【作者】 张宁；

【导师】 姚继红；

【作者基本信息】 大连医科大学 ， 中西医结合药理学， 2016， 博士

【摘要】 慢性酒精性肝病(Alcoholic liver disease,ALD)是长期大量饮酒所致的慢性肝脏疾病,具有较高的发病率和死亡率,严重影响人们的健康生活水平。根据肝脏病理学改变的不同阶段可将ALD分为:酒精性脂肪肝(Alcoholic fatty liver,AFL)、酒精性肝炎(Alcoholic hepatitis,AH)、酒精性肝纤维化(Alcoholic hepatic fibrosis,AHF)及酒精性肝硬化(Alcoholic cirrhosis,AC)。目前研究显示,慢性酒精性肝病的发病机制十分复杂且治疗手段十分有限,因此,研究和掌握ALD发病机制并探索有效干预措施具有重要临床意义。丹酚酸B(Salvianolic acid B,SalB)是丹参根部提取的主要水溶性成分之一,属于多酚类化合物。研究表明丹酚酸B具有抗氧化、抗炎、抗纤维化及抗癌等多种药理特性,已广泛应用于多种疾病的防治,如肝、肺、肾等疾病。丹酚酸B对肝纤维化动物模型具有显著的保护作用。此外,因其抗炎、抗氧化特性,对心脏及神经方面的疾病也具有明显保护作用。然而丹酚酸B对慢性酒精性肝损伤的保护作用和潜在的分子机制尚未见报道。我们前期研究已证实天然化合物丹酚酸B可影响沉默信息调节因子2相关酶I(Silent Information Regulator 2-related Enzymes1,Sirtuin 1,SIRT1)的蛋白表达。SIRT1是广泛表达于哺乳动物体内的Ⅲ型组蛋白去乙酰化酶,在多种细胞生物学功能中发挥关键的调控作用,如基因转录、衰老、能量代谢、氧化还原平衡、炎症及DNA损伤修复、凋亡。肝细胞核因子1α(Hepatic nuclear factor-1α,HNF-1α)是一种具有同源结构域的转录因子,可与复杂的转录因子网络相互作用进而调控肝脏、肾及胰岛β细胞的相关基因表达。新近研究表明HNF-1α可与C型反应性蛋白(C-reactive protein,CRP)的启动子区结合进而调控其表达。CRP蛋白主要在肝脏中表达,在多种慢性疾病的发生发展进程中产生关键作用。有研究显示,在营养缺失的情况下,SIRT1通过脱去HNF-1α结合位点近端组蛋白H4赖氨酸16位点的乙酰基进而抑制HNF-1α介导的CRP启动子转录活化。碳水化合物反应元件结合蛋白(Carbohydrate response element-binding protein,ChREBP)主要参与糖酵解和脂生成相关基因的转录活化,在ALD的疾病进程中发挥重要的作用。肝脏SIRT1特异性敲除的小鼠ChREBP蛋白表达明显上调,伴随严重的肝脂肪变。此外,本实验室新近研究已证实多酚类双萜化合物鼠尾草酸(Carnosic acid,CA)可通过激活SIRT1/ChREBP信号通路减轻大鼠慢性酒精性脂肪肝病。综上所述,我们提出以下假说:1)SIRT1是慢性酒精性肝损伤的重要调控分子,长期慢性酒精摄入所致SIRT1的表达变化可直接或间接影响炎症相关蛋白CRP及脂代谢相关蛋白ChREBP的表达,进而参与调控ALD的疾病进程;2)丹酚酸B可通过靶向SIRT1发挥其抗慢性酒精性肝病的保护作用。AMP依赖的蛋白激酶(AMP-activated protein kinase,AMPK,丝氨酸/苏氨酸蛋白激酶组成的异源三聚体)是机体能量代谢的主要调控子,可增加NAD+的水平。SIRT1是NAD+依赖的蛋白去乙酰化酶,营养摄入受限或应激反应时NAD+水平升高可激活SIRT1进而参与调控能量代谢及应激反应。研究表明AMPK和SIRT1之间存在相互调控作用,在能量代谢及应激反应的调控中相辅相承。因此,我们推测AMPK可能参与介导丹酚酸B对SIRT1蛋白表达的调控。本课题将从以下三部分展开论述:1)丹酚酸B对长期摄入酒精所致大鼠肝损伤的保护作用;2)SalB通过SIRT1抑制CRP和ChREBP表达减轻大鼠慢性酒精性肝损伤;3)SalB通过AMPKα1激活SIRT1缓解大鼠慢性酒精性肝损伤。我们的研究结果显示,丹酚酸B对大鼠慢性酒精性肝损伤具有显著的保护作用,而肝脏SIRT1表达是其发挥保护作用的关键。丹酚酸B可以通过激活SIRT1进而抑制炎症相关蛋白CRP及脂代谢相关蛋白ChREBP的表达减轻大鼠慢性酒精性肝损伤。本研究有助于为ALD的防治提供新的靶点,同时也为丹酚酸B的临床应用及开发提供新的方向。第一部分丹酚酸B对长期酒精摄入所致肝损伤的保护作用目的:探讨SalB对慢性ALD的保护作用,明确SalB对长期酒精摄入的大鼠肝脏病理学和肝脏功能、血清及肝组织中脂质堆积、炎症因子以及对酒精诱导的HepG2细胞存活率的影响。方法:1.丹酚酸B对大鼠慢性酒精性肝损伤的保护作用研究。将50只健康成年的雄性SD大鼠(180-220g)随机分为以下5组:空白对照组;空白对照+SalB给药组(30 mg/kg/d);模型组;模型+低剂量SalB给药组(15mg/kg/d);模型+高剂量SalB给药组(30 mg/kg/d)。给药方法为灌胃,酒精造模参照Lieber-Decarli方案,酒精浓度前6周5%,最后两周8%。8周后腹主动脉采集血液,分离大鼠肝脏组织标本。检测血清中谷草转氨酶(Aspartate Aminotransferase,AST)、谷丙转氨酶(Alanine Aminotransferase,ALT)以及肝组织中总胆固醇(Total Cholesterol,TC)、甘油三酯(Triglycerides,TG)、白介素6(Interleukin-6,IL-6)、肿瘤坏死因子α(Tumor necrosisα,TNF-α)的活性。2、丹酚酸B对酒精诱导HepG2细胞损伤的保护作用研究。HepG2细胞随机分成五组:空白对照组;EtoH组(EtoH浓度为100 m M);低、中、高剂量丹酚酸B处理组(2μM、4μM、8μM)。在细胞中加入100 m M EtoH孵育48h造模,造模前、造模同时及造模后加入药物进行处理,MTT实验测定细胞存活率。结果:1.与空白对照组相比,模型组肝组织病理学检测结果显示肝脏细胞肿胀变形且存在大泡型和小泡型相间的肝脂肪病变,肝小叶结构紊乱,肝脏炎性细胞浸润明显增加;血清ALT、AST及肝组织TG、TC、IL-6、TNF-α的水平显著增加,表明肝组织存在中度或重度脂肪变,出现严重肝损伤和炎症反应。与模型组相比,丹酚酸B预处理组肝组织病理结果显示肝脏脂质堆积及炎症浸润明显得到改善;血清ALT、AST水平下降;肝组织TG、TC以及IL-1β、TNF-α水平显著降低,提示丹酚酸B可明显减轻长期酒精摄入引起的大鼠肝组织损伤和炎症反应。2.在酒精导致的肝损伤体外模型中,分别在造模前、造模同时及造模后加入丹酚酸B,与模型组相比,三种方式的丹酚酸B处理组HepG2细胞存活率明显提高,在中、高剂量组尤为明显,且呈剂量依赖性。表明丹酚酸B对HepG2细胞酒精损伤具有保护作用。结论:丹酚酸B对长期酒精摄入引起的肝损伤具有显著的保护作用。第二部分SalB通过SIRT1抑制CRP和ChREBP表达减轻大鼠慢性酒精性肝损伤目的:探讨SIRT1/CRP及SIRT1/ChREBP通路在慢性酒精性肝病中的作用,进一步阐明丹酚酸B对慢性酒精性肝病的保护作用机制。方法:1.丹酚酸B对大鼠慢性酒精性肝损伤SIRT1、CRP、HNF-1α、ChREBP蛋白和基因表达的影响。按第一部分方法将正常SD大鼠分组,酒精造模8周结束后分离肝组织标本冷冻备用。采用Western Blot法检测肝组织中SIRT1、CRP、HNF-1α、ChREBP及β-actin的蛋白水平;采用q RT-PCR法检测肝组织CRP及ChREBP的m RNA水平;2.探讨SIRT1/CRP和SIRT1/ChREBP通路在酒精诱导的HepG2细胞损伤中作用及丹酚酸B的保护作用机制。实验1.(1)HepG2细胞随机分为4组:阴性对照组;阴性对照+SalB(8μM)组;转染SIRT1 siRNA组;转染SIRT1 siRNA+SalB(8μM)组。转染后,SalB给药作用3h后提取细胞总蛋白。Western Blot法检测细胞内SIRT1及CRP的蛋白表达情况。(2)HepG2细胞随机分为五组:阴性对照组;阴性对照+EtoH组;阴性对照+EtoH+SalB(8μM)组;EtoH+转染SIRT1 siRNA组;EtoH+转染SIRT1siRNA+SalB(8μM)组。转染后,丹酚酸B给药作用3h后,用EtoH(100 m M)诱导48 h后提取细胞蛋白。Western Blot法检测细胞内SIRT1及ChREBP的蛋白表达情况。实验2.HepG2细胞随机分为5组:空白对照组;SalB(8μM)给药组;EtoH组;EtoH+SalB(8μM)给药组;EtoH+Ex527(10μM)预处理组。Ex527是SIRT1特异性的抑制剂。丹酚酸B或Ex527与细胞共同孵育3h或6h后,用浓度为100 m M的EtoH诱导48h造模。Western Blot法检测细胞内SIRT1、CRP的蛋白表达水平。尼罗红染色法检测细胞内脂质堆积的情况。实验3.HepG2细胞随机分为4组:阴性对照组;阴性对照+SalB(8μM)组;转染HNF-1αsiRNA组;转染HNF-1αsiRNA+SalB(8μM)组。转染后,SalB给药作用6h后,用浓度为100 m M的EtoH诱导48h后提取细胞蛋白及RNA。Western Blot法检测细胞内SIRT1、CRP及HNF-1α的蛋白表达情况。q RT-PCR法检测HepG2细胞HNF-1α的m RNA水平。实验4.HepG2细胞随机分为4组:阴性对照组;阴性对照+RES(10μM)组;转染HNF-1αsiRNA组;转染HNF-1αsiRNA+RES(10μM)组。转染后,RES给药作用6h后,用终浓度为100 m M的EtoH诱导48h后提取细胞蛋白。Western Blot法检测细胞内SIRT1、CRP及HNF-1α的蛋白表达情况。结果:1.体内实验结果显示:与空白对照组相比,模型组大鼠肝组织SIRT1及HNF-1α表达降低,肝组织CRP及ChREBP蛋白表达及m RNA水平显著升高。与模型组相比,丹酚酸B预处理后显著上调SIRT1及HNF-1α的蛋白表达水平,同时CRP及ChREBP的蛋白及m RNA水平明显降低。2.体外实验结果:实验1.(1)与阴性对照组相比,SalB可显著上调SIRT1蛋白表达,抑制CRP蛋白表达;SIRT1基因被干扰后,CRP蛋白表达上调,同时SalB对CRP蛋白表达的抑制作用减弱甚至消失,即SalB对CRP蛋白的调控可能是通过激活SIRT1实现的。(2)与阴性对照组相比,阴性对照+EtoH组SIRT1蛋白表达降低而ChREBP蛋白表达显著升高;与阴性对照+EtoH组相比,SalB预处理可显著上调SIRT1蛋白表达,同时抑制ChREBP蛋白表达;SIRT1基因被干扰后,ChREBP蛋白表达上调,同时SalB对ChREBP的抑制作用减弱甚至消失,表明SalB对ChREBP蛋白的调控作用与SIRT1有关。实验2.与EtoH组相比,SalB预处理可显著上调SIRT1蛋白表达,同时抑制CRP蛋白表达,而采用Ex527特异性的阻断SIRT1的蛋白表达后,CRP蛋白表达显著上调,进一步证实SIRT1介导SalB对CRP蛋白表达的调控。实验3.与阴性对照组相比,SalB可显著上调SIRT1及HNF-1α的蛋白表达,抑制CRP的蛋白表达;HNF-1α基因被干扰后,SalB对SIRT1的激活作用不变,而对CRP的抑制作用明显减弱甚至消失,即HNF-1α参与SalB对SIRT1调节的CRP表达抑制。实验4.与阴性对照组相比,SIRT1激活剂白藜芦醇(Resveratrol,RES)上调HNF-1α蛋白表达,同时抑制CRP蛋白表达;HNF-1α基因被干扰后,RES对SIRT1的激活作用不变,而对CRP蛋白表达的抑制作用明显减弱甚至消失,即SalB对SIRT1/CRP通路的调控主要是通过HNF-1α调节实现的。结论:1.SIRT1/CRP和SIRT1/ChREBP信号通路在大鼠慢性酒精性肝病中发挥重要的调控作用;2.在酒精诱导的ALD中,SalB通过上调SIRT1抑制CRP和ChREBP的蛋白表达,从而发挥抗慢性酒精性肝病的保护作用。第三部分SalB通过AMPKα1激活SIRT1缓解大鼠慢性酒精性肝损伤目的:探讨SalB通过调控AMPKα1激活SIRT1发挥肝保护作用的分子机制。方法:1.SalB对AMPKα1蛋白磷酸化水平的影响。正常SD大鼠酒精造模8周结束后分离肝组织标本冷冻备用。采用western-blot法检测肝组织中AMPKα1、p-AMPKα1及β-actin的蛋白水平;2.探究SalB激活SIRT1的相关分子机制。采用RNA干扰等实验,在体外细胞模型检测丹酚酸B对AMPKα1及p AMPKα1蛋白表达水平的影响,进一步探讨丹酚酸B激活SIRT1的分子机制。1)HepG2细胞随机分为四组:空白组;空白+SalB(8μM)组;EtoH组;EtoH+SalB(8μM)组。丹酚酸B给药作用3h后,用EtoH(100 m M)诱导48 h后提取细胞蛋白。Western Blot法检测细胞内AMPKα1、p-AMPKα1及β-actin的蛋白表达情况。2)HepG2细胞随机分为四组:阴性对照组;阴性对照+SalB(8μM)组;转染AMPKα1 siRNA组;转染AMPKα1 siRNA+SalB(8μM)组。转染后,丹酚酸B给药作用3h后提取细胞蛋白。Western Blot法检测细胞内SIRT1、AMPKα1、p-AMPKα1及β-actin的蛋白表达情况。3)HepG2细胞随机分为五组:阴性对照组;阴性对照+EtoH组;阴性对照+EtoH+SalB(8μM)组;EtoH+转染AMPKα1 siRNA组;EtoH+转染AMPKα1 siRNA+SalB(8μM)组。转染后,丹酚酸B给药作用3h后,用EtoH(100 m M)诱导48 h造模,MTT实验测定细胞存活率。结果:1.体内实验结果显示:慢性酒精性肝病大鼠肝组织中p-AMPKα1的蛋白表达水平明显下调,而给予丹酚酸B预处理后可显著逆转p-AMPKα1蛋白表达下降,且呈一定的剂量依赖性。2.体外实验结果显示:1)在HepG2细胞中,与空白组相比,丹酚酸B给药组可上调p-AMPKα1的蛋白表达,而与模型组相比,丹酚酸B预处理亦可上调p-AMPKα1的蛋白表达;2)HepG2细胞转染AMPKα1 siRNA后,AMPKα1基因的表达被抑制。与阴性对照组相比,丹酚酸B预处理组可显著上调p-AMPKα1、SIRT1的蛋白表达,干扰AMPKα1后,丹酚酸B对p-AMPKα1、SIRT1的上调作用明显减弱甚至消失;3)在酒精诱导的肝损伤体外模型中,与阴性对照组相比,酒精诱导的HepG2细胞存活率明显降低,给予丹酚酸B预处理后,HepG2细胞存活率明显提高;当同时给予酒精诱导时,与空白转染组相比,HepG2细胞转染AMPKα1 siRNA后细胞存活率降低,AMPKα1干扰时再给予丹酚酸B处理,HepG2细胞的存活率与模型转染组相比无明显变化。结论:1.丹酚酸B可引起AMPKα1磷酸化。2.丹酚酸B通过调控AMPKα1磷酸化水平激活SIRT1蛋白表达,进而发挥其抗慢性酒精性肝病的保护作用。更多还原

【Abstract】 Alcohol abuse has been recognized as a major cause of chronic alcoholic liver disease(ALD),which becoming one of the most significant health problems with high morbidity and mortality in recent decades.ALD encompasses a disease spectrum ranging from minimal abnormalities,such as steatosis,to more severe liver disease associated with inflammation,as in alcoholic hepatitis(AH),advanced fibrosis,or cirrhosis.Since the pathogenesis of ALD is very complex and treatment is very limited,a thorough understanding of the mechanisms that regulate hepatic steatosis and inflammation may be clinically relevant for preventing and treating ALD.Salvianolic acid B(SalB)is a major water soluble component extracted from Radix Salvia miltiorrhiza and has been widely used for treating many types of diseases including hepatic,lung and renal diseases.SalB has beneficial effects against hepatic fibrosis in animal models;besides,SalB exerts cardioprotective and neuroprotective activity via anti-oxidative and anti-inflammatory actions.However,the role of SalB in preventing onset and progression of chronic alcoholic liver injury remains unknown.SalB is a natural compound that activates mammalian sirtuins 1(SIRT1),an NAD-dependent class Ⅲ histone deacetylase(HDAC)which plays important roles in many physiological processes,including gene transcription,senescence,energy metabolism,oxidative stress and inflammation.Hepatic nuclear factor-1α(HNF-1α)is a homeodomain transcriptional factor that interacts with a complex network of transcription factors to regulate gene expression in the liver,kidney,and pancreatic β-cells.It has been reported that HNF-1α directly bound to the promoter of CRP gene to modulate its expression.C-reactive protein(CRP)is primarily synthesized in the liver and participates in many chronic diseases.Levels of CRP correlate closely with changes in inflammation/disease activity,radiological damage and progression,and functional disability.Previous studies have demonstrated that SIRT1 inhibits HNF-1α-mediated transcriptional activation of the CRP promoter by deacetylating lysine 16 of histone H4 around the proximal HNF-1α binding sites in response to nutrient restriction.Carbohydrate response element-binding protein(ChREBP)has been reported to have an important role in ALD.It has been reported that mice carrying a liver-specific SIRT1 null mutation exhibited increased ChREBP expression and liver steatosis.Our recently study has demonstrated that carnosic acid(CA)alleviates chronic alcoholic liver injury via regulating the SIRT1/ChREBP pathway in rats.According to the above view,we hypothesized that 1)SIRT1 plays an important role during ALD.The changes of SIRT1 expression due to long time chronic alcohol intake would make difference on the expression of CRP(inflammation related protein)and ChREBP(lipid metabolism related protein),which may be closely involved in the pathogenesis of ALD process;2)SalB protect against ALD through targeting SIRT1.AMP-activated protein kinase(AMPK,a heterotrimeric serine/threonin protein kinase)is an emerging key regulator of whole-body metabolism,and has been shown to increase NAD+ levels.SIRT1 is NAD+-dependent protein deacetylase that sense elevated NAD+ levels in response to changes in nutrient availability or stress and regulate the expression of genes involved in energy metabolism and the stress response.Studies have shown that AMPK and SIRT1 positively regulate each other’s activities,allowing them to coordinate their effects on energy metabolism and the stress response..Thus,we hypothesized that SalB-mediated regulation of SIRT1 may involve AMPK.We carried out three following parts to substantiate the above viewpoints.Part I.SalB exerts protective effects against ALD.Part II.SalB attenuates ALD via SIRT1-mediated inhibition of CRP and ChREBP in rats.Part Ⅲ.SIRT1-mediated the protective effects of SalB against ALD involves AMPK.Based on the above studies,we found that SalB treatment exerts significant protective effects against ALD,during which,SIRT1 plays an extremely important role.SalB exerted anti-steatosis and anti-inflammatory effects against alcoholic liver injury by SIRT1-mediated inhibition of CRP and ChREBP expression.This study aims to provide a new therapeutic targets for ALD,and to provide a new direction for the clinical application and development of the SalB.Part Ⅰ SalB’s protective effects against ALDObjective: To explore whether SalB exerts beneficial effects against ALD.Methods:1.To explore the protective effect of SalB in ALD: Male Sprague-Dawley(SD,SCXK 2008-0002)rats weighing 180-220 g were provided by the Animal Center of Dalian Medical University(Dalian,China).All the animal testing procedures were performed under the guidelines of IAEC(Institutional Animal Ethics Committee)with the approval of IAEC of Dalian Medical University.Rats were raised under standard laboratory condition for approximately one week before experimentation.Fifty rats were randomly separated into five groups: 1)control;2)control + SalB(30 mg/kg/d);3)ethanol;4)ethanol + SalB(15 mg/kg/d);and 5)ethanol + SalB(30 mg/kg/d).Liquid diets were based on the Lieber-De Carli formulation,and ethanol content was gradually increased from 5% in the first six weeks to 8% in the final two weeks.The SalB groups received an intragastric administration every day and the control group was treated with the equal volume of saline.After eight weeks,all the rats were euthanized,and the blood and liver tissues were harvested.The levels of triglyceride(TG),total cholesterol(TC)in the liver and the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),TG and TC in the serum were determined.Besides,the levels of TNF-α and IL-6 in the liver were measured.2.To explore the protective effect of SalB in ethanol-exposed HepG2 cells: The HepG2 cells were divided into five groups: the control group,the EtoH group(100m M ethanol)and the SalB treatment groups(2μM,4μM,8μM).The EtoH group and the SalB pre-treatment or co-treatment or post-treatment groups were treated with EtoH(100m M)for 48 hours.MTT method was used to detect cell survival rate.Results:1.Compared with the control group,rats exposed to ethanol revealed nuclear pleomorphism,increased lipid accumulation and inflammatory cell infiltration.Besides,the serum ALT and AST activity,as well as the liver TG,TC,TNF-α and IL-6 levels were remarkably increased,suggesting an extremely serious hepatic steatosis and inflammation injury;Compared with the EtoH group,SalB treatment markedly decreased serum ALT and AST activity,as well as the liver TG,TC,TNF-α and IL-6 levels in a dose-dependent manner,suggesting a protective effect of SalB during chronic alcoholic liver injury.2.In vitro,the HepG2 cells were exposed to ethanol for 48 h with pre-treantment or co-treament or post-treatment of SalB,the MTT results shows that SalB treatment increased the viability of HepG2 cells exposed to ethanol in a concentration-dependent manner,suggesting a protective effect of SalB in ethanol-exposed HepG2 cells.Conclusion:SalB is effective in protecting the chronic ethanol-induced liver injury.Part Ⅱ SalB attenuates ALD via SIRT1-mediated inhibition of CRP and ChREBP in ratsObjective: To investigate the involvement of SIRT1-CRP and SIRT1-ChREBP pathways in ALD and to furtherly explore the protective mechanisms of SalB in ALD.Methods:1.Salvianolic acid B affects the expressive change of protein and m RNA of SIRT1,CRP,HNF-1α and ChREBP of chronic alcoholic liver injury in rats.After eight weeks,all the rats were euthanized,and the blood and liver tissues were harvested.Western blot analysis was used to test the protein expressions of SIRT1,CRP,HNF-1α,ChREBP and β-actin in the liver.q RT-PCR was used to determine the m RNA expression of CRP and ChREBP in the liver.2.To investigate the involvement of SIRT1-CRP and SIRT1-ChREBP pathways in ethanol-induced HepG2 cells injury and to explore the protective mechanisms of SalB.Experiment Set 1: 1)The HepG2 cells were divided into four groups: negative control group,negative control+SalB(8μM)group,siRNA SIRT1 group and siRNA SIRT1+SalB(8μM)group.The protein expression of SIRT1 and CRP were detected by Western Blotting.2)The HepG2 cells were divided into five groups: negative control group,negative control+EtoH group,negative control+EtoH+SalB(8μM)group,siRNA SIRT1+EtoH group and siRNA SIRT1+EtoH+SalB(8μM)group.Pretreatment with SalB for 3h before transfected with SIRT1 siRNA,and then exposed to ethanol(100 m M)for another 48 h.The protein expression of SIRT1 and ChREBP were detected by Western Blotting.Experiment Set 2: The HepG2 cells were divided into five groups: the control group,the EtoH group,the EtoH+SalB(8μM)group,the EtoH+Ex527(10μM)group and the EtoH+SalB(8μM)+Ex527(10μM)group.The HepG2 cells were treated with SalB or Ex527 for 3h or 6h before exposed to ethanol for another 48 h,the protein expression of SIRT1 and CRP was detected by Western Blotting.The intracellular lipid droplets was detected by Nile red stains.Experiment Set 3: The HepG2 cells were divided into four groups: negative control group,negative control+SalB(8μM)group,siRNA HNF-1α group and siRNA HNF-1α+SalB(8μM)group.The HepG2 cells were treated with SalB for 3h after their transfection with HNF-1α siRNA for 48 h,then exposed to ethanol(100 m M)for another 48 h.The protein expression of SIRT1 and CRP and HNF-1α were detected by Western Blotting and q RT-PCR was used to determine the expression of HNF-1α in HepG2 cells.Experiment Set 4: The HepG2 cells were divided into four groups: negative control group,negative control+RES(10μM)group,siRNA HNF-1α group and siRNA HNF-1α+RES(10μM)group.The HepG2 cells were treatment with RES for 6h after transfected with HNF-1α siRNA for 48 h,then exposed to ethanol(100 m M)for another 48 h.The protein expression of SIRT1 and CRP and HNF-1α were detected by Western Blotting.Results:(1)In vivo,compared with the control group,the hepatic SIRT1 and HNF-1α protein levels were remarkably reduced in ethanol group,while the hepatic CRP and ChREBP protein levels were increased in ethanol group;Compared with the ethanol group,SalB treatment triggered an increase in both SIRT1 and HNF-1α expression,whereas the levels of CRP and ChREBP were markedly down-regulated.(2)In vitro:Experiment Set 1: 1)Compared with the negative control group,SalB treatment triggered an increase in SIRT1 expression and markedly down-regulated the expression of CRP;Upon SIRT1 small interfering RNA(siRNA)transfection,CRP expression was extremely increased.Besides,SalB-mediated SIRT1 up-regulation and CRP inhibition were mostly abrogated.These results suggested that SalB-mediated down-regulation of CRP is partly through activating SIRT1.2)Compare with the negative control group,SIRT1 expression was down-regulated in the negative control+EtoH group,whereas the expression of ChREBP was up-regulated;Compared with the negative control+EtoH group,SalB pretreatment markedly increased SIRT1 levels and decreased ChREBP levels,whereas siRNA-mediated SIRT1 knockdown abrogated both increases in SIRT1 and decreases in ChREBP expression.These results suggested that SalB-mediated down-regulation of ChREBP is partly through activating SIRT1.Experiment Set 2: HepG2 cells exposed to ethanol for 48 h exhibited a decrease in SIRT1 and an increase in CRP expression,SalB treatment significantly up-regulated SIRT1 and down-regulated CRP,whereas the SalB-mediated protection was significantly blocked by Ex527.These results further suggested that SalB-mediated down-regulation of CRP is activated by SIRT1.Experiment Set 3: Compared with the negative control group,SalB treatment increased SIRT1 expression and decreased CRP expression in ethanol-exposed HepG2 cells,while SalB-mediated down-regulation of CRP was abrogated by HNF-1α siRNA transfection,which had no effects on SIRT1 expression.Experiment Set 4: Compared with the negative control group,RES treatment up-regulated both SIRT1 and HNF-1α expression,and down-regulated CRP expression in ethanol-exposed HepG2 cells,whereas HNF-1α siRNA transfection abrogated both increases in HNF-1α and decreases in CRP expression,and had no effects upon SIRT1 expression.Conclusion:1.SIRT1-CRP and SIRT1-ChREBP pathways play an important role in development of ALD.2.SalB exerts protective effects against ethanol-induced chronic liver injury via SIRT1-mediated inhibition of CRP and ChREBP.Part Ⅲ SIRT1-mediated the protective effects of SalB against ALD involves phosphorylated AMPKα1Objective: To explore whether SalB-mediated up-regulation of SIRT1 is associated with AMPKα1 regulation.Methods:1.SalB regulates the phosphorylation levels of AMPKα1.After eight weeks,all the rats were euthanized,and the blood and liver tissues were harvested.Western blotting analysis was used to test the expressions of AMPKα1,p-AMPKα1 and β-actin in the liver.2.To investigate the mechanism of which SalB-mediated up-regulation of SIRT1.1)The HepG2 cells were divided into four groups: control group,control+SalB(8μM)group,EtoH group and EtoH+SalB(8μM)group.The protein expression of AMPKα1,p-AMPKα1 and β-actin were detected by Western Blotting.2)The HepG2 cells were divided into four groups: negative control group,negative control+SalB(8μM)group,siRNA AMPKα1 group and siRNA AMPKα1+SalB(8μM)group.Pretreatment with SalB for 3h before transfected with AMPKα1 siRNA,and then e xposed to ethanol(100 m M)for another 48 h.The protein expression of SIRT1,AMPKα1,p-AMPKα1 and β-actin were detected by Western Blotting.3)The HepG2 cells were divided into five groups: negative control group,negative control+EtoH group,negative control+EtoH+SalB(8μM)group,siRNA AMPKα1+EtoH group and siRNA AMPKα1+EtoH+SalB(8μM)group.Pretreatment with SalB for 3h before transfected with AMPKα1 siRNA,and then exposed to ethanol(100 m M)for another 48 h.MTT method was used to detect cell survival rate.Results:1.In vivo,compare with the control group,the hepatic p-AMPKα1 protein levels were remarkably reduced in ethanol group;Consistent with the ethanol group,SalB treatment triggered an increase in p-AMPKα1 expression.2.In vitro: 1)Compared with the control group,SalB treatment triggered an increase in p-AMPKα1 expression,besides,compared with the ethanol group,SalB treatment triggered an increase in p-AMPKα1 expression.2)Compared with the negative control group,SalB pretreatment markedly increased SIRT1 and p-AMPKα1 levels,whereas siRNA-mediated AMPKα1 knockdown abrogated increasement both in SIRT1 and p-AMPKα1 expression.3)In ethanol-exposed HepG2 cells,compare with the negative control group,the viability of HepG2 cells exposed to ethanol were decreased.Compared with the negative control+EtoH group,SalB pretreatment markedly increased the viability of HepG2 cells exposed to ethanol,whereas siRNA-mediated AMPKα1 knockdown reverse the trend.Conclusion:1.SalB up-regulates the expression of p-AMPKα1.2.SalB activates the expression of SIRT1 through regulating the phosphorylation levels of AMPKα1,which make great contributions to its protection against ALD.更多还原