丙泊酚减轻低氧诱导的大鼠肾上腺嗜铬细胞瘤细胞系PC12炎性反应和细胞凋亡

Propofol attenuates hypoxia-induced inflammation and apoptosis in rat pheochromocytoma cell line PC12

目的研究丙泊酚是否通过抑制miR-141-3p的表达,减轻低氧诱导的肾上腺嗜铬细胞瘤细胞系PC12炎性反应和细胞凋亡的分子机制。方法将PC12细胞分为对照组、低氧组、(5、10、20)μmol/L丙泊酚+低氧组、anti-miR-con+低氧组、anti-miR-141-3p+低氧组、miR-con+20μmol/L丙泊酚+低氧组、miR-141-3p+20μmol/L丙泊酚+低氧组。流式细胞仪检测PC12细胞的凋亡;Western blot检测活化的含半胱氨酸天冬氨酸蛋白水解酶-3(cleaved caspase-3)蛋白表达;相关试剂盒检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性;活性氧荧光探针DCFH-DA法测定活性氧(ROS)含量;ELISA检测肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6含量;RT-qPCR检测miR-141-3p表达。结果与对照组比较,低氧组PC12细胞的凋亡率、cleaved caspase-3蛋白表达水平、MDA、ROS、TNF-α、IL-1β、IL-6含量和miR-141-3p表达量均升高,SOD活性减弱(P<0.05)。与低氧组比较,5、10、20μmol/L丙泊酚+低氧组PC12细胞的凋亡率、cleaved caspase-3蛋白表达水平、MDA含量、ROS、TNF-α、IL-1β、IL-6含量和miR-141-3p表达量均随丙泊酚浓度的增加呈降低趋势,SOD活性随丙泊酚浓度的增加呈升高趋势(P<0.05)。Anti-miR-141-3p+低氧组PC12细胞的miR-141-3p表达量、cleaved caspase-3蛋白表达水平、MDA、ROS、TNF-α、IL-1β、IL-6含量和细胞凋亡率均比anti-miR-con+低氧组降低,SOD活性比anti-miR-con+低氧组增加(P<0.05)。miR-141-3p+20μmol/L丙泊酚+低氧组PC12细胞的miR-141-3p表达量、cleaved caspase-3蛋白表达水平、MDA含量、ROS、TNF-α、IL-1β、IL-6含量和细胞凋亡率均高于miR-con+20μmol/L丙泊酚+低氧组,SOD活性低于miR-con+20μmol/L丙泊酚+低氧组(P<0.05)。结论丙泊酚可能通过抑制miR-141-3p的表达,减轻低氧诱导的PC12细胞炎性反应、氧化应激和细胞凋亡。

Objective To investigate whether propofol inhibits the expression of miR-141-3p and reduces the molecular mechanism of hypoxia-induced inflammation and apoptosis of pheochromocytoma cell line PC12.Methods PC12 cells were divided into control group,hypoxia group,(5,10,20)μmol/L propofol+hypoxia group,anti-miR-con+hypoxia group,anti-miR-141-3p+hypoxia group,miR-con+20μmol/L propofol+hypoxia group,miR-141-3p+20μmol/L propofol+hypoxia group.Flow cytometry was used to detect the apoptosis of PC12 cells;Western blot was employed to determine the expression of activated cleaved caspase-3(cleaved caspase-3)protein,and the kits were implemented to monitor malondialdehyde(MDA)content and superoxide dismutase(SOD)activity;Reactive oxygen species fluorescent probe DCFH-DA method to determine reactive oxygen species(ROS)content;ELISA kits to assay tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6 content,RT-qPCR to detect the expression of miR-141-3p.Results Compared with the control group,the apoptosis rate,cleaved caspase-3 protein expression level,MDA,ROS,TNF-α,IL-1β,IL-6 content and miR-141-3p expression of PC12 cells in hypoxia group were all increased,while SOD activity weakened(P<0.05).Compared with the hypoxia group,the apoptosis rate,cleaved caspase-3 protein expression level,MDA content,ROS,TNF-α,IL-1β,IL-6 content and miR-141-3p expression of PC12 cells in 5,10,20μmol/L propofol+hypoxia group decreased with the increase of propofol concentration,and SOD activity increased with the increase of propofol concentration(P<0.05).The expression of miR-141-3p,cleaved caspase-3 protein expression,MDA,ROS,TNF-α,IL-1β,IL-6 content and apoptosis rates of PC12 cells in the anti-miR-141-3p+hypoxia group were lower than those in the anti-miR-con+hypoxia group,and the SOD activity was higher than that in the anti-miR-con+hypoxia group(P<0.05).The miR-141-3p expression,cleaved caspase-3 protein expression level,MDA content,ROS,TNF-α,IL-1β,IL-6 content and cell apoptosis rate of PC12 cells in miR-141-3p+20μmol/L propofol+hypoxia group were higher than miR-con+20μmol/L propofol+hypoxia group,while SOD activity was lower than those from miR-con+20μmol/L propofol+hypoxia group(P<0.05).Conclusions Propofol can alleviate the inflammatory response,oxidative stress and apoptosis of PC12 cells induced by hypoxia by inhibiting the expression of miR-141-3p.