摘要
目的探讨miR-146调控Toll样受体2(TLR2)通路拮抗脂多糖(LPS)对滋养细胞损伤及其机制。方法体外培养HTR-8/Svneo细胞,试验分为5组:对照组、LPS组(0.1 ng/L LPS)、LPS+miR-146 NC组、LPS+miR-146 mimics组、LPS+miR-146mimics+Pam3CSK4(TLR2激活剂)组,转染24 h后,收集上清液用酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白介素1β(IL-1β)、白介素10(IL-10)浓度,通过实时定量聚合酶链反应(qRT-PCR)法检测miR-146表达,通过流式细胞术检测细胞凋亡情况,免疫印迹法(WB)检测凋亡细胞相关蛋白BCL2-相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)基因、裂解半胱氨酸蛋白酶3(cleaved-Caspase 3)及TLR2通路相关蛋白表达。结果与对照组相比,LPS组中miR-146表达、Bcl-2蛋白含量显著降低(P<0.05);TNF-α、IL-1β、IL-10浓度,细胞凋亡率,Bax、cleaved-Caspase 3、TLR2、髓样分化因子88(MyD88)、磷酸化核转录因子-κB(p-NF-κB)/核转录因子-κB(NF-κB)蛋白含量显著升高(P<0.05)。与LPS组、LPS+miR-146NC组相比,LPS+miR-146mimics组miR-146表达、Bcl-2蛋白含量显著升高(P<0.05);TNF-α、IL-1β、IL-10浓度,细胞凋亡率,Bax、cleaved-Caspase 3、TLR2、MyD88、p-NF-κB/NF-κB显著降低。与LPS+miR-146 mimics组相比,LPS+miR-146 mimics+Pam3CSK4组TLR2、MyD88、p-NF-κB/NF-κB表达显著升高(P<0.05)。结论miR-146过表达可抑制脂多糖诱导的滋养细胞炎症损伤及过度凋亡,可能是通过抑制TLR2通路活化实现的。
Objective Objective to investigate the effect of miR-146 on toll like receptor 2(TLR2)pathway and the mechanism of lipopolysaccharide(LPS)induced trophoblast injury.Methods Human trophoblastic HTR-8/Svneo cells were cultured in vitro and divided into five groups:control group,LPS group(0.1 ng/L LPS),LPS+miR-146 NC group,LPS+miR-146 mimics group and LPS+miR-146 mimics+Pam3 CSK4(TLR2 activator)group.After 24 hours of transfection,the supernatant was collected and the concentration of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-10(IL-10)were detected by enzyme linked immunosorbent assay(ELISA),the expression of miR-146 was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR),the cell apoptosis was detected by flow cytometry,and Western blotting(WB)was used to detect the expressions of apoptotic cell related proteins BCL2-associated X(Bax),B-cell lymphoma-2(Bcl-2),Caspase-3 and TLR2 pathway related proteins.Results Compared with the control group,the expression of miR-146 and the content of Bcl-2 protein in LPS group decreased significantly(P<0.05),while the levels of TNF-α,IL-1β,IL-10,apoptotic rate,Bax,cleaved-Caspase 3,TLR2,myeloid differentiation factor88(MyD88),phosphorylated nuclear transcription factorκB(p-NF-κB)/nuclear transcription factorκB(NF-κB)protein content increased significantly(P<0.05);compared with LPS group and LPS+miR-146 NC group,the expression of miR-146 and the content of Bcl-2 protein in LPS+miR-146 mimics group increased significantly(P<0.05),while the concentration of TNF-α,IL-1β,IL-10,apoptotic rate,Bax,cleaved-Caspase 3,TLR2,MyD88,p-NF-κB/NF-κB protein content decreased significantly.Compared with LPS+miR-146 mimics group,TLR2,MyD88 and p-NF-κB/NF-κB expression in LPS+miR-146 mimics+Pam3 CSK4 group were significantly higher(P<0.05).Conclusion Overexpression of miR-146 can inhibit LPS induced inflammatory injury and excessive apoptosis of trophoblasts,which may be achieved by inhibiting the activation of TLR2 pathway.
作者
努尔比也·地里夏提
祖丽菲娅·阿布力克木
热米拉·艾尔肯
NU ER BI YE·Dilxiati;ZU LI FEI YA·Abulikemu;RE MI LA·Aierken(Department of Obstetrics,People’s Hospital of Xinjiang Uygur Autonomous Region,Urumqi,Xinjiang 830001,China)
出处
《中国优生与遗传杂志》
2021年第8期1060-1065,共6页
Chinese Journal of Birth Health & Heredity
