Notch1干扰调控肺血管内皮细胞凋亡作用机制研究

Mechanism by which Notch1 interference regulates the apoptosis of pulmonary microvascular endothelial cells

目的探讨缺口受体1(Notch1)干扰调控肺血管内皮细胞凋亡的作用机制。方法2022年1-12月利用Notch1干扰siRNA转染人肺微血管内皮细胞(HPMEC);利用Cell Counting Kit-8(CCK8)检测各组细胞活力;采用流式细胞术测定活性氧(ROS)水平;采用流式细胞术测定细胞凋亡情况;采用Western blot检测Notch1 siRNA对HPMEC细胞中Notch1、B细胞淋巴瘤-2基因(Bcl-2)、胱天蛋白酶-3(Caspase-3)蛋白表达。结果与空白对照组、siNC组比较,si-Notch1组HPMEC细胞中Notch1 mRNA的表达水平均显著降低,差异均有统计学意义(t=11.25、9.47,均P<0.05)。脂多糖(LPS)+Notch1 siRNA组HPMEC细胞OD值和Bcl-2蛋白表达水平显著高于LPS组和LPS+siRNA对照组(t=11.26、11.68,均P<0.05),而ROS生成量、细胞的凋亡率(t=11.68、11.87,均P<0.05),Notch1、Caspase-3蛋白表达水平显著低于LPS组和LPS+siRNA对照组,差异均有统计学意义(t=5.08、6.60、3.84、5.83,均P<0.05)。结论Notch1干扰可能通过调控ROS水平干预肺血管内皮细胞的凋亡及下调Notch1、Caspase-3蛋白表达和上调Bcl-2蛋白表达,从而干预慢性阻塞性肺疾病(COPD)疾病。

Objective To investigate the mechanism of how Notch1 interference regulates the apoptosis of pulmonary vascular endothelial cells.Methods During January to December 2022,human pulmonary microvascular endothelial cells were transfected with Notch1 siRNA,and the cell viability in each group was evaluated using the Cell Counting Kit-8 assay.The level of reactive oxygen species was determined using flow cytometry,while cell apoptosis was assessed using the same technique.After treatment with Notch1 siRNA,the protein expression levels of Notch1,Bcl-2,and Caspase-3 in the human pulmonary microvascular endothelial cells were detected using western blot assay.Results The expression level of Notch1 mRNA in human pulmonary microvascular endothelial cells was significantly lower in the blank control and si-Notch1 groups than that in the siNC group(t=11.25,9.47,both P<0.05).Additionally,the optical density value and Bcl-2 protein expression level in the lipopolysaccharide(LPS)+Notch1 siRNA group were significantly higher than those in the LPS and LPS+siRNA groups(t=11.26,11.68,both P<0.05).The level of reactive oxygen species and the apoptosis rate of cells were significantly lower in the LPS+Notch1 siRNA group compared with the LPS and LPS+siRNA groups(t=11.68,11.87,both P<0.05).Furthermore,the protein expression levels of Notch1 and Caspase-3 were also significantly lower in the LPS+Notch1 siRNA group compared with the LPS and LPS+siRNA groups(t=5.08,6.60,3.84,5.83,all P<0.05).Conclusion Notch1 interference may interference in the apoptosis of human pulmonary microvascular endothelial cells through regulating the level of reactive oxygen species,downregulating the protein expression of Notch1 and Caspase-3,and upregulating the protein expression of Bcl-2.These actions may contribute to the treatment of chronic obstructive pulmonary disease.