痛泻要方对肝郁脾虚型UC大鼠结肠组织Claudin-1、ZO-1及血清COX-2、iNOS表达的影响

樊俐慧;朱向东;杨霞;王志刚

文章摘要

樊俐慧;朱向东;杨霞;王志刚.痛泻要方对肝郁脾虚型UC大鼠结肠组织Claudin-1、ZO-1及血清COX-2、iNOS表达的影响[J].中医药信息,2023,40(8):15-22

痛泻要方对肝郁脾虚型UC大鼠结肠组织Claudin-1、ZO-1及血清COX-2、iNOS表达的影响

Effect of Tongxie Yaofang on Claudin－1 and ZO－1 in Colon Tissues and COX－2 and iNOS in Serum of IC Rats with Liver Depression and Spleen Deficiency

投稿时间：2022-09-27 录用日期：2022-10-12

DOI：10.19656/j.cnki.1002-2406.20230803

中文关键词: 痛泻要方溃疡性结肠炎密封蛋白1 紧密连接蛋白1 环氧合酶－2 诱导型一氧化氮合酶

英文关键词: Tongxie Yaofang Ulcerative colitis Claudin－1 ZO－1 COX－2 iNOS

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作者	单位
樊俐慧;朱向东;杨霞;王志刚

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中文摘要:

目的：探讨痛泻要方对肝郁脾虚型溃疡性结肠炎（ulcerative colitis, UC）模型大鼠血清中环氧合酶-2(cyclooxygenase-2, COX-2)、诱导型一氧化氮合酶（induced nitric oxide synthase, iNOS）含量及结肠组织中密封蛋白1(Claudin-1)、紧密连接蛋白1(ZO-1)基因及蛋白表达的影响。方法：100只Wistar大鼠用病-证结合法复建肝郁脾虚型UC模型，按随机数字表法将造模组大鼠分为模型组（10 g/kg蒸馏水灌胃），美沙拉嗪组（0.4 g/kg美沙拉嗪灌胃），痛泻要方高、中、低剂量组（20.8、10.4、5.2 g/kg痛泻要方灌胃），每组20只。另取20只正常大鼠作为空白组（10 g/kg蒸馏水灌胃），各组灌胃体积均为10 mL/kg，连续21 d。药物治疗结束后，取血清及结肠组织，ELISA法检测血清中COX-2、iNOS含量；观察结肠组织大体形态及损伤情况，HE染色观察病理表现，RT-qPCR法检测Claudin-1、ZO-1 mRNA表达，免疫组化法和Western blot法检测Claudin-1、ZO-1平均光密度值及蛋白表达。结果：光镜观察显示，模型组大鼠结肠黏膜充血、水肿，炎性细胞浸润，细胞间紧密连接断裂；经痛泻要方干预后，大鼠结肠黏膜病理变化明显改善，细胞间紧密连接正常。与空白组比较，模型组大鼠血清中COX-2、iNOS含量明显升高（P <0.01）;Claudin-1、ZO-1 mRNA、平均光密度值和蛋白表达明显降低（P <0.01）。与模型组比较，美沙拉嗪组COX-2、iNOS含量降低（P <0.01）；痛泻要方高、中剂量组COX-2、iNOS含量显著降低（P <0.01），痛泻要方低剂量组COX-2、i NOS含量降低（P <0.05）；美沙拉嗪组及痛泻要方高、中剂量组Claudin-1、ZO-1 mRNA平均光密度值及蛋白表达量显著升高（P <0.01）；痛泻要方低剂量组Claudin-1、ZO-1 mRNA表达量与模型组比较，差异无统计学意义（P> 0.05）；痛泻要方低剂量组Claudin-1、ZO-1平均光密度值升高（P <0.05），蛋白表达升高（P <0.01,P <0.05）。结论：痛泻要方治疗肝郁脾虚型UC的作用可能与上调关键蛋白Claudin-1、ZO-1的表达量及降低炎症细胞因子COX-2、iNOS的含量有关。

英文摘要:

Objective: To investigate the effect of Tongxie Yaofang on the contents of cyclooxygenase-2(COX-2) and induced nitric oxide synthase(i NOS) in serum and gene and protein expressions of Claudin-1 and zonula occludens-1(ZO-1) in colon tissues of rats with ulcerative colitis(UC) of Liver depression and Spleen deficiency type. Methods: 100 Wistar rats were used to reconstruct the UC model of Liver depression and Spleen deficiency by disease-syndrome combination method. The successful modeling rats were randomly divided into the model group(10 g/kg distilled water), the Mesalazine group(0. 4 g/kg Mesalazine), and the Tongnxie Yaofang groups of highdose, medium-dose and low-dose(20. 8 g/kg, 10. 4 g/kg, 5. 2 g/kg), with 20 rats in each group. Another 20 normal rats were taken as the blank control(10 g/kg distilled water). The gavage volume of each group was 10 ml/kg. After 21 days of corresponding intervention, the serum contents of COX-2 and iNOS were detected by ELISA; the general morphology and injury of colon tissues were observed, and HE staining was used to observe the pathological manifestations. RT-q PCR was used to detect the m RNA expressions of Claudin-1 and ZO-1.Immunohistochemistry and Western blot were used to detect the average optical density and the protein expressions of Claudin-1 and ZO-1. Results: Light microscopic observation showed that the colonic mucosa was in congested and edema state, there was inflammatory cell infiltration, and the tight junction between cells was broken in the model group. After the intervention of Tongxie Yaofang, the pathological changes of rat colonic mucosa were significantly improved and the tight junction between cells was normal. Compared with those in the blank group, the serum contents of COX-2 and i NOS increased significantly(P < 0. 01), and the m RNA and protein expressions of Claudin-1 and ZO-1, as well as the average optical density decreased significantly in the model group(P <0. 01). Compared with those in the model group, the contents of COX-2 and iNOS decreased in the Mesalazine group(P < 0. 01); the contents of COX-2 and i NOS significantly decreased in the Tongxie Yaofang groups of high-dose and medium-dose(P < 0. 01), and the contents of COX-2 and iNOS decreased in the low-dose group as well(P < 0. 05). Compared with those in the model group, the mRNA expressions of Claudin-1 and ZO-1, as well as the average optical density increased significantly in the Mesalazine group and the Tongxie Yaofang groups of high-dose and medium-dose(P < 0. 01); there were no statistical differences in the m RNA expressions of Claudin-1 and ZO-1 between the model group and the Tongxie Yaofang group of low-dose(P > 0. 05); the average optical density increased in the Tongxie Yaofang group of low-dose(P < 0. 05). The protein expressions of Claudin-1 and ZO-1 increased significantly in the Mesalazine group and the Tongxie Yaofang groups of high-dose and medium-dose(P < 0. 01), and the protein expressions of Claudin-1 and ZO-1 increased in the Tongxie Yaofang group of low-dose(P < 0. 01, P < 0. 05). Conclusion: The mechanism of Tongxie Yaofang in treating UC of Liver depression and Spleen deficiency may be related to up-regulating the expression of key proteins of Claudin-1 and ZO-1 and reducing the contents of inflammatory cytokines of COX-2 and iNOS.

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