丹酚酸A调控LncRNA TSPOAP1-AS1缓解H_(2)O_(2)诱导脑微血管内皮细胞凋亡及血管新生能力的影响

Danshensuan A regulates the effect of LncRNA TSPOAP1⁃AS1 on alleviating H2O2 induced apoptosis and angiogenesis in brain microvascular endothelial cells

目的 探讨丹酚酸A调控lncRNA TSPAOP1-AS1缓解H2O2诱导脑微血管内皮细胞凋亡及血管新生能力的影响。方法 选取2021年1月-2023年3月在我院收治的80例高血压脑出血患者和健康体检者,分别设为研究组和对照组。将对数生长的HBMECs分为9组,即Control组、H_(2)O_(2)组、SAA-L组、SAA-M组、SAA-H组、si-TSPOAP1-AS1组、si-NC组和pcDNA-TSPOAP1-AS1组。RT-PCR法检测患者血清及细胞中TSPOAP1-AS1的相对表达水平;分别采用CCK-8法、流式细胞仪和Matrigel体外成管试验检测细胞存活率、凋亡率及血管生成能力;ELISA检测细胞氧化应激;蛋白质印迹法检测细胞中Caspase-3、Bax、Bcl-2蛋白表达。结果 与对照组(1.00±0.09)相比,研究组患者血清中TSPOAP1-AS1相对表达量(4.36±0.37)明显升高(P<0.01)。与Control组比较,H2O2组细胞中TSPOAP1-AS1相对表达水平(2.65±0.23)、细胞凋亡率(18.29±1.65)、细胞中MDA含量(40.81±3.72)和Caspase-3蛋白(0.78±0.08)、Bax蛋白(0.82±0.09)表达均明显增加,细胞存活率(35.36±3.65)、小管形成数目(85.32±7.68)、细胞中SOD活性(15.48±1.63)、GSH-Px含量(9.43±0.82)及Bcl-2蛋白(0.21±0.02)表达均明显降低(P<0.01);与H2O2组相比,SAA-H组和si-TSPOAP1-AS1组细胞中TSPOAP1-AS1相对表达水平(1.21±0.12)(1.30±0.13)、细胞凋亡率(5.31±0.50)(6.01±0.62)、细胞中MDA含量(8.95±0.82)(9.39±0.92)和Caspase-3蛋白(0.32±0.04)(0.35±0.04)、Bax蛋白(0.40±0.05)(0.45±0.05)表达均明显降低,细胞存活率(87.21±7.64)(82.31±7.50)、小管形成数目(198.75±18.32)(180.31±17.64)、细胞中SOD活性(60.27±5.89)(55.26±5.24)、GSH-Px含量(40.63±4.17)(38.76±3.27)及Bcl-2蛋白(0.70±0.08)(0.68±0.07)表达均明显升高(P<0.01)。与SAA-H组相比,pcDNA-TSPOAP1-AS1组细胞中TSPOAP1-AS1相对表达水平(2.49±0.20)、细胞凋亡率(16.90±1.60)、细胞中MDA含量(39.01±3.83)和Caspase-3蛋白(0.72±0.08)、Bax蛋白表(0.78±0.08)达均明显增加,细胞存活率(40.47±2.89)、小管形成数目(90.15±8.62)、细胞中SOD活性(20.16±2.11)、GSH-Px含量(11.56±1.08)及Bcl-2蛋白(0.28±0.03)表达均明显降低(P<0.01)。结论 丹酚酸A可促进H2O2诱导的HBMECs存活和血管生成,抑制细胞凋亡,减轻氧化应激反应,其作用机制可能和抑制lncRNA TSPOAP1-AS1相关。

Objective Exploring the effect of Danshensuan A regulating lncRNA TSPAOP1-AS1 on alleviat-ing H_(2)O_(2) induced apoptosis and angiogenesis in brain microvascular endothelial cells.Methods A total of 80 pa-tients with ischemic stroke admitted to our hospital from January 2021 to March 2023 were selected as the study group,and an additional 80 healthy individuals who came to our hospital for health check-ups during the same peri-od were included as the control group.Logarithmic growth of HBMECs was divided into 9 groups:Control group,H2O2 group,SAA-L group,SAA-M group,SAA-H group,EDA group,si-TSPOAP1-AS1 group,si-NC group,and pcDNA-TSPOAP1-AS1 group.The relative expression levels of TSPOAP1-AS1 in serum and cells were detected us-ing RT-PCR.Cell viability was assessed by CCK-8 assay.Flow cytometry was used to detect cell apoptosis.Matrigel tube formation assay was performed to evaluate angiogenesis ability.ELISA was used to measure cellular oxidative stress.Western blotting was conducted to examine the protein expression of Caspase-3,Bax,and Bcl-2 in cells.Re-sults Compared to the control group(1.00±0.09),the relative expression level of TSPOAP1-AS1 in the serum of patients in the study group(4.36±0.37)was significantly increased(P<0.01).Compared to the Control group,the H2O2 group showed significant increases in TSPOAP1-AS1 relative expression levels in cells(2.65±0.23),cell apop-tosis rate(18.29±1.65),MDA content in cells(40.81±3.72),and protein expression of Caspase-3(0.78±0.08)and Bax(0.82±0.09),as well as significant decreases in cell survival rate(35.36±3.65),tube formation(85.32±7.68),SOD activity in cells(15.48±1.63),GSH-Px content(9.43±0.82),and protein expression of Bcl-2(0.21±0.02)(P<0.01).Compared with the H2O2 group,the relative expression levels of TSPOAP1-AS1 in cells of the SAA-H and si-TSPOAP1-AS1 groups(1.21±0.12)(1.30±0.13),apoptosis rate(5.31±0.50)(6.01±0.62),cellular MDA content(8.95±0.82)(9.39±0.92)and expression of Caspase-3 protein(0.32±0.04)(0.35±0.04)and Bax protein(0.40±0.05)(0.45±0.05)were significantly reduced,and the cell survival rate(87.21±7.64)(82.31±7.50),the number of tubule formation(198.75±18.32)(180.31±17.64),SOD activity(60.27±5.89)(55.26±5.24),GSH-Px content(40.63±4.17)(38.76±3.27),and Bcl-2 protein(0.70±0.08)(0.68±0.07)expression were significantly higher in the cells(P<0.01).Compared to the SAA-H group,the pcDNA-TSPOAP1-AS1 group showed significant in-creases in TSPOAP1-AS1 relative expression levels in cells(2.49±0.20),cell apoptosis rate(16.90±1.60),MDA content in cells(39.01±3.83),and protein expression of Caspase-3(0.72±0.08)and Bax(0.78±0.08),as well as significant decreases in cell survival rate(40.47±2.89),tube formation(90.15±8.62),SOD activity in cells(20.16±2.11),GSH-Px content(11.56±1.08),and protein expression of Bcl-2(0.28±0.03)(P<0.01).Conclu-sion Danshensuan A can promote the survival and angiogenesis of HBMECs induced by H2O2,inhibit cell apopto-sis,and alleviate oxidative stress response.Its mechanism of action may be related to the inhibition of lncRNA TSPOAP1-AS1.