| 黄盛琦,卢爱妮,王德龙,等.基于MAPK/ERK通路研究芍药汤对溃疡性结肠炎黏膜损伤修复作用机制[J].浙江中医药大学学报,2022,46(12):1301-1309, 1319. |
| 基于MAPK/ERK通路研究芍药汤对溃疡性结肠炎黏膜损伤修复作用机制 |
| Study on the Repair Mechanism of Shaoyao Decoction on Mucosal Barrier in Ulcerative Colitis Based on MAPK/ERK Pathway |
| DOI：10.16466/j.issn1005-5509.2022.12.001 |
| 中文关键词: 芍药汤 溃疡性结肠炎 结肠黏膜损伤 claudin-2 ZO-1 HIF-1α MAPK/ERK 信号通路 |
| 英文关键词: Shaoyao Decoction ulcerative colitis colonic mucosal injury claudin-2 ZO-1 HIF-1α MAPK/ERK signaling pathway |
| 基金项目:国家自然科学基金项目(81573871、81774198) |
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| 中文摘要: |
| [目的] 探讨芍药汤通过有丝分裂原活化蛋白激酶/细胞外信号调节激酶(mitogen-activated protein kinase/extracellular signal-regulated kinase，MAPK/ERK)通路对溃疡性结肠炎(ulcerative colitis，UC)结肠黏膜损伤的修复作用和机制。[方法] 经结肠缓慢推注5%三硝基苯磺酸(trinitrobenzene sulfonic acid，TNBS)0.09 mL+无水乙醇0.06 mL，复制大鼠UC模型，将48只无特定病原体(specific pathogen free，SPF)SD大鼠随机分为正常组，模型组，芍药汤高、中、低剂量组与美沙拉嗪组。各组灌胃干预 ，评价疾病活动指数(disease activity index，DAI)，观察组织病理变化并进行组织学活动指数(histological activity index，HAI)评分；以实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction，Real-time qPCR)及免疫印迹法检测结肠组织中屏障保护相关指标的mRNA及蛋白表达水平。建立脂多糖(lipopolysaccharide，LPS)小肠上皮细胞IEC-6损伤模型，以噻唑蓝比色法(dimethylthiahiazo，MTT)筛选药物最佳干预浓度，将细胞分为空白组、LPS组和芍药汤组，划痕实验观察细胞迁移率；免疫印迹法检测细胞闭锁小带蛋白-1(zonula occludens-1，ZO-1)蛋白表达水平。 [结果] 体内实验显示，与正常组比较，模型组大鼠肠黏膜结构缺失，黏膜下层和固有层可见明显的炎性细胞浸润；DAI、HAI评分显著升高(P＜0.01)，紧密连接蛋白-2(claudin-2)、磷酸化p38MAPK(phosphorylated-p38MAPK，p-p38MAPK)、磷酸化ERK1/2(phosphorylated-ERK1/2，p-ERK1/2)蛋白表达显著下调(P＜0.001，P＜0.01，P＜0.001)；肠三叶因子(intestinal trefoil factor，ITF)的mRNA表达显著降低(P＜0.05)，缺氧诱导因子-1α(hypoxia inducible factor-1α，HIF-1α)蛋白与mRNA表达显著升高(P＜0.01)。与模型组比较，芍药汤各组病理损伤明显改善，DAI、HAI评分明显降低(P＜0.05，P＜0.01)，芍药汤各组claudin-2、p-p38 MAPK、p-ERK1/2蛋白表达水平显著上调(P＜0.05，P＜0.01，P＜0.001)，ITF mRNA表达量显著上调(P＜0.01)；HIF-1α蛋白与mRNA表达水平显著下调(P＜0.01)。体外实验显示，与空白组比较，LPS可明显抑制IEC-6细胞迁移(P＜0.01)，下调ZO-1蛋白表达(P＜0.05)。与LPS组比较，芍药汤组细胞迁移率明显上升(P＜0.05，P＜0.01)，ZO-1蛋白表达明显上调(P＜0.05)。[结论] 芍药汤对UC大鼠结肠黏膜损伤具有修复作用，其机制可能与激活MAPK/ERK通路，上调claudin-2、ZO-1，下调HIF-1α蛋白表达，促进肠黏膜上皮细胞迁移有关。 |
| 英文摘要: |
| [Objective] To explore the repairing effect and mechanism of Shaoyao Decoction on colonic mucosal injury in ulcerative colitis(UC) through mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK) pathway. [Methods] UC rat model was established by slowly injecting 5% trinitrobenzene sulfonic acid(TNBS) 0.09 mL and absolute ethanol 0.06 mL into rat colon. Forty-eight specific pathogen free(SPF) SD rats were randomly divided into normal group， model group， high， medium and low dose Shaoyao Decoction groups and mesalazine group. The disease activity index(DAI)， histopathological changes and histological activity index(HAI) were observed after intragastric intervention for 7 days. The mRNA and protein expression levels of barrier protection related indicators in colon tissue were detected by Real-time quantitative polymerase chain reaction(Real-time qPCR) and Western blot. Lipopolysaccharide(LPS) was used to establish the injury model of intestinal epithelial cell IEC-6. The optimal concentration of drugs was selected by dimethylthiahiazo(MTT). IEC-6 cells were divided into blank group， LPS group and Shaoyao Decoction group. The cell migration rate was observed by scratch test; the protein expression of zonula occludens-1(ZO-1) was detected by Western blot. [Results] In vivo experiments showed that compared with normal group， the intestinal mucosal structure of model group was missing， and there was obvious inflammatory cell infiltration in the submucosa and lamina propria. The scores of DAI and HAI were significantly increased(P＜0.01)， claudin-2， phosphorylated-p38MAPK(p-p38MAPK)， phosphorylated-ERK1/2(p-ERK1/2) protein expression were significantly down regulated(P＜0.001， P＜0.01， P＜0.001)， the mRNA expression of intestinal trefoil factor(ITF) was significantly reduced(P＜0.05)， the expression of protein and mRNA of hypoxia inducible factor-1α(HIF-1α) increased significantly(P＜0.01). Compared with model group， the pathological injury of Shaoyao Decoction group was significantly relieved， the DAI and HAI scores were significantly reduced(P＜0.05， P＜0.01)， the expression levels of claudin-2， p-p38MAPK and p-ERK1/2 protein were significantly increased(P＜0.05， P＜0.01， P＜0.001)， and the expression of ITF mRNA was significantly increased(P＜0.01)； the protein and mRNA expression levels of HIF-1α were significantly down regulated(P＜0.01). In vitro experiments showed that LPS could significantly inhibit IEC-6 cell migration compared with blank group(P＜0.01)， the expression of ZO-1 protein was significantly down-regulated(P＜0.05). Compared with LPS group， the cell migration rate of Shaoyao Decoction group was significantly increased(P＜0.05， P＜0.01)， and the expression of ZO-1 protein was significantly up-regulated(P＜0.05). [Conclusion] Shaoyao Decoction can repair colonic mucosal injury in UC rats. Its mechanism may be related to the activation of MAPK/ERK pathway， up regulation of claudin-2， ZO-1 and down-regulation of HIF-1 α protein expression， promotion of intestinal mucosal epithelial cells migration. |
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