Liu Yu-Hui, Rong Zi-Ling, Zhu Hong-Yang, Li Yu-Ting, You Yu
Jiangxi University of Chinese Medicine Nanchang 330004, China.
Gastroenterology Department of the First Affiliated Hospital of Nanchang University Nanchang 330006, China.
Zhongguo Zhong Yao Za Zhi. 2022 Nov;47(21):5863-5871. doi: 10.19540/j.cnki.cjcmm.20220630.401.
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.
本研究通过NOD样受体热蛋白结构域3（NLRP3）信号通路，阐明了参苓白术散治疗溃疡性结肠炎（UC）小鼠模型的机制。适应性喂养3天后，将70只SPF级BALB/c小鼠随机分为7组：正常组、模型组（葡聚糖硫酸钠，DSS）、美沙拉嗪组（DSS + 5-氨基水杨酸，5-ASA）、NLRP3抑制剂组（DSS + MCC950）以及高、中、低剂量参苓白术散组（DSS + 高、中、低剂量参苓白术散），每组10只小鼠。正常组自由饮用双蒸水，其余各组自由饮用含DSS的水以建立急性UC模型。造模同时开始灌胃给药，持续1周。实验期间，记录并评分每组小鼠的一般精神状态和疾病活动度。实验结束后，采集结肠和血清样本。通过苏木精-伊红（HE）染色观察结肠组织的病理变化。采用酶联免疫吸附测定（ELISA）法测定结肠组织中白细胞介素-18（IL-18）和髓过氧化物酶（MPO）水平以及血清中白细胞介素-1β（IL-1β）水平。采用免疫荧光（IF）和免疫组织化学（IHC）方法检测结肠组织中NLRP3和IL-18的表达。采用蛋白质免疫印迹法检测结肠组织中NLRP3、凋亡相关斑点样蛋白（ASC）、半胱天冬酶-1（caspase-1）及下游炎性细胞因子的蛋白水平。与正常组相比，UC造模增加了疾病活动指数（DAI）、结肠病理损伤评分、血清中IL-1β水平以及结肠组织中IL-18和MPO水平（P<0.05，P<0.01）。此外，造模导致结肠出现明显病理变化，并上调了结肠中NLRP3、caspase-1、ASC、前体IL-1β、裂解型IL-1β、前体IL-18和裂解型IL-18的表达（P<0.01）。与模型组相比，给予相应药物可降低DAI、病理损伤评分、血清中IL-1β水平以及结肠组织中IL-18和MPO水平，并下调结肠中NLRP3、caspase-1、ASC、前体IL-1β、裂解型IL-1β、前体IL-18和裂解型IL-18的蛋白水平（P<0.05，P<0.01）。根据既往研究和本研究结果，我们得出结论：参苓白术散可通过调节与NLRP3信号通路相关的蛋白和炎性细胞因子的表达，减轻DSS诱导的UC的炎症反应和肠道损伤。
