摘要
目的研究糖化低密度脂蛋白(Gly-LDL)对人脐静脉内皮细胞(HUVECs)的炎症损伤及Toll样受体4(TLR4)、缺氧诱导因子-1α(HIF-1α)表达的影响,并探讨其可能的机制。方法体外培养HUVECs,实验分为对照组、阳性对照组[50 mg/L普通低密度脂蛋白(n-LDL)]、Gly-LDL(50、75、100 mg/L)组。CCK-8法测定细胞增殖情况;划痕实验测定细胞迁移能力;ELISA法测定肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)分泌水平;qRT-PCR测定TLR4、HIF-1α、TNF-α、IL-6 mRNA水平;Western blot测定TLR4、HIF-1α、TNF-α、IL-6蛋白水平;分别用TLR4、HIF-1α的si-RNA干预Gly-LDL对HUVECs的作用,实验分为对照组、模型组(Gly-LDL 100 mg/L)、si-TLR4组(Gly-LDL 100 mg/L+si-TLR4)、TLR4空载组(Gly-LDL 100 mg/L+si-NC1)、si-HIF-1α组(Gly-LDL 100 mg/L+si-HIF-1α)和HIF-1α空载组(Gly-LDL 100 mg/L+si-NC2)。Western blot法检测TLR4、HIF-1α蛋白表达水平,验证TLR4、HIF-1α相互作用关系。结果Gly-LDL(50、75、100 mg/L)组均可抑制HUVECs的增殖及迁移能力(P<0.01),刺激HUVECs中炎症细胞因子TNF-α、IL-6、ICAM-1、VCAM-1分泌量增加(P<0.001),上调TLR4、HIF-1α、TNF-α、IL-6 mRNA和蛋白水平(P<0.05),并呈浓度依赖性。敲除TLR4后,与模型组相比,TLR4、HIF-1α蛋白表达均下调(P<0.001)。敲除HIF-1α后,与模型组相比,仅HIF-1α蛋白表达下调(P<0.001),而TLR4蛋白在Gly-LDL作用下表达上调。结论Gly-LDL可能通过上调TLR4/HIF-1α炎症信号通路抑制HUVECs的增殖与迁移,上调炎症细胞因子表达,导致血管内皮损伤。
Objective To investigate the effects of glycated low density lipoprotein(Gly-LDL)on the growth of human umbilical vein endothelial cells(HUVECs)and the expression of toll like receptoR4(TLR4)and hypoxia inducible factor-1α(HIF-1α),and to explore its possible mechanism.Methods HUVECs were cultured in vitro and divided into control group,positive control group[50 mg/L normal low density lipoprotein(n-LDL)],low concentration,medium concentration and high concentration Gly-LDL(50,75,100 mg/L)groups.Respectively,the effects of different concentrations of Gly-LDL on survival rate of HUVECs were detected by CCK-8;The motility of HUVECs undeRdifferent treatments were detected by wound healing assays;The level of inflammatory cytokine,such as tumoRinducing factor-α(TNF-α),interleukin-6(IL-6),intercellulaRadhesion molecule-1(ICAM-1)and vasculaRcell adhesion molecule-1(VCAM-1)were detected by ELISA;The mRNA levels of TLR4,HIF-1α,TNF-αand IL-6 were detected by qRT-PCR;Protein expressions of TLR4,HIF-1α,TNF-αand IL-6 were detected by Western blot;Respectively,si-RNA of TLR4 and HIF-1αwas used to intervene the effects of Gly-LDL on HUVECs.The experiment was divided into control group,model group(Gly-LDL 100 mg/L),si-TLR4 group(Gly-LDL 100 mg/L+si-TLR4),TLR4 unloading group(Gly-LDL 100 mg/L+si-NC1),si-HIF-1αgroup(Gly-LDL 100 mg/L+si-HIF-1α)and HIF-1αunloading group(Gly-LDL 100 mg/L+si-NC2).Protein expressions of TLR4 and HIF-1αwere detected by Western blot to verify the interaction between TLR4 and HIF-1α.Results The survival rate and migration rate of HUVECs were inhibited in Gly-LDL(50 mg/L,75 mg/L,100 mg/L)group(P<0.01),the inflammatory cytokines,such as TNF-α,IL-6,ICAM-1,VCAM-1 increased by Gly-LDL function on HUVECs(P<0.001),and the mRNA and protein levels of TLR4,HIF-1α,TNF-αand IL-6 increased by Gly-LDL in a dose dependent manner.AfteRTLR4 was knocked out,the proteins expression of TLR4 and HIF-1αwere down-regulated compared with model group(P<0.05),but afteRHIF-1αwas knockout,only the protein expressi
作者
邱军辉
刘美志
孙杜桑
潘婷
赵维维
沙雯君
鲁郡
雷涛
Qiu Junhui;Liu Meizhi;Sun Dusang;Pan Ting;Zhao Weiwei;Sha Wenjun;Lu Jun;Lei Tao(Shanghai Putuo Central School of Clinical Medicine,Anhui Medical University,Shanghai 200062;Dept of Endocrinolog,Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200062;School of Medical and Life Sciences,Chengdu University of Traditional Chinese Medicine,Chengdu 610000)
出处
《安徽医科大学学报》
CAS
北大核心
2022年第10期1614-1620,共7页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81774083)
上海市医学重点专科建设项目(编号:ZK2019B16)
上海市普陀区卫生健康系统临床特色专科建设项目(编号:2020tszk01)。
