Studies On The Reversal Mechanisms Of Curcumin On Alcoholic Liver Fibrosis

Background and AimsAlcoholic liver disease(ALD)threatens the health of human beings for a long time.At the early stage,its typical characteristic is hepatocyte steatosis.With the development of the disease,the injured hepatocytes will undergo necroptosis,which may trigger constant inflammatory responses.This stage is called alcoholic steatohepatitis.Subsequently,hepatic stellate cell(HSC)become activation with the stimulation of inflammatory factors.Activated HSC secretes excessive extracellular matrix components,which is believed as a core event during the progress of hepatic fibrosis.In this stage,alcoholic steatohepatitis progresses into hepatic fibrosis.Recent years have revealed that nuclear factor erythroid-2 related factor 2(Nrf2)inactivation is closely associated with the development of ALD.Nrf2 is thought to be a core of the evolution of disease.It has been a focus of research both at home and abroad that developing drug candidates for ALD from active ingredients of Chinese medicine.We have researched the effects of curcumin on hepatic fibrosis for years.We have preliminarily identified the antifibrotic activity of curcumin in both in vivo and in vitro systems,where inhibition of HSC activation is the main mechanism.In addition,it is reported that curcumin plays a role in alcohol-caused liver injury.Therefore,we propose hypothesis that curcumin improves alcohol-induced liver steatosis through activating Nrf2,resulting in improved alcoholic fatty liver.Curcumin improves alcoholic steatohepatitis through inhibiting hepatocyte necroptosis.Further,curcumin improves alcoholic hepatic fibrosis through inhibiting HSC activation.Methods and ResultsIn the present study,Sirius red staining and immunofluorescence were initially performed to detect the expression of Nrf2 and NAD(P)H:quinone oxidoreductase 1(NQO1)in liver tissues from alcoholic patients,which showed that the expression and activity of Nrf2 in hepatocytes and HSC were reduced following the aggravated hepatic fibrosis.The rat model of ALD was established and curcumin was utilized as a therapeutic strategy.We found that curcumin alleviated alcohol-caused liver injury,liver inflammation,and liver steatosis.Curcumin also significantly enhanced the expression and activity of Nrf2 in liver.In vitro study showed that curcumin obviously alleviated injury and steatosis of L02 cells,which was accompanied by enhanced expression of Nrf2 and famesoid X receptor(FXR).Nrf2 overexpression plasmids,Nrf2 siRNA,GW4064,and Z-Guggulsterone were used to investigate the role of Nrf2 and FXR in the protective effects of curcumin.Results showed that Nrf2 overexpression plasmids and GW4064,mimic curcum,could enhance the effect of curcumin on inhibition of hepatocyte steatosis.However,Nrf2 siRNA and Z-Guggulsterone had contrary effects.Nrf2 knockdown mice was established by injected with Nrf2 short hairpin RNA(shRNA)lentivirus through caudal vein.ALD model was further established.Results showed that Nrf2 knockdown aggravated the effects of alcohol on liver injury,inflammation,and steatosis.Mouse model of ALD was established and curcumin was utilized as a therapeutic drug.Results showed that curcumin could dose-dependently inhibited alcohol-caused hepatocyte necroptosis.ALD model was further established in Nrf2 knockdown mice and curcumin was adiministrated.Results showed that curcumin inhibited hepatocyte necroptosis in wild type mice but not in Nrf2 knockdown mice.Nrf2 knockdown aggravated the inductive effect of alcohol on the expression of p53 and abrogated the inhibitory effect of curcumin on p53,which was consistent with in vitro experiments.Although Nrf2 siRNA abolished the effect of curcumin on the expression of genes associated with necroptosis,p53 siRNA could restore the effect of curcumin.The phenotype of HSC was detected after being transfected with Nrf2 overexpression plasmids or Nrf2 siRNA.Results showed that Nrf2 overexpression plasmids promoted the production of lipid and regulated the expression of genes associated with lipid metabolism and activation.While Nrf2 siRNA had contrary effects.Hepatic fibrosis model was established in Nrf2 knockdown mice by intraperitoneally injected with CCl4.Results showed that Nrf2 knockdown aggravated the effects of CCl4 on liver injury,inflammation,and fibrosis.Noteworthily,Nrf2 knockdown enhanced the expression of genes associated with HSC activation.AG490 and Stattic,inhibitors of Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway,obviously restored the lipocyte phenotype of HSC.Nrf2 overexpression plasmids inhibited JAK2/STAT3 signaling pathway and the expression of suppressor of cytokine signaling 3(SOCS3).Curcumin restored the lipocyte phenotype of activated HSC and enhanced the transcriptional activity of Nrf2.In vitro study further revealed that Nrf2 overexpression plasmids facilitated the effect of curcumin on reversion of phenotype of activated HSC,while Nrf2 siRNA abrogated the effect of curcumin.Curcumin lose its protective effects on hepatic fibrosis in Nrf2 knockdown mice.The culture supernatant of hepatocytes treated with ethanol stimulated HSC activation,which was abolished by transfected with high mobility group box-1 protein(HMGB1)siRNA in hepatocytes.Conclusions These findings suggested that curcumin alleviated alcohol-induced steatosis and necroptosis via activation of Nrf2 in hepatocytes;curcumin restored lipocyte phenotype of activated HSC and inhibited its activation via activation of Nrf2 in HSC;curcumin inhibited HMGB1-mediated HSC activation via inhibiting release of HMGB1 from necroptotic hepatocytes;curcumin inhibited evolution of alcoholic fatty-alcoholic steatohepatitis-hepatic fibrosis.The present study confirmed the critical role of Nrf2 in ALD and systematically revealed the potential regulatory mechanisms,which provided strong evidence for developing curcumin into a clinical drug for ALD.