Tetrandrine stimulates the apoptosis of hepatic stellate cells and ameliorates development of fibrosis in a thioacetamide rat model

AIM: To investigate the therapeutic effect of tetrandrine on liver fibrosis induced by thioacetamide in rats in vivo and in vitro. METHODS: In vitro study: we investigated the effect of tetrandrine on the apoptosis of rat hepatic stellate cells transformed by simian virus 40 (T-HSC/Cl-6), which retains the features of activated cells. In vivo study: hepatic fibrosis was induced in rats by thioacetamide. Tetrandrine was given orally to rats at doses of 5, 10 or 20 mg/kg for 4 wk compared with intraperitoneal injection of interferon-г. RESULTS: In vitro study: 5, 10 or 25 μg/mL of tetrandrine-induced activation of caspase-3 in t-HSC/Cl-6 cells occurred dose-dependently. In vivo study: tetrandrine treatment as well as interferon-г significantly ameliorated the development of fibrosis as determined by lowered serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (T-Bil) and the levels of liver hydroxyproline (Hyp), hyaluronic acid (HA), laminin (LN) and also improved histological findings. The effects of tetrandrine at the concentration of 20 mg/kg were better than the other concentration groups. CONCLUSION: Tetrandrine promotes the apoptosis of activated HSCs in vitro . Tetrandrine administration can prevent liver fibrosis and liver damage induced bythioacetamide in rats in vivo, indicating that it might exert a direct effect on rat HSCs.

Gentiopicroside ameliorates LKB1/AMPK-dependent alcoholic hepatosteatosis via P2x7R-NLRP3 inflammasome

OBJECTIVE Regulating P2x7R-NLRP3 inflammasome activation might be a potential therapeutic strategy to treat alcoholic hepatosteatosis.We investigated whether this process would be modulated by gentiopicroside(GPS),which is attributed to the bitterness of gentian root extract.METHODS An in vivo model was established by intragastrically treating mice with ethanol,and an in vitro model was created by treating HepG2 cells with ethanol or treating RAW 264.7 macrophages and murine bone marrow-derived macrophages(BMDMs) with lipopolysaccharides(LPS) plus adenosine triphos.phate(ATP).RESULTS In alcoholic hepatosteatotic mice model,GPS decreased serum aminotrans.ferase and triglyceride accumulation.GPS regulated sterol regulatory element-binding protein-1(Srebp1),peroxisome proliferators-actived receptors α(PPARα) and acetyl CoA carboxylase(ACC) expression via elevating liver kinase B1(LKB1)/AMP-activated Kinase(AMPK).Suppression of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),caspase-1 and expression by GPS resulted in the inhibition of interleukin-1β(IL-1β) production.In ethanol-exposed HepG2 cells,GPS reduced lipo.genesis and promoted lipid oxidation via P2x7R-NLRP3 inflammasome activation.P2x7R silencing enhanced AMPK activity,and reduced Srebp1 expression in ethanol-treated hepatocytes.GPS down.regulated P2x7R-mediated inflammatory response to extracellular ATP in LPS-primed RAW 264.7 macro.phages and BMDMs.Additionally,P2x7R deficiency attenuated IL-1β cleavage in RAW 264.7 macro.phages,and GPS further suppressed IL-1β cleavage.CONCLUSION Activation of LKB1/AMPK signaling by GPS might be mediated by P2x7R-NLRP3 inflammasome,suggesting a therapeutic utility of P2x7R blockade in alcoholic hepatosteatosis treatment.

Studies on the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells

OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were treated with SRT1720(10μmol·L^(-1))and AICAR(500μmol·L^(-1))prior to ethanol(50 mmol·L^(-1)) for 24 and 48 h.Cell viability was analyzed by methyl thiazolyl tetrazolium assay.SIRT1,AMPK and p-AMPK m RNA levels for 24 h and 48 h were analyzed by RT-PCR,SIRT1,AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot.RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells.SRT1720 and AICAR attenuated collagen-I,α-smooth muscle actin(α-SMA)levels,activated liver kinase B-1(LKB1)and AMPK phosphorylation in ethanol treated LX-2 cells.Meanwhile,SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells.Furthermore,SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1(SREBP-1)to regulate fatty acid synthesis.CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.

Pleurotus citrinopileatus alleviates alcohol-induced fatty liver via upregulating AMPK signaling

OBJECTIVE The current study was designed to investigate the anti-steatosis effect of Pleurotus citrinopileatus extract(PC) and the underlying mechanism in vivo and in vitro.METHODS Acute and chronic alcoholic hepatosteatosis murine models and ethanol-treated HepG2 cells were applied.RESULTS In vitro,the anti-steatosis effect of PC was further confirmed via Nile red staining in HepG2 cells treated with ethanol.Both of acute and chronic alcohol-induced mice hepatosteatosis model,PC decreased serum aminotransferase and triglyceride accumulation.Upregulated sterol-regulatory element binding protein1(Srebp1),purinergic ligand-gated ion channel 7 receptor(P2 X7 R) and downregulated sirtuin1(SIRT1),adenosine 5′-monophosphate(AMP)-activated protein kinase α(AMPKα) caused by acute and chronic alcohol intake were modulated by PC.In ethanol-exposed HepG2 cells,PC reduced lipid accumulation in a concentration-dependent manner and exhibited superior ability in controlling lipid accumulation compared with metformin.CONCLUSION PC could abolish hepatic lipid accumulation through regulating SIRT1-AMPKα signaling in acute and chronic alcohol-induced hepatic steatosis.

Toll-like receptor 4-related regulation of Ornithogalum caudatum extract on inflammatory responses in LPS activated macrophages

OBJECTIVE To investigate toll-like receptor 4(TLR4)-related the regulation of Ornithogalum caudatum extract(OCE) on inflammatory responses in lipopolysaccharide(LPS) activated macro.phages.METHODS Primary peritoneal macrophage,Raw 264.7,and THP-1 were incubated in 96-well plate for 24 h and treated with OCE of the concentration of 0-400 μg/ml for 4 h.The viability of cells was measured by MTT assay.Specific concentrations of OCE were added into the medium of primary peri.toneal macrophage,Raw 264.7,and THP-1,respectively,then following with lipopolysaccharides(LPS).Cells were harvested and the total cellular protein and nuclear protein were extracted,and the protein content was determined using BCA protein assay Kit.The expressions of TLR4,inducible nitric oxide synthase(iNOS),cyclooxygenase 2(COX-2),α-inhibitor of NF-κB(IκB-α) and nuclear factor-κB(NF-κB) were assayed by Western blot.The expressions of interleukin-1α(IL-1α),interleukin-1β(IL-1β),interleukin-18(IL-18),and tumor necrosis factor-α(TNF-α) were measured by RT-PCR.RESULTS The results of MTT showed that OCE has no cytotoxicity in Raw 264.7 cells between 1.56 μg/ml and 400 μg/ml.Compared with normal group,the expressions of TLR4,iNOS,COX-2,NF-κB and IL-1α,IL-1β,IL-18,TNF-α,the level of nitric oxide(NO) were significantly increased by LPS stimulation,while OCE pretreat.ment reduced these increase induced by LPS.However,OCE pretreatment reversed the reduction of IκB-α after LPS stimulation.CONCLUSION OCE might suppress TLR4 expression and block the inflamma.tion process of NF-κB and iNOS,further decrease the expression of COX-2 and inhibit the release of inflammatory factors.

Hepato-protective effect of thymoquinone against acetaminophen induced liver injury is associated with regulation of JNK and AMPK signaling pathway

OBJECTIVE To investigate the hepato-protective mechanism of thymoquinone(TQ) on the development of acetaminophen(APAP)-induced liver injury.METHODS In vivo,male kunming mice were injected with a single dose of 300 mg·kg^(-1) APAP.Some mice were pretreated with TQ(5 or 20 mg·kg^(-1))and N-acetylcysteine(NAC,300 mg·kg^(-1))2 h before APAP injection.Mice were euthanized at 2 h,6 h,12 h after APAP treatment.In vitro,human Chang liver cells were incubated with 3.125,6.25 or 12.5μmol·L^(-1) TQ,10μmol·L^(-1) SP600125 and 500μmol·L^(-1) AICAR in the presence of APAP for 24 h.Cell viability were analyzed by MTT assay,protein expressions were assessed by Western blot.RESULTS TQ pretreatment significantly reduced serum aminotransferase and increased hepatic glutathione(GSH)and glutathione peroxidase(GSH-PX)activities,while significantly inhibited interleukin-1β(IL^(-1)β)levels.TQ significantly inhibited c-Jun N-terminal kinase(JNK),extracellular signal regulated kinase(ERK)and P38 phosphorylation induced by APAP.Moreover,TQ inhibited phosphatidylinositol 3-kinase(PI3K)/mammalian target of rapamycin(m TOR)signaling activation and activated AMPK phosphorylation induced by APAP.In addition,TQ inhibited signal transducer and activator of transcription 3(STAT3)phosphorylation on APAP-induced liver injury.In vitro,APAP enhanced JNK phosphorylation and attenuated AMPK phosphorylation in Chang liver cel s,and these effects were blocked by pretreatment with TQ,SP600125(JNK inhibitor)and AICAR(AMPK activator).CONCLUSION Our findings suggest that TQ may actively prevent APAP-induced liver injury,and this effect may be mediated by JNK and AMPK signaling pathways.

Dihydroquercetin ameliorates alcoholic liver steatosis through P2x7R-NLRP3 inflammasome pathway

OBJECTIVE Dihydroquercetin(TAX) is the most abundant dihydroflavone found in on.ions,milk thistle and Douglas fir bark.We investigated whether TAX could inhibit the lipid accumulation in alcoholic liver steatosis in vivo and in vitro.METHODS An in vivo model was established by intragas.trically treating mice with ethanol,and an in vitro model was created by treating HepG2 cells with etha.nol.RESULTS TAX regulated Sterol Regulatory Element-binding Protein-1(SREBP1) and Acetyl CoA Carboxylase(ACC) expression via elevating Liver Kinase B1(LKB1)/AMP-activated Kinase(AMPK)phosphorylation.Also,TAX upregulated SIRT1 expression,which suppressed by ethanal intake.Suppression of Purinergic 2X7 receptor(P2x7R),nucleotide-binding oligomerization domain-like re.ceptor protein 3(NLRP3) and Cysteine protease-1(caspase-1) cleavage by TAX resulted in the inhibi.tion of Interleukin-1β(IL-1β) production and release.Additionally,TAX reduced lipogenesis and pro.moted lipid oxidation via the regulation of AMPK and ACC in ethanol-treated steatotic HepG2 cell.TAX downregulated IL-1β cleavage response to Lipopolysaccharides(LPS) plus adenosine triphosphate(ATP) stimulation in HepG2 cells.P2x7R deficiency attenuated lipid accumulation with increasing AMPK activity and decreasing SREBP1 expression in ethanol-treated HepG2 cells.CONCLUSION Our data showed that TAX exhibited the inhibitory properties on lipogenesis and hepatoprotective ca.pacity,indicating that TAX has therapeutic potential for preventing alcoholic liver steatosis.