基于TLR4信号通路探讨右美托咪啶对内毒素诱导的急性肺损伤纤维化大鼠细胞外基质重塑的作用

Effect of dexmedetomidine on extracellular matrix remodeling in rats with endotoxin-induced acute lung injury and fibrosis based on TLR4 signal pathway

目的:基于TLR4/MyD88/NF-κB信号通路探讨右美托咪啶对内毒素诱导的大鼠急性肺纤维化的保护作用。方法:采用随机数字表法将大鼠分为4组:对照组、模型组、低浓度右美托咪啶组、高浓度右美托咪啶组。模型组和低、高浓度右美托咪啶组大鼠气管内联合腹腔内注射内毒素诱导肺损伤纤维化,其中低、高浓度右美托咪啶组在建模前30 min分别腹腔注射右美托咪啶15μg/kg和25μg/kg。测定肺组织湿重/干重(W/D),同时进行HE染色和Masson染色观察大鼠肺组织病理变化及纤维化情况。ELISA法检测大鼠TNF-α、IL-1β、IL-6水平,RT-PCR检测整合素α5、Fn mRNA的表达水平,免疫荧光染色检测大鼠肺组织α-SMA的表达,Western blot法检测MyD88、NF-κB、p-NF-κB、TLR4蛋白表达。结果:与对照组相比,模型组大鼠W/D升高,TNF-α、IL-1β、IL-6水平明显升高,肺组织中MyD88、p-NF-κB、TLR4蛋白表达明显升高。与模型组相比,低、高浓度右美托咪啶组大鼠W/D降低,TNF-α、IL-1β、IL-6水平明显降低,肺组织中MyD88、p-NF-κB、TLR4蛋白表达降低。Fn mRNA表达的变化与整合素α5 mRNA较为相似,低、高浓度右美托咪啶组整合素α5、Fn mRNA的表达在相应时点较模型组均明显降低。与模型组相比,低、高浓度右美托咪啶组肺泡炎症细胞明显减少,纤维化减轻,肺组织病理学损伤明显减轻,免疫荧光化学染色显示α-SMA在纤维化病变区域表达减少,且具有浓度依赖性。结论:TLR4/MyD88/NF-κB信号通路可能参与了内毒素诱导的大鼠急性肺损伤纤维化过程,右美托咪啶可能通过抑制TLR4/MyD88/NF-κB通路来减轻炎症和肺损伤反应。

Objective:Based on TLR4/MyD88/NF-κB signaling pathway to explore the protective effect of dexmedetomidine on endotoxin-induced acute pulmonary fibrosis in rats.Methods:The rats were divided into 4 groups by random number table:control group,model group,low-concentration dexmedetomidine group,high-concentration dexmedetomidine group.Rats in model group,low-concentration dexmedetomidine group and high-concentration dexmedetomidine group were intratracheally combined with intraper‐itoneal injection of endotoxin to induce lung injury and fibrosis.Low-concentration dexmedetomidine group and high-concentration dex‐medetomidine group were injected intraperitoneally with 15μg/kg and 25μg/kg dexmedetomidine 30 min before modeling.The wet weight/dry weight(W/D)of lung tissue was measured,and HE staining and Masson staining were performed at the same time to ob‐serve the pathological changes and fibrosis of rat lung tissue.The levels of TNF-α,IL-1β,and IL-6 in rats were detected by ELISA,the expression levels of integrinα5 and Fn mRNA were detected by RT-PCR,the expression ofα-SMA in rat lung tissue was detected by immunofluorescence staining,and MyD88,NF-κB,p-NF-κB,TLR4 protein expressions were detected by Western blot method.Results:Compared with control group,the W/D of rats in model group was increased,the levels of TNF-α,IL-1βand IL-6 were sig‐nificantly increased,and the protein expressions of MyD88,p-NF-κB and TLR4 in lung tissue were significantly increased.Compared with model group,the W/D of rats in low-concentration dexmedetomidine group and high-concentration dexmedetomidine group were decreased,the levels of TNF-α,IL-1β,and IL-6 were significantly decreased,and the protein expressions of MyD88,p-NF-κB and TLR4 in lung tissues decreased.The change of Fn mRNA expression was similar to that of integrinα5 mRNA.The expressions of integ‐rinα5 and Fn mRNA in low-concentration dexmedetomidine group and high-concentration dexmedetomidine group were significantly lower than those in model group at the corresponding time points.Compared with model group,alveolar inflammatory cells in low-con‐centration dexmedetomidine group and high-concentration dexmedetomidine group were significantly reduced,fibrosis was reduced,and lung tissue pathological damage was significantly reduced.Immunofluorescence chemical staining showed that the expression ofα-SMA in the fibrotic lesion area was reduced,and it was concentration-dependent.Conclusion:The TLR4/MyD88/NF-κB signaling pathway may be involved in the process of rat acute lung injury fibrosis induced by endotoxin,and dexmedetomidine may inhibit the TLR4/MyD88/NF-κB pathway to reduce inflammation and lung injury reaction.