A triterpenoid-enriched extract of bitter melon leaves alleviates hepatic fibrosis by inhibiting inflammatory responses in carbon tetrachloride-treated mice.

Liver fibrosis is a progression of chronic liver disease characterized by excess deposition of fibrillary collagen. The aim of this study was to investigate the protective effect of a triterpenoid-enriched extract (TEE) from bitter melon leaves against carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice. Male ICR mice received TEE (100 or 150 mg kg-1) by daily oral gavage for one week before starting CCl4 administration and throughout the entire experimental period. After intraperitoneal injection of CCl4 for nine weeks, serum and liver tissues of the mice were collected for biochemical, histopathological and molecular analyses. Our results showed that TEE supplementation reduced CCl4-induced serum aspartate aminotransferase and alanine aminotransferase activities. Histopathological examinations revealed that CCl4 administration results in hepatic fibrosis, while TEE supplementation significantly suppressed hepatic necroinflammation and collagen deposition. In addition, TEE supplementation decreased α-smooth muscle actin (α-SMA)-positive staining and protein levels of α-SMA and transforming growth factor-ß1. TEE-supplemented mice had lower mRNA expression levels of interleukin-6, tumor necrosis factor-α, and toll-like receptor 4. Moreover, TEE (150 mg kg-1) supplementation significantly reduced intrahepatic inflammatory Ly6C+ monocyte infiltration. We demonstrated that TEE could ameliorate hepatic fibrosis by regulating inflammatory cytokine secretion and α-SMA expression in the liver to reduce collagen accumulation.

Cordycepin Ameliorates Nonalcoholic Steatohepatitis by Activation of the AMP-Activated Protein Kinase Signaling Pathway.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease, especially nonalcoholic steatohepatitis (NASH), has become a major cause of liver transplantation and liver-associated death. NASH is the hepatic manifestation of metabolic syndrome and is characterized by hepatic steatosis, inflammation, hepatocellular injury, and different degrees of fibrosis. However, there is no US Food and Drug Administration-approved medication to treat this devastating disease. Therapeutic activators of the AMP-activated protein kinase (AMPK) have been proposed as a potential treatment for metabolic diseases such as NASH. Cordycepin, a natural product isolated from the traditional Chinese medicine Cordyceps militaris, has recently emerged as a promising drug candidate for metabolic diseases. APPROACH AND RESULTS: We evaluated the effects of cordycepin on lipid storage in hepatocytes, inflammation, and fibrosis development in mice with NASH. Cordycepin attenuated lipid accumulation, inflammation, and lipotoxicity in hepatocytes subjected to metabolic stress. In addition, cordycepin treatment significantly and dose-dependently decreased the elevated levels of serum aminotransferases in mice with diet-induced NASH. Furthermore, cordycepin treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration, and hepatic fibrosis in mice. In vitro and in vivo mechanistic studies revealed that a key mechanism linking the protective effects of cordycepin were AMPK phosphorylation-dependent, as indicated by the finding that treatment with the AMPK inhibitor Compound C abrogated cordycepin-induced hepatoprotection in hepatocytes and mice with NASH. CONCLUSION: Cordycepin exerts significant protective effects against hepatic steatosis, inflammation, liver injury, and fibrosis in mice under metabolic stress through activation of the AMPK signaling pathway. Cordycepin might be an AMPK activator that can be used for the treatment of NASH.

20S-Protopanaxatriol Ameliorates Hepatic Fibrosis, Potentially Involving FXR-Mediated Inflammatory Signaling Cascades.

Ginseng has been used as a functional food and tonic for enhancing immune power. Here, the potential protective effect of 20S-protopanaxatriol (M4), the metabolite of protopanaxatriol, against hepatic fibrosis is investigated, which could provide nutritional interventions for disease treatment. M4 could inhibit extracellular matrix (ECM) deposition and reduce the levels of proinflammatory cytokines such as caspase 1, interleukin 1 ß (IL-1ß), interleukin 1 receptor type 1 (IL1R1), and interleukin 6 (IL-6). M4 also significantly increased the expression of farnesoid X receptor (FXR), suppressed the purinergic ligand-gated ion channel 7 receptor (P2X7r) signaling pathway, and works as an FXR agonist, GW4064. In thioacetamide (TAA)-induced mice, M4 could attenuate the histopathological changes and significantly regulate the expression levels of FXR and P2X7r. M4 ameliorated TAA-induced hepatic fibrosis due to the reduction of P2X7r secretion, inhibition of hepatic stellate cell (HSCs) activation, and inflammation, which were all associated with FXR activation. Hence, M4 might be useful a nutritional preventive approach in antihepatic fibrosis and antihepatic inflammation.

A Double-Blind, Randomized Placebo-Controlled Trial of Probiotic Lactobacillus casei Shirota in Stable Cirrhotic Patients.

Background: In cirrhosis, a pathological gut microbiome has been linked with immune dysfunction. A pilot study of probiotic Lactobacillus casei Shirota (LcS) in alcoholic cirrhosis demonstrated significant improvement in neutrophil function. This study aimed to evaluate the efficacy of LcS on neutrophil function and significant infection rates in patients with cirrhosis. Methods: 92 cirrhotic patients (Child-Pugh score ≤10) were randomized to receive LcS or placebo, three times daily for six months. Primary end-points were incidence of significant infection and neutrophil function. Secondary end-points were cytokine profile, endotoxin, bacterial DNA positivity, intestinal permeability and quality of life. Results: Rates of infection, decompensation or neutrophil function did not differ between placebo and probiotic groups. LcS significantly reduced plasma monocyte chemotactic protein-1 and, on subgroup analysis, plasma interleukin-1ß (alcoholic cirrhosis), interleukin-17a and macrophage inflammatory protein-1ß (non-alcoholic cirrhosis), compared with placebo. No significant differences in intestinal permeability, bacterial translocation or metabolomic profile were observed. Conclusion: LcS supplementation in patients with early cirrhosis is safe. Although no significant infections were observed in either group, LcS improved cytokine profile towards an anti-inflammatory phenotype, an effect which appears to be independent of bacterial translocation.

Huagan tongluo Fang improves liver fibrosis via down-regulating miR-184 and up-regulating FOXO1 to inhibit Th17 cell differentiation.

BACKGROUND: The purpose of this research is to reveal the improvement effect and potential mechanism of Huagan tongluo Fang (HGTLF) on liver fibrosis. METHODS: A mouse model of liver fibrosis induced by CCl4 was established to analyze the effect of HGTLF on liver fibrosis. The expression changes of miRNA after HGTLF stimulation were detected by qRT-PCR. After interference with miR-184 in Th17 cells, the concentration of IL-17A in cell culture supernatants was detected by ELISA and the proportion of Th17 cells was analyzed by flow cytometry. The relationship between miR-184 and FOXO1 was verified by online software and dual-luciferase reporter system. After HGTLF treatment of Th17 cells overexpressing miR-184, the protein level of FOXO1 was detected by Western blot. RESULTS: HGTLF could significantly improve liver fibrosis in mice. By qRT-PCR, miR-184 was most significantly expressed after HGTLF drug stimulation, and miR-184 was considered to be the major RNA involved in Th17 cell differentiation. Interference with miR-184 in Th17 cells inhibited the differentiation of Th17 cells. By online software and dual-luciferase reporter system assay, the direct interaction of miR-184 with FOXO1 was confirmed. After HGTLF treatment of Th17 cells overexpressing miR-184, FOXO1 protein levels were significantly up-regulated and inhibited the differentiation of Th17 cells, which was reversed by miR-184 inhibitors. The Vivo experiments also confirmed the improvement effect of HGTLF on liver fibrosis in mice. CONCLUSION: Our results indicated that HGTLF could improve liver fibrosis via down-regulating miR-184 and up-regulating of FOXO1 to inhibit Th17 cell differentiation.

Research Progress in Astragalus Membranaceus and Its Active Components on Immune Responses in Liver Fibrosis.

The interaction between immune cells and hepatic stellate cells (HSCs) can modulate the development of hepatic fibrosis. It can also regulate hepatic fibrosis and liver cirrhosis caused by excessive deposition of extracellular matrix (ECM). This article reviews the action mechanism of immune cells on liver fibrosis and the effect of Astragalus membranaeus and its active components on immune cells. In-depth study of interaction between immune cells and HSCs on the pathogenesis of liver fibrosis, and the regulatory effect of Astragalus membranaeus and its active components on immune mechanism will provide new insights in the treatment of liver fibrosis.

Research progress on the anti-hepatic fibrosis action and mechanism of natural products.

Hepatic fibrosis is the most common pathological feature of most chronic liver diseases, and its continuous deterioration gradually develops into liver cirrhosis and eventually leads to liver cancer. At present, there are many kinds of drugs used to treat liver fibrosis. However, Western drugs tend to only target single genes/proteins and induce many adverse reactions. Most of the mechanisms and active ingredients of traditional Chinese medicine (TCM) are not clear, and there is a lack of unified diagnosis and treatment standards. Natural products, which are characterized by structural diversity, low toxicity, and origination from a wide range of sources, have unique advantages and great potential in anti-liver fibrosis. This article summarizes the work done over the previous decade, on the active ingredients in natural products that are reported to have anti-hepatic fibrosis effects. The effective anti-hepatic fibrosis ingredients identified can be generally divided into flavonoids, saponins, polysaccharides and alkaloids. Mechanisms of anti-liver fibrosis include inhibition of liver inflammation, anti-lipid peroxidation injury, inhibition of the activation and proliferation of hepatic stellate cells (HSCs), modulation of the synthesis and secretion of pro-fibrosis factors, and regulation of the synthesis and degradation of the extracellular matrix (ECM). This review provides suggestions for the development of anti-hepatic fibrosis drugs.

The immunoreactivity of TGF-b1 in non-alcoholic fatty liver disease.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease which becomes a rapidly growing health problem in the Western countries. The development of the disease is most often connected to obesity. NAFLD is also considered as the hepatic manifestation of metabolic syndrome. Transforming growth factor b1 (TGF-b1) plays an important role in the pathogenesis of liver fibrosis, being involved in activation of hepatic stellate cells, stimulation of collagen gene transcription, and suppression of matrix metalloproteinase expression. The objective of the study was to evaluate by immunohistochemistry the expression of TGF-b1 in the liver tissue of NAFLD patients and correlate it with anthropometric, biochemical and routine histological parameters. MATERIAL AND METHODS: The study group consisted of 48 patients with diagnosed NAFLD. Liver steatosis, NAFLD Activity Score (NAS) and METAVIR score of fibrosis were evaluated in liver biopsies. The immunoreactivity of TGF-b1 was evaluated semi-quantitatively separately in portal, septal, lobular hepatocytic and lobular sinu-soidal liver compartments. The results were analyzed in regard to patients' clinical and biochemical parameters. RESULTS: Neither steatosis nor NAS correlated with TGF-b1 expression in any liver compartment, whereas METAVIR score of fibrosis was associated with increased immunoreactivity of TGF-b1 in most of the studied liver compartments. TGF-b1 immunoreactivity showed positive correlation with patients' age and its expression in septal compartment disclosed positive correlation with body mass index, and waist and hip circumference. Hyaluronic acid serum level was positively and iron concentration was negatively associated with TGF-b1 ex-pression in the selected consecutive liver compartments. CONCLUSIONS: The immunohistochemical expression of TGF-b1 may be complementary to routine methods of liver fibrosis evaluation.

Prevention of hepatic stellate cell activation using JQ1- and atorvastatin-loaded chitosan nanoparticles as a promising approach in therapy of liver fibrosis.

Preventing hepatic stellate cell (HSC) activation represents a promising approach to resolve liver fibrosis. Several drugs have been reported to delay/prevent HSCs activation, however with limited clinical benefits. The latter may be in part attributed to the limited ability of such drugs in targeting more than one pathway of HSC activation. Added to that, is their inability of reaching their target cell in sufficient amounts to induce a therapeutic effect. In this work, chitosan NPs were loaded with JQ1 and atorvastatin, two drugs that have been reported to prevent HSCs activation, however via different mechanisms. NPs were then modified with different densities of retinol (Rt) for active targeting of HSCs. The NP HSCs targeting ability as a function of Rt density was assessed in vitro on primary HSCs and in vivo in carbon tetrachloride (CCl4) induced fibrotic mouse models. In vitro NPs modified with a low Rt density (LRt-NPs) showed ≈2 folds enhanced HSCs uptake in comparison to unmodified NPs, whereas NPs modified with a high Rt density (HRt-NPs) showed ≈0.8 folds change in uptake relative to unmodified NPs. Similarly, in vivo LRt-NPs showed higher accumulation in fibrotic livers in comparison to healthy livers whereas HRt-NPs and unmodified NPs showed lower accumulation in fibrotic livers relative to healthy controls respectively. Finally, the ability of drug-loaded NPs in preventing HSCs activation was assessed by monitoring the reduction in α-smooth muscle actin (α-SMA) expression by Western blot. NPs loaded with both JQ1 and atorvastatin showed reduction in α-SMA expression. In addition, a synergistic reduction in α-SMA was observed when cells were co-treated with JQ1 and atorvastatin loaded NPs.

Evolving Role of Vitamin D in Immune-Mediated Disease and Its Implications in Autoimmune Hepatitis.

Vitamin D has immunomodulatory, anti-inflammatory, antioxidant, and anti-fibrotic actions that may impact on the occurrence and outcome of immune-mediated disease. The goals of this review are to describe the nature of these expanded roles, examine the implications of vitamin D deficiency in autoimmune hepatitis, and identify opportunities for future investigation. Abstracts were identified in PubMed by multiple search terms. Full-length articles were selected for review, and secondary and tertiary bibliographies were developed. Vitamin D receptors are expressed on the principal cell populations involved in the innate and adaptive immune responses. Macrophages and dendritic cells can produce 1,25-dihydroxyvitamin D within the microenvironment. This active form of vitamin D can inhibit immune cell proliferation, promote an anti-inflammatory cytokine profile, expand regulatory T cells, enhance glucocorticoid actions, increase glutathione production, and inhibit hepatic stellate cells. Vitamin D deficiency has been commonly present in patients with immune-mediated liver and non-liver diseases, and it has been associated with histological severity, advanced hepatic fibrosis, and non-response to conventional glucocorticoid therapy in autoimmune hepatitis. Vitamin D analogues with high potency, low calcemic effects, and independence from hepatic hydroxylation are possible interventions. In conclusion, vitamin D has properties that could ameliorate immune-mediated disease, and vitamin D deficiency has been a common finding in immune-mediated liver and non-liver diseases, including autoimmune hepatitis. Loss of vitamin D-dependent homeostatic mechanisms may promote disease progression. Vitamin D analogues that are independent of hepatic hydroxylation constitute an investigational opportunity to supplement current management of autoimmune hepatitis.

Is there a future for andrographolide to be an anti-inflammatory drug? Deciphering its major mechanisms of action.

Andrographis paniculata has long been part of the traditional herbal medicine system in Asia and in Scandinavia. Andrographolide was isolated as a major bioactive constituent of A. paniculata in 1951, and since 1984, andrographolide and its analogs have been scrutinized with modern drug discovery approach for anti-inflammatory properties. With this accumulated wealth of pre-clinical data, it is imperative to review and consolidate different sources of information, to decipher the major anti-inflammatory mechanisms of action in inflammatory diseases, and to provide direction for future studies. Andrographolide and its analogs have been shown to provide anti-inflammatory benefits in a variety of inflammatory disease models. Among the diverse signaling pathways investigated, inhibition of NF-κB activity is the prevailing anti-inflammatory mechanism elicited by andrographolide. There is also increasing evidence supporting endogenous antioxidant defense enhancement by andrographolide through Nrf2 activation. However, the exact pathway leading to NF-κB and Nrf2 activation by andrographolide has yet to be elucidated. Validation and consensus on the major mechanistic actions of andrographolide in different inflammatory conditions are required before translating current findings into clinical settings. There are a few clinical trials conducted using andrographolide in fixed combination formulation which have shown anti-inflammatory benefits and good safety profile. A concerted effort is definitely needed to identify potent andrographolide lead compounds with improved pharmacokinetics and toxicological properties. Taken together, andrographolide and its analogs have great potential to be the next new class of anti-inflammatory agents, and more andrographolide molecules are likely moving towards clinical study stage in the near future.

Preventive effects of Ophiocordyceps sinensis mycelium on the liver fibrosis induced by thioacetamide.

Thioacetamide (TAA), usually used as a fungicide to control the decay of citrus products, itself is not toxic to the liver, but its intermediates are able to increase oxidative stress in livers and further cause fibrosis. Ophiocordyceps sinensis mycelium (OSM) which contains 10% polysaccharides and 0.25% adenosine decreased (P < 0.05) the lipid accumulation and increased (P < 0.05) antioxidative capacity in livers of thioacetamide (TAA) injected rats. Meanwhile, the increased (P < 0.05) liver sizes, serum alanine transaminase (AST) and aspartate transaminase (ALT) values in thioacetamide (TAA)-injected rats were ameliorated (P < 0.05) by OSM supplementation. Moreover, the levels of proinflammatory cytokines, such as the tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), were also reduced (P < 0.05). The fibrosis phenomena in pathological (Masson's trichrome and H&E stainings) and immunohistochemical [α-smooth actin (αSMA) and CD86/ED1] observations in TAA-treated rats were reduced (P < 0.05) by OSM cotreatment. The protective effect of OSM against TAA-induced liver inflammation/fibrosis may be via downregulations (P < 0.05) of TGF-ß pathways and NFκB which further influenced (P < 0.05) the expressions of fibrotic and inflammatory genes (i. e., αSMA, Col1α, COX2). Therefore, OSM shows preventive effects on the development of TAA-induced hepatic fibrosis.

Ascites' neutrophil function is significantly impaired in patients with decompensated cirrhosis but can be restored by autologous plasma incubation.

Systemic immune cell dysfunction is a typical feature of liver diseases and increases the risk of bacterial infection, especially spontaneous bacterial peritonitis. We evaluated functional properties of neutrophil granulocytes in blood and ascites of patients both with and without decompensated cirrhosis. We collected blood and ascites samples from 63 patients with cirrhosis and eight without cirrhosis. Phagocytosis activity (PA) and oxidative burst activity (OBA) were evaluated after ex vivo stimulation with E. coli, while fluorescence signals were measured by flow cytometry. Ascites' neutrophil function tests were repeated after incubation with autologous plasma. Ascites' neutrophils showed an impaired PA and OBA (median blood PA 98.1% (86.8-99.8) vs. ascites' PA 50.5% (0.4-97.3), p < 0.0001; median blood OBA 98.7% (27.5-100) vs. ascites' OBA 27.5% (0.3-96.7), p < 0.0001). Patients with non-cirrhotic ascites showed higher PA but equally suppressed OBA. Ascites' neutrophil function could be partially restored after incubation with autologous plasma (median increase PA: 22.5% (-49.7 - +93.2), p = 0.002; OBA: 22.8% (-10.4 - +48.8), p = 0.002). Ascites' neutrophils of patients with cirrhosis are functionally impaired, but could be partially restored after incubation with plasma. Further investigations are needed to identify the factors in ascites that are associated with neutrophils' function.

Randomised clinical trial: the effects of a multispecies probiotic vs. placebo on innate immune function, bacterial translocation and gut permeability in patients with cirrhosis.

BACKGROUND: Probiotics may correct intestinal dysbiosis and proinflammatory conditions in patients with liver cirrhosis. AIM: To test the effects of a multispecies probiotic on innate immune function, bacterial translocation and gut permeability. METHODS: In a randomised, double blind, placebo-controlled study, stable cirrhotic out-patients either received a daily dose of a probiotic powder containing eight different bacterial strains (Ecologic Barrier, Winclove, Amsterdam, The Netherlands) (n = 44) or a placebo (n = 36) for 6 months and were followed up for another 6 months. RESULTS: We found a significant but subclinical increase in neutrophil resting burst (2.6-3.2%, P = 0.0134) and neopterin levels (7.7-8.4 nmol/L, P = 0.001) with probiotics but not with placebo. Probiotic supplementation did not have a significant influence on neutrophil phagocytosis, endotoxin load, gut permeability or inflammatory markers. Ten severe infections occurred in total; one during intervention in the placebo group, and five and four after the intervention has ended in the probiotic and placebo group, respectively. Liver function showed some improvement with probiotics but not with placebo. CONCLUSIONS: Probiotic supplementation significantly increased serum neopterin levels and the production of reactive oxygen species by neutrophils. These findings might explain the beneficial effects of probiotics on immune function. Furthermore, probiotic supplementation may be a well-tolerated method to maintain or even improve liver function in stable cirrhosis. However, its influence on gut barrier function and bacterial translocation in cirrhotic patients is minimal.

Cultured Mycelium Cordyceps sinensis allevi¬ates CCl4-induced liver inflammation and fibrosis in mice by activating hepatic natural killer cells.

AIM: Recent evidence shows that cultured mycelium Cordyceps sinensis (CMCS) effectively protects against liver fibrosis in mice. Here, we investigated whether the anti-fibrotic action of CMCS was related to its regulation of the activity of hepatic natural killer (NK) cells in CCl4-treated mice. METHODS: C57BL/6 mice were injected with 10% CCl4 (2 mL/kg, ip) 3 times per week for 4 weeks, and received CMCS (120 mg·kg(-1)·d(-1), ig) during this period. In another part of experiments, the mice were also injected with an NK cell-deleting antibody ASGM-1 (20 µg, ip) 5 times in the first 3 weeks. After the mice were sacrificed, serum liver function, and liver inflammation, hydroxyproline content and collagen deposition were assessed. The numbers of hepatic NK cells and expression of NKG2D (activation receptor of NK cells) on isolated liver lymphocytes were analyzed using flow cytometry. Desmin expression and cell apoptosis in liver tissues were studied using desmin staining and TUNEL assay, respectively. The levels of α-SMA, TGF-ß, RAE-1δ and RAE-1ε in liver tissues were determined by RT-qPCR. RESULTS: In CCl4-treated mice, CMCS administration significantly improved liver function, attenuated liver inflammation and fibrosis, and increased the numbers of hepatic NK cells and expression level of NKG2D on hepatic NK cells. Furthermore, CMCS administration significantly decreased desmin expression in liver tissues, and increased TUNEL staining adjacent to hepatic stellate cells. Injection with NK cell-deleting ASGM-1 not only diminished the numbers of hepatic NK cells, but also greatly accelerated liver inflammation and fibrosis in CCl4-treated mice. In CCl4-treated mice with NK cell depletion, CMCS administration decelerated the rate of liver fibrosis development, and mildly upregulated the numbers of hepatic NK cells but without changing NKG2D expression. CONCLUSION: CMCS alleviates CCl4-induced liver inflammation and fibrosis via promoting activation of hepatic NK cells. CMCS partially reverses ASGM-1-induced depletion of hepatic NK cells.

Enhanced LL-37 expression following vitamin D supplementation in patients with cirrhosis and spontaneous bacterial peritonitis.

BACKGROUND & AIMS: The morbidity and mortality of spontaneous bacterial peritonitis (SBP) are high among patients with cirrhosis; however, the mechanisms of SBP pathogenesis are poorly understood. This study aimed to determine the role of the vitamin D-LL-37 pathway in the pathogenesis and treatment in patients with cirrhosis and SBP. METHODS: Serum 25-hydroxyvitamin D concentrations of 119 patients with chronic liver diseases were tested. Vitamin D receptor (VDR) and LL-37 in peritoneal leucocytes of cirrhotic and ascitic patients with SBP were detected and compared with those without SBP. Then the peritoneal macrophages of non-infected patients were cultured and activated by lipopolysaccharide (LPS) to analyse the changes of VDR and LL-37 expressions after incubation with vitamin D. RESULTS: Vitamin D deficiency or insufficiency was found in all of patients with cirrhosis. LPS inhibited VDR and LL-37 expression in peritoneal macrophages [1.3-fold decrease (P = 0.003) and 20-fold decrease (P = 0.010) respectively]. However, vitamin D could reverse the inhibition of both VDR and LL-37 [1.5-fold increase (P = 0.001) and 2000-fold increase (P < 0.001) respectively]. The effect of the incubation time following vitamin D supplementation was significant for LL-37 expression, with a peak expression found at 36 h (P < 0.001). CONCLUSIONS: When vitamin D levels were low, bacteria inhibited VDR and LL-37 responses in peritoneal macrophages as a mechanism to evade antibacterial defence. Vitamin D supplementation could up-regulate peritoneal macrophage VDR and LL-37 expressions, which resulted in an enhanced immunological defence against SBP in patients with cirrhosis and ascites.

Theophylline, an old drug with multi-faceted effects: Its potential benefits in immunological liver injury in rats.

AIM: A low dose of theophylline enhances histone deacetylase activity leading to inhibition of proinflammatory transcription, and inhibits lung fibroblast proliferation. The present work investigated the effect of lowdose theophylline on biochemical and histological pictures of liver tissues in rats with immunological hepatic injury induced by concanavalin A (Con A). MAIN METHODS: Ratswere assigned to control vehicle,model (Con A) and theophylline groups. Half of the animals in each group were sacrificed at the end of the 4th week and the other half were sacrificed at the end of the 8th week. KEY FINDINGS: There was a time-dependent increase in the liver injury parameters by the end of the 4th and 8th weeks in the Con A treated group. Theophylline (20 mg/kg/day), produced a significant decrease in serum liver enzymes (ALT, AST), serum interferon gamma (IFN-γ) levels and the hepatic transforming growth factor-ß (TGF-ß) level. A significant decrease in liver tissue hydroxyproline content together with reduction in portal hypertension at the end of the 8th week was detected compared to the Con A group. Theophylline treated rats exhibited a significant decrease in hepatic vacuolation, apoptosis, leucocyte infiltration, and accumulation of collagen fibers in comparison to the Con A group. In addition, significant decreases in the area percentage of fibrosis and the area percentage of caspase +ve cells were reported compared to the Con A group. SIGNIFICANCE: Theophylline effectively reduced the inflammation of liver tissues and alleviated the liver damage by decreasing IFN-γ and TGF-ß in liver tissues of rats with immunological hepatic injury.

Dietary Supplementation of Genistein Alleviates Liver Inflammation and Fibrosis Mediated by a Methionine-Choline-Deficient Diet in db/db Mice.

Nonalcoholic fatty liver disease is a complex disorder which includes simple steatosis, steatohepatitis, fibrosis and ultimately cirrhosis. Previous studies have reported that genistein, a soy phytoestrogen, attenuates steatohepatitis induced in obese and type 2 diabetic models. Here we investigated the effect of dietary genistein supplementation (0.05%) on nonalcoholic steatohepatitis (NASH) development induced by a methionine-choline-deficient (MCD) diet in db/db mice. MCD-diet-fed mice exhibited a significantly lower body weight and a higher degree of steatohepatitis with increased oxidative stress, steatosis, inflammation, stellate cell activation, and mild fibrosis. Although genistein did not inhibit hepatic steatosis, we observed that oxidative stress, endoplasmic reticulum stress, and AMP-dependent kinase inactivation were alleviated by genistein. Genistein also down-regulated the augmented gene expressions associated with hepatic inflammation and fibrosis. Therefore, these results suggest that genistein may protect MCD-diet-mediated NASH development by suppressing lipid peroxidation, inflammation, and even liver fibrosis in db/db mice.

A Targeted Multiple Antigenic Peptide Vaccine Augments the Immune Response to Self TGF-ß1 and Suppresses Ongoing Hepatic Fibrosis.

Transforming growth factor (TGF)-ß1 expression is induced upon liver injury, and plays a critical role in hepatic fibrosis. Antibodies against TGF-ß1 represent a novel approach in the treatment of hepatic fibrosis. However, TGF-ß1 is not a suitable antigen due to immunological tolerance. In the current study, we synthesized a multiple antigenic peptide (MAP) vaccine against the dominant B-cell epitope of TGF-ß1. The immunogenicity and potential therapeutic effects of this vaccine were examined using a rat model of hepatic fibrosis. Dominant B-cell epitopes of TGF-ß1 were identified using bioinformatic program. An MAP vaccine corresponding to the 90-98 amino acid domain of TGF-ß1 and containing four dendritic arms was synthesized using a 9-fluorenylmethoxycarbonyl solid phase method. Hepatic fibrosis which was induced in male Sprague-Dawley rats received a high-fat diet and ethanol (1.8 g/kg). Starting from the third week, rats were exposed to 40 % carbon tetrachloride (CCl4; 150 µl/100 g body weight twice weekly, initially 200 µl/100 g) treatment for a duration of 8 weeks. Rats received the MAP vaccine (100 µg) or Freund's adjuvant at weeks 1, 3, 5. A group of rats receiving the fibrosis-inducing regimen alone and a group of healthy rats (receiving an olive oil vehicle alone) were included as controls. At the conclusion of the experiment, serum titre of TGF-ß1 antibody was measured using ELISA and a standard liver functional test panel was examined. The extent of hepatic fibrosis was determined by measuring hydroxyproline content in the liver as well as hematoxylin-eosin (HE) and van Gieson (VG) staining. The expression of TGF-ß1 and alpha-smooth muscle actin (α-SMA) was examined using immunohistochemistry, and presented as positive staining cells. The MAP purity was >90 % upon reverse phase high-performance liquid chromatography, with apparent molecular weight at 4.77 kDa. Serum TGF-ß1 antibody titre was 1:256. The fibrosis-inducing treatment produced significant liver damage, as reflected by increases in liver functional test, HE and VG staining. The MAP vaccine attenuated such damage, as reflected by decreased alanine aminotransferase, aspartate aminotransferase, total bilirubin, and hepatic hydroxyproline (116.78 ± 23.76 vs. 282.71 ± 136.94 IU/L; 319.78 ± 82.48 vs. 495.29 ± 137.13 IU/L; 2.02 ± 0.27 vs. 4.01 ± 0.52 µmol/L; 263.67 ± 41.18 vs. 439.14 ± 43.29 µg/g vs. in model rats, respectively; p < 0.01), as well as fibrosis extent by HE and VG staining. The MAP vaccine reduced TGF-ß1 and α-SMA expression in rats (0.325 ± 0.059 vs. 0.507 ± 0.044 IOD/area; 0.318 ± 0.058 vs. 0.489 ± 0.029 IOD/area vs. model rats, respectively; p < 0.05). The TGF-ß1 MAP vaccine could generate sufficient antibody that suppresses the development of hepatic fibrosis.

IL-30 (IL27p28) attenuates liver fibrosis through inducing NKG2D-rae1 interaction between NKT and activated hepatic stellate cells in mice.

UNLABELLED: Chronic hepatic diseases, such as cirrhosis, hepatocellular carcinoma, and virus-mediated immunopathogenic infections, affect billions of people worldwide. These diseases commonly initiate with fibrosis. Owing to the various side effects of antifibrotic therapy and the difficulty of diagnosing asymptomatic patients, suitable medication remains a major concern. To overcome this drawback, the use of cytokine-based sustained therapy might be a suitable alternative with minimal side effects. Here, we studied the therapeutic efficacy and potential mechanisms of interleukin (IL)-30 as antifibrosis therapy in murine liver fibrosis models. CCl4 or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) 0.1% (wt/wt) Purina 5015 Chow (LabDiet, St. Louis, MO) was fed for 3 weeks to induce liver fibrosis. Either control vector (pCtr) or pIL30 was injected hydrodynamically once per week. A significant decrease in collagen deposition and reduced expression of alpha-smooth muscle actin (α-SMA) protein indicated that IL-30-based gene therapy dramatically reduced bridging fibrosis that was induced by CCl4 or DDC. Immunophenotyping and knockout studies showed that IL-30 recruits natural-killer-like T (NKT) cells to the liver to remove activated hepatic stellate cells (HSCs) significantly and ameliorate liver fibrosis. Both flow cytometric and antibody-mediated neutralization studies showed that liver NKT cells up-regulate the natural killer group 2, member D (NKG2D) ligand and bind with the NKG2D ligand, retinoic acid early inducible 1 (Rae1), and positively activated HSCs to ameliorate liver fibrosis. Furthermore, adoptive transfer of liver NKT cells in T-cell-deficient mice showed reduction of fibrosis upon IL-30 administration. CONCLUSIONS: Highly target-specific liver NKT cells selectively remove activated HSCs through an NKG2D-Rae1 interaction to ameliorate liver fibrosis after IL-30 treatment.