C23 ameliorates carbon tetrachloride-induced liver fibrosis in mice

BACKGROUND C23,an oligo-peptide derived from cold-inducible RNA-binding protein(CIRP),has been reported to inhibit tissue inflammation,apoptosis and fibrosis by binding to the CIRP receptor;however,there are few reports on its role in liver fibrosis and the underlying mechanism is unknown.AIM To explore whether C23 plays a significant role in carbon tetrachloride(CCl4)-induced liver fibrosis.METHODS CCl4 was injected for 6 weeks to induce liver fibrosis and C23 was used beginning in the second week.Masson and Sirius red staining were used to examine changes in fiber levels.Inflammatory factors in the liver were detected and changes inα-smooth muscle actin(α-SMA)and collagen I expression were detected via immu-nohistochemical staining to evaluate the activation of hematopoietic stellate cells(HSCs).Western blotting was used to detect the activation status of the trans-forming growth factor-beta(TGF-β)/Smad3 axis after C23 treatment.RESULTS CCl4 successfully induced liver fibrosis in mice,while tumor necrosis factor-alpha(TNF-α),IL(interleukin)-1β,and IL-6 levels increased significantly and the IL-10 level decreased significantly.Interestingly,C23 inhibited this process.On the other hand,C23 significantly inhibited the activation of HSCs induced by CCl4,which inhibited the expression ofα-SMA and the synthesis of collagen I.In terms of mechanism,C23 can block Smad3 phosphorylation significantly and inhibits INTRODUCTION At present there is no specific and effective drug for treating liver fibrosis caused by acute or chronic injury.Although preclinical research has made breakthroughs,their suitability as clinical treatments is still unknown.The activation of hepatic stellate cells(HSCs)caused by chronic inflammation is a key process in the development of liver fibrosis and activated HSCs expressα-smooth muscle actin(α-SMA)and transdifferentiate into myofibroblasts with proliferation,migration and secretion abilities,synthesizing the extracellular matrix to deposit in the hepatocyte space and subse-quently forming liver fibrosis[1].Although therapeutic strategies have improved due to past few efforts there is no ideal treatment for hepatic fibrosis[2].Extracellular cold inducible RNA binding protein(CIRP)has been shown to play a role in various acute and chronic inflammatory diseases by promoting tissue inflammation and apoptosis and inducing fibrosis through its receptor Toll-like receptor 4(TLR4)[3].C23 is a recognized competitive inhibitor of CIRP that can competitively bind to CIRP receptors and reduce tissue damage in inflammatory diseases[4].C23 has been shown to significantly reduce serum tumor necrosis factor-alpha(TNF-α),IL(interleukin)-6 and IL-1βlevels.In addition,it can reduce tissue TLR4,TNF-α,IL-6 and IL-1βlevels and inhibit the colocalization of CIRP and TLR4,which plays a significant role in systemic inflammation[5].Re-search has shown that CIRP induces the inflammatory phenotype of lung fibroblasts in a TLR4-dependent manner[6].On the other hand,CIRP is associated with markers of fibrosis andα-SMA is significantly positively correlated with CIRP.Cirp-/-mice exhibit attenuated expression ofα-SMA and collagen(COL1A1 and COL3A1),decreased hydroxyproline content,decreased histological fibrosis scores,and improved pulmonary hypertension[7].C23 inhibited the release of TNF-α,the degradation of IκB and the nuclear translocation of NF-κB in CIRP-stimulated macrophages in a dose-dependent manner and C23 treatment significantly increased the serum levels of lactic dehydrogenase,alanine ami-notransferase,IL-6,TNF-αand IL-1βin septic CLP mice[8].Based on previous research we hypothesized that C23 might alleviate liver fibrosis by inhibiting acute and chronic inflammation.As a selective hepatotoxic chemical carbon tetrachloride(CCl4).can induce inflammation and activate HSCs,promoting liver fibrosis.This study reveals the role and mechanism of C23 in CCl4-induced liver fibrosis in mice.at room temperature for 30 minutes.The gray value of each group was calculated after chemiluminescence.

Yinhuang granule alleviates carbon tetrachloride-induced liver fibrosis in mice and its mechanism

BACKGROUND Liver fibrosis is a formidable global medical challenge,with no effective clinical treatment currently available.Yinhuang granule(YHG)is a proprietary Chinese medicine comprising Scutellariae Radix and Lonicerae Japonicae Flos.It is frequently used for upper respiratory tract infections,pharyngitis,as well as acute and chronic tonsillitis.AIM To investigate the potential of YHG in alleviating carbon tetrachloride(CCl4)-induced liver fibrosis in mice.METHODS To induce a hepatic fibrosis model in mice,this study involved intraperitoneal injections of 2 mL/kg of CCl4 twice a week for 4 wk.Meanwhile,liver fibrosis mice in the low dose of YHG(0.4 g/kg)and high dose of YHG(0.8 g/kg)groups were orally administered YHG once a day for 4 wk.Serum alanine/aspartate aminotransferase(ALT/AST)activity and liver hydroxyproline content were detected.Sirius red and Masson's trichrome staining assay were conducted.Realtime polymerase chain reaction,western-blot and enzyme-linked immunosorbent assay were conducted.Liver glutathione content,superoxide dismutase activity level,reactive oxygen species and protein carbonylation amount were detected.RESULTS The administration of YHG ameliorated hepatocellular injury in CCl4-treated mice,as reflected by decreased serum ALT/AST activity and improved liver histological evaluation.YHG also attenuated liver fibrosis,evident through reduced liver hydroxyproline content,improvements in Sirius red and Masson's trichrome staining,and lowered serum hyaluronic acid levels.Furthermore,YHG hindered the activation of hepatic stellate cells(HSCs)and ameliorated oxidative stress injury and inflammation in liver from CCl4-treated mice.YHG prompted the nuclear accumulation of nuclear factor erythroid 2-related factor 2(Nrf2)and upregulated the expression of Nrf2-dependent downstream antioxidant genes.In addition,YHG promoted mitochondrial biogenesis in liver from CCl4-treated mice,as demonstrated by increased liver adenosine triphosphate content,mitochondrial DNA levels,and the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha and nuclear respiratory factor 1.CONCLUSION YHG effectively attenuates CCl4-induced liver fibrosis in mice by inhibiting the activation of HSCs,reducing inflammation,alleviating liver oxidative stress damage through Nrf2 activation,and promoting liver mitochondrial biogenesis.

Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells

BACKGROUND Liver fibrosis is the common pathological process associated with the occurrence and development of various chronic liver diseases.At present,there is still a lack of effective prevention and treatment methods in clinical practice.Hepatic stellate cell(HSC)plays a key role in liver fibrogenesis.In recent years,the study of liver fibrosis targeting HSC autophagy has become a hot spot in this research field.Angiotensin-converting enzyme 2(ACE2)is a key negative regulator of reninangiotensin system,and its specific molecular mechanism on autophagy and liver fibrosis needs to be further explored.AIM To investigate the effect of ACE2 on hepatic fibrosis in mice by regulating HSC autophagy through the Adenosine monophosphate activates protein kinases(AMPK)/mammalian target of rapamycin(mTOR)pathway.METHODS Overexpression of ACE2 in a mouse liver fibrosis model was induced by injection of liver-specific recombinant adeno-associated virus ACE2 vector(rAAV2/8-ACE2).The degree of liver fibrosis was assessed by histopathological staining and the biomarkers in mouse serum were measured by Luminex multifactor analysis.The number of apoptotic HSCs was assessed by terminal deoxynucleoitidyl transferase-mediated dUTP nick-end labeling(TUNEL)and immunofluorescence staining.Transmission electron microscopy was used to identify the changes in the number of HSC autophagosomes.The effect of ACE2 overexpression on Wu Y et al.ACE2 improves liver fibrosis through autophagy WJG https://www.wjgnet.com 4976 September 7,2023 Volume 29 Issue 33 autophagy-related proteins was evaluated by multicolor immunofluorescence staining.The expression of autophagy-related indicators and AMPK pathway-related proteins was measured by western blotting.RESULTS A mouse model of liver fibrosis was successfully established after 8 wk of intraperitoneal injection of carbon tetrachloride(CCl4).rAAV2/8-ACE2 administration reduced collagen deposition and alleviated the degree of liver fibrosis in mice.The serum levels of platelet-derived growth factor,angiopoietin-2,vascular endothelial growth factor and angiotensin II were decreased,while the levels of interleukin(IL)-10 and angiotensin-(1-7)were increased in the rAAV2/8-ACE2 group.In addition,the expression of alpha-smooth muscle actin,fibronectin,and CD31 was down-regulated in the rAAV2/8-ACE2 group.TUNEL and immunofluorescence staining showed that rAAV2/8-ACE2 injection increased HSC apoptosis.Moreover,rAAV2/8-ACE2 injection notably decreased the number of autophagosomes and the expression of autophagy-related proteins(LC3I,LC3II,Beclin-1),and affected the expression of AMPK pathway-related proteins(AMPK,p-AMPK,p-mTOR).CONCLUSION ACE2 overexpression can inhibit HSC activation and promote cell apoptosis by regulating HSC autophagy through the AMPK/mTOR pathway,thereby alleviating liver fibrosis and hepatic sinusoidal remodeling.

Intra-portal transplantation of bone marrow stromal cells ameliorates liver fibrosis in mice

BACKGROUND: Bone marrow cells can differentiate into hepatocytes in a suitable microenvironment. This study was undertaken to investigate the effects of transplanted bone marrow stromal cells (BMSCs) on liver fibrosis in mice. METHODS: BMSCs were harvested and cultured from male BALB/c mice, then transplanted into female syngenic BALB/c mice via the portal vein. After partial hepatectomy, diethylnitrosamine (DEN) was administered to induce liver fibrosis. Controls received BMSCs and non-supplemented drinking water, the model group received DEN with their water, and the experimental group received BMSCs and DEN. Mice were killed after 3 months, and ALT, AST, hyaluronic acid (HA), and laminin (LN) in serum and hydroxyproline (Hyp) in the liver were assessed. Alpha-smooth muscle actin (alpha-SMA) in the liver was assessed by immunohistochemistry. Bone marrow-derived hepatocytes were identified by fluorescent in situ hybridization (FISH) in liver sections. RESULTS: BMSCs were shown to differentiate into hepatocyte-like phenotypes after hepatocyte growth factor treatment in vitro. Serum ALT, AST, HA, and LN were markedly reduced by transplanted BMSCs. Liver Hyp content and alpha-SMA staining in mice receiving BMSCs were lower than in the model group, consistent with altered liver pathology. FISH analysis revealed the presence of donor-derived hepatocytes in the injured liver after cross-gender mouse BMSC transplantation. After three months, about 10% of cells in the injured liver were bone marrow-derived. CONCLUSION: BMSCs transplanted via the portal vein can convert into hepatocytes to repair liver injury induced by DEN, restore liver function, and reduce liver fibrosis.

Transplantation of fetal liver epithelial progenitor cells ameliorates experimental liver fibrosis in mice

AIM： To investigate the effect of transplanted fetal liver epithelial progenitor （FLEP） cells on liver fibrosis in mice. METHODS： FLEP cells were isolated from embryonal day （ED） 14 BALB/c mice and transplanted into female syngenic BALB/c mice （n = 60）. After partial hepatectomy （PH）, diethylnitrosamine （DEN） was administered to induce liver fibrosis. Controls received FLEP cells and non-supplemented drinking water, the model group received DEN-spiked water, and the experimental group received FLEP cells and DEN. Mice were killed after 1, 2, and 3 mo, and alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, hyaluronic acid （HA）, and laminin （LN） in serum, and hydroxyproline （Hyp） content in liver were assessed. Alpha-smooth muscle actin （α-SMA） of liver was tested by immunohistochemistry. Transplanted male mice FLEP cells were identified by immunocytochemistry for sty （sex determination region for Y chromosome） protein. RESULTS： Serum ALT, AST, HA, and LN were markedly reduced by transplanted FLEP cells. Liver Hyp content and （α-SMA staining in mice receiving FLEP cells were lower than that of the model group, which was consistent with altered liver pathology. Transplanted cells proliferated and differentiated into hepatocytes and bile duct epithelial cells with 30%-50% repopulation in the liver fibrosis induced by DEN after 3 mo. CONCLUSION： Transplanted FLEP cells proliferate and differentiate into hepatocytes and bile duct epithelial cells with high repopulation capacity in the fiberized liver induced by DEN, which restores liver function and reduces liver fibrosis.

Leptin administration exacerbates thioacetamide-induced liver fibrosis in mice

AIM： To investigate the effects of leptin administration on liver fibrosis induced by thioacetamide （TAA）. METHODS： Twenty-four male C57BI/6 mice were randomly allocated into four groups, which were intra-peritoneally given saline （2 mL/kg）, leptin （1 mg/kg）, TAA （200 mg/kg）, TAA （200 mg/kg） plus leptin （1 mg/kg） respectively, thrice a week. All mice were killed after 4 wk. The changes in biochemical markers, such as the levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in serum and superoxide dismutase （SOD）, malondialdehyde （MDA） in liver were determined. For histological analysis, liver tissues were fixed with 10% buffered formalin, embedded with paraffin. Hematoxylin-eosin （HE） staining and picric acid-Sirius red dyeing were performed. The level of α1（I） procollagen mRNA in liver tissues was analyzed by RT-PCR. RESULTS： Apparent liver fibrosis was found in TAA group and TAA plus leptin group. Compared to saline group, the levels of ALT and AST in serum and MDA in liver increased in TAA group （205.67±27.69 U/L vs 50.67±10.46 U/L, 177.50±23.65 U/L vs76.33±12.27 U/L, 2.60±0.18 nmol/mg pro vs 1.91±0.14 nmol/mg pro, P〈0.01） and in TAA plus leptin group （256.17±22.50 U/L vs 50.67±10.46 U/L, 234.17±27.37 U/L vs76.33±12.27 U/L, 2.97±0.19 nmol/mg pro vs 1.91±0.14 nmol/mg pro, P〈0.01）. The level of SOD in livers decreased （51.80±8.36 U/mg pro vs 81.52±11.40 U/mg pro, 35.78±6.11 U/mg pro vs81.52± 11.40 U/mg pro, P〈0.01） and the level of α1（I） procollagen mRNA in liver tissues also increased （0.28±0.04 vs0.11± 0.02, 0.54±0.07 vs0.11±0.02, P〈0.01）. But no significant changes were found in leptin group and saline group. Compared to TAA group, ALT, AST, MDA, and α1（I） procollagen mRNA and grade of liver fibrosis in TAA plus leptin group increased （256.17±22.50 U/L vs 205.67± 27.69 U/L, P〈0.05; 234.17±27.37 U/L vs 177.50±23.65 U/L, P〈0.05; 2.97±0.19 nmol/mg pro vs 2.60±0.18 nmol/mgpro, P〈0.05, 0.54±0.07 vs0.28±0.04, P〈0.01, 3.17 vs 2.00, P〈0.05）, and the level of SOD in liver decreased （35.78±6.11 U/rag pro vs 51.80±8.36 U/rag pro, P〈0.05）. There were similar changes in the degree of type I collagen deposition confirmed by picric acid-Sirius red dyeing. CONCLUSION： Leptin can exacerbate the degree of TAA - induced liver fibrosis in mice. Leptin may be an important factor in the development of liver fibrosis.

cytoplasmic domain of tissue factor promotes liver fibrosis in mice

To evaluate the role of tissue factor (TF) and protease activated receptor (PAR)-2 in liver fibrosis. METHODSUsing CCl4 administration for eight weeks, we induced hepatic fibrosis in wild-type C57BL/6 mice and in mice with deletion of the cytoplasmic signalling domain of TF (TF§CT/§CT), deletion of PAR-2 (PAR-2-/-) and combined deletion of TF signalling domain and PAR-2 (TF§CT/§CT/PAR-2-/-). Hepatic fibrosis area was assessed by quantitative imaging of picrosirius red staining. Hepatic collagen content was assessed by hydroxyproline levels. Hepatic stellate cells (αSMA positive) and hepatic macrophages (CD68 positive) were identified by immunohistochemistry. Hepatic gene expression was determined by PCR and liver TGFβ1 content by ELISA. RESULTSCCl4 treated mice with deletion of the PAR-2 gene (PAR-2-/-) and the cytoplasmic domain of TF (TF§CT/§CT) developed significantly less hepatic fibrosis, characterised by reduced liver fibrosis area and hydroxyproline content, compared to control wildtype mice treated with CCl4. The observed reduction in histological fibrosis was accompanied by a significant decrease in the hepatic content of TGFβ, the prototypic fibrogenic cytokine, as well as fewer activated hepatic stellate cells and hepatic macrophages. Deletion of the TF cytoplasmic signalling domain reduced hepatic fibrosis to levels similar to those observed in mice lacking PAR-2 signalling but combined deletion provided no added protection against fibrosis indicating a lack of mutual modulating effects that have been observed in other contexts such as angiogenic responses. CONCLUSIONTissue factor cytoplasmic domain is involved in TF-PAR-2 signalling initiating hepatic fibrosis and is a potential therapeutic target, as its deletion would not impact coagulation.

Dynamic Observation of Liver Fibrosis in Mice Infected with Schistosoma japonica

Summary： The expression of TNF-α in the liver at different periods post Schistosoma japonica infection and the effect on liver fibrosis after supplementary injection of these cytokines were investigated. The mice infected with schistosome cercariae were divided into 3 groups., normal control group, TNF-α-untreated infection group and TNF-α-treated infection group. ABC immunohistochemistry and pathologic image multimedia quantification system were applied to dynamically detect the activity of TNF-α. The results showed that the levels of TNF-α in the liver in TNF-α-untreated infection group were slowly decreased with prolongation of infection time （from 8th, 11th, 14th to 18th week）, while in the TNF-α-treated infection group, those were increased significantly after intraperitoneal injection of TNF-α at 6th week after infection. At first to 8th week after the final injection of TNF-α, the intrahepatic TNF-α levels in the TNF-α-treated infection group were significantly higher than in the other two groups （P〈0.01）, and the granulomatous inflammation and fibrosis in the liver were also milder in the normal control group. It was concluded that at the early stage of Schistosoma japonica infection mouse liver mainly released Thl cytokine and TNF-α from Thl activated macrophages. Six weeks after infection （post egg deposition）, exogenous supplement with intraperitoneal injection of TNF-α could induce the enhanced expression of Thl cytokines and alleviate the liver granulomatous inflammation and fibrosis.

Growth and Reproduction Characteristics of TLR4 Knockout Mice Used for Liver Fibrosis Experiments

[Objectives]This study was conducted to investigate the similarity and differences between TLR4 knockout mice and C57 BL/6 mice used in liver fibrosis research in terms of growth rate and reproduction ability.[Methods]Twenty TLR4 knockout mice and C57 BL/6 mice,half male and half female,were selected to compare the growth rates of body weight and body length of mice from the 4th to 12th weeks;and 20 pairs of male and female mice of the same strain were compared for the number of baby mice of the second litter.[Results]The growth rates of body weight and body length of the TLR4 knockout mice were significantly lower than those of C57 BL/6 mice(P<0.05)(except for the 4th and 5th weeks when there was no significant difference in body length);and in terms of reproductive ability,the TLR4 knockout mice were significantly lower than the C57 BL/6 mice(the ratio of the total number of baby mice in the second litter of the two strains,72∶147).[Conclusions]Knockout of the TLR4 gene has a significant impact on the growth and reproduction of mice.

Liver fibrosis in mice treated with Yue-Ju-Bao-He pills

Background:The aim of this study was to investigate the protective effect and antioxidant mechanism of Yue-Ju-Bao-He pills(YJBH)on liver fibrosis induced by carbon tetrachloride in mice.Methods:Thirty C57BL/6 mice were randomly divided into cntrol group,model group,and YJBH group.The control group received 0.2 mL olive oil intraperitoneally injected twice per week for six weeks.Four weeks after injection,the control group received oral treatment of 0.2 mL normal saline once per day for three weeks.The model and YJBH groups received 20%carbon tetrachloride olive oil solution(2 mL/kg)intraperitoneal injections twice per week for 6 weeks to induce liver fibrosis.After 4 weeks of carbon tetrachloride injections,the model and YJBH groups received oral treatment of either 0.2 mL normal saline or YJBH(0.9 mg/kg)once per day for 3 weeks,respectively.After three weeks of treatment,liver sections from mice were used for hematoxylin-eosin staining and Masson’s Trichrome staining to observe the status of the liver tissue.The therapeutic effect of YJBH on liver fibrosis was studied by determining alanine transaminase and aspartate aminotransferase and pathological changes in livers.Determining the malondialdehydelevel,superoxide dismutase,and glutathione peroxidase activities in liver homogenates were done to observe antioxidant mechanisms.Results:Compared with the control group,the body weight of mice in the model group decreased significantly and the liver index increased.After YJBH treatment,the body weight of mice increased and the liver index decreased compared with the model group.In addition,liver function indexes aspartate aminotransferase and alanine transaminase were significantly improved.The level of malondialdehyde in liver tissue was significantly decreased,and the expression of glutathione peroxidase and superoxide dismutase were increased.The pathological examination showed that liver cell injury of YJBH group was significantly reduced by the infiltration of inflammatory cells into collagen fibers.Conclusion:YJBH can effectively delay the progress of liver fibrosis induced by carbon tetrachloride,and the mechanism may be related to oxidative stress.

Human platelets inhibit liver fibrosis in severe combined immunodeficiency mice

AIM:To investigate the role of human platelets in liver fibrosis.METHODS:Severe combined immunodeficiency(SCID)mice were administered CCl4and either phosphate-buffered saline(PBS group)or human platelet transfusions(hPLT group).Concentrations of hepatocyte growth factor(HGF),matrix metallopeptidases(MMP)-9,and transforming growth factor-β(TGF-β)in the liver tissue were compared between the PBS and the hPLT groups by enzyme-linked immunosorbent assay(ELISA)and Western blotting.The effects of a human platelet transfusion on liver fibrosis included the fibrotic area,hydroxyproline content,and-smooth muscle actin(α-SMA)expression,which were evaluated by picrosirius red staining,ELISA,and immunohistochemical staining using an anti-mouse-SMA antibody,respectively.Phosphorylations of mesenchymal-epithelial transition factor(Met)and SMAD3,downstream signals of HGF and TGF-β,were compared between the two groups by Western blotting and were quantified using densitometry.Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling.Furthermore,the accumulation of human platelets in the liver 2 h after platelet transfusion was compared between normal and fibrotic livers by immunohistochemical staining using an anti-human CD41 antibody.RESULTS:The fibrotic area and hydroxyproline content in the liver were both significantly lower in the hPLT group when compared to the PBS group(fibrotic area,1.7%±0.6%vs 2.5%±0.6%,P=0.03;hydroxyproline content,121±26 ng/g liver vs 156±47 ng/g liver,P=0.04).There was less α-smooth muscle actin staining in the hPLT group than in the PBS group(0.5%±0.1%vs 0.8%±0.3%,P=0.02).Hepatic expression levels of mouse HGF and MMP-9were significantly higher in the hPLT group than in the PBS group(HGF,109±13 ng/g liver vs 88±22 ng/g liver,P=0.03;MMP-9,113%±7%/GAPDH vs 92%±11%/GAPDH,P=0.04).In contrast,the concentration of mouse TGF-β in the liver tissue was significantly lower in the hPLT group than in the PBS group(22±5ng/g liver vs 39±6 ng/g liver,P=0.02).Phosphorylation of Met was more prevalent in the hPLT group than in the PBS group(37%±4%/GAPDH vs 20%±8%/GAPDH,P=0.03).Phosphorylation of SMAD3was weaker in the hPLT group than in the PBS group(60%±12%/GAPDH vs 84%±12%/GAPDH,P=0.1),although this difference was not significant.Furthermore,a lower rate of hepatocyte apoptosis was observed in the hPLT group than in the PBS group(5.9%±1.7%vs 2.9%±2.1%,P=0.02).Significant human platelet accumulation was observed in the fibrotic liver tissues,whereas few platelets accumulated in the normal liver.CONCLUSION:Human platelets inhibit liver fibrosis in SCID mice.Increased concentration of HGF in the liver suppresses hepatic stellate cell activation,induces MMPs,and inhibits hepatocyte apoptosis.

Protective effects of a polymethoxy flavonoids-rich Citrus aurantium peel extract on liver fibrosis induced by bile duct ligation in mice

Objective: To evaluate the possible protective effect of Citrus aurantium peel extract(CAE) against apoptosis in cholestatic liver fibrosis induced by bile duct ligation in mice. Methods: Male ICR mice were divided to 5 groups: 1) Control group(Sham-operated mice), 2) Cholestatic liver injury group induced by bile duct ligation(BDL), 3) BDL mice treated with silymarin(200 mg/kg) for 4 weeks, 4) BDL mice treated with 50 mg/kg CAE for 4 weeks, 5) BDL mice treated with 200 mg/kg CAE for 4 weeks. Mice were sacrificed and liver fibrosis was evaluated by serum and hepatic tissue biochemistry tests and liver histopathological examination. Effects of CAE on inflammation and apoptosis gene regulation were investigated through real-time PCR. CAE effect on lipid metabolism related signaling was determined by western blot analysis. Results: In BDL mice, administration of CAE for 4 weeks markedly attenuated liver fibrosis based on histopathological alteration. Serum and hepatic tissue biochemistry results revealed that CAE(50 and 200 mg/kg) decreased the levels of alanine transaminase, aspartate transaminase, gamma glutamyl transferase, total bilirubin, nitric oxide, and thiobarbituric acid reactive substances. Real-time PCR and western blot analysis showed that CAE regulated inflammation, apoptosis, and lipid metabolism factors increased by BDL. Interleukin family, tumor necrosis factor α, and related apoptosis factors m RNA levels were increased by BDL treatment. However, these increases were suppressed by CAE administration. In addition, CAE effectively increased phosphorylation of AMPactivated protein kinase, nuclear factor E2-related factor 2, and related cytoprotective proteins. Conclusions: CAE can efficiently regulate BDL-induced liver injury with antioxidant, antiinflammatory, and antiapoptotic activities.

Effects of elafibranor on liver fibrosis and gut barrier function in a mouse model of alcohol-associated liver disease

BACKGROUND Alcohol-associated liver disease(ALD)is a leading cause of liver-related morbidity and mortality,but there are no therapeutic targets and modalities to prevent ALD-related liver fibrosis.Peroxisome proliferator activated receptor(PPAR)α and δ play a key role in lipid metabolism and intestinal barrier homeostasis,which are major contributors to the pathological progression of ALD.Meanwhile,elafibranor(EFN),which is a dual PPARαand PPARδagonist,has reached a phase III clinical trial for the treatment of metabolic dysfunctionassociated steatotic liver disease and primary biliary cholangitis.However,the benefits of EFN for ALD treatment is unknown.AIM To evaluate the inhibitory effects of EFN on liver fibrosis and gut-intestinal barrier dysfunction in an ALD mouse model.METHODS ALD-related liver fibrosis was induced in female C57BL/6J mice by feeding a 2.5% ethanol(EtOH)-containing Lieber-DeCarli liquid diet and intraperitoneally injecting carbon tetrachloride thrice weekly(1 mL/kg)for 8 weeks.EFN(3 and 10 mg/kg/day)was orally administered during the experimental period.Histological and molecular analyses were performed to assess the effect of EFN on steatohepatitis,fibrosis,and intestinal barrier integrity.The EFN effects on HepG2 lipotoxicity and Caco-2 barrier function were evaluated by cell-based assays.RESULTS The hepatic steatosis,apoptosis,and fibrosis in the ALD mice model were significantly attenuated by EFN treatment.EFN promoted lipolysis and β-oxidation and enhanced autophagic and antioxidant capacities in EtOH-stimulated HepG2 cells,primarily through PPARαactivation.Moreover,EFN inhibited the Kupffer cell-mediated inflammatory response,with blunted hepatic exposure to lipopolysaccharide(LPS)and toll like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)signaling.EFN improved intestinal hyperpermeability by restoring tight junction proteins and autophagy and by inhibiting apoptosis and proinflammatory responses.The protective effect on intestinal barrier function in the EtOH-stimulated Caco-2 cells was predominantly mediated by PPARδ activation.CONCLUSION EFN reduced ALD-related fibrosis by inhibiting lipid accumulation and apoptosis,enhancing hepatocyte autophagic and antioxidant capacities,and suppressing LPS/TLR4/NF-κB-mediated inflammatory responses by restoring intestinal barrier function.

Angiotensin-converting enzyme 2 alleviates liver fibrosis through the renin-angiotensin system

The present letter to the editor is related to the study titled‘Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells’.Angiotensin-converting enzyme 2 can alleviate liver fibrosis by regulating autophagy of hepatic stellate cells and affecting the renin-angiotensin system.

Staging liver fibrosis with various diffusion-weighted magnetic resonance imaging models

BACKGROUND Diffusion-weighted imaging(DWI)has been developed to stage liver fibrosis.However,its diagnostic performance is inconsistent among studies.Therefore,it is worth studying the diagnostic value of various diffusion models for liver fibrosis in one cohort.AIM To evaluate the clinical potential of six diffusion-weighted models in liver fibrosis staging and compare their diagnostic performances.METHODS This prospective study enrolled 59 patients suspected of liver disease and scheduled for liver biopsy and 17 healthy participants.All participants underwent multi-b value DWI.The main DWI-derived parameters included Mono-apparent diffusion coefficient(ADC)from mono-exponential DWI,intravoxel incoherent motion model-derived true diffusion coefficient(IVIM-D),diffusion kurtosis imaging-derived apparent diffusivity(DKI-MD),stretched exponential model-derived distributed diffusion coefficient(SEM-DDC),fractional order calculus(FROC)model-derived diffusion coefficient(FROC-D)and FROC model-derived microstructural quantity(FROC-μ),and continuous-time random-walk(CTRW)model-derived anomalous diffusion coefficient(CTRW-D)and CTRW model-derived temporal diffusion heterogeneity index(CTRW-α).The correlations between DWI-derived parameters and fibrosis stages and the parameters’diagnostic efficacy in detecting significant fibrosis(SF)were assessed and compared.RESULTS CTRW-D(r=-0.356),CTRW-α(r=-0.297),DKI-MD(r=-0.297),FROC-D(r=-0.350),FROC-μ(r=-0.321),IVIM-D(r=-0.251),Mono-ADC(r=-0.362),and SEM-DDC(r=-0.263)were significantly correlated with fibrosis stages.The areas under the ROC curves(AUCs)of the combined index of the six models for distinguishing SF(0.697-0.747)were higher than each of the parameters alone(0.524-0.719).The DWI models’ability to detect SF was similar.The combined index of CTRW model parameters had the highest AUC(0.747).CONCLUSION The DWI models were similarly valuable in distinguishing SF in patients with liver disease.The combined index of CTRW parameters had the highest AUC.

Ginsenoside Rb1 induces hepatic stellate cell ferroptosis to alleviate liver fibrosis via the BECN1/SLC7A11 axis

Liver fibrosis is primarily driven by the activation of hepatic stellate cells(HSCs),a process associated with ferroptosis.Ginsenoside Rb1(GRb1),a major active component extracted from Panax ginseng,inhibits HSC activation.However,the potential role of GRb1 in mediating HSC ferroptosis remains unclear.This study examined the effect of GRb1 on liver fibrosis both in vivo and in vitro,using CCl4-induced liver fibrosis mouse model and primary HSCs,LX-2 cells.The findings revealed that GRb1 effectively inactivated HSCs in vitro,reducing alpha-smooth muscle actin(a-SMA)and type I collagen(Col1A1)levels.Moreover,GRb1 significantly alleviated CCl4-induced liver fibrosis in vivo.From a mechanistic standpoint,the ferroptosis pathway appeared to be central to the antifibrotic effects of GRb1.Specifically,GRb1 promoted HSC ferroptosis both in vivo and in vitro,characterized by increased glutathione depletion,malondialdehyde production,iron overload,and accumulation of reactive oxygen species(ROS).Intriguingly,GRb1 increased Beclin 1(BECN1)levels and decreased the System Xc-key subunit SLC7A11.Further experiments showed that BECN1 silencing inhibited GRb1-induced effects on HSC ferroptosis and mitigated the reduction of SLC7A11 caused by GRb1.Moreover,BECN1 could directly interact with SLC7A11,initiating HSC ferroptosis.In conclusion,the suppression of BECN1 counteracted the effects of GRb1 on HSC inactivation both in vivo and in vitro.Overall,this study highlights the novel role of GRb1 in inducing HSC ferroptosis and promoting HSC inactivation,at least partly through its modulation of BECN1 and SLC7A11.

Angiotensin-converting enzyme 2 and AMPK/mTOR pathway in the treatment of liver fibrosis:Should we consider further implications?

This editorial contains comments on the article by Zhao et al in print in the World Journal of Gastroenterology.The mechanisms responsible for hepatic fibrosis are also involved in cancerogenesis.Here,we recapitulated the complexity of the renin-angiotensin system,discussed the role of hepatic stellate cell(HSC)autophagy in liver fibrogenesis,and analyzed the possible implications in the development of hepatocarcinoma(HCC).Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers definitively contribute to reducing hepatic fibrogenesis,whereas their involvement in HCC is more evident in experimental conditions than in human studies.Angiotensin-converting enzyme 2(ACE2),and its product Angiotensin(Ang)1-7,not only regulate HSC autophagy and liver fibrosis,but they also represent potential targets for unexplored applications in the field of HCC.Finally,ACE2 overexpression inhibits HSC autophagy through the AMP-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)pathway.In this case,Ang 1-7 acts binding to the MasR,and its agonists could modulate this pathway.However,since AMPK utilizes different targets to suppress the mTOR downstream complex mTOR complex 1 effectively,we still need to unravel the entire pathway to identify other potential targets for the therapy of fibrosis and liver cancer.

Elafibranor alleviates alcohol-related liver fibrosis by restoring intestinal barrier function

We discuss the article by Koizumi et al published in the World Journal of Gastroenterology.Our focus is on the therapeutic targets for fibrosis associated with alcohol-related liver disease(ALD)and the mechanism of action of elafibranor(EFN),a dual agonist of peroxisome proliferator-activated receptorα(PPARα)and peroxisome PPARδ(PPARδ).EFN is currently in phase III clinical trials for the treatment of metabolic dysfunction-associated fatty liver disease and primary biliary cholangitis.ALD progresses from alcoholic fatty liver to alcoholic steatohepatitis(ASH),with chronic ASH eventually leading to fibrosis,cirrhosis,and,in some cases,hepatocellular carcinoma.The pathogenesis of ALD is driven by hepatic steatosis,oxidative stress,and acetaldehyde toxicity.Alcohol consumption disrupts lipid metabolism by inactivating PPARα,exacerbating the progression of ALD.EFN primarily activates PPARα,promoting lipolysis andβ-oxidation in ethanol-stimulated HepG2 cells,which significantly reduces hepatic steatosis,apoptosis,and fibrosis in an ALD mouse model.Additionally,alcohol disrupts the gut-liver axis at several interconnected levels,contributing to a proinflammatory environment in the liver.EFN helps alleviate intestinal hyperpermeability by restoring tight junction protein expression and autophagy,inhibiting apoptosis and inflammatory responses,and enhancing intestinal barrier function through PPARδactivation.

Encapsulating taurine into liposomes:A promising therapeutic for liver fibrosis

We summarize the mechanism by which taurine(Tau)inhibits autophagy and induces iron apoptosis in hepatic stellate cells.Tau interacts with autophagy regulates multifunctional proteins,microtubule-associated protein 1 light chain 3 Beta,and autophagy-related gene 5 to inhibit autophagy,binds to ferritin heavy chain 1 and nuclear receptor coactivator 4 to trigger ferritin autophagy,and interacts with glutathione peroxidase 4 to promote iron apoptosis.There is a solid rationale for developing Tau-based therapies targeting autophagy and ferroptosis regulation.From a pharmaceutical point of view,there are certain requirements for Tau protein delivery systems,such as loading efficiency,stability,and targeting.Nanomaterials should also contain a hydrophilic motif similar to Tau to optimize loading efficiency.Since Tau is a hydrophilic molecule with high water solubility,liposomes,micelles,and amphiphilic polymer nanoparticles may represent a superior choice.The nanostructure of the liposome includes a water region and a lipid membrane to sequester hydrophilic and hydrophobic drugs,respectively,whereas Tau is expected to be loaded into the water region.In addition,a representative method of actively targeting hematopoietic stem cells is introduced.A Tau-based method for the treatment of liver fibrosis is proposed based on the formulation of common liposomes(lecithin plus cholesterol).

Evaluating short-term and long-term liver fibrosis improvement in hepatitis C patients after DAA treatment

Despite achieving a high cure rate with the direct-acting antivirals(DAAs)in hepatitis C treatment,further research is needed to identify additional benefits of the DAA therapy.The current study evaluated liver fibrosis improvement in 848 hepatitis C patients treated with DAAs,who also achieved sustained virologic response.By the fibrosis-4(FIB-4)index,patients were categorized based on their baseline fibrosis levels,and the improvement in fibrosis was analyzed in both short-term(9-26 weeks)and long-term(≥36 weeks)follow-up.The results showed a significant decrease in the FIB-4 index,indicating an improvement in liver fibrosis,in 63.0%and 67.6%of the patients during the short-term and long-term follow-up,respectively.Short-term improvement was associated with factors including ribavirin usage,blood cholinesterase levels,alanine transaminase levels,albumin levels,and the baseline FIB-4 index,while long-term improvement was associated with factors such as aspartate transaminase levels,total protein level,and the baseline FIB-4 index.The current study emphasizes the importance of continuous assessment and post-treatment monitoring of liver fibrosis,which will provide crucial insights for enhancing patient care in hepatitis C management.