异氟醚对Aβ25-35诱导大鼠PC12细胞氧化应激损伤的影响及海藻糖的保护作用

Effect of isoflurane on oxidative stress injury induced by Aβ25-35 and protective effects of trehalose in PC12 cells of rats

目的:探讨吸入麻醉药异氟醚对β淀粉样蛋白25-35(Aβ25-35)诱导大鼠PC12细胞氧化应激损伤的影响,阐明海藻糖对其可能的预防及保护作用。方法:将大鼠PC12细胞随机分为正常对照组(Control组)、异氟醚组(Iso组)、Aβ25-35组(Aβ组)、异氟醚+Aβ25-35组(Iso+Aβ组)、异氟醚+Aβ25-35+海藻糖组(Iso+Aβ+Tre组)和海藻糖组(Tre组)。正常对照组,PC12细胞给予正常细胞培养基培养;Iso组,PC12细胞给予2%异氟醚;Aβ组,PC12细胞给予10μmol·L-1 Aβ25-35;Iso+Aβ组,PC12细胞给予2%异氟醚和10μmol·L-1 Aβ25-35;Iso+Aβ+Tre组,PC12细胞给予2%异氟醚、10μmol·L-1 Aβ25-35和200mmol·L-1海藻糖;Tre组,PC12细胞给予200 mmol·L-1海藻糖。采用MTT法检测细胞存活率;Hoechst 33342荧光染色检测细胞凋亡率;二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)荧光法检测细胞中活性氧(ROS)水平;化学发光法测定细胞中丙二醛(MDA)水平,超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)及过氧化氢酶(CAT)活性。结果:与正常对照组比较,Iso组、Aβ组和Iso+Aβ组PC12细胞凋亡率明显升高(P<0.05或P<0.01),细胞存活率明显降低(P<0.05或P<0.01),细胞中ROS和MDA水平(P<0.05或P<0.01)明显升高,细胞中SOD、GSH-Px和CAT活性明显降低(P<0.05或P<0.01);与Iso和Aβ组比较,Iso+Aβ组细胞凋亡率明显升高(P<0.05),但细胞存活率明显降低(P<0.05),细胞中ROS、MDA水平(P<0.05)明显升高,细胞中SOD和GSH-Px和CAT活性明显降低(P<0.05);与Iso+Aβ组比较,Iso+Aβ+Tre组细胞凋亡率(P<0.05)明显降低,细胞存活率明显升高(P<0.05),细胞中ROS和MDA水平明显降低(P<0.05),细胞中SOD、GSH-Px和CAT活性明显升高(P<0.05)。结论:吸入麻醉药异氟醚能够加剧Aβ25-35诱导PC12细胞氧化应激损伤和细胞凋亡,海藻糖能够通过抗氧化和抗凋亡作用拮抗异氟醚的细胞毒性。

Objective To explore the effect of isoflurane on the oxidative stress injury induced by beta-amyloid protein（Aβ）25-35 in PC12cells of the rats,and to clarify the possible protective effects of trehalose on this injury.Methods The PC12 cells of rats were randomly divided into normal control group（Control group,treated with normal cell culture medium）,isoflurane group（Iso group,treated with 2% isoflurane）,Aβ25-35group（Aβgroup,treated with 10μmol·L-1 Aβ25-35）,isoflurane ＋ Aβ25-35group（Iso＋Aβgroup,treated with 2%isoflurane and 10μmol·L-1 Aβ25-35）,isoflurane＋ Aβ25-35 ＋ trehalose group（Iso＋Aβ＋Tre group,treated with 2%isoflurane,10μmol·L-1 Aβ25-35 and 200 mmol·L-1 trehalose）,and trehalose group（Tre group,treated with 200 mmol·L-1 trehalose）.The survival rates of the PC12 cells were detected by MTT assay;the apoptotic rates of the PC12 cells in various groups were determined by Hoechst 33342staining;the levels of intracellular reactive oxygen species（ROS）of the PC12 cells were measured by DCFH-DA fluorescence assay;the levels of malondialdehyd（MDA）and the activities of superoxide dismutase（SOD）,glutathion peroxidase（GSHPx）,and catalase（CAT）of the PC12 cells were detected by commercial kits.Results Compared with control group,the apoptotic rates of the cells were increased and the survival rates were decreased,and the levels of ROS and MDA and the activities of SOD,GSH-Px,and CAT in the PC12 cells were significantly decreased（P〈0.05 or P〈0.01）in Aβgroup,Iso group and Iso＋Aβgroup.Compared with Iso group or Aβgroup,the apoptotic rates were increased,the survival rates were decreased,the levels of ROS and MDA were increased,and the activities of SOD,GSH-Px,and CAT in the PC12 cells in Iso＋Aβgroup were decreased（P〈0.05）.Compared with Iso＋Aβgroup,the survival rates were increased and the apoptotic rates were decreased and the levels of ROS and MDA were decreased,and the activities of SOD,GSH-Px,and CAT in the PC12 cells in Iso＋Aβ＋Tre group were increased（P〈0.05）.Conclusion Isoflurane could aggravate the oxidative stress injury and the apoptosis induced by Aβ25-35 in the PC12 cells of the rats,and trehalose could alleviate the cytotoxicity of isoflurane via inhibition of oxidation and apoptosis.