摘要
目的:探讨抵当汤及其拆方对脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVECs)炎症反应的影响及作用机制。方法:将体外培养的HUVECs随机分为空白血清组、LPS组、LPS+抵当汤组、LPS+植物药组、LPS+动物药组;抑制Toll样受体4(TLR4)后分为TAK-242组、抵当汤TAK-242组、植物药TAK-242组、动物药TAK-242组,给予相应处理后,采用酶联免疫吸附测定(ELISA)检测细胞上清中高速泳动族蛋白B1(HMGB1)、肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)含量;蛋白质免疫印迹法检测HUVECs中HMGB1、TLR4和核因子κB p65的蛋白表达水平,实时定量PCR(RT-qPCR)法检测HMGB1、TLR4和核因子κB p65的基因表达情况,免疫荧光法测定HMGB1、TLR4和核因子κBp65的荧光强度。结果:与A组比较,LPS组细胞上清液及HUVECs中HMGB1、IL-6、TNF-α、TLR4和核因子κB p65表达均增强(均P<0.01)。与LPS组比较,LPS+抵当汤组细胞上清液及HUVECs中的HMGB1、IL-6、TNF-α、TLR4和核因子κBp65的表达均减弱(P<0.05,P<0.01)。与LPS+抵当汤组比较,LPS+植物药组和LPS+动物药组细胞上清液及HUVECs中的HMGB1、IL-6、TNF-α、TLR4和核因子κBp65表达显著增强(P<0.05,P<0.01)。LPS+植物药组中的HMGB1、IL-6和TNF-α表达低于LPS+动物药组(P<0.05,P<0.01),TLR4和核因子κBp65表达高于LPS+动物药组(P<0.05)。抑制TLR4后,与LPS组比较,TAK-242组的HMGB1、TLR4、核因子κBp65表达减弱(P<0.05,P<0.01)。与TAK-242组比较,抵当汤TAK-242组各指标表达低于TAK-242组(P<0.05,P<0.01)。与抵当汤TAK-242组比较,植物药TAK-242组和动物药TAK-242组各指标表达水平降低(P<0.05,P<0.01)。结论:抵当汤及其拆方可通过抑制细胞炎症反应对HUVECs发挥保护和改善作用,其作用机制可能与抑制HMGB1/TLR4/核因子κB信号通路活化有关。
Objective:To investigate the effects and mechanisms of Dizhi Decoction and its modified formulas on inflammation induced by lipopolysaccharide(LPS)in human umbilical vein endothelial cells(HUVECs).Methods:HUVECs cultured in vitro were randomly divided into a control group,an LPS group,an LPS+Dizhi Decoction group,an LPS+herbal medicine group,and an LPS+animal medicine group.After inhibiting Toll-like receptor 4(TLR4),the groups were further divided into a TAK-242 group,a Dizhi Decoction TAK-242 group,a herbal medicine TAK-242 group,and an animal medicine TAK-242 group.After respective treatments,enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of high mobility group box 1(HMGB1),tumor necrosis factor-alpha(TNF-α),and interleukin-6(IL-6)in the cell supernatants.Western blot was used to determine the protein expression levels of HMGB1,TLR4,and nuclear factor kappa B p65(NF-κB p65)in HUVECs.Real-time quantitative PCR(RT-qPCR)was used to measure the gene expression levels of HMGB1,TLR4,and NF-κB p65.Immunofluorescence was used to assess the fluorescence intensity of HMGB1,TLR4,and NF-κB p65.Results:Compared with the A group,the LPS group showed increased levels of HMGB1,IL-6,TNF-α,TLR4,and NF-κB p65 in both the cell supernatants and HUVECs(all P<0.01).Compared with the LPS group,the LPS+Dizhi Decoction group had reduced levels of HMGB1,IL-6,TNF-α,TLR4,and NF-κB p65 in both the cell supernatants and HUVECs(P<0.05,P<0.01).Compared with the LPS+Dizhi Decoction group,the LPS+herbal medicine group and the LPS+animal medicine group showed significantly increased levels of HMGB1,IL-6,TNF-α,TLR4,and NF-κB p65 in both the cell supernatants and HUVECs(P<0.05,P<0.01).The LPS+herbal medicine group had lower levels of HMGB1,IL-6,and TNF-αthan the LPS+animal medicine group(P<0.05,P<0.01),but higher levels of TLR4 and NF-κB p65(P<0.05).After TLR4 inhibition,compared with the LPS group,the TAK-242 group showed reduced levels of HMGB1,TLR4,and NF-κB p65(P<0.05,P<0.01).Compared with the TAK-242 group,the Dizhi Decoction TAK-242 group had lower levels of these indicators(P<0.05,P<0.01).Compared with the Dizhi Decoction TAK-242 group,the herbal medicine TAK-242 group and animal medicine TAK-242 group showed lower levels of these indicators(P<0.05,P<0.01).Conclusion:Dizhi Decoction and its modified formulas can protect and improve HUVECs by inhibiting cellular inflammation.The mechanism of action may be related to the inhibition of HMGB1/TLR4/NF-κB signaling pathway activation.
作者
王思婷
张玥
程志新
刘效敏
张玉冬
刘明
刘政
范国帅
WANG Siting;ZHANG Yue;CHENG Zhixin;LIU Xiaomin;ZHANG Yudong;LIU Ming;LIU Zheng;FAN Guoshuai(First Clinical Medicine School,Shandong University of Traditional Chinese Medicine,Ji′nan 250014,China;Department of Peripheral Vascular Diseases,Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Ji′nan 250014,China;Department of Sports Injury,Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Ji′nan 250014,China)
出处
《世界中医药》
CAS
北大核心
2024年第16期2404-2412,共9页
World Chinese Medicine
基金
国家自然科学基金项目(81774311)
山东省自然科学基金创新发展联合基金项目(ZR202306170002)。
