α7nAChR抑制MyD88依赖性Zonulin释放改善脑梗死大鼠肠道炎症和肠屏障损伤

α7nAChR ameliorating intestinal inflammation and intestinal barrier damage in rats with cerebral infarction by inhibiting MyD88-dependent Zonulin release

  • 摘要: 目的:观察α7型烟碱乙酰胆碱受体(α7nAChR)激动剂PNU282987对脑梗死大鼠肠道炎症和肠屏障损伤的影响。方法:将36只雄性SD大鼠随机分为假手术(sham)组、中脑动脉栓塞(MCAO)组和PNU282987组。MCAO组和PNU282987组采用Longa改良线栓法制备MCAO模型,PNU282987组腹腔注射1 mg/kg PNU282987,连续7 d。苏木精—伊红(HE)染色观察肠道黏膜病理改变,酶联免疫吸附试验(ELISA)检测结肠组织肿瘤坏死因子(TNF-α)、白细胞介素(IL-6)水平及血清二胺氧化酶(DAO)、D-乳酸(DLA)、内毒素(ET)水平,免疫组化法检测结肠组织Toll样受体2(TLR2)蛋白表达,western blotting法检测结肠组织紧密连接关键蛋白咬合蛋白(Occludin)、闭合蛋白(Claudin-1)、连蛋白(Zonulin)、带状闭合蛋白(ZO-1)、TLR2、髓样分化因子88(MyD88)、核因子-κB(NF-κB)和磷酸化NF-κB(p-NF-κB)蛋白表达。结果:与sham组比较,MCAO组大鼠肠上皮脱落、肠绒毛排列紊乱及黏膜下层炎性浸润,血清DAO、DLA、ET水平及结肠组织TNF-α、IL-6含量显著增高,结肠组织Occludin、Claudin-1、ZO-1蛋白表达显著下调,Zonulin、TLR2、MyD88、p-NF-κB蛋白表达显著上调(均P<0.05)。与MCAO组比较,PNU282987组大鼠肠上皮损伤减轻,血清DAO、DLA、ET水平及结肠组织TNF-α、IL-6含量显著降低,结肠组织Occludin、Claudin-1、ZO-1蛋白表达显著上调,Zonulin、TLR2、MyD88、p-NF-κB蛋白表达显著下调(均P<0.05)。结论:PNU282987可以显著改善脑梗死大鼠肠道炎症及肠屏障损伤,其机制可能与抑制TLR2/MyD88/NF-κB信号通路有关。

     

    Abstract: Objective: To observe the effect of the α7-type nicotinic acetylcholine receptor(α7nAChR) agonist PNU282987 on intestinal inflammation and intestinal barrier damage in rats with cerebral infarction. Methods: A total of 36 male SD rats were randomly divided into three groups: sham group, middle cerebral artery occlusion(MCAO) group, and PNU282987 group. MCAO group and PNU282987 group were prepared by Longa modified sature-embolus method, and PNU282987 group was intraperitoneally injected with 1 mg/kg PNU282987 for 7consecutive days. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in intestinal mucosa. Enzyme-linked immunosorbent assay(ELISA) was performed to detect the levels of tumor necrosis factor(TNF)-α and interleukin(IL)-6 in colon tissue, and the diamine oxidase(DAO), D-lactate(DLA), endotoxin(ET)levels in serum. The expression of toll-like receptor 2(TLR2) protein in colon tissues was detected by immunohistochemistry. Western blotting was used to detect the expression of the key tight junction proteins in colon tissue, including occlusal proteins(Occludin), closed protein-1(Claudin-1), Zonulin, zona occludens(ZO-1), TLR2,myeloid differentiation factor 88(MyD88), nuclear factor-κB(NF-κB) and phosphorylated NF-κB(p-NF-κB) proteins. Results: Compared with the sham group, the MCAO group showed intestinal epithelial desquamation, disordered arrangement of intestinal villi, and inflammatory infiltration in the submucosal layer of the intestine. The levels of DAO, DLA, ET in serum, and TNF-α and IL-6 contents in colon tissue were significantly increased,while the expression of Occludin, Claudin-1, and ZO-1 proteins in colon tissue was significantly decreased, and the expression of Zonulin, TLR2, MyD88, and p-NF-κB proteins was significantly increased(all P<0.05). Compared with the MCAO group, the PNU282987 group showed reduced intestinal epithelial damage, significantly decreased levels of serum DAO, DLA, ET, and TNF-α and IL-6 contents in colon tissue, significantly increased expression of Occludin, Claudin-1, and ZO-1 proteins in colon tissue, and significantly decreased expression of Zonulin, TLR2, MyD88, and p-NF-κB proteins(all P<0.05). Conclusion: PNU282987 can significantly improve intestinal inflammation and intestinal barrier damage in rats with cerebral infarction, and its mechanism may be related to inhibition of TLR2/MyD88/NF-κB signaling pathway.

     

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