Salvianolic Acid B Prevents Against Chronic Alcoholic Liver Injury Via SIRT1-Mediated Inhibition Of CRP And ChREBP In Rats

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 ? histone deacetylase(HDAC)which plays important roles in many physiological processes,including gene transcription,senescence,energy metabolism,oxidative stress and inflammation.Hepatic nuclear factor-1?(HNF-1?)is a homeodomain transcriptional factor that interacts with a complex network of transcription factors to regulate gene expression in the liver,kidney,and pancreatic ?-cells.It has been reported that HNF-1? 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-1?-mediated transcriptional activation of the CRP promoter by deacetylating lysine 16 of histone H4 around the proximal HNF-1? 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 ?.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 ? 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-? 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(2?M,4?M,8?M).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-? 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-? 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 ? 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-1? 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-1?,ChREBP and ?-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(8?M)group,siRNA SIRT1 group and siRNA SIRT1+SalB(8?M)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(8?M)group,siRNA SIRT1+EtoH group and siRNA SIRT1+EtoH+SalB(8?M)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(8?M)group,the EtoH+Ex527(10?M)group and the EtoH+SalB(8?M)+Ex527(10?M)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(8?M)group,siRNA HNF-1? group and siRNA HNF-1?+SalB(8?M)group.The HepG2 cells were treated with SalB for 3h after their transfection with HNF-1? siRNA for 48 h,then exposed to ethanol(100 m M)for another 48 h.The protein expression of SIRT1 and CRP and HNF-1? were detected by Western Blotting and q RT-PCR was used to determine the expression of HNF-1? in HepG2 cells.Experiment Set 4: The HepG2 cells were divided into four groups: negative control group,negative control+RES(10?M)group,siRNA HNF-1? group and siRNA HNF-1?+RES(10?M)group.The HepG2 cells were treatment with RES for 6h after transfected with HNF-1? siRNA for 48 h,then exposed to ethanol(100 m M)for another 48 h.The protein expression of SIRT1 and CRP and HNF-1? were detected by Western Blotting.Results:(1)In vivo,compared with the control group,the hepatic SIRT1 and HNF-1? 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-1? 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-1? 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-1? expression,and down-regulated CRP expression in ethanol-exposed HepG2 cells,whereas HNF-1? siRNA transfection abrogated both increases in HNF-1? 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 ? SIRT1-mediated the protective effects of SalB against ALD involves phosphorylated AMPK?1Objective: To explore whether SalB-mediated up-regulation of SIRT1 is associated with AMPK?1 regulation.Methods:1.SalB regulates the phosphorylation levels of AMPK?1.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 AMPK?1,p-AMPK?1 and ?-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(8?M)group,EtoH group and EtoH+SalB(8?M)group.The protein expression of AMPK?1,p-AMPK?1 and ?-actin were detected by Western Blotting.2)The HepG2 cells were divided into four groups: negative control group,negative control+SalB(8?M)group,siRNA AMPK?1 group and siRNA AMPK?1+SalB(8?M)group.Pretreatment with SalB for 3h before transfected with AMPK?1 siRNA,and then e xposed to ethanol(100 m M)for another 48 h.The protein expression of SIRT1,AMPK?1,p-AMPK?1 and ?-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(8?M)group,siRNA AMPK?1+EtoH group and siRNA AMPK?1+EtoH+SalB(8?M)group.Pretreatment with SalB for 3h before transfected with AMPK?1 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-AMPK?1 protein levels were remarkably reduced in ethanol group;Consistent with the ethanol group,SalB treatment triggered an increase in p-AMPK?1 expression.2.In vitro: 1)Compared with the control group,SalB treatment triggered an increase in p-AMPK?1 expression,besides,compared with the ethanol group,SalB treatment triggered an increase in p-AMPK?1 expression.2)Compared with the negative control group,SalB pretreatment markedly increased SIRT1 and p-AMPK?1 levels,whereas siRNA-mediated AMPK?1 knockdown abrogated increasement both in SIRT1 and p-AMPK?1 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 AMPK?1 knockdown reverse the trend.Conclusion:1.SalB up-regulates the expression of p-AMPK?1.2.SalB activates the expression of SIRT1 through regulating the phosphorylation levels of AMPK?1,which make great contributions to its protection against ALD.