Hepatic Fibrosis Formation Research Articles

N6-methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis via targeting miR-144-3p/Et-1 axis

Kupffer cells (KCs), as residents and sentinels of the liver, are involved in the formation of hepatic fibrosis (HF). However, the biological functions of circular RNAs (circRNAs) in KCs to HF have not been determined. In this study, the expression levels of circRNAs, microRNAs, and messenger RNAs (mRNAs) in KCs from a mouse model of HF mice were investigated using microarray and circRNA-Seq analyses. circDcbld2 was identified as a candidate circRNA in HF, as evidenced by its up-regulation in KCs. Silver staining and mass spectrometry showed that Wtap and Igf2bp2 bind to cirDcbld2. The suppression of circDcbld2 expression decreased the KC inflammatory response and oxidative stress and inhibited hepatic stellate cell (HSCs) activation, attenuating mouse liver fibrogenesis. Mechanistically, Wtap mediated the N6-methyladenosine (m6A) methylation of circDcbld2, and Igf2bp2 recognized m6A-modified circDcbld2 and increased its stability. circDcbld2 contributes to the occurrence of HF by binding miR-144-3p/Et-1 to regulate the inflammatory response and oxidative stress. These findings indicate that circDcbld2 functions via the m6A/circDcbld2/miR-144-3p/Et-1 axis and may act as a potential biomarker for HF treatment.

Abstract 15537: The Role of Liver Epsins in Nonalcoholic Steatohepatitis

Introduction: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that is associated with high risks of cardiovascular diseases. Epsin is an evolutionarily conserved protein family that is involved in clathrin-mediated endocytosis. We found that both Epsin1/2 expressions in the liver are significantly up-regulated by the Western Diet (WD) induced NASH mice model, as well as in the liver biopsy of NASH patients. Hypothesis: We hypothesize that the expressions of Epsin1 and Epsin2 in the liver are required for NASH progression. Methods: We conditionally deleted Epsin1/2 using Albumin-Cre (Liver-DKO). We then induced a mouse model of NASH by feeding both WT and Liver-DKO mice a WD for 52 weeks. We used single cell multiome, combined with molecular approaches to study the mechanisms. We tested a strategy to deliver Epsin1/2 siRNAs encapsulated nanoparticles (NPs) targeting the liver via intravenous injection to treat diet induced NASH in mice. Results: We found WD-fed Liver-DKO mice have significant reductions in hepatic fibrosis formation and less lipid accumulation, and this was associated with better glucose and insulin tolerance, as well as lower serum aspartate transaminase (AST) and alanine aminotransferase (ALT) compared with WD-fed WT mice. Through analysis of the single cell multiome from the WT and Liver-DKO livers, we found more Kupffer cell in WT NASH mice compared with DKO. Interestingly, monocyte-derived inflammantory Kupffer cell (KCs) are enriched in WD-fed WT mice, however, embryonic-derived KCs are the enriched in liver DKO, and associate with dramatically reduced total KCs population. We also found that HNF4a was dramatically reduced in WD-fed WT mice, but its expression was preserved in WD-fed Liver-DKO mice, suggesting the loss of hepatocyte identity was inhibited by Epsins deletion. Therapeutically, we found significant resolution of hepatic fibrosis after knockdown of Epsin1/2 siRNA NP treatment. Conclusions: Liver specific deletion of Epsin1/2 significantly inhibits NASH progression by suppression of inflammation that protects hepatocytes from severe chronic injury. Our preclinical data suggests specially targeting Epsin1 and Epsin2 in the liver could be a promising therapeutic strategy for NASH treatment.

Protective effect of Anneslea fragrans ethanolic extract against CCl4-induced liver injury by inhibiting inflammatory response, oxidative stress and apoptosis.

Anneslea Fragrans Wall. (AF) is a medicinal and edible plant distributed in China. Its leaves and bark generally used for the treatments of diarrhea, fever, and liver diseases. While its ethnopharmacological application against liver diseases has not been fully studied. This study was aimed to evaluate the hepatoprotective effect of ethanolic extract from A. fragrans (AFE) on CCl4 induced liver injury in mice. The results showed that AFE could effectively reduce plasma activities of ALT and AST, increase antioxidant enzymes activities (SOD and CAT) and GSH level, and decrease MDA content in CCl4 induced mice. AFE effectively decreased the expressions of inflammatory cytokines (IL-1β, IL-6, TNF-α, COX-2 and iNOS), cell apoptosis-related proteins (Bax, caspase-3 and caspase-9) and increased Bcl-2 protein expression via inhibiting MAPK/ERK pathway. Additionally, TUNEL staining, Masson and Sirius red staining, immunohistochemical analyses revealed that AFE could inhibit the CCl4-induced hepatic fibrosis formation via reducing depositions of α-SMA, collagen I and collagen III. Conclusively, the present study demonstrated that AFE had an hepatoprotective effect by MAPK/ERK pathway to inhibit oxidative stress, inflammatory response and apoptosis in CCl4-induced liver injury mice, suggesting that AFE might be served as a hepatoprotective ingredient in the prevention and treatment of liver injury.

Diallyl trisulfide plays an antifibrotic role by inhibiting the expression of Bcl-2 in hepatic stellate cells.

Hepatic fibrosis is an important early stage in the evolution of liver cirrhosis, and specific medicine and therapeutic measures are unavailable to date. Hepatic stellate cells (HSCs) are the main cells involved in the formation of hepatic fibrosis, and induction of the apoptosis of HSCs is an important strategy for the treatment of hepatic fibrosis. Diallyl trisulfide (DATS) is a natural product and is the main active ingredient in garlic. However, the exact molecular mechanisms underlying HSC apoptosis induced by DATS are not well understood. This study aimed to analyze the efficiency and mechanism of DATS in hepatic fibrosis. Different concentrations (25, 50, 100, and 200 μM) of DATS were used to treat HSCs. Changes in cell morphology and formation of apoptotic bodies were observed under an inverted microscope and an electric microscope. Bcl‐2 signaling involving Bax, Caspase‐3, Caspase‐6, Caspase‐8, Caspase‐9, p53, Apaf‐1, and Cyto‐c in fibrosis were examined, which is a critical step in the evaluation of antihepatic fibrosis agents. We also evaluated the effect of DATS on the cellular morphology of HSCs and apoptosis‐related factors under different Bcl‐2 expression states. Our results suggest that DATS regulates hepatic fibrosis by blocking the Bcl‐2 signaling pathway and upregulating the Bax/Bcl‐2 ratio.

Combined Treatment with Hydroxyurea and a Phosphodiesterase 9 (PDE9) Inhibitor Decreases Hepatic Damage in an Animal Model of Sickle Cell Anemia

Sickle cell anemia (SCA) is associated with recurrent vaso-occlusive processes and chronic inflammation, which may result in severe liver damage, affecting patient morbidity and mortality. The phosphodiesterase 9 (PDE9) enzyme degrades intracellular cyclic guanylate monophosphate (cGMP) and is highly expressed in the hematopoietic cells of SCA patients (Almeida et al., 2008, Br J Haematol). The inhibition of this enzyme induces fetal hemoglobin production in erythroid cells and, in a mouse model of inflammatory SCA vaso-occlusion, prevents acute vaso-occlusive processes and improves animal survival when used together with hydroxyurea (HU) (Almeida et al., 2012 Blood). As PDE9 inhibitors (PDE9i) may represent a strategy to specifically amplify the activation of the nitric oxide/guanylyl cyclase pathway by HU in hematopoietic cells, we aimed to determine whether the administration of a PDE9i (BAY73-6691) and/or HU for 4 weeks can modulate chronic liver inflammation and intrahepatic fibrosis in an animal model of SCA. Chimeric SCA and control (CON) mice were generated from the bone marrow transplantation of Berkeley transgenic SCA mice and C57BL/6 mice, respectively, to irradiated C57BL/6 mice. Three months after transplantation, phenotyped SCA mice received a five-times weekly dose of vehicle (0.9% sodium chloride, n=5), PDE9i (1 mg/Kg BAY73-6691, n=3), HU (50 mg/Kg, n=3) or PDE9i plus HU (n=3) by gavage for 4 weeks. Fibrosis was visualized in liver sections by second harmonic generation (SHG) using confocal microscopy (LSM780 microscope, Carl Zeiss). NKT cell (CD16/32+CD3e+) quantification, as a percentage of liver granulocytes, was determined by flow cytometry. After surgical exposure of the liver, the vaso-occlusive processes were analyzed by intravital confocal microscopy. In contrast to vehicle-treated CON mice, vehicle-treated SCA animals presented extensive accumulation of thick fibers of collagen, characteristic of fibrosis, at 4 months post-transplantation. Collagen fibers were generally localized near blood vessels and were associated with a marked inflammatory infiltrate (Fig. 1). Vehicle-treated SCA mice also presented a significantly higher number of NKT cells in the liver, compared to CON mice (0.39% ± 0.10%, 0.17% ± 0.04%, respect., P<0.05). In association with these findings, intravital microscopy demonstrated increased neutrophil (neu) recruitment in liver sinusoids, compared to CON mice (12.7 ± 2.0 and 5.2 ± 1.5 neu/0.262 mm2, P<0.001), usually in association with platelet aggregates, characteristic of vaso-occlusive processes (Fig. 1). Neither PDE9i nor HU, when administered alone, prevented the accumulation of thick fibers of collagen in the liver, or the formation of hepatic fibrosis and inflammatory infiltrates in SCA animals (Fig. 1). In contrast, the treatment of SCA mice with both PDE9i plus HU for 4 weeks markedly reduced fibrosis and the organization of liver collagen, as well as the inflammatory infiltrate, compared to SCA-vehicle animals (Fig. 1). All three treatments significantly diminished NKT cell numbers in SCA livers (0.13% ± 0.02, 0.14% ± 0.01, 0.14% ± 0.07 for PDE9i, HU and PDE9i+HU, respectively, P<0.05, compared to SCA-vehicle). In contrast, none of the treatments were capable of inhibiting neu recruitment in the liver sinusoids (20.1 ± 3.0, 15.0 ± 2.7, 12.5 ± 3.6 neu/0.262 mm2, for PDE9i, HU and PDE9i+HU, respect., P>0.05, compared to SCA-vehicle), possibly due to the extremely small size of these vessels. Additionally, the formation of platelet-neutrophil heterocellular aggregates in sinusoids were not observed to be modulated by these agents (Fig. 1). In conclusion, while the oral treatment of SCA mice with PDE9i and/or HU for four weeks failed to significantly abrogate leukocyte recruitment in liver sinusoids, at the concentrations employed, the use of HU plus PDE9i in SCA mice effectively diminished the extensive hepatic damage, including fibrosis, that results from chronic liver inflammation, representing a potential pharmacological approach for the management of liver damage in SCA. [Display omitted] DisclosuresConran:Bayer AG: Research Funding.

Correlational analysis of alternation of clinical features of intrahepatic lymphocyte subsets with HBV virology and liver fibrosis in HBV-Tg composite CCl(4) mice model

Objective: To observe the alteration of clinical features of intrahepatic lymphocyte subsets in C57BL/6N-TG (1.28HBV)/Vst hepatitis B virus (HBV) transgenic mice composite carbon tetrachloride (CCl(4)) with intraperitoneal injection under the background of hepatitis B to induce liver fibrosis mice model, and analyze their correlation with serum HBV DNA and liver tissue hydroxyproline (Hyp) content. Methods: HBV-Tg mice were intraperitoneally injected with 10% CCl(4) to induce the rapid formation of hepatic fibrosis. Serum HBV DNA, HBsAg, HBeAg levels and liver tissue HBsAg expressional conditions were used to evaluate the virological characteristics of mice model. The degree of hepatic inflammation and fibrosis in mice were observed by HE, Sirius Red staining and liver tissue hydroxyproline (Hyp) content. Intrahepatic T lymphocyte, B lymphocyte, CD4+T lymphocyte, CD8+T lymphocyte, natural killer (NK) cell and natural killer T (NKT) cells distribution were observed by flow cytometry. One-way analysis of variance was used for intergroup data comparison, and LSD was used for pairwise comparison. Pearson's correlation analysis was used to analyze the correlation between the above lymphocyte subsets and serum HBV DNA and liver tissue Hyp content. Results: Serum HBsAg, HBeAg and liver tissue HBsAg had equal positive expression in the HBV-Tg composite CCl(4) mice model group, and the serum HBV DNA load was > 1 × 10(6) IU / ml. Compared with the wild-type control group, liver tissue Hyp content of the composite model group was significantly higher [(196.39 ± 38.14) μg /g and (347.67 ± 59.53) μ g/g, P < 0.01). The degree of inflammation and fibrosis in liver tissues was aggravated, and the proportion of all intrahepatic CD4+T, NK and NKT cells was significantly reduced (P < 0.01), while the proportion of CD8+T lymphocytes (30.58% ± 2.89% vs. 46.50% ± 2.24%, P < 0.01) and B lymphocytes (28.82% ± 2.24% vs. 37.10% ± 8.59%, P < 0.05) was significantly increased. Serum HBV DNA level was positively correlated with the proportion of intrahepatic T lymphocytes (r = 0.413, P < 0.05), and negatively correlated with the proportion of NK cells (r = -0.419, P < 0.05). Liver tissue Hyp content was negatively correlated with the proportion of all CD4+T lymphocytes (r = -0.871), NK cells (r = -0.716), and NKT cells (r = -0.876) (all P < 0.01), and positively correlated with the proportion of all CD8 + T lymphocytes (r = 0.852), and B lymphocytes (r = 0.593) (all P < 0.01). Conclusion: HBV-Tg composite CCl4 mice model can induce positive HBV virological indicators, liver inflammation and fibrosis in mice model of hepatitis B coexisting with fibrosis. This model has the features of immune disorder of liver lymphocyte similar to human disease, and the immune disorder of intrahepatic lymphocytes is correlated with HBV viral load and liver fibrosis degree.

Activation of Nrf2/AREs-mediated antioxidant signalling, and suppression of profibrotic TGF-β1/Smad3 pathway: a promising therapeutic strategy for hepatic fibrosis — A review

Hepatic fibrosis (HF) is a wound-healing response that occurs during chronic liver injury and features by an excessive accumulation of extracellular matrix (ECM) components. Activation of hepatic stellate cell (HSC), the leading effector in HF, is responsible for overproduction of ECM. It has been documented that transforming growth factor-β1 (TGF-β1) stimulates superfluous accumulation of ECM and triggers HSCs activation mainly via canonical Smad-dependent pathway. Also, the pro-fibrogenic TGF-β1 is correlated with generation of reactive oxygen species (ROS) and inhibition of antioxidant mechanisms. Moreover, involvement of oxidative stress (OS) can be clearly elucidated as a fundamental event in liver fibrogenesis. Nuclear factor erythroid 2-related factor 2-antioxidant response elements (Nrf2-AREs) pathway, a group of OS-mediated transcription factors with diverse downstream targets, is associated with the induction of diverse detoxifying enzymes and the most pivotal endogenous antioxidative system. More specifically, Nrf2-AREs pathway has recently assigned as a new therapeutic target for cure of HF. The overall goal of this review will focus on recent findings about activation of Nrf2-AREs-mediated antioxidant and suppression of profibrotic TGF-β1/Smad3 pathway in the liver, providing an overview of recent advances in transcriptional repressors that dislocated during HF formation, and highlighting possible novel therapeutic targets for liver fibrosis.

Effect of the transdifferentiation of BECs into myofibroblasts on the pathogenesis of secondary cholestatic hepatic fibrosis.

The present study investigated the effect of the transdifferentiation of bile duct epithelial cells (BECs) into myofibroblasts on the pathogenesis of secondary cholestatic hepatic fibrosis and examined the underlying mechanisms. A total of 60 male rats with hepatic fibrosis were randomly divided into two groups: A secondary cholestatic hepatic fibrosis model group induced by ligation of the bile duct (BDL) and a sham group, which only underwent segregation of the choledochus. Rats in the BDL group were dynamically observed after week 1, 2, 3 and 4 post-BDL, and the remaining rats were sacrificed after week 5 to determine histological changes and hydroxyproline content. The cellular co-localization of cytokeratin (CK)7/α-smooth muscle actin (SMA) or α-SMA/desmin was detected by immunofluorescence staining and laser confocal microscopy, while the protein expression levels of CK7, α-SMA and desmin were determined by western blot analysis. Sirius red staining was also performed and quantified. The results revealed a significant correlation between the protein expression of CK7 and α-SMA (r=0.9692, P<0.01). Furthermore, a predominant correlation between the number of cells stained for CK7/α-SMA and collagen deposition in liver tissues was identified, while the correlation of cells with co-localized α-SMA and desmin was less pronounced. The transdifferentiation of BECs into myofibroblasts may be a key pathological factor in secondary cholestatic hepatic fibrosis formation.

Live imaging of collagen deposition during experimental hepatic schistosomiasis and recovery: a view on a dynamic process

Hepatic fibrosis is the central cause of chronic clinical pathology resulting from infection by the blood flukes Schistosoma japonicum or S. mansoni. Much has been elucidated regarding the molecular, cellular and immunological responses that correspond to the formation of the granulomatous response to trapped schistosome eggs. A central feature of this Th2 response is the deposition of collagen around the periphery of the granuloma. To date, traditional histology and transcriptional methods have been used to quantify the deposition of collagen and to monitor the formation of the hepatic granuloma during experimental animal models of schistosomiasis. We have investigated the dynamic nature of granuloma formation through the use of a transgenic mouse model (B6.Collagen 1(A) luciferase mice (B6.Coll 1A-luc+)). With this model and whole-animal bioluminescence imaging, we followed the deposition of collagen during an active schistosome infection with Chinese and Philippines geographical strains of S. japonicum and after clearance of the adult parasites by the drug praziquantel. Individual mice were re-imaged over the time course to provide robust real-time quantitation of the development of chronic fibrotic disease. This model provides an improved method to follow the course of hepatic schistosomiasis-induced hepatic pathology and effectively supports the current dogma of the formation of hepatic fibrosis, originally elucidated from static traditional histology. This study demonstrates the first use of the B6.Coll 1A-luc+ mouse to monitor the dynamics of disease development and the treatment of pathogen-induced infection with the underlying pathology of fibrosis.Schistosome flukes induce pathology due to trapped parasite eggs, producing a granulomatous-immune response delineated by fibrosis and the deposition of collagen in the liver. The authors present whole animal imaging for the quantitation of hepatic collagen deposition during the early stages of pathology.

Effective components of traditional Chinese medicine for regulating TGF-Beta1/Smads signaling pathway in hepatic fibrosis

Hepatic fibrosis is a liver damage healing response affected by a variety of factors; its formation is associated with multiple cytokines and a variety of signaling pathways. Transforming growth factor beta1( TGF-β1) is one of the strongest fibrosis cytokines known,and involves almost all the key links in hepatic fibrosis. TGF-β1/Smads signal pathway is the most classical pathway for TGF-β1 to play its role in promoting fibrosis as well as one of the most important signaling pathways of hepatic fibrosis formation. Studies for the signal pathway have made a series of scientific research achievements in recently years. Traditional Chinese medicine has the advantages of " multiple ingredients,multiple targets and less side effects",and is widely used in the clinical treatment of hepatic fibrosis.Effective components of traditional Chinese medicine are monomer compounds,which are extracted and purified from traditional Chinese medicine. Nowadays,the molecular biology studies of effective traditional Chinese medicine have become a hotspot. Modern advanced technology and methods can be used to directly clarify the targets and the signaling pathways,reveal the mechanism of traditional Chinese medicine in treating diseases,and promote the modernization and international development of traditional Chinese medicine industry. This review summarized the structure,function and application of TGF-β1/Smads signaling pathway in the progress of anti-hepatic fibrosis,and analyzed the action mode and possible mechanism of various effective components of traditional Chinese medicine in regulating TGF-β1/Smads signaling pathway and intervening the treatment of hepatic fibrosis in the past five years,so as to put forward new ideas for innovating new targeted traditional Chinese medicine for hepatic fibrosis.

β-Actin: Not a Suitable Internal Control of Hepatic Fibrosis Caused by Schistosoma japonicum.

Schistosomiasis japonica is a significant health problem that leads to morbidity and mortality of humans. It is characterized by hepatic granulomatous response and fibrosis caused by eggs deposition in the liver. β-actin, a traditional housekeeping gene, is widely used as an internal control to normalize gene and protein expression. However, β-actin expression can fluctuate upon the treatment with pharmacological agents or under some physiological and pathological conditions. In this study, we found that the expressions of both β-actin mRNA and protein increased significantly with hepatic fibrosis formation after 6 weeks infection with Schistosoma japonicum and kept high level during the progression of hepatic fibrosis, while the levels of β-Tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) remained stable. The dynamic change of β-actin was similar with the profibrogenic factors, including α-SMA, Collagen I, and Collagen III. We employed immunofluorescence staining and further showed that the expression level of β-actin was positively correlated with α-SMA. What is more, there was a positive correlation between the level of β-actin mRNA and the content of hydroxyproline in liver. This study provides evidences that β-actin is variable and unsatisfied for application as an internal control in hepatic fibrosis induced by S. japonicum infection.

Preliminary study on molecular detection of polysaccharide from Amusium pleuronectes and its intervention to hepatic fibrosis in rats infected with Schistosoma japonicum

To detect the molecular characterization of polysaccharide purified from Amusium pleuronectes, so as to investigate its role of intervention to the formation of hepatic fibrosis caused by Schistosoma japonicum infection. The crude polysaccharide from A. pleuronectes was extracted and further purified, and the molecular weight and monosaccharide composition were determined by the high pressure size exclusion chromatography and PMP pre-column derivatization method, respectively. A total of 50 female BALB/c mice were randomly divided into five groups：A (normal group), B (experimental group), C (polysaccharide group), D (praziquantel), and E (polysaccharide + praziquantel group). The mice in B, C, D, or E groups were attacked on the abdominal skin by using the cercariae of S. japonicum (30 ± 2 for each mouse) respectively. After 8 weeks, the mice in C, D, and E groups were administrated by polysaccharide and/or praziquantel, and the mice in B group were instead of saline. All the livers and sera were collected after 16 weeks. HE staining was employed for the livers, and serum IFN-γ and IL-13 were measured by using ELISA kits. The molecular weight of purified polysaccharide from A. pleuronectes was 11.7 kDa. Compared with A and B groups, the serum levels of IFN-γ in C, D, and E groups were significantly increased (F = 63.525, P < 0.01). However, the serum levels of IL-13 in C, D, and E groups were significantly decreased (F = 99.788, P < 0.01) compared with that in B group. HE staining showed that the egg nodules and hepatic fibrosis were observed in B, C, D, and E groups. The number of egg nodules and fibrosis degree in E group were milder than those in B group (χ2 = 7.875, P < 0.05). The polysaccharide from A. pleuronectes has an obvious effect in preventing hepatic fibrosis process induced by S. japonicum infection, particularly combining with the administration of praziquantel.

Advances in the regulatory roles of exosomes on pathological process of schistosomiasis hepatic fibrosis

Exosomes are a group of membraneous vesicles generated and released by multi-vesicular bodies or cell membranes in a variety of cell types. Acting as important messages between cells, they participate in almost every physiological and pathological process of living organisms. Exosomes contain specific proteins, mRNA, miRNAs, etc. and mediate intercellular communications, signal transductions and gene expressions effectively. Exosomes are involved in the formation of hepatic fibrosis, which is the typical liver pathological change in the progression of schistosomiasis and is caused by the liver repair and (or) regeneration involving inflammation stimulated by exosomes, activated hepatic stellate cells and other related pathways in reaction to the parasite infection. Exosomes could serve as new markers for schistosomiasis hepatic fibrosis diagnosis and potential targets for its treatment. This paper briefly reviews the latest development of studies on the regulatory roles of exosomes in schistosomiasis hepatic fibrosis, so as to provide ideas for searching new treatment targets of the disease.

Combination effects of alogliptin and pioglitazone on steatosis and hepatic fibrosis formation in a mouse model of non-alcoholic steatohepatitis

This study aimed to evaluate the effects of combination therapy with a dipeptidyl peptidase-4 inhibitor, alogliptin, and a peroxisome proliferator-activated receptor-γ agonist, pioglitazone, in a preclinical model of nonalcoholic steatohepatitis using low-density lipoprotein receptor-knockout mice fed a modified choline-deficient l-amino acid-defined diet. Monotherapy with either alogliptin (10–200 mg/kg) or pioglitazone (6–20 mg/kg) significantly decreased hepatic triglyceride content and fibrosis. The concomitant treatment of alogliptin (30 mg/kg), pioglitazone (20 mg/kg) also decreased hepatic triglyceride and hepatic collagen-I mRNA at greater extent compared to monotherapy. Hepatic expression of CD11b mRNA and monocyte chemoattractant protein-1 were also reduced by the concomitant treatment. These results suggest that via an anti-inflammatory potential in addition to anti-metabolic effects, the combination therapy of alogliptin and pioglitazone may provide therapeutic benefits to type 2 diabetes patients with nonalcoholic steatohepatitis, which will be proven in controlled clinical trials.

Neovascularization aggravated hepatic fibrosis in combined hypoxia NASH rat model

Objective: To optimize the construction of combined hypoxia NASH rat model on the basis of preliminary work, and to explore the role of neovascularization in the process of hepatic fibrosis. Methods: 32 rats were divided randomly to four groups that were null control group(A group ), hypoxia group(B group), high fat diet group(C group ) and high fat diet plus hypoxia group (D group ),treated with null , Intraperitoneal injection of NaNO(2), high fat diet and high fat diet plus Intraperitoneal injection of NaNO(2) respectively. Every group was observed for 16 weeks, B and D group was treated with Intraperitoneal injection of NaNO(2) 20 mg/kg.d at the laster 8 weeks. Liver histology NASH activity score(NAS) and Fibro score(FibroS), biochemical index were detected in this combined hypoxia NASH rat model(D group), meanwhile the changes of HIF1α, inflammatory factor and neovascularization were measured by ELISA, realtime PCR and immunohistochemistry. Results: Liver tissue NAS > 4 was seen in C and D group. D group showed NASH characteristics, including significantly steatosis at liver acinar 3 area(mostly a microvesicular type fat droplets mixed with macrovesicular type), hepatocyte balloon degeneration, obvious lobular inflammation, while fibrosis score increased significantly, including visible hepatic sinusoid fibrosis, fibrosis around portal vein, and bridging fibrosis in a considerable portion of the rats. Compared with C group, biochemical indicators of aspartate aminotransferase (AST), HIF1α, neovascularization-related VEGFA, VEGFR2 mRNA level increased obviously and the expression of immunohistochemistry VEGFR2, CD34 enhanced markedly in D group(p < 0.05). Conclusion: A combined hypoxia NASH rat model can be established throught feeding 16 weeks' high-fat diet then intraperitoneal injection of NaNO(2) 20 mg / kg.d at the laster 8 weeks, meanwhile chronic hypoxia can accelerate this combined hypoxia NASH model liver fibrosis process. In this process neovascularization promoted the formation of hepatic fibrosis in this model.

The Role of the Apelin/APJ System in the Regulation of Liver Disease.

Apelin is an endogenous peptide that is a ligand for the APJ receptor (angiotensin II receptor like-1, AT-1). The apelin/APJ system is distributed in diverse periphery organ tissues. It has been shown that the apelin/APJ system plays various roles in physiology and pathophysiology of many organs. It regulates cardiovascular development or cardiac disease, glycometabolism and fat metabolism as well as metabolic disease. The apelin/APJ system participates in various cell activities such as proliferation, migration, apoptosis or inflammation. However, apelin/APJ function in the liver is still under investigation. In the liver, the apelin-APJ system could play an inhibitory role in liver regeneration and promote Fas-induced apoptosis. It may participate in the formation of hepatic fibrosis or cirrhosis, and even cancer. In this review, we summarize the role of the apelin/APJ system in liver disease.

Egg antigen p40 of Schistosoma japonicum promotes senescence in activated hepatic stellate cells via SKP2/P27 signaling pathway

Schistosomiasis is characterized by egg deposition, granulomatous inflammatory reaction and then subsequent hepatic fibrosis formation. Activated HSCs are regarded as the main effector cells in the progression of liver fibrosis and induction of senescence in hepatic stellate cells (HSCs) is vital to the reversion of hepatic fibrosis. Our previous work has showed that S. japonicum egg antigen p40 (Sjp40) could promote HSCs senescence via a STAT3/p53/p21 mechanism. In this paper, the major aim was to explore whether there are other signaling pathways in the process of Sjp40-induced HSCs aging and the underlying effect of SKP2/P27 signal pathway in this procedure. We observed the Sjp40-induced decrease of α-SMA and the senescence of LX-2 cells, and Sjp40 could upregulate P27 and downregulate the protein level of SKP2. The senescence induced by Sjp40 might be reversed in LX-2 cells that treated with P27-specific siRNA or with SKP2-special over-expression plasmid. In addition, we also demonstrated that the decreased expression of P-Rb and α-SMA induced by Sjp40 were partly restored by SKP2-overexpression. These data suggest that Sjp40 might inhibit HSCs activation by promoting cellular senescence via SKP2/P27 signaling pathway, which put forward novel mechanism in the treatment of liver fibrosis.

ЗНАЧЕНИЕ ПОКАЗАТЕЛЕЙ ИММУНОСУПРЕССИИ И ЭЛАСТОМЕТРИИ ПЕЧЕНИ В ТАКТИКЕ ЛЕЧЕНИЯ КО-ИНФЕКЦИИ ВИЧ/HCV

Objective: Correction of the main treatment regimen for HIV/HCV co-infection, taking into account the degree of immunosuppression based on indices of CD4+ cells in 1 μl of blood and the severity of hepatic fibrosis tissue according to the elastometry of the liver Methods: A complex examination of 21 patients with HIV/HCV co-infection was carried out, according to anamnestic data who are the users of long-term injecting drugs. The diagnosis of HIV infection was confirmed by the positive result of the rapid test, ELISA, (enzyme-linked immunosorbent assay), immunoblotting. Hepatitis C virus is diagnosed by detection by ELISA of serum anti-HCV. To determine the degree of liver fibrosis, the method of elastometry on the FibroScan apparatus (France) was used on the basis of the Institute of Gastroenterology. The results were evaluated on a scale of the degree of fibrosis of liver METAVIR from F0 (healthy liver) to F4 (hepatic cirrhosis). The number of CD4+ lymphocytes in 1 μl of blood was determined by flow cytometry. Results: With a targeted comprehensive examination of 21 patients with HIV/HCV co-infection, pronounced oscillations in the results of elastometry of the hepatic tissue (from F0 to F4), regardless of the number of CD4+ cells (24 to 809 cells/μl) were found. Conclusion: The results obtained indicate that there is no direct correlation between the degree of immunosuppression in CD4+ indices and the process of formation of hepatic fibrosis in patients with co-infection with HIV/HCV, which is a significant additional diagnostic criterion in choosing the priority of therapy. Keywords: Elastometry, immunosuppression, co-infection, HIV, HCV.

Vitamin D and S-farnesylthiosalicylic acid have a synergistic effect on hepatic stellate cells proliferation.

Hepatic stellate cells (HSCs) have a key role in the formation of hepatic fibrosis. The active form of vitamin D, 1,25(OH)2D3, has been found to have antiproliferative and antifibrotic effects in various tissues including liver. Farnesylthiosalicylic acid (FTS), a novel Ras antagonist, was also found to inhibit hepatic fibrosis. The purpose of this study was to examine the antiproliferative and antifibrotic effects of the combined treatment of 1,25(OH)2D3 and FTS on primary cultured HSCs. Primary HSCs, isolated from rat's livers, were treated with 1,25(OH)2D3, FTS or a combination of both. Proliferation was assessed by bromodeoxyuridine. Expression of p-ERK, ERK, Ras-GTP, total-Ras, CyclinD1 and fibrotic markers was measured by western blotting analysis and real-time PCR. Cytotoxicity was assessed by lactate dehydrogenase method. The combined treatment inhibited HSCs proliferation by threefold. The effect was synergistic and non-cytotoxic. In concordance, the combined treatment suppressed CyclinD1 expression by ~2-fold, whereas 1,25(OH)2D3 or FTS alone showed a significantly lower inhibitory effect. The effect of the combined treatment on CyclinD1 expression was mediated via Ras-GTP and p-ERK signal transduction pathway. The effect on fibrotic markers showed that 1,25(OH)2D3 decreased collagen Iα1 expression by ~40%, FTS by ~50% and the combined treatment by ~60%. 1,25(OH)2D3 inhibited tissue inhibitor of metalloproteinases-1 (TIMP-1) expression by 20%. FTS alone or 1,25(OH)2D3 + FTS inhibited TIMP-1 expression by 60%. FTS inhibited transforming growth factor-β (TGF-β) expression by 25%, while 1,25(OH)2D3 had no effect. Although the combination of 1,25(OH)2D3 and FTS did not demonstrate an additive antifibrotic effect, it showed a synergistic antiproliferative effect on primary HSCs. Therefore, the combined treatment may have a potential therapeutic value in the initiation of fibrotic process.

Bevacizumab Attenuates Hepatic Fibrosis in Rats by Inhibiting Activation of Hepatic Stellate Cells

Angiogenesis is a fundamental part of the response to tissue injury, which is involved in the development of hepatic fibrosis. Vascular endothelial growth factor plays an important role in angiogenesis. The expression of VEGF is increased during hepatic fibrogenesis and correlates with the micro-vessel density. In this study, we investigated the effects of bevacizumab, an anti-angiogenetic drug, on the formation of hepatic fibrosis. We found that bevacizumab could attenuate the development of hepatic fibrosis and contribute to the protection of liver function. Bevacizumab was also found to downregulate the expression α-SMA and TGF-β1, which have been reported to be profibrogenic genes in vivo. We also observed that the expression of VEGF increased significantly during the development of hepatic fibrosis and CCl4 was found to induce hepatocytes to secrete VEGF, which led to the activation and proliferation of HSCs. Bevacizumab was also found to block the effects of the hepatocytes on the activation and proliferation of HSCs. Our results suggest that bevacizumab might alleviate liver fibrosis by blocking the effect of VEGF on HSCs. Bevacizumab might be suitable as a potential agent for hepatic fibrosis therapy.