仙茅苷介导PERK/ATF4/CHOP通路减轻UC大鼠肠黏膜病理改变的作用与机制探讨

Role and mechanism of curculigoside mediated PERK/ATF4/CHOP pathway in alleviating intestinal mucosal pathological changes in UC rats

目的 观察仙茅苷治疗溃疡性结肠炎(UC)大鼠的作用,并探讨其对蛋白激酶R样内质网激酶(PERK)/活化转录因子4(ATF4)/增强子结合蛋白同源蛋白(CHOP)通路的调控作用。方法 取61只SD大鼠以三硝基苯磺酸(TNBS)法诱导建立UC模型,按随机数字表分为仙茅苷低、中、高剂量组(25、50、100 mg/kg仙茅苷溶于生理盐水),美沙拉嗪组(500 mg/kg美沙拉嗪溶于生理盐水)、PERK抑制剂组(GSK2606414 1.0 mg/kg溶于生理盐水)、模型组(生理盐水),另取10只健康SD大鼠记为正常组(生理盐水),生理盐水体积均为1 ml/100 g大鼠体质量,均灌胃每天1次,连续10 d。给药结束后次日,评价疾病活动指数(DAI);气相色谱法(GC)测定两组大鼠5 h尿液中乳果糖(L)与甘露醇(M)排泄率(L/M)比值;双抗体夹心法测定血清糖皮质激素浓度,酶联免疫法测定血清白介素-6(IL-6)、干扰素(IFN-γ)、白介素-8(IL-8)和肿瘤坏死因子-α(TNF-α)水平;苏木素-伊红(HE)染色观察肠黏膜病理改变;实时-逆转录荧光定量聚合酶链反应(RT-qPCR)检测肠黏膜组织PERK、ATF4、CHOP、Bcl2关联X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)信使核糖核酸(mRNA)表达;免疫印迹法(WB)检测肠黏膜组织PERK、ATF4、CHOP、Bax、Bcl-2蛋白表达及磷酸化PERK(p-PERK)水平。结果 肉眼和HE染色观察证实建模成功;与正常组比较,模型组L/M,DAI和肠黏膜病理评分,血清糖皮质激素浓度和血清IL-6、IFN-γ、IL-8和TNF-α水平,PERK、ATF4、CHOP、Bax mRNA与蛋白表达,p-PERK水平均升高(P<0.05),Bcl-2 mRNA及蛋白表达均下降(P<0.05);与模型组比较,仙茅苷3个剂量组、美沙拉嗪组、PERK抑制剂组L/M,DAI和肠黏膜病理评分,血清糖皮质激素浓度和血清IL-6、IFN-γIL-8和TNF-α水平,PERK、ATF4、CHOP、Bax mRNA与蛋白表达,p-PERK水平均下降(P<0.05),Bcl-2 mRNA及蛋白表达均升高(P<0.05);糖皮质激素浓度、PERK、ATF4、CHOP、Bax、Bcl-2 mRNA及蛋白表达,p-PERK水平仙茅苷低剂量组与美沙拉嗪组,仙茅苷中剂量组与美沙拉嗪组、PERK抑制剂组其它指标差异均无统计学意义(P>0.05),其余每2组比较差异均有统计学意义(P<0.05);模型组结肠黏膜病理严重改变,仙茅苷低剂量组有所减轻,仙茅苷中剂量组、美沙拉嗪组和PERK抑制剂组均明显减轻,仙茅苷高剂量组显著减轻。结论 仙茅苷可改善UC大鼠肠黏膜屏障功能、控制疾病活动度、减轻病理改变,推测与抑制PERK/ATF4/CHOP通路有关。

Objective To observe the therapeutic effect of curculioside on rats with ulcerative colitis(UC), and to explore its regulatory effect on the protein extracellular regulated kinases(PERK)/activated transcription factor 4(ATF4)/enhancer-binding protein(C/EBP) homologous protein(CHOP) pathway. Methods Sixty-one SD rats were induced by trinitrobenzene sulfonic acid(TNBS) to establish UC model. According to the random number table, they were divided into low, medium and high dose curculioside groups(25, 50 and 100 mg/kg citronelioside dissolved in normal saline), mesalazine group(500 mg/kg mesalazine dissolved in normal saline), PERK inhibitor group(GSK2606414 1.0 mg/kg dissolved in normal saline), and model group(normal saline), and another 10 healthy SD rats were recorded as the normal group(normal saline), and the volume of normal saline was 1ml/100g the body weight of rats, gavaged once a day for 10 consecutive days. On the day after the end of administration, disease activity index(DAI) was evaluated. The ratio of lactulose(L) to mannitol(M) excretion rate(L/M) in the 5-hour urine of two groups of rats was determined by Gas chromatography(GC). Double antibody sandwich method was used to measure serum glucocorticoid concentration, and enzyme-linked immunosorbent assay was used to detect serum interleukin-6(IL-6), interferon-γ(IFN-γ), interleukin-8(IL-8) and tumor necrosis factor-α(TNF-α) levels. Hematoxylin eosin(HE) staining was used to observe the pathological changes of the intestinal mucosa. Real time reverse transcription fluorescence quantitative polymerase chain reaction(RT-qPCR) was used to detect the expression of PERK, ATF4, CHOP, Bcl2 associated X protein(Bax), B lymphoblastoma 2(Bcl-2) messenger RNA(mRNA) in intestinal mucosa. Immunoblotting(WB) was used to detect the expression of PERK, ATF4, CHOP, Bax, and Bcl-2 proteins in intestinal mucosal tissue, as well as the levels of phosphorylated PERK(p-PERK). Results Visual and HE staining observations confirmed successful modeling. Compared with the normal group, the model group had L/M, DAI and intestinal mucosal pathological score, serum glucocorticoid concentration, and serum IL-6, IFN-γ, IL-8 and TNF-α levels, mRNA and protein expressions of PERK, ATF4, CHOP, Bax, and p-PERK increased(P<0.05), while the mRNA and protein expressions of Bcl-2 decreased(P<0.05). Compared with the model group, the L/M, DAI and intestinal mucosal pathological scores, serum glucocorticoid concentration, and serum IL-6, IFN-γ, IL-8 and TNF-αlevels, mRNA and protein expressions of PERK, ATF4, CHOP, Bax, and p-PERK of the three dose curculioside groups, mesalazine group, and PERK inhibitor group decreased(P<0.05), while the mRNA and protein expressions of Bcl-2 increased(P<0.05). There were no significant differences in glucocorticoid concentration, PERK, ATF4, CHOP, Bax, Bcl-2 mRNA and protein expressions, p-PERK levels between the low dose curculioside group and the mesalazine group, between the medium dose curculioside group and the PERK inhibitor group(P>0.05), and there were significant differences between each other two groups(P<0.05). The pathological changes of colonic mucosa in the model group were serious. The low dose curculioside group was relieved, the medium dose curculioside group, the Mesalazine group and the PERK inhibitor group were significantly relieved, and the high-dose group of citronelloside was significantly relieved. Conclusion Curculioside can improve intestinal mucosal barrier function, control disease activity, and alleviate pathological changes in UC rats, which is speculated to be related to the inhibition of the PERK/ATF4/CHOP pathway.