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目的 :探讨血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)对多巴胺能细胞损伤的影响及其机制。方法 :体外培养CATH.a细胞,鱼藤酮和(或)AngⅡ处理细胞24 h。采用噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide,MTT)法检测细胞存活率,蛋白印迹技术检测血管紧张素Ⅱ-1型受体(angiotensinⅡtype 1 receptor,AT1R)和血管紧张素Ⅱ-2型受体(angiotensinⅡtype 2receptor,AT2R)蛋白表达,流式细胞仪检测细胞内活性氧(reactive oxygen species,ROS)含量,荧光定量PCR检测尼克酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶亚基gp91phox和p67phox基因表达,免疫荧光检测gp91phox蛋白表达。结果:AngⅡ通过AT1R导致鱼藤酮诱导的CATH.a细胞存活率降低(P<0.05)。AngⅡ使NADPH氧化酶亚基gp91phox和p67phox表达增加(P<0.05),并呈剂量依赖性促进细胞内ROS产生。AT1R阻滞剂氯沙坦和NADPH氧化酶抑制剂夹竹桃麻素使AngⅡ诱导的ROS产生减少(P<0.01)。结论:AngⅡ作用于AT1R,通过NADPH氧化酶产生ROS,促进鱼藤酮诱导的多巴胺能细胞损伤,参与帕金森病的发生与发展。
Objective: To investigate the effect of angiotensin Ⅱ (AngⅡ) on the damage of dopaminergic cells and its mechanism. Methods: CATH.a cells, rotenone and (or) Ang Ⅱ cells were cultured in vitro for 24 h. Cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Western blotting was used to detect the expression of angiotensin Ⅱ type 1 receptor (AT1R) and angiotensin Ⅱ type 2 receptor , AT2R) were detected by flow cytometry. The content of reactive oxygen species (ROS) in the cells was detected by flow cytometry. The gp91phox of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit was detected by fluorescence quantitative PCR. And p67phox gene expression, gp91phox protein expression was detected by immunofluorescence. Results: AngⅡ through AT1R caused rotenone-induced CATH.a cell survival rate decreased (P <0.05). AngⅡ increased the expression of gp91phox and p67phox of NADPH oxidase (P <0.05), and promoted the intracellular ROS production in a dose-dependent manner. Losartan, an AT1R blocker, and apocynin, a NADPH oxidase inhibitor, decreased Ang II-induced ROS production (P <0.01). CONCLUSION: AngⅡ acts on AT1R and produces ROS through NADPH oxidase, which promotes the injury of rotenone-induced dopaminergic cells and is involved in the occurrence and development of Parkinson’s disease.