木香烃内酯通过调控LncRNA LINC01116/miR-9-5p的表达来减轻过氧化氢诱导的大鼠脑微血管内皮细胞损伤

Costunolide reduces hydrogen peroxide-induced damage to rat brain microvascular endothelial cells by regulating the expression of LncRNA LINC01116/miR-9-5p

目的探讨木香烃内酯(Cos)对过氧化氢(H2O2)诱导的大鼠脑微血管内皮细胞的氧化应激、凋亡的影响及其对长链非编码RNA LINC01116(LncRNA LINC01116)/微小RNA-9-5p(miR-9-5p)的调控作用。方法原代分离培养大鼠脑微血管内皮细胞,并随机分成Con组、H2O2组、Cos-L组、Cos-M组、Cos-H组、Cos-H+pcDNA组、Cos-H+pcDNA-LINC01116组;采用化学比色法检测丙二醛(MDA)、还原型谷胱甘肽(GSH)的水平及超氧化物歧化酶(SOD)的活性;流式细胞术检测细胞凋亡率;实时荧光定量聚合酶链反应(qRT-PCR)检测LINC01116、miR-9-5p的表达水平;双荧光素酶报告实验验证LINC01116与miR-9-5p的靶向关系;蛋白免疫印迹法(Western blot)检测B淋巴细胞瘤-2相关蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)的表达水平。结果 H2O2处理后MDA的水平显著升高(P<0.05),GSH水平与SOD活性显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),Bax蛋白水平显著升高(P<0.05),Bcl-2蛋白水平显著降低(P<0.05),LINC01116的表达水平显著升高(P<0.05),miR-9-5p的表达水平显著降低(P<0.05);Cos处理后MDA的水平显著降低(P<0.05),GSH水平与SOD活性显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),Bax蛋白水平显著降低(P<0.05),Bcl-2蛋白水平显著升高(P<0.05),LINC01116的表达水平显著降低(P<0.05),miR-9-5p的表达水平显著升高(P<0.05),且Cos-L组、Cos-M组、Cos-H组上述指标的水平比较均有明显差异(P<0.05);双荧光素酶报告实验证实LINC01116可靶向结合miR-9-5p;LINC01116过表达可减弱木香烃内酯对H2O2诱导的大鼠脑微血管内皮细胞凋亡及氧化应激的作用。结论木香烃内酯可能通过抑制LINC01116的表达及促进miR-9-5p的表达来抑制H2O2诱导的大鼠脑微血管内皮细胞的氧化应激及细胞凋亡,从而减轻细胞损伤。

Objective To explore the effect of Costunolide(Cos) on H2O2-induced oxidative stress and apoptosis in rat brain microvascular endothelial cells and its regulatory effect on LncRNA LINC01116/miR-9-5 p.Methods Primary cultured rat brain microvascular endothelial cells were randomly divided into Con group, H2O2 group, Cos-L group, Cos-M group, Cos-H group, Cos-H + pcDNA group, Cos-H + pcDNA-LINC01116 group. The level of MDA and GSH and the activity of SOD were detected by chemical colorimetry. Flow cytometry was used to detect the apoptosis rate. qRT-PCR was used to detect the expression levels of LINC01116 and miR-9-5 p. The dual luciferase report experiment verified the targeting relationship between LINC01116 and miR-9-5 p. Western blot was used to detect the expression levels of Bax and Bcl-2.Results After H2O2 treatment, the level of MDA was significantly increased(P<0.05), the level of GSH and the activity of SOD were significantly decreased(P<0.05), cell apoptosis rate was significantly increased(P<0.05), the level of Bax protein was significantly increased(P<0.05), the level of Bcl-2 protein was significantly reduced(P<0.05), the expression level of LINC01116 was significantly increased(P<0.05), the expression level of miR-9-5 p was significantly reduced(P<0.05). After Cos treatment, the level of MDA was significantly reduced(P<0.05), the level of GSH and the activity of SOD were significantly increased(P<0.05), cell apoptosis rate was significantly reduced(P<0.05), the level of Bax protein was significantly reduced(P<0.05), the level of Bcl-2 protein was significantly increased(P<0.05), the expression level of LINC01116 was significantly reduced(P<0.05), the expression level of miR-9-5 p was significantly increased(P<0.05), and there were statistically significant differences in the above parameters among Cos-L group, Cos-M group and Cos-H group(P<0.05). The dual luciferase report experiment confirmed that LINC01116 could target and bind to miR-9-5 p. LINC01116 overexpression could attenuate the effect of costunolide on H2O2-induced apoptosis and oxidative stress in rat brain microvascular endothelial cells.Conclusion Costunolide might inhibit H2O2-induced oxidative stress and apoptosis in rat brain microvascular endothelial cells by inhibiting the expression of LINC01116 and promoting the expression of miR-9-5 p to reduce cell damage.