摘要
目的:探究热炎宁合剂(RYN)抗脂多糖(LPS)诱导的大鼠急性肺损伤(ALI)作用及机制。方法:将50只SD大鼠随机分为5组:对照组、模型组、地塞米松组(3.5 mg·kg^(–1)·d^(–1))、RYN低剂量组(8 mL·kg^(–1)·d^(–1))和RYN高剂量组(16 mL·kg^(–1)·d^(–1))。制备LPS诱导的大鼠ALI模型。在不同时间点监测大鼠肛温变化;采用苏木素-伊红(HE)染色观察大鼠肺泡和支气管病理情况;免疫组化检测肺组织白细胞介素-1β(IL-1β)和NOD样受体热蛋白结构域相关蛋白3(NLRP3)的表达和分布;酶联免疫吸附分析(ELISA)检测血清中C反应蛋白(CRP)、IL-1β和肿瘤坏死因子-α(TNF-α)水平。采用转录组测序分析筛选差异表达基因并绘制热图、火山图和韦恩图,并进行基因本体(GO)通路富集分析。采用Alexa Fluor 680 C2马来酰亚胺荧光探针检测肺组织中巯基(-SH)的水平,免疫荧光共定位检测肺组织中Kelch样ECH关联蛋白1(Keap1)和核因子红细胞系2相关因子2(Nrf2)的表达和互相结合关系。采用蛋白质免疫印迹(Western blot)法检测肺组织中Nrf2蛋白质的表达和亚磺酸(-SOH)水平。结果:RYN能显著降低LPS诱导的大鼠体温升高(P<0.05,P<0.01),改善ALI大鼠肺泡和支气管的病理损伤(P<0.05,P<0.01),降低大鼠血清中CRP(P<0.05)、IL-1β(P<0.01)、TNF-α(P<0.05)的水平,以及IL-1β(P<0.05)、NLRP3(P<0.05,P<0.01)的表达和分布。转录组结果显示,RYN组与其他组比较基因差异有统计学意义(P<0.05),GO通路富集分析发现差异mRNA与氧化还原相关信号通路相关。RYN上调ALI肺组织中-SH的水平(P<0.05),相应地,降低其-SOH(P<0.05)水平,RYN还能抑制Keap1和Nrf2结合(P<0.05,P<0.01)并促进Nrf2(P<0.05,P<0.01)的表达。结论:RYN通过调节-SH和-SOH水平,抑制Keap1和Nrf2的结合,并进一步激活Nrf2的表达,抑制LPS诱导的氧化应激,发挥抗LPS诱导的大鼠ALI的作用。
Objective:To investigate the therapeutic effect and mechanism of Reyanning mixture(RYN)for lipopolysaccharide(LPS)-induced acute lung injury(ALI)in rats.Methods:Fifty SD rats were randomly assigned into five groups:control group,model group,dexamethasone group(3.5 mL·kg^(–1)·d^(–1)),and RYN low-dose group(8 mL·kg^(–1)·d^(–1))and RYN high-dose group(16 mL·kg^(–1)·d^(–1)).The rat model of LPS-induced ALI was established.Rectal temperatures were measured at different time points.Hematoxylin-eosin staining(HE)was used to observe the alveolar and bronchial pathology in rats;immunohistochemistry was used to detect the expression and distribution of interleukin-1β(IL-1β)and NOD-like receptor heat protein structural domain-related protein 3(NLRP3)in lung tissues;and enzyme-linked immunosorbent assay(ELISA)was used to detect the serum levels of C-reactive protein(CRP),IL-1βand tumor necrosis factor-α(TNF-α)levels in serum.Transcriptional sequencing was performed to identify differentially expressed genes,generate heat maps,volcano plots,and Venn diagrams,followed by gene ontology(GO)pathway enrichment analysis.Alexa Fluor 680 C2 maleimide fluorescent probe was used to detect the level of sulfhydryl(-SH)in lung tissues,and immunofluorescence co-localization was used to detect the expression and inter-binding relationship of Kelch-like ECH-associated protein 1(Keap1)and nuclear factor erythroid lineage 2-related factor(Nrf2)in lung tissues.Protein immunoblotting(Western blot)was used to detect the expression and sulfenic acid(-SOH)level of Nrf2 protein in lung tissues.Results:RYN attenuated LPS-induced increases in body temperature(P<0.05,P<0.01),ameliorated the pathological damage to alveoli and bronchi(P<0.05,P<0.01),and decreased the serum levels of CRP(P<0.05),IL-1β(P<0.01),and TNF-α(P<0.05)as well as the expression and distribution of IL-1β(P<0.05)and NLRP3(P<0.05,P<0.01).The transcriptome results showed statistically significant(P<0.05)genetic differences in the RYN group compared with the other groups,and GO pathway enrichment analysis revealed that the differential mRNAs were associated with redox-related signaling pathways.RYN up-regulated the-SH level(P<0.05)and correspondingly decreased the-SOH level(P<0.05)in the lung tissue of ALI rats.Further studies demonstrated that RYN inhibited the interaction between Keap1 and Nrf2(P<0.05,P<0.01),thereby promoting Nrf2 expression(P<0.05,P<0.01).Conclusion:RYN inhibits LPS-induced oxidative stress and exerts anti-LPS-induced protection against rat ALI by regulating the levels of-SH and-SOH,inhibiting the binding of Keap1 and Nrf2,and further activating the expression of Nrf2.
作者
张婷婷
赵慧梅
杜玮
施建羽
张海宴
宋忠兴
史鑫波
刘红娜
刘海静
周瑞
ZHANG Tingting;ZHAO Huimei;DU Wei;SHI Jianyu;ZHANG Haiyan;SONG Zhongxing;SHI Xinbo;LIU Hongna;LIU Haijing;ZHOU Rui(Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,Shaanxi University of Chinese Medicine,Xianyang 712046,China;Shaanxi Institute for Food and Drug Control,Xi'an 712065,China;Tsing Hua De Ren Xi'an Happiness Pharmaceutical Co.,Ltd.,Xi'an 710043,China)
出处
《中国现代中药》
2025年第7期1300-1311,共12页
Modern Chinese Medicine
基金
陕西省秦创原“科学家+工程师”队伍建设项目(2023KXJ-128)
2023年度陕西中医药大学科技创新团队项目(2023-CXTD-04)
陕西省重点研发计划项目。
