School of Basic Medicine, Gansu university of Chinese Medicine, Lanzhou 730000, China.
Department of Thoracic Surgery, Gansu Provincial Cancer Hospital, Lanzhou 730000, China.
J Tradit Chin Med. 2022 Aug;42(4):565-575. doi: 10.19852/j.cnki.jtcm.20220608.001.
To investigate the therapeutic effect of Sishen Wan (, SSW) on ulcerative colitis (UC) induced by dinitrobenzene sulfonic acid and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB (TLR2/IRAK4/NF-κB) sig-naling pathway in colonic tissue.
In this study, 120 Sprague-Dawley rats were randomly divided into blank and model groups. The experimental UC model in rats was established by subcutaneous injection of hydrocortisone + senna gavage for 21 d + dinitrobenzene sulfonic acid (DNBS)/ ethanol solution enema. The successful model rats were randomly divided into the model group; mesalazine (0.36 g/kg) group; and high-, medium-, and low- dose SSW (24, 12, and 6 g/kg) groups. The model and blank groups were gavaged with equal volumes of distilled water once a day for 21 d. The general condition of the rats was observed, and the body mass, fecal properties, and occult blood were recorded for calculating the disease activity index (DAI) score. The colonic tissue of the rats was collected, and its general morphology and pathological form were noted for obtaining the colonic mucosal injury index (CMDI) score. Hematoxylin-eosin staining was used to view the pathological changes of the colon tissue in each group, apoptosis of the cells was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and quantitative real-time polymerase chain reaction was used to measure the expressions of TLR2, myeloid differentiation primary response gene 88 (MyD88), IRAK4, and NF-κB p65 mRNA in the colon tissue. The expressions of TLR2, MyD88, IRAK4, and NF-κB p65 protein were detected using western blotting and immunohistochemistry assay, and the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the colon tissue were determined using enzyme linked immunosorbent assay.
Compared with the blank group, the general condition of the model group was relatively poor. The DAI and CMDI scores of the model group increased significantly (< 0.01), the glands and intestinal mucosa disappeared partially, and several inflammatory cells infiltrated and gathered in the mucosal layer and base layer of the rats in the model group. Furthermore, the cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of rats in the model group increased significantly (< 0.01). The levels of IL-1β and TNF-α increased significantly in the colon tissue of rats in the model group (< 0.01). After treatment with SSW, compared with the model group, the general condition of the UC rats improved. Moreover, the DAI and CMDI scores of the UC rats decreased significantly (< 0.05), and the pathological changes in the colon tissue of the UC rats tended to be normal. The cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of the UC rats decreased gradually ( < 0.01), and the levels of IL-1β and TNF-α decreased significantly (< 0.01).
SSW can improve the general condition and alleviate the intestinal mucosal injury of UC model rats. Additionally, SSW can inhibit the TLR2/IRAK4/ NF-κB signaling pathway, but further studies are required to confirm it.
观察四神丸对二硝基苯磺酸诱导的溃疡性结肠炎（UC）大鼠的治疗作用，并探讨其对结肠组织中 Toll 样受体 2/白细胞介素 1 受体相关激酶 4/核因子-κB（TLR2/IRAK4/NF-κB）信号通路的影响。
将 120 只 SD 大鼠随机分为空白组和模型组。采用氢化可的松+番泻叶灌胃+二硝基苯磺酸（DNBS）/乙醇溶液灌肠建立大鼠实验性 UC 模型，成功造模的大鼠随机分为模型组、美沙拉嗪（0.36 g/kg）组、四神丸高、中、低剂量（24、12、6 g/kg）组，模型组和空白组给予等体积蒸馏水灌胃，连续 21 d。观察大鼠一般情况，记录体质量、粪便性状、粪便隐血，计算疾病活动指数（DAI）评分；取大鼠结肠组织，观察其大体形态和病理形态，计算结肠黏膜损伤指数（CMDI）评分；HE 染色观察各组大鼠结肠组织的病理变化，末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记法（TUNEL）检测细胞凋亡，实时荧光定量聚合酶链反应（qPCR）检测 TLR2、髓样分化因子 88（MyD88）、IRAK4、NF-κB p65 mRNA 在结肠组织中的表达；Western blot 和免疫组化法检测 TLR2、MyD88、IRAK4、NF-κB p65 蛋白的表达，酶联免疫吸附法（ELISA）检测大鼠结肠组织中白细胞介素 1β（IL-1β）和肿瘤坏死因子-α（TNF-α）的水平。
与空白组比较，模型组大鼠一般情况较差，DAI 和 CMDI 评分均显著升高（P<0.01），大鼠结肠腺体和肠黏膜部分消失，黏膜层和黏膜下层可见较多炎细胞浸润聚集；结肠组织 TLR2、MyD88、IRAK4、NF-κB p65 mRNA 及蛋白表达水平均显著升高（P<0.01），IL-1β和 TNF-α 水平均显著升高（P<0.01）。与模型组比较，四神丸各剂量组大鼠一般情况改善，DAI 和 CMDI 评分均显著降低（P<0.05），结肠组织病理变化趋于正常，TLR2、MyD88、IRAK4、NF-κB p65 mRNA 及蛋白表达水平均逐渐降低（P<0.01），IL-1β和 TNF-α 水平均显著降低（P<0.01）。
四神丸可改善 UC 模型大鼠的一般情况，减轻其肠道黏膜损伤，其作用机制可能与抑制 TLR2/IRAK4/NF-κB 信号通路有关。
