基于TLR4/MyD88/NF-κB通路探讨金双歧对慢性肾脏病大鼠肠黏膜屏障的影响

Effects of golden bifid on intestinal mucosal barrier of rats with chronic kidney disease based on TLR4/MyD88/NF-κB signaling pathway

摘要:

目的 探讨金双歧对慢性肾脏病大鼠肠黏膜屏障的作用及机制。方法 30只大鼠随机分为对照组、模型组和金双歧组，每组10只。模型组和金双歧组以阿霉素、腺嘌呤复制慢性肾脏病模型。模型复制成功后，金双歧组采用金双歧治疗4周。测定各组大鼠的24 h尿蛋白及血肌酐、尿素氮、D-乳酸、内毒素、白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）水平。HE染色观察各组大鼠的肾脏、结肠病理变化，Masson染色观察各组大鼠肾脏纤维化改变，透射电镜观察各组大鼠结肠黏膜超微结构，免疫荧光法检测各组大鼠结肠Occludin蛋白表达，Western blotting检测各组大鼠结肠Toll样受体4（TLR4）、髓样分化因子88（MyD88）、核因子κB P65（NF-κB P65）蛋白表达，实时荧光定量聚合酶链反应（qRT-PCR）检测各组大鼠结肠TLR4、MyD88、NF-κB P65 mRNA表达。结果 与对照组比较，模型组、金双歧组大鼠的肌酐、尿素氮、24 h尿蛋白、肾间质损伤评分均升高（P <0.05），肾间质纤维化相对面积增加（P <0.05）；模型组与金双歧组大鼠的上述指标比较，差异无统计学意义（P >0.05）。与对照组比较，模型组、金双歧组大鼠的结肠黏膜病理损伤评分升高（P <0.05），金双歧组结肠黏膜病理损伤评分低于模型组（P <0.05）；与对照组比较，模型组、金双歧组大鼠结肠黏膜超微结构损害明显、Occludin蛋白表达减少，金双歧组较模型组超微结构有所恢复，Occludin蛋白表达增加。与对照组比较，模型组、金双歧组大鼠血清D-乳酸、内毒素、IL-6、IL-1β、TNF-α水平均升高（P <0.05），金双歧组较模型组均降低（P <0.05）。与对照组比较，模型组、金双歧组结肠TLR4、MyD88、NF-κB P65蛋白及mRNA表达均升高（P <0.05），金双歧组较模型组均下降（P <0.05）。结论 金双歧对病理损伤重的肾脏无明显保护作用，金双歧可能通过抑制TLR4/MyD88/NF-κB通路活化，改善慢性肾脏病所致肠黏膜屏障结构及功能障碍，减轻系统炎症反应。

Abstract:

Objective To explore the effects of Golden bifid on the intestinal mucosal barrier of rats with chronic kidney disease and the mechanism.Methods Thirty rats were randomly divided into normal control group (NOR), model group (CKD), and golden bifid group (JSQ). Besides NOR group, the other rats were treated with doxorubicin and adenine to establish chronic kidney disease model. The rats in JSQ group were received respectively golden bifid preparations for four weeks. The determination of 24-hours urinary protein level, serum creatinine, blood urea nitrogen, endotoxin, D-lactic acid, IL-6, IL-1β, and TNF-ɑ were measured. Kidney and colon tissues were harvested for HE. Ultrastructure of colon tissue was observed transmission electron microscopy. The expression and location of TJ protein Occludin in the epithelium were investigated by immunofluorescence microscopy. The expression of TLR4/MyD88/NF-κB signaling pathway protein were measured by western blot. The expression of TLR4, MyD88, NF-κB P65 mRNA were measured by qRT-PCR.Results The serum creatinine, blood urea nitrogen, 24-hours urinary protein level, pathological scoring of renal injury, renal interstitial fibrosis relative area ratio in CKD and JSQ group were significantly higher than Nor group (P < 0.05). But there is no obvious difference between CKD and JSQ group. Compared with the NOR group, the CKD group had severe pathological damage of intestinal mucosa, significant damage of colonic ultrastructure, and significantly decreased expression of Occludin protein, but the JSQ group recovered partly. Compared with NOR group, The concentration of D-lactic acid, endotoxin, IL-6, IL-1β, TNF-ɑ in CKD group were all significantly higher (P < 0.05), but the JSQ group recovered partly. Compared with NOR group, the protein and mRNA expressions of TLR4, MyD88, and NF-κB P65 in the colon of the CKD group were significantly increased, but the JSQ group was decreased partly.Conclusion Gold bifid has no obvious protective effect on the kidney with severe pathological damage. Gold bifid may improve the structure and function of intestinal mucosal barrier caused by chronic kidney disease and reduce systemic inflammation by inhibiting the activation of TLR4/MyD88/NF-κB pathway.