同型半胱氨酸通过诱导miR-33激活NF-κB途径上调RAW264.7源性泡沫细胞TNF-α、IL-6的表达

Homocysteins upregulates the expression of tumor necrosis factor α,interleukin-6 in RAW264.7-derived foam cells by inducing miR-33 to activate nuclear factor kappa B pathway

目的研究同型半胱氨酸(Hcy)是否通过诱导miR-33激活核因子κB(NF-κB)途径上调肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)的表达,促进炎症反应,加剧动脉粥样硬化(As)。方法RAW264.7源性巨噬细胞,经氧化型低密度脂蛋白(ox-LDL)诱导为泡沫细胞后,分别将miR-33mimics和miR-33 inhibitor转染入细胞内后每组给予5.0mmol/L的Hcy干预;油红“O”染色确定泡沫细胞模型是否诱导成功;Western blot和实时定量PCR测定NF-κB、TNF-α、IL-6的蛋白和mRNA表达;高效液相色谱检测细胞内胆固醇含量。结果油红“O”染色后含脂滴的泡沫细胞被染成红色;与其它组相比,miR-33 mimics组的NF-κB、TNF-α、IL-6的蛋白和mRNA表达均增高且细胞内的胆固醇含量增加(P<0.05);miR-33 inhibitor组的NF-κB、TNF-α、IL-6的蛋白和mRNA表达均降低且细胞内的胆固醇含量降低(P<0.05);空白对照组、miR-33 mimics对照组和miR-33 inhibitor对照组间相比,所有检测指标均无统计学差异(P>0.05)。结论Hcy通过诱导miRNA-33激活NF-κB途径上调TNF-α和IL-6的表达,增加炎症反应,促进动脉粥样硬化。

Aim To investigate whether homocysteine ( Hcy) upregulates the expression of tumor necrosis factor α( TNF-α) and interleukin-6 ( IL-6) by inducing activation of nuclear factor kappa B ( NF-κB) pathway by miR-33,promotes inflammatory lesions and exacerbates atherosclerosis ( As) . Methods RAW264.7-derived macrophages were induced by oxidized low-density lipoprotein ( ox-LDL) as foam cells,and miR-33 mimics /miR-33 inhibitors were transfected into cells. Each group received 5.0 mmol /L of Hcy intervention; oil red“O”staining was used to determine whether the foam cell model was successfully induced; Western blot and real-time quantitative PCR was used to determine the protein and mRNA expression of NF-κB,TNF-α,IL-6; the cellular cholesterol content was analyzed by HPLC. Results The successfully induced foam cells were stained with oil red“O”,and the nuclei containing lipid droplets were stained red; compared with other group,the expression of NF-κB,TNF-α and IL-6 protein and mRNA,the intracellular cholesterol content was significantly increased in the miR-33 mimics group ( P<0.05) . No difference was observed in the above indexes between blank group,miR-33 mimics-NC group and miR-33 inhibitor-NC group ( P>0.05) . Conclusion Hcy can upregulate the expression of TNF-α and IL-6 by inducing miR-33 to activate NF-κB pathway,increase inflammatory reaction and promote atherosclerosis ( As) .