Studies On The Pharmacological Mechanism Of Dihydroartemisinin On Alcoholic Fatty Liver

[background]Oxidative stress and excessive lipid accumulation occupies a vital status in the pathogenesis of alcoholic liver disease(ALD).Experimental studies have shown that the lipid accumulation of of hepatocytes could produce lipotoxicity.Lipotoxicity can cause activation of JNK and the endoplasmic reticulum(ER)stress,and then induce mitochondrial dysfunction,including activation of mitochondrial apoptosis pathway,leading to cell death.It is well documented that that alcohol can cause generation of reactive oxygen species(ROS)in the liver.Oxidative stress and abnormal accumulation of toxic lipid species is thought to be the main causes of unfolded protein response(UPR)and ER stress.Therefore,pharmacological agents,which protect hepatocyte against lipoapoptosis,may be a new strategy for the treatment of ALD.A large number of studies have shown that lipin-1 plays a key role in the regulation of hepatocyte steatosis caused by ALD,and is closely related to liver damage caused by alcohol.The results of our previous study showed that Dihydroartemisinin(DHA),a natural derivatives of artemisinin,had a significant pharmacological effect on ALD.DHA can effectively relieve the steatosis and liver injury of ALD rats.The aim of this study was to determine the effect of DHA on alcohol-induced hepatocyte lipoapoptosis and to elucidate the molecular mechanism underlying its effects.It provides a new way of thinking and a clear direction for the study of the mechanism of DHA.[method]This paper focuses on two parts of the experiment in vivo and in vitro.1.in vitro studyThe L02 cell line,immortalized human hepatocyte,was used in this study.Oil red O staining and Nile red staining was conducted for hepatic lipid accumulation.Activities of serum and hepatic total cholesterol(TC)and triglyceride(TG)were detected using commercial assay kits.The number of apoptotic cells was stained by TUNEL and fluorescein isothiocyanate(FITC)-labeled annexin V/and propidium iodide(PI)double staining in hepatocytes.Cell viability was determined using a CCK-8 kit.The homeostasis of hepatocyte mitochondria was detected by JC-1 staining,and the protein expression and activation of the target genes in hepatocytes were detected by western blot and immunofluorescence staining.2.in vivo study42 male ICR mice were randomly divided into 6 groups(7 mice/group).Mice in group 1,2,3,4,5 and 6 were respectively administrated with Ad.Fc plus saline,Ad.Fc plus alcohol(56%,v/v,lml/100g),Ad.Fc,alcohol plus DHA,Ad.pLipinl(adenovirus encoding mouse lipinl plasmid,2.5×107 pfu/g)plus alcohol,Ad.pLipinl,alcohol plus DHA,and Ad.Fc,alcohol plus Rapamycin.The hematoxylin and eosin staining was performed to observe microstructures of mice,and the expression of proinflammatory factors in the liver of mice was observed by immunohistochemical staining.Activities of serum aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),lactate dehydrogenase(LDH),TC,TG were detected using commercial assay kits.Interleukin 6,interleukin 8,tumor necrosis factor alpha,C/EBP homologous protein(CHOP)in mice serum were detected by Enzyme-linked immunosorbent assay(ELISA).Western blot and immunofluorescence staining method was performed to detect protein expression of target genes in liver tissue in mice.[results]In vitro studies,data showed that DHA could inhibit alcohol-induced steatosis in a dose dependent manner,downregulating fatty acid synthesis and upregulating the expression of?-oxidation-related genes.TUNEL staining showed that DHA could significantly inhibited the apoptosis of hepatocytes induced by alcohol,which could regulate the lipoapoptosis.Results of western blot demonstrated that activation of ER stress-JNK/CHOP-mitochondria cascade was inhibited by DHA in ethanol-treated hepatocytes.JC-1 staining also showed that DHA alleviated mitochondrial dysfunction caused by lipotoxicity.The results showed that DHA alleviated the alcohol-induced apoptosis in hepatocyte,which might be related to the inhibition of mTOR activation,and DHA can inhibit lipin-1 expression in a dose-dependent manner and regulate the nuclear translocation of lipin-1.Therefore DHA alleviated lipoapoptosis and improved the hepatocyte injury caused by alcohol,which might be related to regulation of mTOR on lipin-1.In vivo study,data showed that compared to the normal control,the fluorescence intensity of lipin-1 in model group were significantly increased The results showed that the ratio of Lpinl?/? in the liver of alcohol-treated mice was significantly elevated compared to controls.This tendency was enhanced in lipinl ?-overexpression mice but weakened in mice receiving DHA and Rapamycin.Serological and immunohistochemical results showed that DHA could significantly improve alcohol induced liver injury and inflammatory infiltration in mice,which could be weakened by lipin-1 beta overexpression and synergistic with Rapamycin.Oil red O staining was used to detect TC and TC contents.The results showed that lipin-1 overexpression weakens DHA's effect on hepatic steatosis,while Rapamycin improves liver fat metabolism disorder similar to DHA.The results of Western blot detection of genes related to fatty acid synthesis and fatty acid decomposition have also been found.Mechanism study found that lipin-1 beta can weaken the effect of DHA on the dysfunction of mitochondria and endoplasmic reticulum induced by lipotoxicity.Therefore,DHA attenuates the hepatocyte apoptosis induced by lipotoxicity,which is related to inhibiting mTOR activation and regulating lipin-1.[Conclusion]In vitro,results showed that DHA could improve the steatosis of hepatocytes induced by ethanol,and it could also improve the ethanol-induced hepatocyte injury by decreasing the apoptosis-related pathways induced by lipotoxicity,and this effect was related to the inhibitory effect of DHA on mTOR activation.In vivo studies have shown that DHA and Rapamycin could obviously inhibit the expression of lipin-1 ? and regulate the nucleocytoplasmic shutting oflipin-1.Our results proved that DHA regulated lipin-1 expression and nuclear translocation through inhibiting activation of mTOR,to improve liver injury by reducing inflammatory infiltration induced by alcohol in mice,and alleviate hepatocyte apoptosis induced by lipotoxicity by relieving steatosis of alcoholic fatty liver.