Injured Hepatocytes Research Articles

HGF/heparin-immobilized decellularized liver matrices as novel hepatic patches for hepatocyte regeneration in an acute liver injury model

Although liver cirrhosis is a common condition and cause of death, treatment methods remain limited; for example, instead of facilitating hepatocyte regeneration, they may only alleviate or resolve symptoms. Notably, studies have indicated that liver fibrosis is reversible; moreover, prompt and appropriate treatment can prevent liver fibrosis from developing into liver cirrhosis. In this study, a hepatocyte growth factor (HGF)/heparin-immobilized decellularized liver matrix (HGF/heparin-DLM) was fabricated for hepatocyte regeneration in a model of acute d-galactosamine–induced liver injury. Various initial concentrations (0.2, 0.4, 0.6, 0.8, 1 mg/mL) were used for the immobilization of heparin on DLM films. The amounts of immobilized heparin on the DLM films at these concentrations were 4.7 ± 1.1, 12.2 ± 2.7, 18.2 ± 5.1, 21.9 ± 3.8, and 29.1 ± 1.1 μg/cm2, respectively. The relative viability and albumin synthesis of the hepatocytes on the HGF/heparin-DLM films were 19% and 26% greater than those of the hepatocytes cultured in regular petri dishes on day 3, respectively. The LDH and albumin of the injured hepatocytes on the HGF/heparin-DLM films were 95% lower and almost 2.7 times higher than those cultured in regular petri dishes on day 5, respectively. In sum, the HGF/heparin-DLM films were effective in hepatocyte culturing and repairing d-galactosamine–induced hepatocyte damage. Thus, they have potential for future clinical applications.

Interleukin-17 in Liver Disease Pathogenesis.

Interleukin 17A (IL-17A)-producing T helper 17 (Th17) cells were identified as a subset of T helper cells that play a critical role in host defense against bacterial and fungal pathogens. Th17 cells differentiate from Th0 naïve T-cells in response to transforming growth factor β1 (TGF-β1) and IL-6, the cytokines which also drive development of liver fibrosis, require activation of transcription factor retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt). IL-17A signals through the ubiquitously expressed receptor IL-17RA. Expression of IL-17RA is upregulated in patients with hepatitis B virus/hepatitis C virus (HBV/HCV) infections, nonalcoholic steatohepatitis (NASH), alcohol-associated liver disease (AALD), hepatocellular carcinoma (HCC), and experimental models of chronic toxic liver injury. The role of IL-17 signaling in the pathogenesis of NASH- and AALD-induced metabolic liver injury and HCC will be the focus of this review. The role of IL-17A-IL-17RA axis in mediation of the cross-talk between metabolically injured hepatic macrophages, hepatocytes, and fibrogenic myofibroblasts will be discussed.

Pathology of Hepatic Iron Overload

Pathology of Hepatic Iron Overload

Vitamin E pretreated Wharton’s jelly-derived mesenchymal stem cells attenuate CCl4-induced hepatocyte injury in vitro and liver fibrosis in vivo

Oxidative microenvironment in fibrotic liver alleviates the efficacious outcome of mesenchymal stem cells (MSCs)-based cell therapy. Recent evidence suggests that pharmacological pretreatment is a rational approach to harness the MSCs with higher therapeutic potential. Here, we investigated whether Vitamin E pretreatment can boost the antifibrotic effects of Wharton’s jelly-derived MSCs (WJMSCs). We used rat liver-derived hepatocytes injured by CCl4 treatment in co-culture system with Vitamin E pretreated-WJMSCs (Vit E-WJMSCs) to evaluate the hepatoprotective effect of Vit E-WJMSCs. After 24 h of co-culturing, we found that Vit E-WJMSCs rescued injured hepatocytes as hepatocyte injury-associated medium (AST, ALT, and ALP) and mRNA (Cyp2e1, Hif1-α, and Il-1β) markers reduced to normal levels. Subsequently, CCl4-induced liver fibrosis rat models were employed to examine the antifibrotic potential of Vit E-WJMSCs. After 1 month of cell transplantation, it was revealed that Vit E-WJMSCs transplantation ceased fibrotic progression, as evident by improved hepatic architecture and functions, more significantly in comparison to naïve WJMSCs. In addition, Vit E-WJMSCs transplantation decreased the expressions of fibrosis-associated gene (Tgf-β1, α-Sma, and Col1α1) markers in the liver parenchyma. Intriguingly, the results of tracing experiments discovered that more WJMSCs engrafted in the Vit E-WJMSCs treated rat livers compared to naïve WJMSCs treated livers. These findings implicate that pretreatment of WJMSCs with Vitamin E improves their tolerance to hostile niche of fibrotic liver; thereby further enhancing their efficacy for hepatic fibrosis.

From the Editor's Desk…

From the Editor's Desk…

Mesenchymal Stem Cell-Derived Exosomes and Other Extracellular Vesicles as New Remedies in the Therapy of Inflammatory Diseases.

There is growing evidence that mesenchymal stem cell (MSC)-based immunosuppression was mainly attributed to the effects of MSC-derived extracellular vesicles (MSC-EVs). MSC-EVs are enriched with MSC-sourced bioactive molecules (messenger RNA (mRNA), microRNAs (miRNAs), cytokines, chemokines, immunomodulatory factors) that regulate phenotype, function and homing of immune cells. In this review article we emphasized current knowledge regarding molecular mechanisms responsible for the therapeutic effects of MSC-EVs in attenuation of autoimmune and inflammatory diseases. We described the disease-specific cellular targets of MSC-EVs and defined MSC-sourced molecules, which were responsible for MSC-EV-based immunosuppression. Results obtained in a large number of experimental studies revealed that both local and systemic administration of MSC-EVs efficiently suppressed detrimental immune response in inflamed tissues and promoted survival and regeneration of injured parenchymal cells. MSC-EVs-based anti-inflammatory effects were relied on the delivery of immunoregulatory miRNAs and immunomodulatory proteins in inflammatory immune cells (M1 macrophages, dendritic cells (DCs), CD4+Th1 and Th17 cells), enabling their phenotypic conversion into immunosuppressive M2 macrophages, tolerogenic DCs and T regulatory cells. Additionally, through the delivery of mRNAs and miRNAs, MSC-EVs activated autophagy and/or inhibited apoptosis, necrosis and oxidative stress in injured hepatocytes, neurons, retinal cells, lung, gut and renal epithelial cells, promoting their survival and regeneration.

Inhibiting alpha subunit of eukaryotic initiation factor 2 dephosphorylation protects injured hepatocytes and reduces hepatocyte proliferation in acute liver injury

AimTo investigate the impact of alpha subunit of eukaryotic initiation factor 2 (eIF2α) phosphorylation on liver regeneration.MethodsMale BALB/c mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce liver injury. Human hepatocyte LO2 cells were incubated with thapsigargin to induce endoplasmic reticulum (ER) stress. Salubrinal, integrated stress response inhibitor (ISRIB), and DnaJC3 overexpression were used to alter eIF2α phosphorylation levels.ResultsCCl4 administration induced significant ER stress and eIF2α phosphorylation, and increased hepatocyte proliferation proportionally to the extent of injury. Inhibiting eIF2α dephosphorylation with salubrinal pretreatment significantly mitigated liver injury and hepatocyte proliferation. In LO2 cells, thapsigargin induced significant eIF2α phosphorylation and inhibited proliferation. Inhibiting eIF2α dephosphorylation partly restored cell proliferation during ER stress.ConclusionsIn acute liver injury, inhibiting eIF2α dephosphorylation protects injured hepatocytes and reduces hepatocyte proliferation.

Decellularized liver matrix as substrates for rescue of acute hepatocytes toxicity

More and more scholars regard the lesion of liver cirrhosis as a series of progressive clinical stages. Besides, liver cirrhosis is a late stage of scarring (fibrosis) of the liver, and has long been a common cause of death for the global adult population, thus, the treatment of liver cirrhosis is a key point investigated in the biomedical field. Here, we propose a novel hypothesis; if decellularized liver matrix (DLM) is possible injected into the injurant-induced fiberized liver via the hepatic portal vein to thoroughly repair the liver, it may be an effective therapeutic method for liver fibrosis or even liver cirrhosis. This study mixed rat DLM with gelatin-hydroxyphenylpropionic acid (Glt-HPA) to form a three-dimensional structure to simulate the in vivo liver environment, and cultured the primary rat hepatocytes in it. Afterward, the hepatocytes were treated using D-galactosamine (GaIN), CHCl3 , and CCl4 -containing medium to simulate the toxin-mediated liver fibrosis in vitro. Finally, they were cultured in a DLM-containing medium to observe the viability and functions of the damaged hepatocytes, and the hypothesis of this research was proved, meaning that Glt-HPA-DLM acting on damaged hepatocytes may repair them. Results have shown that Glt-HPA-DLM was effective for hepatocytes culture and repaired injured hepatocytes from GaIN, CHCl3 , and CCl4 (albumin synthesis was increased by 219, 108, and 12%, respectively, whereas relative lactate dehydrogenase activity was reduced by 38, 68, and 67%, after 5 days of culture, separately). This research shows promising effects against hepatic fibrosis and may have potential for liver cirrhosis in vivo.

Cell Type-specific Gene Expression Profiling in the Mouse Liver.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Parathyroid hormone-related protein activates HSCs via hedgehog signalling during liver fibrosis development

Recently, we showed that parathyroid hormone-like hormone (PTHLH), a cytokine-like polyprotein, is critical for extracellular matrix (ECM) deposition through the activation of hepatic stellate cells (HSCs). Here, we show that N-terminal PTHLH is secreted into the supernatant of injured hepatocytes, its expression is positively correlated with liver fibrosis severity based on mice liver biopsies, and it is primarily expressed in the cytoplasm of hepatocytes along the fibrous septa of fibrotic livers. PTHLH overexpression in mice was achieved through adeno-associated virus-mediated gene delivery (AAV9-PTHLH), and liver fibrosis was induced with carbon tetrachloride (CCl4). We observed that AAV9-PTHLH induced spontaneous development of liver fibrosis and increased sensitivity to CCl4. PTHLH increased Hedgehog (Hh) pathway activation in a PTH1R-dependent manner, and the effect of PTHLH was primarily mediated by protein kinase C (PKC) θ. PTHLH-mediated PTH1R-PKC θ pathway activation is a key event in the profibrotic Hh-dependent activation of HSCs.

Cadherin-11 contributes to liver fibrosis induced by carbon tetrachloride.

Background and aimsLiver fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) leading to impaired function and cirrhosis. Previous reports support a role for cadherin-11 (CDH11) in regulating the development of dermal and pulmonary fibrosis. In the current report, the extent to which CDH11 modulates the development of liver fibrosis induced by carbon tetrachloride (CCL4) was assessed.MethodsWild type (WT) and CDH11 deficient (CDH11-/-) mice were treated with CCl4 or vehicle control for 8 weeks to induce liver fibrosis. Liver fibrosis was assessed by histology, collagen content, and RTPCR of fibrotic mediators.ResultsLivers from WT mice treated with CCl4 had increased levels of CDH11 which localized to injured hepatocytes, hepatic stellate cells, and macrophages. Interestingly, CDH11-/- mice had decreased histological evidence of liver fibrosis, collagen deposition, α-smooth muscle actin (α-SMA) accumulation, and mRNA levels of fibrotic mediators such as Col1-α1, Snail, TGF-β and IL-6.ConclusionsThese data demonstrate that CDH11 is increased during liver fibrosis, is an important regulator of liver fibrosis induced by CCL4 and suggest that CDH11 may be a potential therapeutic target for liver fibrosis.

Human liver regeneration following massive hepatic necrosis: Two distinct patterns.

Massive hepatic necrosis is a rare but often fatal complication of various liver injuries. Nevertheless, some patients can survive by spontaneous hepatic regeneration. It is known that surviving hepatocytes and/or progenitor cells can participate in this process but the mechanism of hepatic recovery is vague. We examined 13 explanted human livers removed for acute liver failure. Combined immunohistochemistry, digital image analysis, and three-dimensional reconstruction of serial sections were applied. Two patterns of regeneration could be distinguished. In livers with centrilobular necrosis, the surviving injured periportal hepatocytes started to proliferate and arrange into acinar structures and expressed α-fetoprotein. If the injury wiped out almost all hepatocytes, large areas of parenchymal loss were invaded by an intense ductular reaction. The cells at the distal pole of the ductules differentiated into hepatocytes and formed foci organized by the branches of the portal vein. The expanding foci often containing complete portal triads were arranged around surviving central veins. Their fusion eventually could be an attempt to re-establish the hepatic lobules. Regeneration of human livers following massive hepatic necrosis can occur in two ways-either through proliferation of α-fetoprotein-positive acinary-arranged hepatocytes or through ductular progenitor cells, with the latter being less efficient. Further investigation of these regenerative pathways may help identify biomarkers for likelihood of complete regeneration and hence have therapeutic implications.

MFGE8 protects against CCl4 -induced liver injury by reducing apoptosis and promoting proliferation of hepatocytes.

Milk fat globule-EGF factor 8 (MFGE8) has been reported to play various roles in acute injury and inflammation response. However, the role of MFGE8 in liver injury is poorly investigated. The present research was designed to clarify the expression and function of MFGE8 in carbon tetrachloride (CCl4 )-induced liver injury. Using serum cytokine arrays, we selected a promising cytokine MFGE8 as the candidate in the process of hepatitis-fibrosis-hepatocellular carcinoma (HCC) progression, based on the elevated expression in both hepatic fibrosis and HCC models. We validated the increased expression of MFGE8 in liver tissues and serum samples of acute and chronic CCl4 -induced mice. Immunohistochemistry staining of mouse liver tissues indicated that elevated MFGE8 expression was mainly derived from the injured hepatocytes. In addition, MFGE8 expression in the supernatant of primary hepatocytes was accumulated with prolongation of culture time, and CCl4 treatment further increased the expression of MFGE8. Moreover, a strong correlation between serum MFGE8 expression and liver transaminase activities suggested that MFGE8 may be a novel candidate in liver injury. Intriguingly, mice pretreated with MFGE8 were protected from CCl4 -induced liver injury through antiapoptosis role in the early stage and proproliferation role in the late stage. MFGE8 reduced apoptosis by inhibiting the activation of IRE1α/ASK1/JNK pathway and promoted proliferation by phosphorylation of ERK and AKT. Moreover, serum MFGE8 expression was increased in hepatitis patients while decreased in liver cirrhosis patients. All the results suggest MFGE8 as a novel marker and promising therapeutic agent of liver injury.

Hepatocyte Injury Induced by Contrast-Enhanced Diagnostic Ultrasound.

Contrast-enhanced diagnostic ultrasound (US) has a potential to induce localized biological effects. The potential for contrast-enhanced diagnostic US bioeffects in liver were researched, with guidance from a report by Yang et al (Ultrasonics 2012; 52:1065-1071). Contact and standoff scanning was performed for 10 minutes with a diagnostic US phased array at 1.6 MHz during bolus injection or infusion of a contrast agent at a high dose. The impact of the imaging on rat liver was investigated by measuring enzyme release, microvascular leakage, and staining of injured hepatocytes. The results showed liver enzyme release at 30 minutes, indicating liver injury, and elevated extraction of Evans blue dye, indicating microvascular leakage. In addition, Evans blue and trypan blue vital-staining methods revealed scattered stained cells within the US scan plane. For the Evans blue method, fluorescent cell counts in frozen sections were greatest for standoff exposure with contrast infusion. The count decreased strongly with depth for bolus injection, which was probably reflective of the high attenuation noted for this agent delivery method. The results qualitatively confirmed the report by Yang et al and additionally showed hepatocyte vital staining. Research is needed to determine the threshold for the effects and the contrast agent dose response.

COL6A3-derived endotrophin links reciprocal interactions among hepatic cells in the pathology of chronic liver disease.

Extracellular matrix dysregulation is associated with chronic liver disease. CollagenVI-alpha3 chain (COL6A3) is a biomarker for hepatic fibrosis and poor prognosis of hepatocellular carcinoma (HCC), but its function in liver pathology remains unknown. High levels of COL6A3 and its cleaved product, endotrophin (ETP) in tumor-neighboring regions are strongly associated with poor prognosis in HCC patients. Here, we report that the high levels of ETP in injured hepatocytes induce JNK-dependent hepatocyte apoptosis and activate nonparenchymal cells to lead further activation of hepatic inflammation, fibrosis, and apoptosis. Nevertheless ETP per se showed limited phenotypic changes in normal liver tissues. Furthermore, inhibition of ETP activity by utilizing neutralizing antibodies efficiently suppressed the pathological consequences in chronic liver diseases. Our results implicate ETP mechanistically as a crucial mediator in reciprocal interactions among various hepatic cell populations in the pathogenesis of chronic liver disease, and it could be a promising therapeutic target particularly in individuals with high local levels of COL6A3. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Crocetin promotes autophagy in injured rat hepatocytes induced by lipopolysaccharide and D-galactosamine in vitro

To observe the effect of crocetin on autophagy in rat hepatocytes exposed to lipopolysaccharide (LPS) and D-galactosamine (D-gal) and explore the mechanism. Cultured rat hepatocytes were exposed to LPS (1 mg/L) and Dgal (60 mg/L) to induce cell injury and treated with crocetin, 3MA, or crocetin+3MA. Twelve hours after the treatments, the cells were examined for levels of ALT, AST and LDH in the supernatant using ELISA. LC3 fluorescence in the cells following immunofluorescence staining was observed using fluorescence microscopy. Autophagosomes in the cells were observed by transmission electron microscopy, and the cellular expressions of LC3, p62 and SIRT1 were detected using Western blotting. The levels of ALT, AST and LDH in the hepatocytes were elevated after LPS- and D-gal-induced injury, reached the highest levels after 3MA treatment, but were decreased significantly by crocetin treatment. LC3 fluorescence increased obviously in the injured hepatoctyes, and the increment was the most obvious in crocetin-treated cells; LC3 fluorescence was decreased significantly after 3MA treatment. Cell injury induced obvious increase in autophagy in the hepatocytes, and the number of autophagosomes increased significantly after crocetin treatment but was reduced significantly after 3MA treatment. The cell injury caused an obvious up-regulation of LC3 and SIRT1 expression and down-regulated p62 expression. LC3 and SIRT1 expression levels were the highest and the expression of p62 was the lowest in cells with crocetin treatment. 3MA treatment significantly reduced the expression of LC3 and SIRT1 and increased the expression of p62 in the injured cells. Autophagy is increased in injured rat hepatocytes, and crocetin can promote autophagy in the injured cells to reduce further cell injury.

Therapeutic effects of serum extracellular vesicles in liver fibrosis

ABSTRACTThe lack of approved therapies for hepatic fibrosis seriously limits medical management of patients with chronic liver disease. Since extracellular vesicles (EVs) function as conduits for intercellular molecular transfer, we investigated if EVs from healthy individuals have anti-fibrotic properties. Hepatic fibrogenesis or fibrosis in carbon tetrachloride (CCl4)- or thioacetic acid-induced liver injury models in male or female mice were suppressed by serum EVs from normal mice (EVN) but not from fibrotic mice (EVF). CCl4-treated mice undergoing EVN therapy also exhibited reduced levels of hepatocyte death, inflammatory infiltration, circulating AST/ALT levels and hepatic or circulating pro-inflammatory cytokines. Hepatic histology, liver function tests or circulating proinflammatory cytokine levels were unaltered in control mice receiving EVN. As determined using PKH26-labelled EVN, principal target cells included hepatic stellate cells (HSC; a normally quiescent fibroblastic cell that undergoes injury-induced activation and produces fibrosis during chronic injury) or hepatocytes which showed increased EVN binding after, respectively, activation or exposure to CCl4. In vitro, EVN decreased proliferation and fibrosis-associated molecule expression in activated HSC, while reversing the inhibitory effects of CCl4 or ethanol on hepatocyte proliferation. In mice, microRNA-34c, -151-3p, -483-5p, -532-5p and -687 were more highly expressed in EVN than EVF and mimics of these microRNAs (miRs) individually suppressed fibrogenic gene expression in activated HSC. A role for these miRs in contributing to EVN actions was shown by the ability of their corresponding antagomirs to individually and/or collectively block the therapeutic effects of EVN on activated HSC or injured hepatocytes. Similarly, the activated phenotype of human LX-2 HSC was attenuated by serum EVs from healthy human subjects and contained higher miR-34c, -151-3p, -483-5p or -532-5p than EVs from hepatic fibrosis patients. In conclusion, serum EVs from normal healthy individuals are inherently anti-fibrogenic and anti-fibrotic, and contain microRNAs that have therapeutic actions in activated HSC or injured hepatocytes.Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; CCl4: carbon tetrachloride; CCN2: connective tissue growth factor; E: eosin; EGFP: enhanced green fluorescent protein; EVs: extracellular vesicles; EVF: serum EVs from mice with experimental hepatic fibrosis; EVN: serum EVs from normal mice; H: hematoxylin; HSC: hepatic stellate cell; IHC: immunohistochemistry; IL: interleukin; MCP-1: monocyte chemotactic protein-1; miR: microRNA; mRNA: messenger RNA; NTA: nanoparticle tracking analysis; PCNA: proliferating cell nuclear antigen; qRT-PCR: quantitative real-time polymerase chain reaction; SDS-PAGE: sodium dodecyl sulphate – polyacrylamide gel electrophoresis; αSMA: alpha smooth muscle actin; TAA: thioacetic acid; TG: transgenic; TGF-β: transforming growth factor beta; TEM: transmission electron microscopy; TNFα: tumour necrosis factor alpha.

Apple Cider Vinegar (as a Prophetic Medicine Remedy) Exerts Tissue-protective Effects in Streptozotocin-induced Diabetes in Animals

Background: Diabetes mellitus (DM) was reported to be related to occurrence of both biochemical and histopathological changes in hepatic tissue. DM-induced tissue damage and decreased immunity may favor carcinogenesis and tumor development in pediatric practice and adults. In this study, we aimed at investigating the hepatoprotective and tissue-protective effects of apple cider (ACV) against DM-induced hepatic derangements over a relatively long period (30 days). Effects were evaluated using streptozotocin (STZ)-induced DM in rats as an experimental model. Materials and methods: DM was experimentally caused by injecting one dose of STZ (65 mg/kg given through intraperitoneal injection. Thirty wistar rats were categorized into three experimental groups: control group, STZ-treated group and STZ plus ACV treated group 2 ml/kg BW). Animals were sacrificed 30 days post treatment. Results: Our study revealed that ACV administration induced hepatoprotective effects in diabetic rats. Biochemically, ACV reduced significantly serum glycemic values (glucose, total cholesterol, low density lipoprotein-cholesterol while high density lipoprotein cholesterol significantly increased). STZ-induced DM in rats was associated with marked fatty changes hepatocytes cytoplasm evidenced by accumulation of lipid droplets and inflammatory changes e.g. lymphocytic infiltration. That was confirmed by Electron microscopic evaluation that showed aggregations of polymorphic mitochondria, loss of mitochondrial cristae and condensed mitochondrial matrices. Hepatocytes cytoplasm showed vacuolations with a large number of variable-sized lipid droplets. Hepatocytes rough endoplasmic reticulum got distorted and fragmented into smaller stacks. ACV proved effective in reversing all these metabolic changes. ACV-treated hepatocytes exhibited minimal STZ-induced toxic effects. ACV enhanced recovery of the injured hepatocytes and normalized serum biochemical values. In prophetic medicine, Prophet Muhammad (Allah's apostle peace be upon him) strongly advised eating and considered it as the best edible as narrated in the prophetic hadith: vinegar is the best edible. Conclusion: ACV is promising as a food additive or as a part of diet in diabetic patients particularly in early stages of DM. ACV slows down DM progress and causes hepatoprotection against the metabolic damages resulting from STZ- induced diabetes mellitus

Pathogenesis of NASH: How Metabolic Complications of Overnutrition Favour Lipotoxicity and Pro-Inflammatory Fatty Liver Disease.

Overnutrition, usually with obesity and genetic predisposition, lead to insulin resistance, which is an invariable accompaniment of nonalcoholic fatty liver disease (NAFLD). The associated metabolic abnormalities, pre- or established diabetes, hypertension and atherogenic dyslipidemia (clustered as metabolic syndrome) tend to be worse for nonalcoholic steatohepatitis (NASH), revealing it as part of a continuum of metabolic pathogenesis. The origins of hepatocellular injury and lobular inflammation which distinguish NASH from simple steatosis have intrigued investigators, but it is now widely accepted that NASH results from liver lipotoxicity. The key issue is not the quantity of liver fat but the type(s) of lipid molecules that accumulate, and how they are "packaged" to avoid subcellular injury. Possible lipotoxic mediators include free (unesterified) cholesterol, saturated free fatty acids, diacylglycerols, lysophosphatidyl-choline, sphingolipids and ceramide. Lipid droplets are intracellular storage organelles for non-structural lipid whose regulation is influenced by genetic polymorphisms, such as PNPLA3. Cells unable to sequester chemically reactive lipid molecules undergo mitochondrial injury, endoplasmic reticulum (ER) stress and autophagy, all processes of interest for NASH pathogenesis. Lipotoxicity kills hepatocytes by apoptosis, a highly regulated, non-inflammatory form of cell death, but also by necrosis, necroptosis and pyroptosis; the latter involve mitochondrial injury, oxidative stress, activation of c-Jun N-terminal kinase (JNK) and release of danger-associated molecular patterns (DAMPs). DAMPs stimulate innate immunity by binding pattern recognition receptors, such as Toll-like receptor 4 (TLR4) and the NOD-like receptor protein 3 (NLRP3) inflammasome, which release a cascade of pro-inflammatory chemokines and cytokines. Thus, lipotoxic hepatocellular injury attracts inflammatory cells, particularly activated macrophages which surround ballooned hepatocytes as crown-like structures. In both experimental and human NASH, livers contain cholesterol crystals which are a second signal for NLRP3 activation; this causes interleukin (IL)-1β and IL18 secretion to attract and activate macrophages and neutrophils. Injured hepatocytes also liberate plasma membrane-derived extracellular vesicles; these have been shown to circulate in NASH and to be pro-inflammatory. The way metabolic dysfunction leads to lipotoxicity, innate immune responses and the resultant pattern of cellular inflammation in the liver are likely also relevant to hepatic fibrogenesis and hepatocarcinogenesis. Pinpointing the key molecules involved pharmacologically should eventually lead to effective pharmacotherapy against NASH.

Pathways of production and delivery of hepatocyte exosomes

Hepatocyte exosomes (ExoHep) are proposed to mediate physiological or pathophysiological signaling in a variety of hepatic target cells. ExoHep were purified from the medium of primary mouse hepatocytes or AML12 cells and characterized as ~100nm nanovesicles that were positive for proteins commonly found in exosomes (CD9, CD81, flotillin) or hepatocytes (asialoglycoprotein receptor). Ethanol treatment of hepatocytes caused increased ExoHep release and increased cellular mRNA expression of components involved in intracellular vesicle trafficking (Rab 5a,b,c, Rab 7a, Rab 27a,b) or exosome biogenesis via the ESCRT (HGS, Alix, STAM1, TSG101, VTA1, YKT6) or ceramide (nSmase2) pathways. RNA interference of HGS, Alix, TSG101 or nSmase 2 caused exosome production by normal or ethanol-treated hepatocytes to be reduced. In mice, in vivo administration of fluorescently-labeled ExoHep resulted in their accumulation in the liver and preferential localization to hepatic stellate cells (HSC) or hepatocytes, the latter of which showed enhanced ExoHep binding when isolated from fibrotic mice. In cell co-cultures, the intercellular transfer of RNA from hepatocytes to hepatocytes or HSC was blocked by the exosome inhibitor GW4869. ExoHep binding to HSC or hepatocytes occurred via mechanisms that involved heparin-like molecules and cellular integrin αv or β1 subunits , and resulted in a reversal of fibrosis-associated gene expression in HSC and of ethanol-induced damage in hepatocytes. These studies provide insight regarding the regulation and/or participation of exosome biogenesis or trafficking components in hepatocytes and show that ExoHep can mediate therapeutic changes in activated HSC or injured hepatocytes that occur downstream of heparin- or integrin-dependent binding interactions.