消旋山莨菪碱在照射诱导肺上皮细胞损伤中的保护作用

Protective effect of racanisodamine on radiation-induced lung epithelial cell injury

目的探讨消旋山莨菪碱(654-2)对X射线诱导小鼠肺上皮细胞损伤的保护作用及潜在机制。方法利用小鼠肺泡上皮细胞MLE-12单次大剂量X线照射建立体外放射性肺损伤(RILI)模型,细胞分组为:对照组(未照射)、照射组(16 Gy照射)、处理1组(16 Gy照射+2μmol/L 654-2)、处理2组(16 Gy照射+10μmol/L 654-2)、抑制剂组(16 Gy照射+10μmol/L 654-2+2μmol/L ML385)。照射后不同时间点收集细胞进行相关检测。细胞衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测细胞衰老;细胞集落形成能力检测观察细胞处理后的恢复能力;蛋白质印迹法检测p21、p16、磷酸化组蛋白H2AX(γH2AX)、核因子E2相关因子2(Nrf2)、Nrf2 Ser40位点磷酸化(p-Nrf2)、p62、血红素氧合酶1(HO-1)、NAD(P)H醌脱氢酶1(NQO1)等蛋白表达;流式细胞术检测细胞凋亡及细胞内总活性氧(ROS)产生;按照相关试剂盒检测谷胱甘肽(GSH)、超氧化物歧化酶(SOD);实时反转录PCR检测检测谷氨酸半胱氨酸连接酶催化亚基(GCLC)、谷氨酸半胱氨酸连接酶修饰亚基(GCLM)等mRNA表达情况。计量资料以xˉ±s表示,两组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果与照射组相比,处理1、2组细胞SA-β-Gal染色阳性细胞比例减少,细胞衰老减轻(P均<0.001)。与照射组相比,处理2组的γH2AX蛋白表达减少(P=0.037),细胞凋亡减少(P=0.026),细胞增殖能力增强(P=0.004),GSH(P=0.002)、SOD(P<0.001)活力提高,且ROS减少(P=0.001)。随着654-2的作用时间延长,MLE-12细胞的总蛋白中Nrf2、p-Nrf2的表达增强,同时Nrf2下游抗氧化蛋白NQO1及HO-1的表达增强,GCLC及GCLM mRNA表达增强。当加入Nrf2小分子抑制剂ML385后,抑制剂组的SA-β-Gal染色阳性细胞数(P=0.145)、ROS(P=0.317)与照射组的差异无统计学意义。结论654-2可以激活Nrf2通路,增强细胞抗氧化能力,抑制细胞衰老,从而发挥对RILI的保护作用。

ObjectiveTo investigate the protective effect of racanisodamine(654-2)on lung epithelial cell injury induced by X-ray in mice and unravel the underlying mechanism.MethodsMouse alveolar epithelial cells MLE-12 were used to establish radiation-induced lung injury(RILI)model in vitro and divided into 4 groups as follows:control(no irradiation),radiation(16 Gy radiation),treatment 1(16 Gy radiation+2μmol/L 654-2),treatment 2(16 Gy radiation+10μmol/L 654-2),and inhibitor(16 Gy radiation+10μmol/L 654-2+2μmol/L ML385),respectively.Cells were sampled at different time points after radiation.Cell senescence was detected with senescence-associatedβ-galactosidase(SA-β-Gal)staining.Cell colony-forming ability was detected to observe the recovery capability of cells after treatment.The expression levels of p21,p16,phosphorylated histone H2AX(γH2AX),nuclear factor erythroid-2 related factor 2(Nrf2),Nrf2 Ser40 site phosphorylation(p-Nrf2),p62,heme oxygenase-1(HO-1)and NAD(P)H quinone oxidoreductase 1(NQO1)proteins were measured by Western blot.Cell apoptosis and the production of intracellular reactive oxygen species(ROS)were determined by flow cytometry.The expression levels of glutathione(GSH)and superoxide dismutase(SOD)were detected according to the manufectuer instructions.The expression levels of glutamate-cysteine ligase catalytic subunit(GCLC)and glutamate-cysteine ligase modifier subunit(GCLM)mRNA were determined by real time reverse transcription PCR.Measurement data were expressed as Mean±SD.Comparison between two groups was conducted by independent sample t-test,and comparison among multiple groups was performed by one-way ANOVA.ResultsCompared with the radiation group,the proportion of cells with positive staining of SA-β-Gal was significantly lower and cell senescence were alleviated in the treatment 1 and 2 groups(all P<0.001).Compared with the radiation group,the expression level ofγH2AX protein was significantly down-regulated(P=0.037),cell apoptosis rate was significantly decreased(P=0.026),the proliferation capacity of MLE-12 was enhanced(P=0.004),GSH(P=0.002)and SOD(P<0.001)activity was enhanced and ROS production was declined(P=0.001)in the treatment 2 group.The expression levels of Nrf2 and p-Nrf2 in total protein were up-regulated over the time of 654-2 intervention.Meanwhile,the expression levels of antioxidant proteins of NQO1 and HO-1 were up-regulated and that of GCLC and GCLM mRNA was also up-regulated.There were no significant differences in the number of cells with positive staining of SA-β-Gal(P=0.145)and ROS production(P=0.317)between the inhibitor and radiation groups after supplement of ML385,small-molecule inhibitor of Nrf2.Conclusion654-2 can activate the Nrf2 pathway,enhance cell antioxidant capacity and inhibit cell senescence,thereby playing a protective role on radiation-induced lung injury.