痛泻要方对溃疡性结肠炎大鼠下丘脑中IL-6，IL-6R表达的影响

目的:观察痛泻要方对溃疡性结肠炎(ulcerative colitis,UC)大鼠下丘脑中白细胞介素-6(IL-6)和白细胞介素-6受体(IL-6R)基因和蛋白表达的影响。方法:将SPF级Wistar大鼠运用复合方法进行溃疡性结肠炎造模后,随机分为5组:模型组,痛泻要方低、中、高剂量(2.75,5.5,11 g·kg~(-1))组,美沙拉嗪(5-ASA)组。药物处理大鼠21 d后麻醉处死,肉眼观察结肠大体形态损伤并进行评分。分离各组大鼠血清,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测血清中IL-6含量,分离大鼠下丘脑组织并提取总RNA后,实时荧光定量PCR(Real-time polymerase chain reaction,Real-time PCR)法检测IL-6和IL-6R基因表达,免疫组织化学染色法检测下丘脑组织IL-6和IL-6R的分布及蛋白表达量。结果:ELISA结果显示,与空白组比较,模型组大鼠血清中IL-6含量明显升高,经过药物处理后,药物组血清中IL-6含量均有所下降,其中以痛泻要方高剂量组效果最为明显(P0.05)。Real-time PCR结果表明,模型组大鼠下丘脑组织中IL-6和IL-6R基因表达量明显高于空白组(P0.05),经药物治疗后,药物组IL-6和IL-6R基因表达均有所降低,其中以5-ASA组和痛泻要方高剂量组降低最为明显(P0.05)。免疫组织化学染色结果表明,与空白组比较,溃疡性结肠炎大鼠IL-6和IL-6R蛋白表达量明显增加(P0.05),药物处理可以降低大鼠下丘脑IL-6和IL-6R蛋白表达量,其中以5-ASA组和痛泻要方高剂量组降低最为明显(P0.05)。结论:痛泻要方对2,4,6三硝基苯磺酸(2,4,6-trinitrobenzene sulfonic acid,TNBS)/乙醇法UC大鼠下丘脑组织中IL-6和IL-6R基因和蛋白的表达具有明显抑制作用,提示痛泻要方治疗溃疡性结肠炎的机制可能与抑制下丘脑中IL-6/IL-6R信号通路的异常活化有关。

Effect of Tongxie Yaofang on IL-6, IL-6R mRNA and Protein Expressions of Hypothalamus Tissues in Rats with Experimental Ulcerative Colitis

Objective: To explore the curative effect of Tongxie Yaofang on ulcerative colitis (UC) in rat hypothalamus cytokine interleukin-6 (IL-6) and interleukin-6 receptors (IL-6R) gene and protein expressions. Method: After the ulcerative colitis model was established by the composite method. SPF Wistar rats were randomly divided into 5 groups: model group, low, middle, high-dose Tongxie Yaofang groups (2.75, 5.5, 11 g·kg^-1), and 5-ASA group. After modeling, rats were given drugs for 21 days. Then, colonic mucosa damage was observed. The levels of IL-6 in the serum tissues were detected by enzyme linked immunosorbent assay (ELISA). The proteins expressions of IL-6 and IL-6R in rat hypothalamus tissues were detected by immunohistochemistry, and their gene expressions were measured by Real-time PCR method. Result: ELISA test results show that compared with control group, model group showed significant increase in content of IL-6 in serum of rats. After drug treatment, the serum IL-6 in drug group were decreased, with the most obvious effect in high-dose Tongxie Yaofang group (P<0.05). Real-time PCR results showed that model group''s hypothalamic IL-6 and IL-6R gene expressions were higher than those of blank group (P<0.05). After treatment with drugs, drug group''s IL-6 and IL-6R gene expressions were reduced, and 5-ASA and high-dose Tongxie Yaofang groups were superior to the other groups (P<0.05). Immunohistochemical staining results showed that compared with control group, IL-6 and IL-6R protein expressions of ulcerative colitis rats increased significantly (P<0.05), drug treatment can reduce their hypothalamic IL-6 and IL-6R protein expressions, and 5-ASA and high-dose Tongxie Yaofang groups were superior to the other groups (P<0.05). Conclusion: Tongxie Yaofang can obviously inhibit IL-6 and IL-6R gene and protein expressions in hypothalamic tissues of the rat UC model by TNBS/ethanol method, suggesting Tongxie Yaofang''s mechanism in the treatment of ulcerative colitis may be correlated with inhibition of abnormal activation of IL-6/IL-6R signaling pathway in hypothalamus.