溴莫尼定对低氧诱导的PC12细胞神经损伤的保护作用

Protective effect of brimonidine against neural injury induced by hypoxia in PC12 cells

目的探讨溴莫尼定对缺氧诱导的PC12细胞神经损伤的保护作用及其机制。方法实验于2018年5月至2019年8月进行,建立缺氧诱导的大鼠肾上腺髓质嗜铬瘤PC12细胞损伤模型,实验分为对照组、缺氧组(缺氧损伤)、缺氧+0.25µmol/L溴莫尼定组、缺氧+0.5µmol/L溴莫尼定组、缺氧+1µmol/L溴莫尼定组。采用MTT法检测各组细胞活力;流式细胞仪检测各组细胞凋亡率;根据检测试剂盒测定乳酸脱氢酶(LDH)、丙二醛、超氧化物歧化酶(SOD)含量;蛋白质印迹法检测细胞中B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)及蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)/信号转导及转录活化因子3(STAT3)信号通路相关蛋白表达。结果缺氧处理后,细胞活力较对照组降低[(0.35±0.04)比(0.69±0.08)](P<0.05),溴莫尼定不同剂量组细胞活力较Hypoxia组升高[(0.37±0.05)、(0.40±0.06)、(0.44±0.05)、(0.53±0.07)、(0.60±0.09)比(0.35±0.04)](P<0.05);Hypoxia组细胞凋亡率增加[(32.89±3.46)%比(3.58±0.34)%](P<0.05),溴莫尼定干预后细胞凋亡率降低[(7.41±0.75)%比(32.89±3.46)%](P<0.05),Bax蛋白表达量降低[(1.48±0.16比3.76±0.31)](P<0.05),而Bcl-2蛋白表达量升高[(0.89±0.12)比(0.29±0.04)](P<0.05);Hypoxia组细胞SOD含量较对照组降低[(50.43±5.36)U/mg比(173.39±18.21)U/mg](P<0.05),溴莫尼定干预后可提高缺氧诱导的细胞SOD含量降低[(143.28±15.43)U/mg比(50.43±5.36)U/mg](P<0.05);Hypoxia组细胞LDH[(62.31±6.82)U/mL比(19.07±1.65)U/mL]、丙二醛含量[(4.29±0.43)µmol/g比(1.35±0.12)µmol/g]较对照组增高(P<0.05),溴莫尼定处理后可降低缺氧引起的LDH[(31.35±3.21)U/mL比(62.31±6.82)U/mL]、丙二醛含量[(1.76±0.19)µmol/g比(4.29±0.43)µmol/g]增加(P<0.05);Hypoxia组细胞Akt、p-Akt、mTOR、p-mTOR、STAT3、p-STAT3表达降低(P<0.05),溴莫尼定处理后可提高细胞Akt、p-Akt、mTOR、p-mTOR、STAT3、p-STAT3的表达(P<0.05);抑制Akt/mTOR/STAT3信号通路可逆转溴莫尼定对缺氧诱导的PC12细胞增殖、凋亡及LDH、丙二醛、SOD水平的影响。结论溴莫尼定可通过激活Akt/mTOR/STAT3信号通路促进缺氧诱导的PC12细胞增殖、抑制细胞凋亡及增强其抗氧化能力进而对缺氧诱导的神经细胞损伤发挥保护作用。

Objective To investigate the protective effect and mechanism of brimonidine against hypoxia-induced neuronal injury in PC12 cells.Methods Experiments were conducted from May 2018 to August 2019,a model of PC12 cell injury induced by hypoxia was established.The experiment was divided into control group,Hypoxia group(hypoxia injury),Hypoxia+0.25 μmol/L brimonidine group,Hypoxia+0.5 μmol/L brimonidine group and Hypoxia+1 μmol/L brimonidine group.Cell viability was measured by methylthiazolyl tetrazolium(MTT).The apoptotic rate of each group was detected by flow cytometry.The contents of lactate dehydrogenase(LDH),malondialdehyde(MDA),and superoxide dismutase(SOD)were determined according to the test kits.The expressions of Bax,Bcl-2 and Akt/mTOR/STAT3 signaling pathway-related proteins were detected by Western blotting.Results After hypoxia treatment,the cell viability(0.35±0.04 vs.0.69±0.08)was lower than that in the control group(P<0.05),and the cell viability in different dose groups of brimonidine(0.37±0.05,0.40±0.06,0.44±0.05,0.53±0.07,0.60±0.09 vs.0.35±0.04)were higher than those in the Hypoxia group(P<0.05);the apoptosis rate in the Hypoxia group was increased[(32.89±3.46)%vs.(3.58±0.34)%](P<0.05).After monidine intervention,the apoptosis rate decreased[(7.41±0.75)%vs.(32.89±3.46)%](P<0.05),and the expression of Bax protein decreased(P<0.05)(1.48±0.16 vs.3.76±0.31),while the expression of Bcl-2 protein increased(0.89±0.12 vs.0.29±0.04)(P<0.05);the SOD content of cells in the Hypoxia group[(50.43±5.36)U/mg vs.(173.39±18.21)U/mg]was higher than that in the control group(P<0.05),and brimonidine could increase the hypoxia-induced SOD content[(143.28±15.43)U/mg vs.(50.43±5.36)U/mg](P<0.05);In the Hypoxia group,LDH[(62.31±6.82)U/mL vs.(19.07±1.65)U/mL],MDA content[(4.29±0.43)μmol/g vs.(1.35±0.12)μmol/g]were higher than those in the control group(P<0.05),brimonidine treatment could reduce the LDH[(31.35±3.21)U/mL vs.(62.31±6.82)U/mL],MDA content[(1.76±0.19)μmol/g vs.(4.29±0.43)μmol/g](P<0.05).The expressions of Akt,p-Akt,mTOR,p-mTOR,STAT3 and p-STAT3 were decreased in Hypoxia group(P<0.05).After treatment with brimonidine,the expressions of Akt,p-Akt,mTOR,p-mTOR and p-STAT3 were increased(P<0.05).Inhibition of Akt/mTOR/STAT3 signaling pathway reversed the effects of brimonidine on hypoxia-induced PC12 cell proliferation,apoptosis,and LDH,MDA,and SOD levels.Conclusion Brimonidine can protect hypoxia-induced neuronal injury by activating Akt/mTOR/STAT3 signaling pathway,promoting hypoxia-induced PC12 cell proliferation,inhibiting apoptosis and enhancing its antioxidant capacity.