| Objective:Long-term excessive drinking results in alcoholic liver disease(ALD),excessive deposition of extracellular matrix and collagen,and extensive liver fibrosis.Liver fibrosis is a frequently-occurring disease and common disease.However,it is still urgent to develop therapeutic drug and improve clinical trials for ALD.In this study,we used EtOH and thioacetamide-induced hepatic fibrosis mouse model to study the anti-hepatic fibrosis and its underlying mechanism of Acanthopanic acid(AA)and Cucurbitacin E(CuE)in vitro and in vivo.Method:(1)Mice were randomly separated into the following six groups:normal group,EtOH group,EtOH+AA-20 group,EtOH+AA-40 group,AA-40 single group and pair-fed group,each group six mouse.Mice in normal group were fed ad libitum and water during the experiment.Mice in the AA groups were daily gavages 20 or 40 mg/kg of AA.Then,mice were allowed free access to the ethanol Lieber-DeCarli diet containing 5%(vol/vol)ethanol for 28 d.And mice in pair-fed group were pair-fed with the isocaloric control diet for 28 d.No any additional food or drinking water was provided for all groups and pair-fed group mice during the experiment.Mice were anesthetized,and blood and tissue samples were collected at 9 h after last feeding.The liver tissue was fixed in 10%formalin or kept at-80 ℃ for subsequent analysis.Hematoxylin eosin staining(H&E),Oil red staining and Nile red staining were used to detect histopathological changes.The expression of sterol regulatory element binding protein(SREBP1)and F4/80 was detected by immunohistochemistry and tissue fluorescence staining.The expressions of a-SMA,collagen I,IL-1 and AMPK upstream and downstream genes of hepatic fibrosis were detected by Western blotting and PCR.Human hepatic stellate cells LX-2 were given AMPKa-specific siRNA transfected 48 h,and then given 12.5 μM AA for 2 h.Western blotting,PCR and immunofluorescence were used to detect the expression of AMP-dependent protein kinase AMPK and SIRT1 protein,an inhibitor of interleukin-1 receptor-related kinase 4,IRAK4 protein expression.LX-2 was pretreated with 12.5 μM AA for 30 min,and 50 mM EtOH combined with 1 μg/mL LPS for 30 min.Western blotting and PCR were used to confirm the effect of AA on the expression of NLRP3 and caspasel in AMPK,IRAK4,inflammatory cytokines and NOD-like receptor family members during LX-2 cell activation.(2)Mice were randomLy separated into the following five groups:normal group,EtOH group,EtOH + AA-20 group,EtOH + AA-40 group,each group six mouse.AAadministration groups were pretreated with single dose of AA(20 or 40 mg/kg body weight,respectively)by oral gavage for 14 consecutive days.At the same time,normal group and EtOH group were administered by gavage with equal volume of saline for 14 consecutive days.Following,except normal group,other groups were exposed to three doses of ethanol(5 g/kg body weight)by gavage within 24 h.All animals were sacrificed at 9 h after the last ethanol dosing,and blood was taken from the carotid artery of the mice by ether anaesthesia.The liver tissue was fixed in 10%formalin or kept at-80℃.The protein expression of Toll-like receptor family was detected by Western blotting,and liver fibrosis and infiltration of inflammatory cytokines were analyzed by staining the pathological sections of liver.Rat hepatic stellate cells were stimulated with 200 mM EtOH and 1 g/mL LPS for 1 h.Different concentrations of acanthopanax acid were added to continue the incubation for 6 h,and the cells were harvested.Fat stain was detected by oil red staining.The expression of IL-6,TNF-α,TGF-β and MCP-1 were detected by ELISA.Western blotting was used to detect the expression of lipoprotein SREBP1,Lipin family proteins and TLR4 signaling pathway related proteins.At the same time,the expression of a-SMA,Lipin 1 and IRAK4 was detected by the cell fluorescence method.(3)Mice were randomLy divided into five groups(8 mice per group):a normal group,a TAA group,two CuE groups(5 or 10 mg/kg)and a curcumin group(Cur,20 mg/kg).Except for the normal group,all mice were intraperitoneally injected with TAA(100 mg/kg)three times in the first week,and TAA(200 mg/kg)for the subsequent four weeks at three times per week.Mice in the CuE groups and Cur group were intragastrically administered CuE(5 and 10 mg/kg)or Cur(20 mg/kg)for five weeks,respectively.H&E method,Masson staining and Sirius red staining analysis of liver fibrosis and infiltration of inflammatory cytokines.Immunohistochemical staining was used to detect the phosphorylation levels of a-SMA and Akt,AMPK in liver fibrosis.In CuE-induced activation of hepatic stellate cells,apoptosis-associated proteins BCL-2,Bax,PARP,Caspase3,phosphorylated protein p-JNK,p-p38,p-ERK and fibrosis markers a-SMA,TIMP-1,Collagen-I protein or mRNA expression.The effect of cucurbitacin E on the phosphorylation of PI3K,AKT and mTOR compared with PI3K inhibitor LY294002 was detected by Western blotting.Results:(1)Chronic alcohol led to elevated levels of ALT,AST and LPS,many fatty degenerations in liver tissue,increased expressions of a-SMA,Collagen I,IRAK and inflammatory cytokines and protein,while SIRT1,p-AMPK,p-LKB1 protein expression decreased.Pretreatment of AA can effectively reduce the expression of these factors.In vitro experiments showed that AA can significantly inhibit the release of IL-1β,IL-1α,IL18,NLRP3,IL-6,Caspase-1 and TNF-a by ethanol combined with LPS and decrease the protein and mRNA of IRAK4 expression.(2)AA can effectively reduce the levels of EtOH/LPS leading to TNFα,TGFβ,IL6 and MCP-1,and inhibit the expression of SREBP1,α-SMA and Collagen I.EtOH/LPS can lead to increased expression of Lipin-1,and the expression of Lipin-1 can be significantly decreased after pretreatment with AA.Pretreatment of AA also significantly decreased TLR4,CD14,IRAK1,IRAK4,TRAF3,p-TAK and NF-κB(p65)protein expression.In addition,EtOH/LPS significantly decreased the protein expression of SIRT1,p-LKB1,p-ACC and PPARa,and increased the expression of PPARy,which was markedly reversed after giving AA.Acute alcohol model,AA can reduce the ALT and AST,relieve fatty degeneration,reduce fat droplet aggregation,inhibition of TLR4,IRAK1,IRAK4,TRAF3,p-TAK1 and NF-κB(p65)protein expression.(3)In the model of liver fibrosis induced by TAA,CuE can reduce TAA-induced phosphorylation of Akt、mTOR、PI3K and P70S6K in vivo,decrease the expression of fibrosis marker α-SMA,TIMP-1 and Collagen-I and increase the phosphorylation of AMPK.Pathologically reduces hepatocyte degeneration and the presence of collagen fibers.Cucurbitacin E inhibited the proliferation of t-HSC cells in a concentration-and time-dependent manner.CuE inhibits the cleavage of caspase 1 and its substrate in a concentration-and time-dependent manner,modulates the ratio of BCL-2 to Bax,increases the expression of cytochrome C,and binds annexin-V and propidium iodide as fluorescent probes Flow cytometry confirmed that 12.5 μM CuE induced t-HSC cells apoptosis.CuE inhibited the phosphorylation of TNF-a-stimulated ERK in a concentration-and time-dependent manner and inhibited the protein and mRNA expression of α-SMA,TIMP-1 and Collagen I.12.5 μM CuE,like AICAR and metformin,significantly up-regulated phosphorylation of AMPK,down-regulated the expression of p-mTOR and inhibited the expression of p-PI3K and p-Akt with the same effect as LY294002.Conclusion:AA and CuE showed good regulation on hepatic fibrosis:1.AA regulates hepatic injury induced by chronic alcohol by regulating LKB1-AMPK signaling pathway and activating SIRT1,especially by inhibiting IRAK1/4 signaling pathway to regulate the body’s fatty acid balance and the occurrence of inflammatory reactions.2.AA can inhibit hepatic fibrosis and lipid deposition in acute alcoholic mice and regulate the activation of alcohol and LPS in vitro to stimulate the activation of HSC-T6 cells.3.CuE can modulate PI3K/Akt,AMPK and mTOR signaling to improve TAA-induced hepatic fibrosis.4.CuE can effectively reduce the hepatic stellate cell activity.CuE inhibited the phosphorylation of PI3K/Akt,activated AMPK phosphorylation,decreased the deposition of extracellular matrix and the protein expression of a-SMA and Collagen I.However,as an effective medicine,they need further study to determine the best and most accurate anti-liver fibrosis drugs. |
PDF Full Text Request