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
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摘要
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: CuSO_4 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,CuSO_4 showed a dose-effect and time-effect relationship with cells( P < 0. 01). With the increase of the dosage and time of CuSO_4,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 CuSO_4-induced cell death( P < 0. 01). The results of MTT showed that the leakage rate of LDH increased significantly after treatment with CuSO_4 compared with the normal group( P < 0. 01),and the rabbit serum of Gandou decoction significantly decreased the LDH leakage rate of CuSO_4-injured cells( P < 0. 05). DCFH-DA fluorescence staining showed that CuSO_4 significantly increased the production of ROS in cells( P < 0. 01). The rabbit serum containing Gandou decoction significantly inhibited CuSO_4-induced intracellular ROS production( P < 0. 01). JC-1 staining showed that CuSO_4 induced a significant decrease in mitochondrial membrane potential in cells( P < 0. 01),while serum containing Gandou decoction inhibited CuSO_4-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.
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: CuSO_4 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,CuSO_4 showed a dose-effect and time-effect relationship with cells( P < 0. 01). With the increase of the dosage and time of CuSO_4,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 CuSO_4-induced cell death( P < 0. 01). The results of MTT showed that the leakage rate of LDH increased significantly after treatment with CuSO_4 compared with the normal group( P < 0. 01),and the rabbit serum of Gandou decoction significantly decreased the LDH leakage rate of CuSO_4-injured cells( P < 0. 05). DCFH-DA fluorescence staining showed that CuSO_4 significantly increased the production of ROS in cells( P < 0. 01). The rabbit serum containing Gandou decoction significantly inhibited CuSO_4-induced intracellular ROS production( P < 0. 01). JC-1 staining showed that CuSO_4 induced a significant decrease in mitochondrial membrane potential in cells( P < 0. 01),while serum containing Gandou decoction inhibited CuSO_4-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.
引文
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[19] Lim J H,Kim H W,Kim M Y,et al. Cinacalcet-mediated activation of the Ca MKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy[J]. Cell Death Dis,2018,9(3):270.
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[21] ZHANG Y,Nicholatos J,Dreier J R,et al. Coordinated regulation of protein synthesis and degradation by m TORC1[J]. Nature,2014,513(7518):440-443.
[22] Ro S H,Semple I A,Park H,et al. Sestrin2 promotes Unc-51-like kinase1mediated phosphorylation of p62/sequestosome-1[J]. FEBS J,2014,281(17):3816-3827.
[23] Kim J,Kundu M,Viollet B,et al. AMPK and m TOR regulate autophagy through direct phosphorylation of Ulk1[J]. Nature Cell Biol,2011,13(2):132-141.
[24] CHANG H P,LU C C,CHIANG J H,et al. Pterostilbene modulates the suppression of multidrug resistance protein1 and triggers autophagic and apoptotic mechanisms in cisplatin-resistant human oral cancer CAR cells via AKT signaling[J]. Int J Oncol,2018,10(1):3892-4298.
[25] LI L,CHEN Y,Gibson S B. Starvation-induced autophagy is regulated by mitochondrial reactive oxygen species leading to AMPK activation[J]. Cell Signal,2013,25(1):50-65.
[2] Ala A,Walker A P,Ashkan K,et al. Wilson's disease[J]. Lancet,2007,369(9559):397-408.
[3] WANG H,CHENG N,DONG J,et al. Serum pentraxin 3is elevated in patients with neurological Wilson's disease[J]. Clinica Chimica Acta,2016,462:178-182.
[4] Banci L,Bertini I,Cantini F,et al. Cellular copper distribution:a mechanistic systems biology approach[J]. Cell Mol Life Sci,2010,67(15):2563-2589.
[5] Papur O S,Akman S A,Terziogˇlu O. Clinical and genetic analysis of pediatric patients with Wilson[J].Turk J Gastroenterol,2015,26:397-403.
[6] CHEN C,SHEN B,XIAO J J,et al. Currently clinical views on genetics of Wilson's disease[J]. Chin Med J(Engl),2015,128(13):1826.
[7] Yamahara K,Yasuda M,Kume S,et al. The role of autophagy in the pathogenesis of diabetic nephropathy[J]. J Diabet Res,2013,doi:1155/2013/193757.
[8] Boya P,Reggiori F,Codogno P. Emerging regulation and functions of autophagy[J]. Nat Cell Biol,2013,15(7):713-720.
[9] Inoki K. m TOR signaling in autophagy regulation in the kidney[J]. Semin Nephrol,2014,34(1):2-8.
[10]丁超,王训.肝豆汤改良方调节高铜诱导神经元损伤的信号调控机制的研究[D].合肥:安徽中医药大学,2014.
[11]徐陈陈,董健健,程楠,等.肝豆汤改良方对Wilson's病模型TX乳鼠神经元内Cyt C/Caspase信号通路的分子调控机制[J].中国实验方剂学杂志,2017,23(6):143-148.
[12]张静,方媛,崔圣伟,等.肝豆汤联合二巯基丙磺酸钠对湿热内蕴型肝豆状核变性患者的影响[J].中国实验方剂学杂志,2017,23(17):190-194.
[13]张静,陈怀珍,艾文龙,等.肝豆汤联合DMPS驱铜治疗对Wilson病湿热内蕴型认知功能障碍的影响[J].中国实验方剂学杂志,2018,24(15):210-215.
[14] Costa A,Scholer-Dahirel A,Mechta-Grigoriou F. The role of reactive oxygen species and metabolism on cancer cells and their microenvironment[J]. Semin Cancer Biol,2014,25:23-32.
[15]刘晓杰,杨威,祁金顺.氧化应激与阿尔茨海默病[J].生理学报,2012,64(1):87-95.
[16] Maillet V,Boussetta N,Leclerc J,et al. LKB1 as a gatekeeper of hepatocyte proliferation and genomic integrity during liver regeneration[J]. Cell Reports,2018,22(8):1994-2005.
[17] Mauro L,Naimo G D,Gelsomino L,et al. Uncoupling effects of estrogen receptorɑon LKB1/AMPK interaction upon adiponectin exposure in breast cancer[J]. FASEB J,2018,32(8):4343-4355.
[18] Deepa S,ZHOU L,Ryu J,et al. APPL1 mediates adiponectin-induced LKB1 cytosolic localization through the PP2A-PKC signaling pathway[J]. Molecular Endocrinol,2011,25(10):1773-1785.
[19] Lim J H,Kim H W,Kim M Y,et al. Cinacalcet-mediated activation of the Ca MKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy[J]. Cell Death Dis,2018,9(3):270.
[20] Inoki K,ZHU T,GUAN K L. TSC2 mediates cellular energy response to control cell growth and survival[J].Cell,2003,115(5):577-590.
[21] ZHANG Y,Nicholatos J,Dreier J R,et al. Coordinated regulation of protein synthesis and degradation by m TORC1[J]. Nature,2014,513(7518):440-443.
[22] Ro S H,Semple I A,Park H,et al. Sestrin2 promotes Unc-51-like kinase1mediated phosphorylation of p62/sequestosome-1[J]. FEBS J,2014,281(17):3816-3827.
[23] Kim J,Kundu M,Viollet B,et al. AMPK and m TOR regulate autophagy through direct phosphorylation of Ulk1[J]. Nature Cell Biol,2011,13(2):132-141.
[24] CHANG H P,LU C C,CHIANG J H,et al. Pterostilbene modulates the suppression of multidrug resistance protein1 and triggers autophagic and apoptotic mechanisms in cisplatin-resistant human oral cancer CAR cells via AKT signaling[J]. Int J Oncol,2018,10(1):3892-4298.
[25] LI L,CHEN Y,Gibson S B. Starvation-induced autophagy is regulated by mitochondrial reactive oxygen species leading to AMPK activation[J]. Cell Signal,2013,25(1):50-65.