丹参酮Ⅱ_A通过激活PPARα减轻4-HNE诱导的肝细胞损伤的机制研究
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摘要
丹参酮Ⅱ_A(tanshinoneⅡ_A,TanⅡ_A)是丹参主要的脂溶性有效成分,能够增强对脂质的代谢功能,降低血清中脂质过氧化物浓度,也具有抗氧化损伤、清除自由基及抑制肝内坏死炎症反应的作用,对肝功能损害预后起到保护作用。因此,研究TanⅡ_A保护肝功能的确切机制可以为TanⅡ_A防治肝损伤提供重要的理论与实验依据。该文主要通过体外细胞实验,研究TanⅡ_A减轻4-羟基壬烯酸(4-hydroxynonenal,4-HNE)诱导的肝细胞氧化损伤的作用及机制。以正常肝细胞系NCTC 1469为细胞模型,建立4-HNE处理的肝细胞氧化损伤模型。通过乳酸脱氢酶(lactate dehydrogenase,LDH)释放及Hoechst染色法检测TanⅡ_A对肝细胞氧化损伤的保护作用; Western blot法检测TanⅡ_A作用前后过氧化物酶增殖激活受体α(peroxisome proliferator-activated receptorα,PPARα)及过氧化物酶体增殖物反应元件(peroxisome proliferator response element,PPRE)的蛋白表达变化; Real-time PCR法检测其下游关键酶脂肪醛脱氢酶(fatty aldehyde dehydrogenase,FALDH)的基因表达变化。结果表明4-HNE可增加肝细胞LDH释放,降低细胞存活率,而TanⅡ_A能逆转4-HNE引起的肝细胞损伤。Western blot及Real-time PCR法检测结果显示4-HNE可显著下调肝细胞内PPARα及其下游FALDH的表达,增加细胞内4-HNE蛋白质水平,而经过TanⅡ_A处理后PPARα及FALDH的表达可显著提高,4-HNE蛋白水平也得到了降低。这揭示TanⅡ_A可逆转脂质氧化产物4-HNE引起的肝细胞损伤,其作用机制可能与激活PPARα特异性结合PPRE元件,激活下游FALDH的表达以及清除4-HNE有关,这可能是TanⅡ_A发挥治疗作用的关键机制之一。
Tanshinone Ⅱ_A( Tan Ⅱ_A),the liposoluble constituents of Salvia miltiorrhiza,can not only ameliorate the lipidic metabolism and decrease the concentration of lipid peroxidation,but also resist oxidation damage,scavenge free radicals and control inflammation,with a protective effect on prognosis after liver function impairment. Therefore,the studies on the exact mechanism of Tan Ⅱ_A in protecting the liver can provide important theoretical and experimental basis for the prevention and treatment effect of Tan Ⅱ_A for liver injury. In the present study,the protective effects and mechanism of Tan Ⅱ_A on 4-hydroxynonenal( 4-HNE)-induced liver injury were investigated in vitro. Normal liver tissues NCTC 1469 cells were used to induce hepatocytes oxidative damages by 4-HNE treatment. The protective effect of Tan Ⅱ_A on hepatocytes oxidative damages was detected by release amount of lactate dehydrogenase( LDH) analysis and hoechst staining. The protein expression changes of peroxisome proliferator-activated receptor α( PPARα) and peroxisome proliferator response element( PPRE) were analyzed by Western blot analysis in NCTC 1469 cells before and after Tan Ⅱ_A treatment. The gene expression changes of fatty aldehyde dehydrogenase( FALDH) were analyzed by Real-time polymerase chain reaction( PCR) analysis. The results showed that 4-HNE increased the release amount of LDH,lowered the cell viability of NCTC 1469 cells,and Tan Ⅱ_A reversed 4-HNE-induced hepatocyte damage. Western blot analysis and RT-PCR analysis results showed that 4-HNE decreased the expression of PPARα and FALDH and increased the expression of 4-HNE. However,the expression of PPARα and FALDH were increased significantly and the expression of 4-HNE was decreased obviously after Tan Ⅱ_A treatment. This study confirmed that the curative effect of Tan Ⅱ_A was obvious on hepatocytes damage,and the mechanism may be associated with activating PPARα and FALDH expression as well as scavenging 4-HNE.
Tanshinone Ⅱ_A( Tan Ⅱ_A),the liposoluble constituents of Salvia miltiorrhiza,can not only ameliorate the lipidic metabolism and decrease the concentration of lipid peroxidation,but also resist oxidation damage,scavenge free radicals and control inflammation,with a protective effect on prognosis after liver function impairment. Therefore,the studies on the exact mechanism of Tan Ⅱ_A in protecting the liver can provide important theoretical and experimental basis for the prevention and treatment effect of Tan Ⅱ_A for liver injury. In the present study,the protective effects and mechanism of Tan Ⅱ_A on 4-hydroxynonenal( 4-HNE)-induced liver injury were investigated in vitro. Normal liver tissues NCTC 1469 cells were used to induce hepatocytes oxidative damages by 4-HNE treatment. The protective effect of Tan Ⅱ_A on hepatocytes oxidative damages was detected by release amount of lactate dehydrogenase( LDH) analysis and hoechst staining. The protein expression changes of peroxisome proliferator-activated receptor α( PPARα) and peroxisome proliferator response element( PPRE) were analyzed by Western blot analysis in NCTC 1469 cells before and after Tan Ⅱ_A treatment. The gene expression changes of fatty aldehyde dehydrogenase( FALDH) were analyzed by Real-time polymerase chain reaction( PCR) analysis. The results showed that 4-HNE increased the release amount of LDH,lowered the cell viability of NCTC 1469 cells,and Tan Ⅱ_A reversed 4-HNE-induced hepatocyte damage. Western blot analysis and RT-PCR analysis results showed that 4-HNE decreased the expression of PPARα and FALDH and increased the expression of 4-HNE. However,the expression of PPARα and FALDH were increased significantly and the expression of 4-HNE was decreased obviously after Tan Ⅱ_A treatment. This study confirmed that the curative effect of Tan Ⅱ_A was obvious on hepatocytes damage,and the mechanism may be associated with activating PPARα and FALDH expression as well as scavenging 4-HNE.
引文
[1]陈玉帅,王炳元,厉有名.非酒精性脂肪性肝病与酒精性肝病2016研究进展[J].中华肝脏病杂志,2017,25(3):181.
[2]袁媛,吴芹,石京山,等.丹参及其主要成分保肝作用的研究进展[J].中国中药杂志,2015,40(4):588.
[3]宋献美,石科,葛文静,等.不同引经药配伍大黄-丹参药对抗大鼠肝纤维化的比较[J].中国实验方剂学杂志,2017,23(20):132.
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[5]Ming S,Xiaorong H,Zuoan H,et al. Effects of tanshinoneⅡA on fibrosis in a rat model of cirrhosis through heme oxygenase-1,inflammation,oxidative stress and apoptosis[J]. Mol Med Rep,2016,13(4):3036.
[6]Yawei L,Yi Tsau H. Inhibitory effect of tanshinoneⅡAon rat hepatic stellate cells[J]. PLoS ONE,2014,9(7):e103229.
[7]夏婷,张瑾,姚佳慧,等.氧化应激在酒精性肝病中作用机制的研究进展[J].中国药理学通报,2017,33(10):1353.
[8]Lei D,Like W,Zhongyan D,et al. Danshen protects against early-stage alcoholic liver disease in mice via inducing PPARαactivation and subsequent 4-HNE degradation[J]. PLoS ONE,2017,12(10):e0186357.
[9]Ronis M J, Mercer K E, Gannon B, et al. Increased4-hydroxynonenal protein adducts in male GSTA4-4/PPAR-alpha double knockout mice enhance injury during early stages of alcoholic liver disease[J]. Am J Physiol Gastrointest Liver Physiol,2016,308:G403.
[10]严瑾,沃兴德,范春雷,等.山楂叶总黄酮对PPARγ-PPRE信号调控系统的影响[J].中国药学杂志,2005(8):625.
[11]陈蕊,刘志军,程悦,等.丹参注射液与保肝药物联合治疗酒精性肝病的Meta分析[J].现代临床医学,2014,40(2):103.
[12]张伟伟,陆茵.丹参抗肿瘤活性成分研究新进展[J].中国中药杂志,2010,35(3):389.
[13]Huiqin Z, Huiyong Y. Role of lipid peroxidation derived4-hydroxynonenal(4-HNE)in cancer:focusing on mitochondria[J]. Redox Biol,2015,4(4):193.
[14]Andringa K K,Udoh U S,Landar A,et al. Proteomic analysis of4-hydroxynonenal(4-HNE)modified proteins in liver mitochondria from chronic ethanol-fed rats[J]. Redox Biol,2014,2:1038.
[15]于晨辉,杜仲燕,高佳,等.4-HNE通过抑制TNF-α介导的NF-κB活化诱导酒精性肝损伤[J].中国病理生理杂志,2013,29(6):1046.
[16]唐小峦,李焕德,袁娟.基于报告基因的PPAR-α激动剂筛选体系的建立[J].中国现代应用药学,2010,27(8):677.
[17]Xiao M,Zhong H,Xia L,et al. Pathophysiology of mitochondrial lipid oxidation:role of 4-hydroxynonenal(4-HNE)and other bioactive lipids in mitochondria[J]. Free Radic Biol Med,2017,10(111):316.
[18]Ren T T,Zhu J J,Zhu L L,et al. The combination of bueberry juice and probiotics ameliorate non-alcoholic steatohepatitis(NASH)by affecting SREBP-1c/PNPLA-3 pathway via PPAR-α[J]. Nutrients,2017,9(3):198.
[19]Huo X,Yang S,Sun X,et al. Protective effect of glycyrrhizic acid on alcoholic liver injury in rats by modulating lipid metabolism[J]. Molecules,2018,23(7):E1623.
[20]Yamada K,Taketani T. Management and diagnosis of mitochondrial fatty acid oxidation disorders:focus on very-long-chain acylCoA dehydrogenase deficiency[J]. J Hum Genet,2019,64(2):73.
[21]Ashibe B,Motojima K. Fatty aldehyde dehydrogenase is up-regulated by polyunsaturated fatty acid via peroxisome proliferatoractivated receptor alpha and suppresses polyunsaturated fatty acid-induced endoplasmic reticulum stress[J]. FEBS J,2009,276(23):6956.
[2]袁媛,吴芹,石京山,等.丹参及其主要成分保肝作用的研究进展[J].中国中药杂志,2015,40(4):588.
[3]宋献美,石科,葛文静,等.不同引经药配伍大黄-丹参药对抗大鼠肝纤维化的比较[J].中国实验方剂学杂志,2017,23(20):132.
[4]薄怀来.中药复方制剂治疗酒精性肝病规律探讨[J].中医临床研究,2017,9(14):3.
[5]Ming S,Xiaorong H,Zuoan H,et al. Effects of tanshinoneⅡA on fibrosis in a rat model of cirrhosis through heme oxygenase-1,inflammation,oxidative stress and apoptosis[J]. Mol Med Rep,2016,13(4):3036.
[6]Yawei L,Yi Tsau H. Inhibitory effect of tanshinoneⅡAon rat hepatic stellate cells[J]. PLoS ONE,2014,9(7):e103229.
[7]夏婷,张瑾,姚佳慧,等.氧化应激在酒精性肝病中作用机制的研究进展[J].中国药理学通报,2017,33(10):1353.
[8]Lei D,Like W,Zhongyan D,et al. Danshen protects against early-stage alcoholic liver disease in mice via inducing PPARαactivation and subsequent 4-HNE degradation[J]. PLoS ONE,2017,12(10):e0186357.
[9]Ronis M J, Mercer K E, Gannon B, et al. Increased4-hydroxynonenal protein adducts in male GSTA4-4/PPAR-alpha double knockout mice enhance injury during early stages of alcoholic liver disease[J]. Am J Physiol Gastrointest Liver Physiol,2016,308:G403.
[10]严瑾,沃兴德,范春雷,等.山楂叶总黄酮对PPARγ-PPRE信号调控系统的影响[J].中国药学杂志,2005(8):625.
[11]陈蕊,刘志军,程悦,等.丹参注射液与保肝药物联合治疗酒精性肝病的Meta分析[J].现代临床医学,2014,40(2):103.
[12]张伟伟,陆茵.丹参抗肿瘤活性成分研究新进展[J].中国中药杂志,2010,35(3):389.
[13]Huiqin Z, Huiyong Y. Role of lipid peroxidation derived4-hydroxynonenal(4-HNE)in cancer:focusing on mitochondria[J]. Redox Biol,2015,4(4):193.
[14]Andringa K K,Udoh U S,Landar A,et al. Proteomic analysis of4-hydroxynonenal(4-HNE)modified proteins in liver mitochondria from chronic ethanol-fed rats[J]. Redox Biol,2014,2:1038.
[15]于晨辉,杜仲燕,高佳,等.4-HNE通过抑制TNF-α介导的NF-κB活化诱导酒精性肝损伤[J].中国病理生理杂志,2013,29(6):1046.
[16]唐小峦,李焕德,袁娟.基于报告基因的PPAR-α激动剂筛选体系的建立[J].中国现代应用药学,2010,27(8):677.
[17]Xiao M,Zhong H,Xia L,et al. Pathophysiology of mitochondrial lipid oxidation:role of 4-hydroxynonenal(4-HNE)and other bioactive lipids in mitochondria[J]. Free Radic Biol Med,2017,10(111):316.
[18]Ren T T,Zhu J J,Zhu L L,et al. The combination of bueberry juice and probiotics ameliorate non-alcoholic steatohepatitis(NASH)by affecting SREBP-1c/PNPLA-3 pathway via PPAR-α[J]. Nutrients,2017,9(3):198.
[19]Huo X,Yang S,Sun X,et al. Protective effect of glycyrrhizic acid on alcoholic liver injury in rats by modulating lipid metabolism[J]. Molecules,2018,23(7):E1623.
[20]Yamada K,Taketani T. Management and diagnosis of mitochondrial fatty acid oxidation disorders:focus on very-long-chain acylCoA dehydrogenase deficiency[J]. J Hum Genet,2019,64(2):73.
[21]Ashibe B,Motojima K. Fatty aldehyde dehydrogenase is up-regulated by polyunsaturated fatty acid via peroxisome proliferatoractivated receptor alpha and suppresses polyunsaturated fatty acid-induced endoplasmic reticulum stress[J]. FEBS J,2009,276(23):6956.