化湿败毒散通过减少胶原交联改善肺纤维化的机制研究

Mechanism study of Huashi Baidu Powder in improving pulmonary fibrosis by reducing collagen cross-linking

目的:探讨中药化湿败毒散对小鼠肺纤维化的治疗作用及其潜在机制。方法:50只C57BL/6J小鼠随机分成正常组、假手术组、博来霉素组、化湿败毒散组(早干预)、化湿败毒散组(晚干预)5组。用博来霉素气管注射,建立小鼠特发性肺纤维化(IPF)模型。使用RNA-Seq筛选各组基因水平的异同,预测其涉及生物学过程和通路;记录小鼠的体质量变化;检测肺功能(呼吸频率和流量);拍摄肺部CT图像;用双光子荧光显微镜观测肺组织胶原形态;HE染色观察组织病理变化;qPCR检测肺组织中缺氧诱导因子1α(HIF1α)、赖氨酰氧化酶(LOX)、赖氨酰氧化酶样蛋白(LOXL)1~LOXL4以及赖氨酸羟化酶2(PLOD2)的mRNA水平;免疫组化染色评估HIF1α、LOXL2、LOXL3、PLOD2在肺组织中的蛋白表达水平。结果:转录组结果表明化湿败毒散主要影响了胶原蛋白合成、细胞外基质相互作用和炎症反应,其机制可能涉及HIF-1通路;化湿败毒散组(早干预)相比于化湿败毒散组(晚干预)可加速体质量恢复;改善肺部胶原交联,减缓纤维化进程;对呼吸频率和流量恢复具有正向调节作用;同时可降低小鼠肺组织中HIF1α、LOX、LOXL3的基因表达水平(P<0.05,P<0.01);下调HIF1α、LOXL2、LOXL3、PLOD2的蛋白表达水平(P<0.01)。结论:化湿败毒散可降低肺纤维化中的胶原交联水平,减缓肺纤维化进程。

Objective:To explore therapeutic effect and potential mechanism of Huashi Baidu Powder(HSBD)on pulmonary fibrosis in mice.Methods:Fifty C57BL/6J mice were randomly divided into five groups:the normal group,sham surgery group,bleomycin group,HSBD group(early intervention),and HSBD group(late intervention).A model of idiopathic pulmonary fibrosis(IPF)in mice was established by tracheal injection of bleomycin.RNA-Seq was used to screen for similarities and differences in gene expression levels among the groups,predicting their involvement in biological processes and pathways.Mouse weight changes were recorded,lung function(respiratory rate and flow)was measured,lung CT images were taken,lung tissue collagen morphology was observed using two-photon fluorescence microscopy,tissue pathological changes were assessed using HE staining,and qPCR was employed to detect mRNA levels of hypoxia-inducible factor 1α(HIF1α),lysyl oxidase(LOX),lysyl oxidase-like subtypes(LOXL)1-LOXL4,and lysyl hydroxylase 2(PLOD2)in lung tissue.Immunohistochemical staining was conducted to evaluate the protein expression levels of HIF1α,LOXL2,LOXL3,and PLOD2 in lung tissue.Results:Transcriptome results indicate that HSBP primarily affects collagen synthesis,extracellular matrix interactions,and inflammatory responses,with its mechanism potentially involving the HIF-1 pathway.Compared to the late intervention group,the early intervention group of HSBP accelerates weight recovery,alleviates lung collagen crosslinking,slows down the fibrotic process,and has a positive regulatory effect on the recovery of respiratory rate and flow.Simultaneously,it can reduce the gene expression levels of HIF1α,LOX,and LOXL3 in mouse lung tissue(P<0.05,P<0.01),also downregulate the protein expression levels of HIF1α,LOXL2,LOXL3,and PLOD2(P<0.01).Conclusion:HSBP can reduce collagen cross-linking levels in pulmonary fibrosis,slowing down the fibrotic process.