氧化应激环境下LncRNA MALAT1对内皮细胞TLR4/MyD88/NF-κB信号通路的影响

Effect of LncRNA MALAT1 on TLR4/MyD88/NF-κB Signaling Pathway in Endothelial Cells under Oxidative Stress

目的探究氧化应激环境下长链非编码RNA肺腺癌转移相关转录物1(LncRNA MALAT1)对内皮细胞Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核因子κB(NF-κB)信号通路的影响。方法本实验时间为2022年10—12月。将人脐静脉内皮细胞(HUVEC)分为A、B、C、D组,分别使用600μmol/L的过氧化氢(H_(2)O_(2))处理0、6、8、10 h,随后采用CCK-8法测定细胞活性以分析H_(2)O_(2)诱导氧化应激细胞模型的最佳干预时间。将HUVEC分为对照组(不做任何处理)、H_(2)O_(2)组(使用600μmol/L的H_(2)O_(2)处理8 h以构建氧化应激细胞模型),采用q-PCR检测LncRNA MALAT1表达水平。将HUVEC分为对照组(不做任何处理)、H_(2)O_(2)组(使用600μmol/L的H_(2)O_(2)处理8 h以构建氧化应激细胞模型)、H_(2)O_(2)+siRNA组(转染LncRNA MALAT1的siRNA后使用600μmol/L的H_(2)O_(2)处理8 h以构建氧化应激细胞模型),采用Western blot法检测TLR4、MyD88、NF-κB表达水平。将HUVEC分为对照组(不做任何处理)、H_(2)O_(2)组(使用600μmol/L的H_(2)O_(2)处理8 h以构建氧化应激细胞模型)、H_(2)O_(2)+siRNA组(转染LncRNA MALAT1的siRNA后使用600μmol/L的H_(2)O_(2)处理8 h以构建氧化应激细胞模型),采用q-PCR检测TLR4、MyD88、NF-κB mRNA表达水平。结果B、C、D组细胞活性均小于A组(P<0.05);C组细胞活性最接近60%,故H_(2)O_(2)诱导氧化应激细胞模型的最佳干预时间为8 h。H_(2)O_(2)组LncRNA MALAT1表达水平低于对照组(P<0.05)。H_(2)O_(2)+siRNA组TLR4、MyD88、NF-κB表达水平高于对照组(P<0.05);H_(2)O_(2)+siRNA组MyD88、NF-κB表达水平高于H_(2)O_(2)组(P<0.05)。H_(2)O_(2)组TLR4、MyD88mRNA表达水平高于对照组(P<0.05);H_(2)O_(2)+siRNA组TLR4、MyD88、NF-κB mRNA表达水平高于对照组、H_(2)O_(2)组(P<0.05)。结论氧化应激环境下,LncRNA MALAT1表达水平降低,进而导致内皮细胞TLR4/MyD88/NF-κB信号通路被激活。

Objective To explore the effect of long chain non-coding RNA metastasis-associated lung adenocarcinoma tran 1(LncRNA MALAT1)on Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-kappa B(NF-κB)in endothelial cells under oxidative stress.Methods This experiment was conducted from October to December 2022.Human umbilical vein endothelial cells(HUVECs)were divided into groups A,B,C and D,and treated with 600μmol/L hydrogen peroxide(H_(2)O_(2))for 0,6,8 and 10 h,respectively.Then the cell activity was determined by CCK-8 method to analyze the optimal intervention time of H_(2)O_(2) to induce oxidative stress cell model.HUVECs were divided into control group(without any treatment)and H_(2)O_(2) group(treated with 600μmol/L H_(2)O_(2) for 8 h to construct oxidative stress cell model),and the expression level of LncRNA MALAT1 was detected by q-PCR.HUVECs were divided into control group(without any treatment),H_(2)O_(2) group(treated with 600μmol/L H_(2)O_(2) for 8 h to construct oxidative stress cell model),H_(2)O_(2)+siRNA group(transfected with LncRNA MALAT1 siRNA and treated with 600μmol/L H_(2)O_(2) for 8 h to construct oxidative stress cell model).The expression levels of TLR4,MyD88 and NF-κB were detected by Western blot.HUVECs were divided into control group(without any treatment),H_(2)O_(2) group(treated with 600μmol/L H_(2)O_(2) for 8 h to construct oxidative stress cell model),H_(2)O_(2)+siRNA group(transfected with LncRNA MALAT1 siRNA and treated with 600μmol/L H_(2)O_(2) for 8 h to construct oxidative stress cell model).The mRNA expression levels of TLR4,MyD88 and NF-κB were detected by q-PCR.Results The cell activity of groups B,C and D was lower than that of group A(P<0.05).The cell activity of group C was the closest to 60%,so the optimal intervention time of H_(2)O_(2) to induce oxidative stress cell model was 8 h.The expression level of LncRNA MALAT1 in H_(2)O_(2) group was lower than that in control group(P<0.05).The expression levels of TLR4,MyD88 and NF-κB in H_(2)O_(2)+siRNA group were higher than those in control group(P<0.05).The expression levels of MyD88 and NF-κB in H_(2)O_(2)+siRNA group were higher than those in H_(2)O_(2) group(P<0.05).The mRNA expression levels of TLR4 and MyD88 in H_(2)O_(2) group were higher than those in control group(P<0.05).The mRNA expression levels of TLR4,MyD88 and NF-κB in H_(2)O_(2)+siRNA group were higher than those in control group and H_(2)O_(2) group(P<0.05).Conclusion Under oxidative stress,the expression level of LncRNA MALAT1 is decreased,which leads to the activation of TLR4/MyD88/NF-κB signaling pathway in endothelial cells.