基于Cer通路探讨肝豆汤对Wilson病模型高铜诱导海马神经元细胞损伤的保护作用
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摘要
目的:探讨肝豆汤调控海马神经元内神经酰胺(Cer)信号通路的分子靶点并观察其相应的调控机制。方法:本实验采用海马神经元HT-22细胞,分为正常组,模型组,10%,15%及20%肝豆汤组5个组,模型组为硫酸铜(CuSO_4)孵育HT-22细胞所得,正常组加入空白含药血清,10%,15%及20%肝豆汤组分别加含不同体积分数(10%,15%,20%)肝豆汤兔血清的培养基培养。噻唑蓝(MTT)比色法检测不同剂量CuSO_4作用不同时间点对HT-22细胞生长和增殖的影响;流式细胞仪检测细胞内活性氧(ROS)释放量的变化;蛋白免疫印迹法(Western blot)检测Cer信号通路相关蛋白表达情况。结果:MTT结果显示CuSO_4在一定时间和浓度范围内对HT-22细胞生长和增殖有明显抑制作用;流式细胞仪检测发现,与正常组比较,模型组ROS荧光强度显著增加(P <0. 01),肝豆汤组较模型组可显著降低细胞内ROS的释放量(P <0. 01)。Western blot检测结果显示与模型组比较,肝豆汤组可明显降低细胞内酸性鞘磷脂水解酶(ASM),Cer,p38丝裂原活化蛋白激酶(p38 MAPK),细胞色素C(Cyt C),半胱氨酸天冬氨酸蛋白酶-9(Caspase-9),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达(P <0. 05,P <0. 01)。结论:高铜可诱导HT-22细胞内线粒体氧化应激并引起Cer信号通路失活,从而诱导海马神经元损伤的发生。肝豆汤可能通过促进过量铜排出而抑制细胞内ASM,Cer,p38 MAPK,Cyt C,Caspase-9,Caspase-3蛋白的表达,从而减轻高铜对海马神经元的损伤作用。肝豆汤可通过减少脑内铜含量,进而调控Cer信号通路达到减轻高铜诱导海马神经元损伤的治疗效果。
Objective: To detect ceramide( Cer) signaling pathway-related proteins expression levels in HT-22 with Gandou decoction( GDD),in order to explore its molecular targets and mechanism in regulating Cer signaling pathway. Method: The experiment was divided into normal group( normal HT-22 cultured by 10% blank rabbit serum),model group( HT-22 cells incubated with CuSO_4),and GDD group( HT-22 cells incubated with CuSO_4,continuously cultured by rabbit serum containing10%,15%,20% GDD). HT-22 cells were incubated with different concentrations of CuSO_4. The cell growth and proliferation were assessed using methyl thiazolyl tetrazolium( MTT) method; flow cytometry was used to analyze the expression of reactive oxygen species( ROS);Western blot was used to detect relevant protein expression of Cer signaling pathway. Result: The results of MTT showed that CuSO_4 inhibited the growth and proliferation of HT-22 cells in a time and concentration-dependent manner; flow cytometry results showed that the model group increased the release of ROS compared with the normal group( P < 0. 01); compared with the model group, GDD could significantly reduce the release of ROS( P < 0. 01). Western blot indicated that GDD concentration-dependently decreased the expressions of acid sphingomyelinase( ASM),Cer,p38 mitogen-activated protein kinase( p38 MAPK),cytochrome C( Cyt C),cysteinyl aspartate specific proteinase( Caspase)-9,Caspase-3 in HT-22 compared with the model group( P <0. 05,P < 0. 01). Conclusion: High copper can induce oxidative stress and deactivate Cer signaling pathway,which led to hippocampal neuron injury. These findings suggest that GDD reduces neurotoxicity induced by copper overload by increasing the copper excretion that inhibits the expressions of ASM,Cer,p38 MAPK,Cyt C,Caspase-9,Caspase-3. GDD reduces neurotoxicity induced by copper overload by decreasing copper levels in brain and then regulating Cer signaling pathway.
Objective: To detect ceramide( Cer) signaling pathway-related proteins expression levels in HT-22 with Gandou decoction( GDD),in order to explore its molecular targets and mechanism in regulating Cer signaling pathway. Method: The experiment was divided into normal group( normal HT-22 cultured by 10% blank rabbit serum),model group( HT-22 cells incubated with CuSO_4),and GDD group( HT-22 cells incubated with CuSO_4,continuously cultured by rabbit serum containing10%,15%,20% GDD). HT-22 cells were incubated with different concentrations of CuSO_4. The cell growth and proliferation were assessed using methyl thiazolyl tetrazolium( MTT) method; flow cytometry was used to analyze the expression of reactive oxygen species( ROS);Western blot was used to detect relevant protein expression of Cer signaling pathway. Result: The results of MTT showed that CuSO_4 inhibited the growth and proliferation of HT-22 cells in a time and concentration-dependent manner; flow cytometry results showed that the model group increased the release of ROS compared with the normal group( P < 0. 01); compared with the model group, GDD could significantly reduce the release of ROS( P < 0. 01). Western blot indicated that GDD concentration-dependently decreased the expressions of acid sphingomyelinase( ASM),Cer,p38 mitogen-activated protein kinase( p38 MAPK),cytochrome C( Cyt C),cysteinyl aspartate specific proteinase( Caspase)-9,Caspase-3 in HT-22 compared with the model group( P <0. 05,P < 0. 01). Conclusion: High copper can induce oxidative stress and deactivate Cer signaling pathway,which led to hippocampal neuron injury. These findings suggest that GDD reduces neurotoxicity induced by copper overload by increasing the copper excretion that inhibits the expressions of ASM,Cer,p38 MAPK,Cyt C,Caspase-9,Caspase-3. GDD reduces neurotoxicity induced by copper overload by decreasing copper levels in brain and then regulating Cer signaling pathway.
引文
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[12] HOU Q,JIN J,ZHOU H,et al. Mitochondrially targeted ceramidespreferentially promote autophagy,retard cell growth,and induce apoptosis[J]. J Lipid Res,2011,52(2):278-288.
[13]张阔军,顾勤兰,王明慧,等.酸性鞘磷脂酶/神经酰胺通路在疾病发生发展中作用机制的研究进展[J].药学进展,2015,39(11):838-845.
[14] Iwayama H,Ueda N. Role of mitochondrial Bax,caspases and MAPKs for ceramide-induced apoptosis in renal proximal tubular cells[J]. Mol Cell Biochem,2013,379(1/2):37-42.
[15]刘哲,李博,李龙江.神经酰胺抗肿瘤作用的研究进展[J].现代口腔医学杂志,2016,30(1):39-40.
[16]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.
[2] 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.
[3]王佳炜,王训.四硫钼酸铵调节Wilson病铜负荷大鼠铜代谢及MAPK相关信号转导机制的研究[D].合肥:安徽中医药大学,2013.
[4]杨任民,韩咏竹,任明山,等.中药治疗肝豆状核变性107例疗效观察[J].中医杂志,1993,34(11):676-677,644.
[5]董健健,韩咏竹,程楠.肝豆汤对Wilson病模型TX小鼠肝细胞内铜代谢通路的分子调控机制[J].中国实验方剂学杂志,2016,22(24):128-133.
[6]程楠,杨任明.中药肝豆汤治疗Wilson病疗效机制的研究进展[J].中国实验方剂学杂志,2019,25(7):49-55.
[7] Pichili V B R,Kakulavarapu V R R,Michael D N. The mitochondrial permeability transition,and oxidative and nitrosative stress in the mechanism of Cutoxicity in cultured neurons and astrocytes[J]. Laboratory Investigation,2008,88:816-830.
[8] Brewer G J. A brand new mechanism for Cu toxicity[J]. J Hepatol,2007,47(4):621-622.
[9]罗磊,钟霞丽,钟才高,等.生物样品中酸性鞘磷脂酶活性测定[J].中国公共卫生,2015,31(4):515-517.
[10]张露勇,罗菲亚,胡培丽,等.神经酰胺通过JNK-c-Jun信号通路诱导胶质瘤细胞自噬性死亡[J].首都医科大学学报,2015,36(2):276-281.
[11] Truman J P,Al Gadban M M,Smith K J,et al. Acid sphingomyeli-nase in macrophage biology[J]. Cell Mol Life Sci,2011,68(20):3293-3305.
[12] HOU Q,JIN J,ZHOU H,et al. Mitochondrially targeted ceramidespreferentially promote autophagy,retard cell growth,and induce apoptosis[J]. J Lipid Res,2011,52(2):278-288.
[13]张阔军,顾勤兰,王明慧,等.酸性鞘磷脂酶/神经酰胺通路在疾病发生发展中作用机制的研究进展[J].药学进展,2015,39(11):838-845.
[14] Iwayama H,Ueda N. Role of mitochondrial Bax,caspases and MAPKs for ceramide-induced apoptosis in renal proximal tubular cells[J]. Mol Cell Biochem,2013,379(1/2):37-42.
[15]刘哲,李博,李龙江.神经酰胺抗肿瘤作用的研究进展[J].现代口腔医学杂志,2016,30(1):39-40.
[16]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.