The Effect And Mechanism Of Berberine On Improving Nonalcoholic Fatty Liver Disease

Background: Non-alcoholic fatty liver disease(NAFLD)is genetic-environment-metabolic stress related diseases.At present,its morbidity exceeds the chronic viral liver diseases,becoming the most common chronic liver diseases worldwide and without targeted therapy for now.The NAFLD spectrum includes simple steatosis,non-alcoholic steatohepatitis,liver fibrosis,cirrhosis and even hepatocellular carcinoma.Sphingosine-1-phosphate(S1P)is one of the metabolic products of the sphingomyelin,it is catalyzed by sphingosine kinase1/2 in cells.S1 P is an important signaling molecule and plays a critical role in regulating cellular inflammatory reaction and lipid metabolism.It can act directly as an intracellular signaling molecule or through G-protein coupled receptors.Sphingosine-1-phosphate receptors(S1PRs)belong to the G protein coupled receptor family,and widely expressed in the liver and intestinal tissue.S1 P binding to the S1 PRs can initiate cell transmembrane signal transduction,regulate cell growth in apoptosis,angiogenesis and other physiological and pathological processes.Berberine(BBR,C20H18NO4),an isoquinoline quaternary alkaloid isolated from many medicinal herbs,has been widely used in Chinese Medicine for more than thousands of years.During the past decades,BBR are widely used for the treatment of several afflictions,such as intestinal complications and infections.What’s more,BBR has reliable efficacy and excellent safety.Currently,the research on the pathogenesis and treatment of NAFLD is a hot and difficult topic in the world.The treatment of NAFLD is mainly to adjust lifestyle and control the combined diseases,such as obesity,hyperlipidemia,hyperglycemia and hypertension.The medicine of NAFLD now is lack of targeting.It thus appears that multiple mechanisms are involved in the therapeutic effect of BBR,However,the functional mechanisms underlying the beneficial effects of BBR are still unknown.Therefore,there are the core issues that studies should clarify the pathogenesis of NAFLD,find and develope the targeted and effective clinical drugs for NAFLD.Aims: 1.To investigate the protective effect of berberine on high fat diet(HFD)-induced hepatic lipotoxicity and inflammatory injury in in vivo and in vitro.2.To explore its potential mechanisms of S1P/S1PR2 regulating the PA induced lipid deposition in hepatocytes,providing new ideas for the clinical targeting drugs.Methods: Thirty male C57BL/6J mice were randomly assigned to three groups,then fed with a standard diet or a HFD or HFD plus berberine(200 mg/kg per day intragastrically)for 12 weeks and record the weight of mice weekly.1.At the end of the 12 th week,the mice were fasted overnight,blood was taken from eyeballs,serum biochemical indices including ALT,AST,TC,TG and glucose were measured by biochemical analyzer.2.HE staining was used to observe liver inflammation,and fatty infiltration was stained with oil red.3.Ileocecal feces were collected and the composition of gut microbiota was analyzed by 16 S r RNA sequencing.Serum level of LPS,S1 P and hepatic levels of LPS,TNF-α and IL-6 were examined by ELISA.4.In vitro,the primary hepatic macrophages of C57BL/6J mice challenged with palmitic acid and LPS,respectively was used to study the effect of berberine.The ultrastructure of macrophages was observed using electron microscope,and the proinflammatory cytokines were examined by real-time PCR.5.Primary mice hepatocyte monolayer cultures were prepared from male C57BL/6J mice,and Hep G2 cells were used in this experiment;four groups were designed: blank control group,0.5 m M PA group,100 n M S1P+0.5 m M PA group and 10 μM JTE013 +0.5 m M PA group,cells were treated for 24 hours,oil red staining was used to specifically stain the intracellular fat.The difference between each group was compared with the absorption of OD.6.RNA and total protein were extracted from the mouse MPH,and the expression of S1PR2 m RNA was detected by RT-PCR,western blotting(WB)detected the protein expression of S1PR2 in the MPH.7.MPH were pre-treated with S1 P or JTE013 for 0.5 h,then PA treated 0.5 h,the expression of ERK1/2 and p-ERK were detected on protein by WB.Results: 1.The weight of HFD-mice increased significantly from sixth weeks,the mice weighs an average of 34.4±3.3 g at the end of the 12 th week.The body weight of the treatment group was significantly lower than that of the control group.2.In vivo,Both ALT,AST,TG,TC and glucose were increased in HFD-mice,the treatment of berberine improved the liver biochemical dysfunction,hyperlipidemia and hyperglycemia in mice fed with HFD(P<0.05).3.HE and oli red O staining of liver tissue shows that the morphology and structure of hepatocytes were intact,and no lipid droplets were found in control group’s hepatocytes;In HFD group,hepatocytes were steatosis,with different sizes of globular lipid droplets in the cytoplasm,inflammatory cells infiltration;Berberine significantly inhibited the HFD-induced hepatic lipid accumulation,and alleviated hepatocytes ballooning and lobular inflammatory cell infiltration.4.16 S r RNA sequencing showed that berberine supplementation restored the abundance of Bacteroidaceae,Bifidobacteriaceae and Desulfovibrionaceae in ileocecus which were disturbed by HFD.5.ELISA tests showed the levels of serum LPS and hepatic LPS,IL-6 were markedly decreased in mice treated with berberine.The levels of serum S1 P were elevated in BBR group(P<0.01).6.In vitro,electron microscope showed that after the treatment of PA,the primary hepatic macrophages had obvious lipid deposition in the cells,lipid droplets in macrophages decreased significantly in BBR group.The results of RT-PCR showed that berberine significantly decreased the expressions of TNF-α and IL-6 in macrophages.7.ELISA results showed that the serum level of S1 P in high fat diet group was significantly higher than that of the control group,and berberine can effectively reduce the level of S1 P in mice.There was no significant change in the level of S1 P of liver tissue(P>0.05).8.The results of oil red O staining and OD detection were all shown PA can obviously induce lipid deposition in the MPH,and lots of irregular lipid droplets appear in the cytoplasm.After JTE013 treatment,the accumulation of lipid droplets were significantly reduced in MPH.9.RT-PCR showed that PA obviously increased the S1PR2 m RNA expression in MPH,however JTE013 clearly reduced the expression of S1PR2 m RNA.WB showed that S1 P and PA stimulated the expression of S1PR2 in MPH,then JTE013 effectively decreased S1PR2 expression compared with the blank control group.10.WB analysis and Image J showed that compared with the control group,PA significantly raised the protein expression of p-ERK in MPH,and JTE013 could inhibit the expression of p-ERK.Conclusion: 1.Berberine can effectively ameliorate the hepatic lipid accumulation and inflammation in mice with experimental NAFLD.2.The mechanism of ameliorate inflammation might be related with regulating gut microbiota,reducing LPS production,and subsequently inhibiting the release of inflammatory cytokines by hepatic macrophages.3.Berberine can significantly reduce the level of S1 P in the serum of C576L/6J mice induced by high fat diet.The mechanism of PA induce the excessive lipid deposition in hepatocytes may be through S1P/S1PR2/ERK related signaling pathway.