肝豆汤对Wilson病模型高铜诱导的SH-SY5Y细胞自噬效应的影响及其作用机制

Effect of Gandou Decoction on Autophagy of SH-SY5Y Cell Induced by High Copper and Its Mechanism

目的:研究肝豆汤对高铜诱导的人神经母细胞瘤(SH-SY5Y)细胞自噬效应的影响及其作用机制,为中医药防治脑型Wilson病(Wilson disease,WD)提供新的治疗靶点和研究思路。方法:噻唑蓝(MTT)比色法筛选硫酸铜(CuSO_4)造模浓度(0,100,200,400,800,1 600μmol·L-1)及时间; MTT比色法筛选含药血清浓度(5%,10%,15%,20%)及时间;乳酸脱氢酶(LDH)释放实验检测细胞LDH漏出率;流式细胞法检测细胞内活性氧(ROS)的含量;荧光染料JC-1检测细胞线粒体膜电位;流式细胞仪对自噬进行定量分析。蛋白免疫印迹法(Western blot)检测肝激酶B1(LKB1),腺苷酸活化蛋白激酶(AMPK),自噬微管相关蛋白轻链3A/B(LC3A/B),哺乳动物雷帕霉素靶蛋白(mTOR),unc-51样激酶1(ULK1),磷酸化ULK(p-ULK),磷酸化AMPK(p-AMPK)蛋白的表达。结果:MTT结果显示,CuSO_4对细胞的损伤呈现一定的量效和时效关系(P <0. 01),随着CuSO_4作用浓度及时间的增加,细胞存活率呈现下降趋势; 10%含肝豆汤兔血清可显著抑制CuSO_4诱导的细胞死亡(P <0. 01)。LDH释放实验显示,与正常组比较,CuSO_4作用细胞后LDH漏出率显著增加(P <0. 01),与模型组比较,含肝豆汤兔血清明显降低CuSO_4损伤细胞的LDH漏出率(P <0. 05)。DCFH-DA荧光染色显示,与正常组比较,CuSO_4可显著增加细胞内ROS生成(P <0. 01),与模型组比较,含肝豆汤兔血清可显著抑制CuSO_4诱导的细胞内ROS产生(P <0. 01)。JC-1染色结果显示,与正常组比较,CuSO_4诱导细胞线粒体膜电位Δψm显著降低(P <0. 01),与模型组比较,含肝豆汤兔血清明显抑制CuSO_4诱导的线粒体膜电位降低Δψm(P <0. 05)。Western blot结果显示,与正常组比较,模型组细胞内LKB1,AMPK,LC3A/B,ULK1及p-AMPK蛋白的表达显著增加,mTOR及p-ULK蛋白的表达显著降低(P <0. 01)。与模型组比较,含肝豆汤兔血清组LKB1,AMPK,LC3A/B,ULK1及p-AMPK蛋白表达显著降低,mTOR及p-ULK蛋白表达显著增加(P <0. 01)。结论:高铜可通过诱导细胞内线粒体氧化应激,上调自噬相关蛋白LKB1,p-AMPK,AMPK,LC3A/B及ULK1的表达,下调自噬相关蛋白mTOR及p-ULK的表达,导致细胞发生自噬性死亡,而肝豆汤可通过调控LKB1/AMPK信号通路,下调自噬相关蛋白LKB1,p-AMPK,LC3A/B,ULK及AMPK的表达,上调自噬相关蛋白及基因mTOR及p-ULK的表达,抑制自噬的发生,阻断高铜诱导的神经元损伤,从而发挥神经保护作用。

Objective:To explore the effect of Gandou decoction on autophagy of SH-SY5 Y cells induced by high copper and its mechanism,in order to provide new therapeutic targets and research ideas for the prevention and treatment of brain-type Wilson disease(WD)with traditional Chinese medicine.Method:CuSO4 model showed a certain dose-effect and time-effect relationship according to methyl thiazolyl tetrazolium(MTT);lactate dehydrogenase(LDH)leakage rate was detected by LDH release assay;flow cytometry method was used to detect intracellular reactive oxygen species(ROS)content.The fluorescent dye JC-1 was used to detect the mitochondrial membrane potential of the cells.Flow cytometry was used to quantify autophagy.The expressions of liver kinase B1(LKB1),AMP-activated protein kinase(AMPK),microtubule-associated protein 1 light chain 3(LC3 A/B),mammalian target of rapamycin(mTOR)and UNC-51-like kinase-1(ULK1),phosphorylation-ULK(p-ULK),phosphorylation-AMPK(p-AMPK)were detected by Western blot.Result:According to MTT results,CuSO4 showed a dose-effect and time-effect relationship with cells(P<0.01).With the increase of the dosage and time of CuSO4,the survival rate of cells showed a downward trend(P<0.01).MTT results showed that 10% of rabbit serum containing Gandou decoction significantly inhibited CuSO4-induced cell death(P<0.01).The results of MTT showed that the leakage rate of LDH increased significantly after treatment with CuSO4 compared with the normal group(P<0.01),and the rabbit serum of Gandou decoction significantly decreased the LDH leakage rate of CuSO4-injured cells(P<0.05).DCFH-DA fluorescence staining showed that CuSO4 significantly increased the production of ROS in cells(P<0.01).The rabbit serum containing Gandou decoction significantly inhibited CuSO4-induced intracellular ROS production(P<0.01).JC-1 staining showed that CuSO4 induced a significant decrease in mitochondrial membrane potential in cells(P<0.01),while serum containing Gandou decoction inhibited CuSO4-induced mitochondrial membrane potential in a dose-dependent manner(P<0.05).The western blot results showed that compared with the normal group,the protein expressions of LKB1,AMPK,LC3 A/B,ULK,p-AMPK in the model group were significantly increased,while the protein expressions of mTOR and p-ULK were significantly decreased(P<0.01).Compared with the model group,the expressions of LKB1,AMPK,LC3 A/B,p-AMPK and ULK were significantly decreased,whereas the protein expressions of mTOR and p-ULK were significantly increased in the rabbit serum group containing Gandou decoction(P<0.01).Conclusion:High copper can induce autophagic apoptosis in SH-SY5 Y cells by inducing intracellular mitochondrial oxidative stress,up-regulating the expressions of autophagy-related proteins LKB1,AMPK,LC3 A/B,ULK,p-AMPK and downregulating the expressions of mTOR and p-ULK.However,Gandou decoction can inhibit the occurrence of autophagy,and cut off high copper-induced neuronal damage by down-regulating the expressions of autophagyrelated proteins LKB1,AMPK,LC3 A/B,ULK,p-AMPK,and up-regulating the expression of mTOR and p-ULK,so as to exert a neuroprotective effect.