肝PC对张氏肝细胞酒精损伤的保护作用与Caveolin-1的关系
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摘要
众所周知,磷脂是构成生物膜的主要成分,摄取磷脂酰胆碱(PC)可改善膜磷脂组分,增加膜流动性和膜酶活性,提高细胞的抗氧化能力。Caveolin-1 (窖蛋白-1, Cav-1)是膜脂筏Caveolae(胞膜窖)的标记蛋白。近年发现,磷脂能够促进Caveolin-1小泡的形成及转运,对Caveolae结构的形成和稳定及细胞膜组装具有重要作用。本室前期工作表明,肝脏磷脂酰胆碱(肝PC)含有丰富的烯醚键,能够抵抗脂质过氧化,对小鼠急性酒精肝损伤具有明显的保护作用;同时发现,肝PC能够促进小鼠肝脏中Cav-1蛋白表达,但作用机制尚不完全清楚。
本文以张氏肝细胞为对象,利用siRNA技术筛选出稳定传代的Cav-1低表达细胞株(Cav-1KD),建立酒精性肝细胞损伤模型;利用MTT、电导法和薄板层析(TLC)等技术检测酒精损伤的Cav-1KD细胞膜特性及磷脂成份变化的影响;利用Western blot技术确定肝PC预保护的酒精肝损伤细胞中氧化应激途径相关蛋白表达的变化,进一步探讨肝PC对肝细胞酒精性损伤的保护作用与Cav-1的关系,旨在了解由Cav-1蛋白在肝脏损伤修复中的作用和信号转导机制。
结果如下:
1.酒精作用导致细胞中超氧化物歧化酶(SOD)水平明显降低(P<0.05),并促进氧化应激通路中TLR4、TNF-α、NF-κB表达增高及p38磷酸化激活,引发张氏肝细胞氧化应激。肝PC显著提高张氏肝细胞SOD活性(P<0.05),降低TLR4、TNF-α、NF-κB、P-p38等蛋白表达水平,对肝细胞起保护作用。
2.利用RNA干扰技术建立张氏肝细胞模型,发现200mmol/L酒精引起Cav-1低表达细胞ALT释放增加(P<0.05),细胞电导率显著增高(P<0.01),膜PC、PE、SM等含量均降低。提示,Cav-1表达下调导致张氏肝细胞对酒精作用的敏感性明显增高。
3.肝PC预保护可以明显提高酒精损伤的Cav-1KD细胞存活率,促进Cav-1及PKCα蛋白表达。
结论:肝PC对张氏肝细胞酒精性损伤的保护作用与Cav-1密切相关。Cav-1表达下调导致张氏肝细胞对酒精作用的敏感性增高。肝PC可以通过改变膜磷脂成分,促进Cav-1介导的PKCα信号通路的激活,提高肝细胞膜的抗氧化能力,对抗酒精性损伤。
Phospholipid is an essential component of cell membrane. Phospholipid uptake can improve membrane fluidity and enzyme activity. Caveolin-1 is a mainly functional protein in Caveolae, and plays an important role in signal transduction. The added phospholipids promote the formation and transition of Caveolin-1vesicles. It is essential for Caveolae formation and the synthesis of new membranes. Our previous study showed that, phosphatidylcholine (PC) is rich in allyl ether which plays an important role in antioxidant activity protection. Moreover, phospholipid can promote the expression of Caveolin-1. But the mechanism is not entirely clear.
Caveolin-1 down-regulated cell line was obtained by siRNA interference with Chang liver cells, and alcohol-injury model was established in order to investigate the role of Caveolin-1 in cell repair. We detected the electrical conductivity and the composition of membrane phospholipid in Chang liver cell and Caveolin-1 down-regulated cells which were alcohol-induced. Western blot analysis was used to detect the expression of Caveolin-1 or oxidative stress-related proteins in Chang liver cells which were pre-protected by PC, further investigated the role of Caveolin-1 in cell repair and the protective mechanism of PC.
Results:
1. Chang liver cells incubated with alcohol induced oxidative stress, leading to the level of superoxide dismutase (SOD) reduced (P<0.05) and the expression of TLR4, TNF-α, NF-κB, p-P38 increased. However, pre-protected by liver Phosphatidylcholine increases the SOD activity (P<0.05) in Chang liver cells and reduce TLR4, TNF-α, NF-κB, P-p38 expression.
2. Caveolin-1 down-regulated cell line was obtained by siRNA interference with Chang liver cells. The release of ALT increased in Caveolin-1 down-regulated cells which were incubated with different alcohol concentrations. And the changes of cell electrical conductivity (P<0.05) and membrane phospholipid composition were significant in Caveolin-1 down-regulated cells.
3. Added PC could increase the level of Caveolin-1 and PKCα.
Conclusion: Liver-derived phospholipids significantly protected the membrane lipid peroxidation caused by alcohol-injury, and the mechanism was closely correlated with the expression of Caveolin-1. Caveolin-1 gene silencing induced the sensitivity increased which incubated with alcohol. The protective effect of liver phospholipid is regulated by phospholipid-PKCαsignaling which involved the role of Cavolin-1.
本文以张氏肝细胞为对象,利用siRNA技术筛选出稳定传代的Cav-1低表达细胞株(Cav-1KD),建立酒精性肝细胞损伤模型;利用MTT、电导法和薄板层析(TLC)等技术检测酒精损伤的Cav-1KD细胞膜特性及磷脂成份变化的影响;利用Western blot技术确定肝PC预保护的酒精肝损伤细胞中氧化应激途径相关蛋白表达的变化,进一步探讨肝PC对肝细胞酒精性损伤的保护作用与Cav-1的关系,旨在了解由Cav-1蛋白在肝脏损伤修复中的作用和信号转导机制。
结果如下:
1.酒精作用导致细胞中超氧化物歧化酶(SOD)水平明显降低(P<0.05),并促进氧化应激通路中TLR4、TNF-α、NF-κB表达增高及p38磷酸化激活,引发张氏肝细胞氧化应激。肝PC显著提高张氏肝细胞SOD活性(P<0.05),降低TLR4、TNF-α、NF-κB、P-p38等蛋白表达水平,对肝细胞起保护作用。
2.利用RNA干扰技术建立张氏肝细胞模型,发现200mmol/L酒精引起Cav-1低表达细胞ALT释放增加(P<0.05),细胞电导率显著增高(P<0.01),膜PC、PE、SM等含量均降低。提示,Cav-1表达下调导致张氏肝细胞对酒精作用的敏感性明显增高。
3.肝PC预保护可以明显提高酒精损伤的Cav-1KD细胞存活率,促进Cav-1及PKCα蛋白表达。
结论:肝PC对张氏肝细胞酒精性损伤的保护作用与Cav-1密切相关。Cav-1表达下调导致张氏肝细胞对酒精作用的敏感性增高。肝PC可以通过改变膜磷脂成分,促进Cav-1介导的PKCα信号通路的激活,提高肝细胞膜的抗氧化能力,对抗酒精性损伤。
Phospholipid is an essential component of cell membrane. Phospholipid uptake can improve membrane fluidity and enzyme activity. Caveolin-1 is a mainly functional protein in Caveolae, and plays an important role in signal transduction. The added phospholipids promote the formation and transition of Caveolin-1vesicles. It is essential for Caveolae formation and the synthesis of new membranes. Our previous study showed that, phosphatidylcholine (PC) is rich in allyl ether which plays an important role in antioxidant activity protection. Moreover, phospholipid can promote the expression of Caveolin-1. But the mechanism is not entirely clear.
Caveolin-1 down-regulated cell line was obtained by siRNA interference with Chang liver cells, and alcohol-injury model was established in order to investigate the role of Caveolin-1 in cell repair. We detected the electrical conductivity and the composition of membrane phospholipid in Chang liver cell and Caveolin-1 down-regulated cells which were alcohol-induced. Western blot analysis was used to detect the expression of Caveolin-1 or oxidative stress-related proteins in Chang liver cells which were pre-protected by PC, further investigated the role of Caveolin-1 in cell repair and the protective mechanism of PC.
Results:
1. Chang liver cells incubated with alcohol induced oxidative stress, leading to the level of superoxide dismutase (SOD) reduced (P<0.05) and the expression of TLR4, TNF-α, NF-κB, p-P38 increased. However, pre-protected by liver Phosphatidylcholine increases the SOD activity (P<0.05) in Chang liver cells and reduce TLR4, TNF-α, NF-κB, P-p38 expression.
2. Caveolin-1 down-regulated cell line was obtained by siRNA interference with Chang liver cells. The release of ALT increased in Caveolin-1 down-regulated cells which were incubated with different alcohol concentrations. And the changes of cell electrical conductivity (P<0.05) and membrane phospholipid composition were significant in Caveolin-1 down-regulated cells.
3. Added PC could increase the level of Caveolin-1 and PKCα.
Conclusion: Liver-derived phospholipids significantly protected the membrane lipid peroxidation caused by alcohol-injury, and the mechanism was closely correlated with the expression of Caveolin-1. Caveolin-1 gene silencing induced the sensitivity increased which incubated with alcohol. The protective effect of liver phospholipid is regulated by phospholipid-PKCαsignaling which involved the role of Cavolin-1.
引文
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[2]刘梅,陆伦根,曾民德.多烯磷脂酰胆碱对肝细胞保护机制的研究进展.肝脏, 2006, 11(1): 43-45.
[3] Vasiliouv, Nebertd W. Analysis and update of the human aldehyde dehydrogenase (ALDH) gene family [J]. Hum Genom-ics, 2005, 2 (2): 138-143.
[4] Fataccioli V. Effects of chronic ethanol administration on rat liver proteasome activities: relationship with oxidative stress [J]. Hepatology, 2004, 34:14-20.
[5] Lin SC, Chung CY, Chiang CL, et al. The influence of propolis ethanol extract on liver microsomal enzymes and glutathione after chronic alcohol administration [J]. Am J ChinMed, 1999, (27): 83-93.
[6] Sen N, Das BB, Ganguly A, et al. Camptothecin induced mitochondrial dysfunction leading to programmed cell death in unicellular hemoflagellate leishmania donovani [J]. Biol Chem, 2004, 11(8): 924-936.
[7] Craig J McClain, et al. Recent advances in alcoholic liver diseaseⅣDysregulated cytokine metabolism in alcoholic liver disease [J]. Am J Physiol Gastrointest Liver Physiol, 2004, 287: 497-502.
[8] Gobejishvili L, Barve S, Joshi-Barve S, et al. Chronic ethanol-mediated decrease in cAMP primes macrophages to enhanced LPS-inducible NF-kappaB activity and TNF expression: relevance to alcoholic liver disease. Am J Physiol Gastrointest Liver Physiol, 2006, 291: 681-688.
[9] NanjiAA, MiaoL, ThomasP, et al. Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat. Gastroenterology, 1997, 112: 943-951.
[10] Kostadinova R, Wahli W, Michalik L. PPARs in diseases: control mechanisms of inflammation. Curr Med Chem, 2005, 12: 2995-3009.
[11]石富成,周景春,孟冬娅等.大豆磷脂对脑梗死治疗作用的研究[J].中华医学杂志, 2001, 81(21): 1310.
[12] Xu YQ, Leo MA, Lieber CS. DLPC attenuated alcohol-induced cytotoxicity in HepG2 cells expressing CYP2E1. Alcohol, 2005, 40: 172-175.
[13] Aleynik MK, Leo MA, Aleynik SI, et al. Polyenylphosphatidylcholine opposes the increase of cytochrome P24502E1 by ethanol and corrects its iron-induced decrease. Alcohol Clin Exp Res, 1999, 23: 96-100.
[14] Aleynik MK, Lieber CS. Dilinoleoylphosphatidylcholine decreases ethanol- induced cytochrome P4502E1. BiochemBiophys Res Commun, 2001, 288:1047-1051.
[15] Mak KM, Wen KF, Ren CL, et al. Dilinoleoylphosphatidylcholine reproduces the antiapoptotic actions of polyenylphosphatidylcholine against ethanol-induced hepatocyte apoptosis. Alcohol Clin Exp Res, 2003, 27: 997-1005.
[16] Osada S, Mizuno K, Saido T C, et al. Phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin. J Biol Chem, 1990, 265(36): 22434-22440.
[17] Irina Treede, Annika Braun, Richard Sparla, et al. Anti-inflammatory Effects of Phosphatidylcholine. The Journal of Biological Chemistry, 2007, 282 (37): 27155–27164.
[18]贾丹,刘翠平,邹伟.缩醛磷脂的功能及信号传导机制研究进展.天然产物研究与开发, 2007, 19: 541-544.
[19] Zou W, Li ZY, Li YL, et al. Overexpression of PEMT2 downregulates the PI3K/Akt signaling pathway in rat hepatoma cells. Biochim Biophys Acta, 2002, 1581:49-56.
[20] Tessitore L, Marengo B, Vance DE, et al. Expression of Phosphatidylethnolamine N- methyItransferase in human hepatoceller carcinomas. Oncology, 2003, 65(2): 152-158.
[21] Aleynik SI, Lieber CS. Polyenylphosphatidylcholine corrects the alcohol-induced hepatic oxidative stress by restoreing S-adenosylmethionine. Alcohol, 2003, 38: 208-212.
[22] Vishnudutt Purohit, Manal F Abdelmalek, Samir Zakhari, et al. Role of S-adenosylmethionine, folate, and betaine in the treatment of alcoholic liver disease. Clinical Nutrition, 2007, 86: 14-24.
[23]王亮,刘翠平,贾丹,邹伟.肝源性磷脂酰胆碱的分离纯化及其对肝癌细胞抑制作用的评价.色谱, 2006, 24, (3): 271-274.
[24] Sergent O, Pereira M, Belhomme C, et al. Role for membrane fluidity in ethanol-induced oxidative stress of primary rat hepatocytes. J Pharmacol Exp Ther, 2005, 1:5.
[25] Lieber CS. CYP2E1: from ASH to NASH. Hepatol Res, 2004, 28: 1-11.
[26] Jokelainen K, Reinke LA, Nanji AA. Nf- kappab activation is associated with free radical generation and eddotoxemia and precedes pathologyical injury in experimental alcoholic liver desease. Cytokine, 2001, 16: 36-39.
[27] Michelsen KS, Wong MH, Shah PK, et al. Lack of toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. Proc Natl Acad Sci USA, 2004, 101: 10679-10684.
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