芍药苷减轻布比卡因诱导背根神经节细胞神经毒性的机制研究

Mechanism of paeoniflorin reducing the neurotoxicity of dorsal root ganglion cells induced by bupivacaine

目的基于细胞凋亡和氧化应激途径探讨芍药苷减轻布比卡因(BV)麻醉诱导背根神经节(DRG)原代神经细胞神经毒性的作用。方法将DRG原代神经细胞随机分成空白对照组、芍药苷组、BV组及芍药苷+BV组。MTT法检测细胞的增殖率;Annexin V-FITC/PI双染色检测细胞凋亡率;Western blot检测凋亡相关蛋白Bax、Bcl-2和活性caspase-3的表达水平;DCFH-DA法检测细胞内活性氧(ROS)水平;ELISA法检测细胞内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)和丙二醛(MDA)水平。结果与空白对照组比较,BV组、芍药苷+BV组中DRG原代神经细胞的增殖率、Bcl-2表达及SOD和GPx含量显著降低,而细胞凋亡率、Bax和活性caspase-3表达、平均荧光强度、MDA含量显著升高,差异均有统计学意义(P<0.05);芍药苷组DRG原代神经细胞的增殖率、凋亡率、Bax、Bcl-2、活性caspase-3表达无显著变化(P>0.05),但平均荧光强度、MDA含量显著降低,SOD、GPx含量显著升高,差异均有统计学意义(P<0.05)。与BV组比较,芍药苷组、芍药苷+BV组中DRG原代神经细胞的增殖率、Bcl-2表达、SOD和GPx含量显著增加,细胞凋亡率、Bax和活性caspase-3表达、平均荧光强度、MDA含量显著降低,差异均有统计学意义(P<0.05)。结论芍药苷可通过抑制细胞凋亡和氧化应激减轻BV诱导的DRG原代神经细胞神经毒性作用,有望成为治疗BV诱导的神经毒性的潜在药物。

Objective To explore the effect of paeoniflorin on alleviating the neurotoxicity of primary dorsal root ganglion(DRG)neurons induced by bupivacaine(BV)anesthesia based on the apoptosis and oxidative stress pathways.Methods The primary neurons of DRG were randomly divided into the blank control group,the paeoniflorin group,the BV group and the paeoniflorin+BV group.The cell proliferation rate was detected by MTT assay,the apoptosis rate was detected by Annexin V-FITC/PI double staining,the expression levels of apoptosis related proteins including Bax,Bcl-2 and active caspase-3 were detected by Western blot,the level of intracellular reactive oxygen species(ROS)was detected by DCFH-DA assay,and the levels of intracellular superoxide dismutase(SOD),glutathione peroxidase(GPx)and malondialdehyde(MDA)were detected by ELISA.Results Compared with the blank control group,the proliferation rates,expression of Bcl-2 and contents of SOD and GPx of DRG primary neurons in the BV group and the paeoniflorin+BV group were significantly reduced,while the apoptosis rates,expression of Bax and active caspase-3,average fluorescence intensities and MDA contents were significantly increased,with statistically significant differences(P<0.05);the DRG primary neurons of the paeoniflorin group had no obvious change in the proliferation rate,apoptosis rate,expression of Bax,Bcl-2 or active caspase-3(P>0.05),while with lower average fluorescence intensity and MDA content,and higher contents of SOD and GPx,with statistically significant differences(P<0.05).Compared with the BV group,the proliferation rates,expression of Bcl-2,contents of SOD and GPx of DRG primary neurons in the paeoniflorin group and the paeoniflorin+BV group were significantly increased,and the apoptosis rates,expression of Bax and active caspase-3,average fluorescence intensities,and MDA contents were significantly reduced,with statistically significant differences(P<0.05).Conclusion Paeoniflorin can alleviate the neurotoxicity of primary DRG neurons induced by BV through inhibiting apoptosis and oxidative stress,which is expected to be a potential drug for the treatment of BV-induced neurotoxicity.