基于NLRP3炎症小体探讨参苓白术散治疗溃疡性结肠炎的作用机制

Mechanism of Shenling Baizhu Powder on treatment of ulcerative colitis based on NLRP3 inflammatory

基于NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermoprotein domain 3,NLRP3)炎症小体信号通路探讨参苓白术散(Shenling Baizhu Powder)对溃疡性结肠炎(ulcerative colitis, UC)模型小鼠的作用机制。70只SPF级BALB/c小鼠适应性喂养3 d后,随机分为7组:正常组、模型组、美沙拉嗪组、NLRP3抑制剂MCC950组以及参苓白术散高、中、低剂量组,每组10只。正常组自由饮用双蒸水,其余各组自由饮用葡聚糖硫酸钠(dextran sodium sulfate, DSS)诱导BALB/c小鼠建立急性UC模型,造模的同时开始灌胃给药,为期1周。实验期间对各组小鼠一般精神状态和疾病活动情况进行记录,并统计评分。实验结束后,收取结肠和血清,使用HE染色法观察结肠组织病理变化,酶联免疫吸附测定法(enzyme linked immunosorbent assay, ELISA)检测小鼠结肠组织中白细胞介素18(interleukin-18,IL-18)、髓过氧化物酶(myeloperoxidase, MPO)和血清中白细胞介素1β(interleukin-1β,IL-1β)水平的变化情况,免疫荧光法、免疫组化法分别检测结肠组织中NLRP3、IL-18的表达情况,蛋白免疫印迹法检测结肠组织蛋白NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein, ASC)、半胱氨酸天冬氨酸蛋白水解酶1(cystein-asparate protease 1,caspase-1)及其相关下游炎症因子的表达水平,以此探讨参苓白术散治疗UC的作用机制。与正常组相比,模型组小鼠疾病活动指数(disease activity index, DAI)评分、结肠病理损伤评分、血清中炎症因子IL-1β、结肠炎症因子IL-18和MPO含量都显著增加(P<0.05,P<0.01);结肠组织出现明显的病理学改变,其炎症因子表达含量升高,结肠中NLRP3、caspase-1、ASC、pro-IL-1β、cleaved-IL-1β、pro-IL-18、cleaved-IL-18蛋白表达显著上调(P<0.01)。与模型组相比,各给药组的DAI评分、病理学损伤评分、血清中炎症因子IL-1β、结肠炎症因子IL-18和MPO的表达均降低,结肠中NLRP3、caspase-1、ASC、pro-IL-1β、cleaved-IL-1β、pro-IL-18、cleaved-IL-18蛋白表达显著下调(P<0.05,P<0.01)。基于前期研究基础结合该实验结果发现,参苓白术散可改善由DSS诱发的UC炎症反应,改善肠道损伤情况,其作用机制可能与调控NLRP3炎症小体信号通路相关蛋白及其相关炎症因子有关。

This study deciphered the mechanism of Shenling Baizhu Powder in treatment of mouse model of ulcerative colitis(UC) via NOD-like receptor thermoprotein domain 3(NLRP3) signaling pathway. After three days of adaptive feeding, 70 SPF-grade BALB/c mice were randomized into 7 groups: normal group, model group(dextran sodium sulfate, DSS), mesalazine group(DSS + 5-aminosalicylic acid, 5-ASA), NLRP3 inhibitor group(DSS + MCC950), and high-, medium-, and low-dose Shenling Baizhu Powder groups(DSS + high-, medium-, and low-dose Shenling Baizhu Powder), with 10 mice per group. The normal group had free access to double distilled water, and the remaining groups had free access to DSS-containing water to establish the acute UC model. Intragastric administration was started at the same time as modeling for one week. During the experiment, the general mental state and disease activity of each group of mice were recorded and scored. After the experiment, colon and serum samples were collected. The pathological changes in colon tissue were observed through hematoxylin-eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the levels of interleukin-18(IL-18) and myeloperoxidase(MPO) in colon tissue and interleukin-1β(IL-1β) in serum. Immunofluorescence(IF) and immunohistochemistry(IHC) methods were employed to examine the expression of NLRP3 and IL-18 in colon tissue. Western blot was employed to measure the protein levels of NLRP3, apoptosis-associated speck-like protein(ASC), cystein-aspartate protease 1(caspase-1), and downstream inflammatory cytokines in colon tissue. Compared with the normal group, the modeling of UC increased the disease activity index(DAI), colon pathological injury score, IL-1β level in serum, and IL-18 and MPO levels in colon tissue(P<0.05, P<0.01). Furthermore, the modeling caused obvious pathological changes and up-regulated the expression of NLRP3, caspase-1, ASC, pro-IL-1β, cleaved-IL-1β, pro-IL-18, and cleaved-IL-18 in the colon(P<0.01). Compared with the model group, the administration of corresponding drugs decreased the DAI, pathological injury score, IL-1β level in serum, and IL-18 and MPO levels in colon tissue, and down-regulated the protein levels of NLRP3, caspase-1, ASC, pro-IL-1β, cleaved-IL-1β, pro-IL-18, and cleaved-IL-18 in the colon(P<0.05, P<0.01). According to the results of previous study and this study, we concluded that Shenling Baizhu Powder can alleviate the inflammatory response and intestinal damage of DSS-induced UC by regulating the expression of the proteins and inflammatory cytokines associated with NLRP3 signaling pathway.