管相关蛋白轻链3β(microtubule-associated protein light chain 3 beta,LC3B)表达水平。结果与模型组比较,血竭散和SASP干预后大鼠结肠组织病理学评分,白细胞介素-1β(interleukin-1 beta,IL-1β)、白细胞介素-18(interleukin-18,IL-18)和肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)表达水平显著降低(P<0.05)。与空白组比较,模型组大鼠结肠组织中p-mTOR表达水平显著降低,Beclin1、LC3B、NLRP3、AIM2、ASC和Caspase-1表达水平显著增加(P<0.05)。血竭散和SASP干预能上调p-mTOR表达水平,下调Beclin1、LC3B、NLRP3、AIM2、ASC和Caspase-1表达水平(P<0.05)。结论血竭散可能通过调控mTOR/Beclin1信号通路抑制肠细胞自噬,减少炎症小体活化,改善克罗恩病大鼠结肠炎。

Objective To investigate the mechanism of action of Xuejie Powder in the treatment of Crohn's disease(CD)by analyzing its effect on autophagy and inflammasomes in colon tissue of CD rats.Methods After a rat model of CD was established by 2,4,6-trinitrobenzene sulfonic acid,36 rats were randomly divided into control group,model group,sulfasalazine(SASP)group,and low-,middle-,and high-dose Xuejie Powder groups,with 6 rats in each group.Hematoxylin and eosin staining was used to observe the histopathological changes of colon tissue;ELISA was used to measure the expression of inflammatory cytokines in colon tissue;quantitative real-time PCR was used to measure the mRNA expression of Beclin1,NOD-like receptor family pyrin domain containing 3(NLRP3),and absent in melanoma 2(AIM2),and Western blotting was used to measure the protein expression of mammalian target of rapamycin(mTOR),p-mTOR,Beclin1,NLRP3,AIM2,ASC,and Caspase-1;immunofluorescence assay was used to measure the expression of microtubule-associated protein light chain 3B(LC3B).Results Compared with the model group,the Xuejie Powder groups and the SASP group had significant reductions in the histopathological score of colon tissue and the expression levels of interleukin-1β,interleukin-18,and tumor necrosis factor-α(P<0.05).Compared with the control group,the model group had a significant reduction in the expression level of p-mTOR in colon tissue and significant increases in the expression levels of Beclin1,LC3B,NLRP3,AIM2,ASC,and Caspase-1(P<0.05).Xuejie Powder and SASP upregulated the expression level of p-mTOR and downregulated the expression levels of Beclin1,LC3B,NLRP3,AIM2,ASC,and Caspase-1(P<0.05).Conclusion Xuejie Powder might inhibit intestinal cell autophagy,reduce the activation of inflammasomes,and improve colitis in CD rats by regulating the mTOR/Beclin1 signaling pathway.