糖痹康对高糖培养雪旺细胞自噬相关蛋白的影响

Effect of Tangbikang on High Glucose-induced Autophagy in Rat Schwann Cells

目的：研究发现细胞自噬是糖尿病周围神经病变（diabetic peripheral neuropathy,DPN）的重要发病机制之一,本研究探讨治疗DPN的中药复方糖痹康（Tangbikang,TBK）对高糖环境下大鼠雪旺细胞（rat Schwann cells,RSC）自噬相关蛋白的影响。方法：利用SD大鼠制备TBK含药血清,并采用50 mmol·L^-1的葡糖糖（Glu）RSC细胞培养基制备高糖RSC细胞模型。实验总共分为6组：正常组（Con,20%正常大鼠血清）,高糖组（Glu,50 mmol·L^-1Glu＋20%正常大鼠血清）;弥可保组（MKB,50 mmol·L^-1Glu＋20%弥可保含药血清）;TBK高剂量组（TBKH,50 mmol·L^-1Glu＋20%TBK含药血清）;TBK中剂量组（TBKM,50 mmol·L^-1Glu＋10%TBK含药血清＋10%正常大鼠血清）;TBK低剂量组（TBKL,50 mmol·L^-1Glu＋2.5%TBK含药血清＋17.5%正常大鼠血清）。干预48 h后,采用细胞增殖实验细胞增殖与活性检测（CCK-8）法检测RSC细胞增殖活性,流式细胞术检测细胞凋亡率,蛋白免疫印迹法（Western blot）检测自噬蛋白（Beclin 1）和微管相关蛋白1轻链3Ⅱ（LC3Ⅱ）的表达水平。结果：与Con组比较,高糖因素使得Glu组RSC增殖活性显著降低（P〈0.01）,细胞凋亡率显著增加（P〈0.01）,且自噬相关标志蛋白Beclin 1和LC3Ⅱ显著下降（P〈0.01）;与Glu组比较,MKB组与TBKH组RSC细胞增殖显著减弱（P〈0.01）,MKB组,TBKH和TBKM组RSC细胞凋亡率显著减小（P〈0.01）,MKB组和TBKH组RSC细胞自噬标志蛋白Beclin 1和LC3Ⅱ的表达水平显著升高（P〈0.01）。结论：TBK能够促进高糖环境下RSC细胞自噬,减少凋亡,具有一定的神经保护作用,该研究为DPN新药研发奠定基础。

Objective：To study the effect of traditional Chinese medicine Tangbikang（TBK） on autophagy in rat Schwann cells with high glucose,in order to verify the autophagy is an important pathogenesis of diabetic peripheral neuropathy（DPN）.Method：The drug-contained serum was prepared by serumpharmacological method.Rat Schwann cells（RSC） were cultured in vitro.The experiment included 6 groups：normal group（Con,20% normal rat serum）,high-glucose group（Glu,50 mmol · L-1 Glu ＋ 20% normal rat serum）,methycobal group（MKB,50 mmol·L^-1 Glu ＋ 20% rat serum containing methycobal）,high-dose TBK group（TBKH,50 mmol·L^-1 Glu ＋ 20% rat serum containing TBK）,medium-dose TBK group（TBKM,50 mmol·L^-1 Glu ＋ 10% rat serum containing TBK ＋ 10% normal rat serum）;and low-dose TBK group（TBKL,50 mmol·L^-1 Glu ＋ 2.5% rat serum containing TBK ＋ 17.5% normal rat serum）.After 48 h,cell viability was detected by cell counting kit-8（CCK-8） method,apoptosis was detected by flow cytometry method,and expressions of autophagy protein Beclin 1 and microtubule-associated protein 1 light Chain 3-Ⅱ（LC3 Ⅱ） were detected by Western blot method.Result：High glucose level activated proliferation of RSC（P〈0.01）,increased apoptosis rate（P〈0.01）,and decreased Beclin 1 and LC3 Ⅱ protein expressions（P〈0.01）;TBK serum promoted RSC cell proliferation（P〈0.01） and apoptosis（P〈0.01）,and up-regulated expressions of Beclin 1 and LC3 Ⅱ proteins（P〈0.01）.Conclusion：TBK could accelerate injured RSC in a high-glucose environment by reducing Schwann cell apoptosis and promoting autophagy.