利多卡因对糖尿病大鼠脊髓神经元的损伤作用

Injury effect of lidocaine on spinal cord neurons of diabetic rats

目的探讨利多卡因对糖尿病大鼠脊髓神经元的损伤作用。方法选择健康SPF级雄性SD大鼠30只、GK大鼠30只,12周龄,体重180~200 g。取大鼠脊髓神经元进行体外分离培养。采用随机数字表法将大鼠脊髓神经元分为四组:SD大鼠脊髓神经元组(C组)、SD大鼠脊髓神经元+利多卡因组(CL组)、GK大鼠脊髓神经元组(GK组)和GK大鼠脊髓神经元+利多卡因组(GKL组)。C组和GK组置于DMEM高糖培养基中,在37℃,5%CO_(2)培养箱内培养24 h,CL组和GKL组在相同条件下加入浓度为2 mmol/L的利多卡因培养24 h。采用Western blot法检测脊髓神经元中自噬相关蛋白PINK1、mTORC1、AMPK、p-AMPK、LC3Ⅱ和LC3Ⅰ含量,DCFH-DA法检测脊髓神经元中活性氧(ROS)含量,免疫荧光法检测脊髓神经元中PINK1和LC3含量。结果与C组比较,CL组、GK组和GKL组脊髓神经元PINK1、LC3含量和p-AMPK/AMPK、LC3Ⅱ/Ⅰ比值均明显升高(P<0.05),mTORC1含量明显降低(P<0.05),ROS含量明显升高(P<0.05)。与GK组比较,GKL组脊髓神经元PINK1、LC3含量和p-AMPK/AMPK、LC3Ⅱ/Ⅰ比值明显升高(P<0.05),mTORC1含量明显降低(P<0.05),ROS含量明显升高(P<0.05)。结论利多卡因诱导GK大鼠脊髓神经元的自噬相关蛋白的表达及ROS的释放,加重对GK大鼠脊髓神经元的损伤。

Objective To explore the injury effect of lidocaine in spinal cord neurons of diabetic rats.Methods Thirty SPF healthy SD rats and thirty GK rats,aged 12 weeks,weighing 180-200 g were selected.The spinal cord neurons of rats were isolated and cultured in vitro,and randomly divided into four groups:spinal cord neurons of SD rats group(group C),spinal cord neurons of SD rats+lidocaine group(group CL),spinal cord neurons of GK rats group(group GK),and spinal cord neurons of GK rats+lidocaine group(group GKL).Spinal cord neurons in groups C and GK were placed in DMEM high glucose medium and cultured in an incubator with 5%CO_(2)at 37℃for 24 hours.Lidocaine at a concentration of 2 mmol/L was added to groups CL and GKL,and then neurons in groups CL and GKL were cultured for 24 hours under the same conditions as groups C and GK.The content of autophagy-related proteins PINK1,mTORC1,AMPK,p-AMPK,LC3Ⅱ,and LC3Ⅰin spinal cord neurons was measured by Western blot.The content of reactive oxygen species(ROS)in spinal cord neurons was detected by DCFH-DA.The content of PINK1 and LC3 in spinal cord neurons was measured by immunofluorescence.Results Compared with group C,the content of PINK1,LC3,and p-AMPK/AMPK,LC3Ⅱ/Ⅰlevel in groups CL,GK and GKL were significantly increased(P<0.05),the content of mTORC1 was significantly decreased(P<0.05),and the content of ROS was significantly increased(P<0.05).Compared with group GK,the content of PINK1,LC3,and p-AMPK/AMPK,LC3Ⅱ/Ⅰlevel in the group GKL were significantly increased(P<0.05),the content of mTORC1 was significantly decreased(P<0.05),and the content of ROS was significantly increased(P<0.05).Conclusion Lidocaine aggravates the damage to spinal cord neurons of GK rats by promoting the expression of autophagy-related proteins and the release of ROS.