miR-21在黄芪甲苷保护ox-LDL诱导的内皮细胞炎症损伤过程中的作用
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摘要
目的探讨miR-21在黄芪甲苷保护氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)炎症损伤中的作用及其机制。方法将体外培养的HUVEC分为对照组(未干预)、模型组(以100μmol/L ox-LDL作用24 h)、治疗-低剂量(L)组、治疗-中剂量(M)组和治疗-高剂量(H)组,其中治疗-L、M和H组细胞以100μmol/L ox-LDL作用24 h后,分别加入10、20和40 mg/L的黄芪甲苷作用2 h。MTT法检测细胞的存活率,ELISA法检测细胞上清液中炎症相关因子TNF-α、IL-6和NF-κB的含量,RT-PCR检测细胞内miR-21和TLR4 mRNA的表达,Western blot检测TLR4蛋白的表达。双荧光素酶报告基因实验检测miR-21和TLR4的靶向关系。转染miR-21模拟物和抑制剂构建miR-21过表达和低表达的HUVEC株,观察miR-21对HUVEC炎症因子分泌和TLR4表达的影响;同时,在给予40 mg/L的黄芪甲苷治疗的基础上,观察转染miR-21抑制剂下调miR-21表达对黄芪甲苷作用下HUVEC炎症因子的影响。结果与对照组相比,模型组、治疗-L组、治疗-M组和治疗-H组细胞存活率和细胞中miR-21表达显著降低,而细胞上清液中TNF-α、IL-6和NF-κB的含量以及细胞中TLR4 mRNA、蛋白的表达显著升高(P<0.05);与模型组相比,治疗-L组、治疗-M组和治疗-H组细胞存活率和miR-21表达逐渐升高,而TNF-α、IL-6和NF-κB的含量以及TLR4 mRNA、蛋白的表达逐渐降低,差异具有统计学意义(P<0.05)。双荧光素酶报告基因实验证实TLR4是miR-21的潜在靶基因。miR-21过表达降低HUVEC内炎症因子的分泌和TLR4的表达,反之,则促进HUVEC内炎症因子的分泌和TLR4的表达;下调miR-21表达后可逆转黄芪甲苷对HUVEC炎症因子的调控。结论黄芪甲苷可减轻ox-LDL诱导的HUVEC炎症损伤,其作用机制可能与上调miR-21靶向抑制TLR4表达有关。
Objective To investigate the role and mechanism of miR-21 in the inflammatory injury of human umbilical vein endothelial cells(HUVEC)induced by astragaloside-protected oxidized low density lipoprotein(ox-LDL). Methods HUVEC cultured in vitro were divided into control group(without intervention),model group(treated with 100 μmol/L ox-LDL for 24 h),treatment-low dose group(L),treatment-medium dose group(M)and treatment-high dose group(H). The cells in the treatment group-L,M and H were treated with 100 μmol/L ox-LDL for 24 h,and then added with 10,20 and 40 mg/L of astragaloside for 2 h. Cell viability was detected by MTT assay,the level of inflammatory related factors TNF-α,IL-6 and NF-κB in cell supernatant were measured by ELISA,the expression of intracellular miR-21 and TLR4 was detected by RT-PCR,the expression of TLR4 protein was tested by Western blot,and the targeting relationship between microRNA-21 and TLR4 was checked by dual luciferase reporter gene assay. In addition,HUVEC with over-and low-expression of miR-21 were constructed by transfecting mimic and inhibitor of miR-21,and the effects of miR-21 on the secretion of inflammatory factors and the expression of TLR4 in HUVEC were observed. At the same time,on the basis of 40 mg/L astragaloside treatment,the effects of down-regulation of miR-21 expression by transfection inhibitor of miR-21 on inflammatory factors in HUVEC under the action of astragaloside were observed. Results Compared with the control group,the cell survival rate and the expression of miR-21 in the model group,treatment-L,treatment-M and treatment-H groups were decreased significantly,while the level of TNF-a,IL-6 and NF-kB in cell supernatant and the expression of TLR4 mRNA and protein in cells were increased significantly(P<0.05). Compared with the model group,the cell viability and the expression of miR-21 were increased gradually,while the contents of TNF-a,IL-6 and NF-kB and the expression of TLR4 mRNA and protein were decreased gradually in treatment-L group,treatment-M group and treatment-H group(P<0.05). Dual luciferase reporter assays confirmed that TLR4 was a potential target gene of miR-21. Overexpression of miR-21 reduced the secretion of inflammatory cytokines and the expression of TLR4 in HUVEC. Conversely,it promoted the secretion of inflammatory cytokines and the expression of TLR4 in HUVEC. Down-regulation of miR-21 expression reversed the regulation of Astragaloside on HUVEC inflammatory factors. Conclusion Astragaloside can alleviate the inflammation injury of HUVEC induced by ox-LDL,and its mechanism may be related to up-regulation of the expression of TLR4 targeting by miR-21.
Objective To investigate the role and mechanism of miR-21 in the inflammatory injury of human umbilical vein endothelial cells(HUVEC)induced by astragaloside-protected oxidized low density lipoprotein(ox-LDL). Methods HUVEC cultured in vitro were divided into control group(without intervention),model group(treated with 100 μmol/L ox-LDL for 24 h),treatment-low dose group(L),treatment-medium dose group(M)and treatment-high dose group(H). The cells in the treatment group-L,M and H were treated with 100 μmol/L ox-LDL for 24 h,and then added with 10,20 and 40 mg/L of astragaloside for 2 h. Cell viability was detected by MTT assay,the level of inflammatory related factors TNF-α,IL-6 and NF-κB in cell supernatant were measured by ELISA,the expression of intracellular miR-21 and TLR4 was detected by RT-PCR,the expression of TLR4 protein was tested by Western blot,and the targeting relationship between microRNA-21 and TLR4 was checked by dual luciferase reporter gene assay. In addition,HUVEC with over-and low-expression of miR-21 were constructed by transfecting mimic and inhibitor of miR-21,and the effects of miR-21 on the secretion of inflammatory factors and the expression of TLR4 in HUVEC were observed. At the same time,on the basis of 40 mg/L astragaloside treatment,the effects of down-regulation of miR-21 expression by transfection inhibitor of miR-21 on inflammatory factors in HUVEC under the action of astragaloside were observed. Results Compared with the control group,the cell survival rate and the expression of miR-21 in the model group,treatment-L,treatment-M and treatment-H groups were decreased significantly,while the level of TNF-a,IL-6 and NF-kB in cell supernatant and the expression of TLR4 mRNA and protein in cells were increased significantly(P<0.05). Compared with the model group,the cell viability and the expression of miR-21 were increased gradually,while the contents of TNF-a,IL-6 and NF-kB and the expression of TLR4 mRNA and protein were decreased gradually in treatment-L group,treatment-M group and treatment-H group(P<0.05). Dual luciferase reporter assays confirmed that TLR4 was a potential target gene of miR-21. Overexpression of miR-21 reduced the secretion of inflammatory cytokines and the expression of TLR4 in HUVEC. Conversely,it promoted the secretion of inflammatory cytokines and the expression of TLR4 in HUVEC. Down-regulation of miR-21 expression reversed the regulation of Astragaloside on HUVEC inflammatory factors. Conclusion Astragaloside can alleviate the inflammation injury of HUVEC induced by ox-LDL,and its mechanism may be related to up-regulation of the expression of TLR4 targeting by miR-21.
引文
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[17]李如月,向晓辉,张斌,等.TLR4信号通路相关miR-NAs在炎症反应调节中的研究进展[J].天津医药,2017,45(7):771-776.
[18]Tian D,Sha Y,Lu J M,et al.MiR-370 inhibits vascular inflammation and oxidative stress triggered by oxidized low-density lipoprotein through targeting TLR4[J].Journal of cellular biochemistry,2018,119(7):6231-6237.
[19]蔡谦谦,周红,张贵婷,等.oxLDL/β2GPI/抗β2GPI抗体复合物促进脐静脉内皮细胞迁移及炎症因子表达[J].细胞与分子免疫学杂志,2017,33(7):865-869.
[20]Ma L,Yang Y,Sun X,et al.Propofol regulates the expression of TLR4 through miR21 in human umbilical vein endothelial cells[J].Molecular Medicine Reports,2017,16(6):9074-9080.
[2]Lin F,Pei L,Zhang Q,et al.Ox-LDL induces endothelial cell apoptosis and macrophage migration by regulating caveolin-1 phosphorylation[J].Journal of cellular physiology,2018,233(10):6683-6692.
[3]Zhang H,Liang B,Li T,et al.Orexin A Suppresses Oxidized LDL Induced Endothelial Cell Inflammation via MAPK p38 and NF-κB Signaling Pathway[J].IUBMB life,2018,70(10):961-968.
[4]Li L,Hou X,Xu R,et al.Research review on the pharmacological effects of astragaloside IV[J].Fundam Clin Pharmacol,2017,31(1):17-36.
[5]张志鑫,李彦杰,秦合伟,等.基于PI3K/Akt/mTOR信号通路调控巨噬细胞自噬探讨黄芪甲苷抗动脉粥样硬化的作用机制[J].中草药,2017,48(17):3575-3581.
[6]魏冰,姜希娟,卢斌,等.ox-LDL对内皮细胞CD40信号表达的影响及黄芪甲苷的抗炎作用[J].天津中医药大学学报,2017,36(4):282-285.
[7]Tang F,Yang T L,Zhang Z,et al.MicroRNA-21suppresses ox-LDL-induced human aortic endothelial cells injuries in atherosclerosis through enhancement of autophagic flux:involvement in promotion of lysosomal function[J].Experimental Cell Research,2017,359(2):374-383.
[8]Yu P,Cheng S,Yang L,et al.Astragaloside IV stimulates angiogenesis after myocardial infarction by regulating microRNA-21 expression[J].International Journal of Clinical and Experimental Medicine,2016,9(5):7818-7827.
[9]Li J,Li A,Li M,et al.Ginkgolic acid exerts an antiinflammatory effect in human umbilical vein endothelial cells induced by ox-LDL[J].Die Pharmazie-An International Journal of Pharmaceutical Sciences,2018,73(7):408-412.
[10]Zhang Y H,He K,Shi G.Effects of MicroRNA-499on the inflammatory damage of endothelial cells during coronary artery disease via the targeting of PDCD4through the NF-ΚB/TNF-αsignaling pathway[J].Cellular Physiology and Biochemistry,2017,44(1):110-124.
[11]Guo J,Liang W,Li J,et al.Knockdown of FSTL1 inhibits oxLDL-induced inflammation responses through the TLR4/MyD88/NF-κB and MAPK pathway[J].Biochemical and biophysical research communications,2016,478(4):1528-1533.
[12]Qin H,Liu P,Lin S.Effects of astragaloside IV on the SDF-1/CXCR4 expression in atherosclerosis of apoE-/-mice induced by hyperlipaemia[J].Journal of the American College of Cardiology,2015,64(16):385154.
[13]马海涛,王辉.黄芪甲苷对过氧化氢诱导损伤的人脐静脉内皮细胞的保护作用[J].郑州大学学报(医学版),2016,51(2):248-251.
[14]You L,Fang Z,Shen G,et al.Astragaloside IV prevents high glucose-induced cell apoptosis and inflammatory reactions through inhibition of the JNK pathway in human umbilical vein endothelial cells[J].Molecular medicine reports,2019,19(3):1603-1612.
[15]李兆欣,刘江月,王其新.Aliskiren抑制LPS诱导HUVECs新生血管的形成及可能机制[J].中国病理生理杂志,2016,32(4):602-609.
[16]Wan C X,Xu M,Huang S H,et al.Baicalein protects against endothelial cell injury by inhibiting the TLR4/NF-κB signaling pathway[J].Molecular medicine reports,2018,17(2):3085-3091.
[17]李如月,向晓辉,张斌,等.TLR4信号通路相关miR-NAs在炎症反应调节中的研究进展[J].天津医药,2017,45(7):771-776.
[18]Tian D,Sha Y,Lu J M,et al.MiR-370 inhibits vascular inflammation and oxidative stress triggered by oxidized low-density lipoprotein through targeting TLR4[J].Journal of cellular biochemistry,2018,119(7):6231-6237.
[19]蔡谦谦,周红,张贵婷,等.oxLDL/β2GPI/抗β2GPI抗体复合物促进脐静脉内皮细胞迁移及炎症因子表达[J].细胞与分子免疫学杂志,2017,33(7):865-869.
[20]Ma L,Yang Y,Sun X,et al.Propofol regulates the expression of TLR4 through miR21 in human umbilical vein endothelial cells[J].Molecular Medicine Reports,2017,16(6):9074-9080.