AGTR1拮抗剂奥美沙坦对HTF凋亡的促进作用及其机制

Promoting effect of AGTR1 blocker olmesartan on the apoptosis of HTF and its mechanism

目的探讨血管紧张素1型受体(AGTR1)拮抗剂奥美沙坦(OMS)对人Tenon囊成纤维细胞(HTF)凋亡的作用及其机制。方法收集于西安交通大学第二附属医院行斜视手术的患者Tenon囊组织,采用组织块法培养原代HTF,vimentin免疫荧光染色及流式细胞术鉴定原代细胞。采用10 ng/ml转化生长因子β2(TGF-β2)诱导HTF建立细胞纤维化模型。将体外培养的细胞分为正常对照组、TGF-β2组、TGF-β2+OMS组和OMS组,各组细胞分别予以普通培养液、含TGF-β2的培养液、含TGF-β2和OMS的培养液、含OMS的培养液培养细胞48 h。Annexin V/PI染色流式细胞术检测细胞凋亡情况,分析细胞早期凋亡率、晚期凋亡率及总凋亡率。Western blot法检测线粒体凋亡途径中procaspase-9、cleaved caspase-9、bax和bcl-2蛋白表达水平。比色法检测细胞乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性。结果成功分离培养原代HTF,所培养细胞呈长梭形,vimentin免疫荧光染色呈阳性,流式细胞术检测所培养原代细胞中vimentin表达阳性率>99%。正常对照组、TGF-β2组、TGF-β2+OMS组和OMS组细胞早期凋亡率、晚期凋亡率、总凋亡率总体比较,差异均有统计学意义(F=24.92、3.96、41.82,均P<0.05),其中TGF-β2+OMS组早期凋亡率和总凋亡率较正常对照组和TGF-β2组明显升高,TGF-β2+OMS组晚期凋亡率较正常对照组明显升高,差异均有统计学意义(均P<0.05)。正常对照组、TGF-β2组、TGF-β2+OMS组和OMS组cleaved caspase-9/procaspase-9、bax、bax/bcl-2总体比较差异均有统计学意义(F=4.40、7.98、4.61,均P<0.05),其中TGF-β2+OMS组bax/bcl-2较正常对照组明显升高,TGF-β2+OMS组cleaved caspase-9/procaspase-9、bax、bax/bcl-2较TGF-β2组明显升高,差异均有统计学意义(均P<0.05)。正常对照组、TGF-β2组、TGF-β2+OMS组、OMS组细胞LDH活性值分别为(783.99±79.97)、(913.16±196.86)、(2529.06±240.21)、(2134.29±138.96)μmol/(min·L),总体比较差异有统计学意义(F=24.95,P<0.05),其中与正常对照组和TGF-β2组比较,TGF-β2+OMS组和OMS组LDH活性值明显升高,差异均有统计学意义(均P<0.05)。正常对照组、TGF-β2组、TGF-β2+OMS组、OMS组细胞SOD活性值分别为(50.35±0.97)、(41.61±4.56)、(28.88±3.26)、(37.61±4.83)μmol/(min·L),总体比较差异有统计学意义(F=5.71,P<0.05),其中TGF-β2+OMS组SOD活性值低于正常对照组和TGF-β2组,OMS组SOD活性值低于正常对照组,差异均有统计学意义(均P<0.05)。结论AGTR1拮抗剂OMS可以有效促进HTF凋亡,bax/bcl-2/caspase-9介导的线粒体凋亡途径及氧化应激途径可能是OMS调控HTF凋亡过程的潜在机制。

Objective To study the effect and mechanism of angiotensin type 1 receptor(AGTR1)blocker olmesartan(OMS)on the apoptosis of human Tenon capsule fibroblasts(HTF).Methods Tenon capsule tissues were obtained from patients during strabismus surgery in the Second Affiliated Hospital of Xi'an Jiaotong University.Primary HTF were cultured by explant culture.Primary cells were identified by vimentin immunofluorescence staining and flow cytometry.The fibrosis model of HTF was established using 10 ng/ml transforming growth factor-β2(TGF-β2).The cells were divided into normal control group cultured in culture medium,TGF-β2 group in culture medium containing TGF-β2,TGF-β2+OMS group in culture medium containing TGF-β2 and OMS,and OMS group in culture medium containing OMS,and were cultured for 48 hours.Cell apoptosis was detected by flow cytometry with annexin V/PI staining.The early apoptosis,late apoptosis,and total apoptosis rates were analyzed.The protein expression of procaspase-9,cleaved caspase-9,bax and bcl-2 in the mitochondrial apoptosis pathway was detected by Western blot.The activity of lactate dehydrogenase(LDH)and superoxide dismutase(SOD)was detected by colorimetry.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University(No.2019-014).Results Primary HTF were successfully isolated and cultured.The cultured cells were long spindle-shaped and positive for vimentin.The expression rate of vimentin in the primary cells was greater than 99%.A statistically statistical difference was found in the early apoptosis rate,late apoptosis rate,and total apoptosis rate among the four groups(F=24.92,3.96,41.82;all at P<0.05).The early and total apoptosis rates were significantly higher in TGF-β2+OMS group than normal control group and TGF-β2 group,and the late apoptosis rate in TGF-β2+OMS group was significantly higher than that of normal control group(all at P<0.05).There were statistically significant differences in cleaved caspase-9/procaspase-9,bax,and bax/bcl-2 among the four groups(F=4.40,7.98,4.61;all at P<0.05).The bax/bcl-2 expression was significantly increased in TGF-β2+OMS group in comparison with normal control group,and the expressions of cleaved caspase-9/procaspase-9,bax,and bax/bcl-2 were significantly elevated in TGF-β2+OMS group compared with TGF-β2 group(all at P<0.05).LDH activity in the normal control group,TGF-β2 group,TGF-β2+OMS group and OMS group was(783.99±79.97),(913.16±196.86),(2529.06±240.21),and(2134.29±138.96)μmol/(min·L),respectively,showing a statistically significant difference(F=24.95,P<0.05).Compared with normal control group and TGF-β2 group,LDH activity in TGF-β2+OMS group was increased,and the differences were statistically significant(both at P<0.05).SOD activity in the normal control group,TGF-β2 group,TGF-β2+OMS group and OMS group was(50.35±0.97),(41.61±4.56),(28.88±3.26),and(37.61±4.83)μmol/(min·L),respectively,showing a statistically significant difference(F=5.71,P<0.05).SOD activity was reduced in TGF-β2+OMS group compared with normal control group and TGF-β2 group,reduced in OMS group compared with normal control group,and the differences were statistically significant(all at P<0.05).Conclusions AGTR1 blocker OMS can promote the apoptosis of HTF effectively.Mitochondrial apoptosis pathway mediated by bax/bcl-2/caspase-9 and oxidative stress pathway are the potential mechanisms that OMS regulates the apoptosis of HTF.