Hepatic Infiltration Research Articles

Procyanidins and its metabolites by gut microbiome improves insulin resistance in gestational diabetes mellitus mice model via regulating NF-κB and NLRP3 inflammasome pathway

Gestational Diabetes Mellitus (GDM) has an effect on the health of pregnant women and fetuses. Procyanidins (PA) is a flavonoid with anti-diabetic activity, but its effects and mechanisms on GDM have not been defined. Herein, we studied further the functions and mechanisms of PA on insulin resistance (IR) in GDM mice, as well as on postpartum and offspring mice. GDM mice model was built by feeding a high-fat-high-sucrose diet, and PA intervention (27.8 mg/kg/d) was performed from 4 weeks before pregnancy to delivery. Intestinal flora deficient (IFD) mice model was established by broad spectrum antibiotics. PA decreased the gestational weight gain, and the levels of fasting blood glucose, insulin, homeostasis model of assessment for IR index, yet increased the levels of HOMA for insulin sensitivity index. Interestingly, in IFD mice the effect of PA on improving IR was significantly weakened. PA inhibited inflammation by decreasing the levels of IL-6, TNF-α, IL-17 and CRP, which also been blocked in the IFD mice. Moreover, PA improved glycometabolism and reduced the secretion of inflammatory factors and hepatic inflammation infiltration of mice at 4 weeks postpartum, but had no significant effect on offspring mice. Mechanistically, PA treatment suppressed the nuclear factor‐κB (NF‐κB) p65 nuclear translocation and nucleotide‐binding domain like receptor protein 3 (NLRP3) inflammasome activation. In vitro studies, 4-hydroxyphenylacetic acid and 3-(4-hydroxyphenyl) propionic acid, main intestinal flora metabolites of PA restrained NF‐κB/NLRP3 activation. In conclusions, PA improved IR via NF-κB/NLRP3 pathway in GDM and postpartum mice, which partly through its metabolites by gut microbiome.

Nuclear Receptor Small Heterodimer Partner (SHP) Regulates Hepatic Macrophage Differentiation Through Posttranscriptional Regulation of PPARg

BackgroundHepatic macrophages, consisting of resident Kupffer cells and infiltrating monocyte‐derived macrophages (MDMs), are central players of innate immunity involved in hepatic inflammation. The significance of macrophage differentiation towards M1‐like pro‐inflammatory or M2‐like anti‐inflammatory phenotypes in liver inflammatory diseases is widely appreciated; however, the mechanisms controlling this process are not well understood. SHP is a negative regulator of inflammatory signaling. We recently found that Shpis downregulated in M1 and upregulated in M2‐like macrophages, linking Shp to the regulation of macrophage differentiation. In this study, we uncovered a novel regulatory network consisting of Shp, miR‐34a, and peroxisome proliferator–activated receptor gamma (Pparg) in hepatic macrophage differentiation.MethodsWe generated transgenic mice by knocking out Shp from the cells of myeloid origin using Lyz2‐Cre (Shp‐MKO represents Shpflox/flox; Lyz2‐Cre positive). The littermates Lyz2‐Cre negative mice were used as wild‐type controls (WT represents Shpflox/flox; Lyz2‐Cre negative). The in vitro M1 and M2 macrophage differentiation was conducted using primary hepatic macrophages isolated from WT and Shp‐MKO mice and RAW 264.7 mouse macrophage cell line overexpressing Shp(RAW‐Shp). Lipopolysaccharide (LPS) was injected into WT and Shp‐MKO mice to induce acute liver inflammation. Hepatic non‐parenchymal cells were isolated by liver perfusion and subjected to Flow Cytometry to determine immune cell heterogeneity. Hepatic infiltrating CD11b+ MDMs were isolated using microbeads followed by determination of inflammatory gene expression by qPCR.ResultsThe in vitro macrophage differentiation study showed that Shp deletion promoted M1‐like but interfered M2‐like macrophage polarization. In contrast, Shp‐overexpression inhibited M1‐like polarization of RAW‐Shp cells. Flow cytometry analysis revealed a significant increase in the infiltration of CD11b+ MDMs into the liver of Shp‐MKO in comparison to WT after LPS injection. Additionally, the percentage of pro‐inflammatory CD11b+Ly6c+MDMs population doubled in Shp‐MKO liver after LPS treatment. Accordingly, LPS injection significantly induced hepatic Tnfa and Ccl2expressions in Shp‐MKO, which supports the overall increase in hepatic infiltration of pro‐inflammatory macrophages observed in Shp‐MKO. On the molecular level, SHP overexpression significantly induced Pparg, the master regulator of M2‐like macrophage polarization. To explore the molecular basis of Shp regulation of Pparg, we performed small RNA sequencing of Shp‐knockout liver. A total of 53 different microRNAs (miRNAs) were upregulated in Shp‐knockout liver. Using miRNA target prediction tool, we found that 5 of these upregulated miRNAs (miR‐34a, mir‐27a, miR‐130b, miR‐673, miR‐540) can target Pparg‐3´‐UTR. We confirmed that miR‐34a‐5p, the negative regulator of Pparg, was increased in Shp deficient macrophages but decreased in Shpoverexpressing macrophages.ConclusionOur study revealed a previously unknown role of SHP in macrophage differentiation. We found that Shp promotes the anti‐inflammatory macrophage differentiation by posttranscriptional regulation of Pparg through inhibition of miR‐34a. Targeting SHP may be useful for developing effective treatment for liver inflammatory diseases.

Inhibition of Ileal Bile Acid Transport is Protective Against Cholestatic Liver Injury in Cyp2c70‐/‐Mice with Humanized Bile Acid Composition

Cyp2c70 is the key murine liver enzyme responsible for the synthesis of 6‐hydroxylated muricholates. Cyp2c70 knockout (2c70‐/‐) mice present with a more human‐like bile acid (BA) composition devoid of protective, hydrophilic muricholic acids and with features of cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be protective in cholestatic models such as the Mdr2‐/‐ mouse. The aims of this study were to determine if IBAT inhibition is protective in 2c70‐/‐ mice and identify the underlying molecular mechanisms.Methods2c70‐/‐ mice (C57Bl/6J) were generated using CRISPR/Cas9 technology. Male and female wild type (WT) and 2c70‐/‐ mice were fed chow or chow containing 0.006% SC‐435 for 9 weeks following weaning at 3 weeks of age. Standard growth parameters were recorded; organs, gallbladder bile and blood were collected at necropsy for histologic, RNA and biochemical analyses.ResultsMale and female 2c70‐/‐ mice maintained on chow diet exhibited increased levels of serum liver injury markers ALT, AST, and BAs compared to WT mice. Evaluation of 2c70‐/‐ liver histology revealed increased hepatic inflammation, macrophage infiltration and bilary cell proliferation, which were markedly reduced with SC‐435 treatment. IBAT inhibition in 2c70‐/‐ mice improved liver histology, and reduced hepatic inflammation and bilary cell proliferation to levels indistinguishable from WT. The liver BA content was increased by 53% in 2c70‐/‐ mice, and was enriched in more hydrophobic BA species with an overall hydrophobicity similar that of humans (Figure 1). Treating 2c70‐/‐ mice with SC‐435 reduced the hepatic BA burden by 61%, but did not affect the overall hydrophobicity. RNA‐Seq and KEGG pathway analysis performed using liver RNA revealed genes involved in hepatic steroid and lipid metabolism (Sult2a1, Sult1e1, Hsd17b7), and detoxification (Cyp2c39, Cyp2c54, Cyp2c59, Cyp3a41a/b) were down‐regulated in 2c70‐/‐ mice and their expression were rescued with SC‐435 treatment. Conversely, genes in pathways related to immune cell activation and inflammation were significantly up‐regulated in 2c70‐/‐ mice and reduced to WT levels with IBAT inhibition (Figure 2).ConclusionsOur findings indicate that reducing the hepatic BA burden without altering the hydrophobic compostion is sufficient to protect 2c70‐/‐ mice from hepatic injury. The findings suggest that there may be a threshold in liver for pathological accretion of hydrophobic BAs and reducing the hepatic BA pool is sufficient to alleviate liver injury, independent of hydrophobicity. Moreover, these data support the inhibition IBAT activity, and other approaches to reduce hepatic return of BAs in the enterohepatic circulation, as therapeutic targets to treat cholestasis.

Receptor-interacting protein kinase-1 ablation in liver parenchymal cells promotes liver fibrosis in murine NASH without affecting other symptoms.

Non-alcoholic steatohepatitis (NASH), a chronic liver disease that emerged in industrialized countries, can further progress into liver fibrosis, cirrhosis, and hepatocellular carcinoma. In the next decade, NASH is predicted to become the leading cause of liver transplantation, the only current interventional therapeutic option. Hepatocyte death, triggered by different death ligands, plays key role in its progression. Previously, we showed that the receptor-interacting protein kinase-1 (RIPK1) in hepatocytes exhibits a protective role in ligand-induced death. Now, to decipher the role of RIPK1 in NASH, Ripk1LPC-KO mice, deficient for RIPK1 only in liver parenchymal cells, and their wild-type littermates (Ripk1fl/fl) were fed for 3, 5, or 12weeks with high-fat high-cholesterol diet (HFHCD). The main clinical signs of NASH were analyzed to compare the pathophysiological state established in mice. Most of the symptoms evolved similarly whatever the genotype, whether it was the increase in liver to body weight ratio, the steatosis grade or the worsening of liver damage revealed by serum transaminase levels. In parallel, inflammation markers followed the same kinetics with significant equivalent inductions of cytokines (hepatic mRNA levels and blood cytokine concentrations) and a main peak of hepatic infiltration of immune cells at 3weeks of HFHCD. Despite this identical inflammatory response, more hepatic fibrosis was significantly evidenced at week 12 in Ripk1LPC-KO mice. This coincided with over-induced rates of transcripts of genes implied in fibrosis development (Tgfb1, Tgfbi, Timp1, and Timp2) in Ripk1LPC-KO animals. In conclusion, our results show that RIPK1 in hepatocyte limits the progression of liver fibrosis during NASH.

HISTOPATHOLOGY OF FASCIOLIASIS-INFESTED COMMERCIAL CATTLE LIVERS (CCL) AT MAJOR MARKETS IN BENIN CITY, NIGERIA

Fascioliasis is an important parasitic foodborne disease responsible for significant public health problems with adverse pathological consequence of human. There are growing concerns about meat hygiene and safety of cattle for commercial purpose. Hence, the present study aimed at randomly reconnoitering some commercial cattle livers (CCL) sold at major markets in Benin City, Nigeria by histopathological examination. Eight hundred and twenty samples of liver tissue with an average weight of 9.25g obtained from 10 major markets in Benin City, Nigeria were examined for fascioliasis infestation between the months of February and August, 2014 using standard histochemical methods. The results showed that 8/820 representing (0.98%) of cattle livers are infested. (Table 1) further revealed that CCL at Aduwawa market had the highest infestation 4/150 representing (2.7%), closely followed by new Benin market 2/110(1.8%); Uselu market 1/80(1.3%) and Oliha market 1/80(1.3%). Gross examination of cattle livers showed multiple patches of necrosis while the histopathology revealed necrosis with dilated hepatic sinusoid, cellular infiltration and inflammatory hepatocyte with marked presence of parasitic infestation. From the preceding, therefore, the hygienic and health status of CCL in Benin City, Nigeria are relatively unsafe. Nonetheless, this is from a histopathology perspective.

Ammonia Scavenger Restores Liver and Muscle Injury in a Mouse Model of Non-alcoholic Steatohepatitis With Sarcopenic Obesity.

Recent studies have revealed that sarcopenia is closely associated with obesity and non-alcoholic steatohepatitis (NASH). However, few attempted to explore the cause-and-effect relationship between sarcopenic obesity and NASH. In this study, we investigated muscular alterations in a rodent NASH model to elucidate their intrinsic relations and explore the potential therapeutic target. Forty-six 8-week-old and twenty 42-week-old male C57BL/6 mice (defined as young and middle-aged mice, respectively) were fed with a high-fat diet (HFD) for 12 or 20 weeks. A subset of young mice was subjected to ammonia lowering treatment by L-ornithine L-aspartate (LOLA). We examined body composition and muscle strength by nuclear magnetic resonance and grip strength meter, respectively. At the end of the 12th week, all HFD-fed mice developed typical steatohepatitis. Meanwhile, sarcopenia occurred in HFD-fed middle-aged mice, whereas young mice only demonstrated decreased grip strength. Until the end of week 20, young mice in the HFD group exhibited significant sarcopenia and obesity phenotypes, including decreased lean body mass and grip strength, and increased body fat mass and percentage body fat. Additionally, plasma ammonia level was markedly increased in HFD-fed mice of both ages at week 20. Plasma ammonia level was negatively associated with muscle strength and myofiber diameter in young mice. LOLA can significantly reduce plasma levels of ammonia, alanine aminotransaminase, aspartate aminotransaminase, and cholesterol in mice fed an HFD. Hepatic infiltration of inflammatory cells and collagen deposition area were significantly decreased in HFD group by LOLA treatment. Meanwhile, LOLA significantly increased lean body mass, grip strength, and average muscle fiber diameter of HFD-fed mice. These findings suggest that the occurrence of NASH precedes sarcopenia in HFD mice, and the steatohepatitis-related hyperammonemia might contribute to the pathogenesis of sarcopenia. LOLA might be an effective drug for both steatohepatitis and sarcopenic obesity.

The preventive effect of recombinant human hepatocyte growth factor for hepatic steatosis in a rat model of short bowel syndrome

PurposeShort bowel syndrome (SBS) patients require total parenteral nutrition (TPN) following massive small bowel resection (SBR), which may cause intestinal failure-associated liver disease (IFALD), a life-threatening complication. Hepatocyte growth factor (HGF) acts as a potent hepatocyte mitogen with anti inflammatory and antioxidant actions. The present study evaluated the effect of recombinant human HGF (rh-HGF) on SBR and subsequent IFALD using a parentally fed rat model of SBS. MethodsRats underwent jugular vein catheterization for continuous TPN and 90% SBR. They were divided into 2 groups: TPN alone (SBS/TPN group: n = 7) or TPN plus the intravenous administration of rh-HGF (0.3 mg/kg/day) (SBS/TPN+HGF group: n = 7). On day 7, their tissues and stool were harvested to evaluate the effects of HGF. ResultsRegarding the histological findings, based on the nonalcoholic fatty liver disease (NAFLD) activity score, the SBS/TPN+HGF group showed significantly less hepatic steatosis and inflammatory cell infiltration than the SBS/TPN group (NAFLD activity score, 4.00 ± 1.83 vs. 1.00 ± 0.82; p < 0.01). The SBS/TPN+HGF group showed a higher expression of Farnesoid X receptor in the liver and lower expression of Toll-like receptor 4 in the ileum than the SBS/TPN group. Regarding the composition of the bacterial gut microbiota, Actinobacteria, Bacteroidetes and Proteobacteria were decreased in the SBS/TPN+HGF group compared with the SBS/TPN group. ConclusionIn our SBS with TPN rat model, rh-HGF administration had a preventive effect against hepatic steatosis and dysbiosis. rh-HGF may therefore be a potentially effective therapeutic agent for SBS and subsequent IFALD. Type of studyExperimental research.

Expression of IL-37 Correlates With Immune Cell Infiltrate and Fibrosis in Pediatric Autoimmune Liver Diseases.

The activation of innate immune mechanisms is key for chronic liver injury. Interleukin-37 (IL-37) is a profound inhibitor of innate and adaptive immune responses, and its overexpression protects mice from liver inflammation and fibrosis. Here, we characterize the hepatic inflammatory infiltrate and expression of IL-37 in children with autoimmune liver diseases. We compared the inflammatory microenvironment of the liver in a retrospective cohort of children with primary sclerosing cholangitis (PSC), autoimmune sclerosing cholangitis (ASC) and autoimmune hepatitis (AIH) by immunohistochemistry. The expression of IL-37 was quantified in liver parenchyma and portal tracts. Double immunofluorescence was used for detection of IL-37 in specific cell types and colocalization with Smad3. AIH is characterized by a dense lymphoplasmacytic infiltrate whereas ASC shows high numbers of granulocytes in portal tracts. IL-37 expression correlates positively with liver inflammation and fibrosis, the number of infiltrating immune cells and serum markers for hepatic inflammation. IL-37 is mainly expressed in hepatocytes, cholangiocytes and infiltrating immune cells. Double staining revealed IL-37 positivity in T helper and regulatory T cells (Treg), Kupffer (KC) and hepatic stellate cells (HSC). IL-37 colocalizes with intranuclear pSmad3L in areas of liver inflammation. Pediatric ASC separates from PSC and AIH by a granulocyte-rich portal infiltrate. Upregulation of IL-37 with liver injury, the expression in Treg as well as KC and HSC and the colocalization of IL-37 with pSmad3L in cholangiocytes and hepatocytes suggest a modulating role to limit hepatic inflammation and fibrosis in pediatric autoimmune liver diseases.

The KDM6A-SPARCL1 axis blocks metastasis and regulates the tumour microenvironment of gastrointestinal stromal tumours by inhibiting the nuclear translocation of p65.

It is urgent to explore the pathogenic mechanism of gastrointestinal stromal tumours (GISTs). KDM6A, a histone demethylase, can activate gene transcription and has not been reported in GISTs. SPARCL1 may serve as a metastasis marker in GIST, but the molecular mechanism remains to be further explored. This study aimed to explore the biological function and molecular mechanism of KDM6A and SPARCL1 in GIST. CCK-8, live cell count, colony formation, wound-healing and Transwell migration and invasion assays were employed to detect the cell proliferation, migration and invasion. A xenograft model and hepatic metastasis model were used to assess the role of KDM6A and SPARCL1 in vivo. KDM6A inhibited the proliferation, migration and invasion of GIST cells. Mechanistically, KDM6A promotes the transcription of SPARCL1 by demethylating histone H3 lysine trimethylation and consequently leads to the inactivation of p65. SPARCL1 affected the metastasis of GIST cells in a mesenchymal-epithelial transition- and matrix-metalloproteinase-dependent manner. SPARCL1 knockdown promoted angiogenesis, M2 polarisation and macrophage recruitment by inhibiting the phosphorylation of p65. Moreover, KDM6A and SPARCL1 inhibited hepatic metastasis and macrophage infiltration in vivo. Our findings establish the critical role of the KDM6A-SPARCL1-p65 axis in restraining the malignancy of GIST.

Impact of moderate dietary protein restriction on glucose homeostasis in a model of estrogen deficiency

The need to consume adequate dietary protein to preserve physical function during ageing is well recognized. However, the effect of protein intakes on glucose metabolism is still intensively debated. During age-related estrogen withdrawal at the time of the menopause, it is known that glucose homeostasis may be impaired but the influence of dietary protein levels in this context is unknown. The aim of the present study is to elucidate the individual and interactive effects of estrogen deficiency and suboptimal protein intake on glucose homeostasis in a preclinical model involving ovariectomy (OVX) and a 13 week period of a moderately reduced protein intake in 7 month-old ageing rats. To investigate mechanisms of action acting via the pancreas-liver-muscle axis, fasting circulating levels of insulin, glucagon, IGF-1, FGF21 and glycemia were measured. The hepatic lipid infiltration and the protein expression of GLUT4 in the gastrocnemius were analyzed. The gene expression of some hepatokines, myokines and lipid storage/oxidation related transcription factors were quantified in the liver and the gastrocnemius. We show that, regardless of the estrogen status, moderate dietary protein restriction increases fasting glycemia without modifying insulinemia, body weight gain and composition. This fasting hyperglycemia is associated with estrogen status-specific metabolic alterations in the muscle and liver. In estrogen-replete (SHAM) rats, GLUT4 was down-regulated in skeletal muscle while in estrogen-deficient (OVX) rats, hepatic stress-associated hyperglucagonemia and high serum FGF21 were observed. These findings highlight the importance of meeting dietary protein needs to avoid disturbances in glucose homeostasis in ageing female rats with or without estrogen withdrawal.

Ameliorative effect of tocotrienol and selenium yeast against the adverse effect of florfenicol in broilers’ liver

The prolonged use of florfenicol can lead to detrimental side effects in poultry. This work focuses on the role of tocotrienol and selenium yeast to mitigate the adverse effects of florfenicol in broilers’ liver. One hundred and fifty, one-day-old Cobb broiler chicks where equally divided in 5 experimental groups according to the following experimental design: Group (1) control group chicks fed to a balanced diet only. Group (2) Chicks treated with florfenicol (20 mg/kg b.w.) per bird for 3 successive days and the florfenicol was administered in other groups by the same dose and for the same period of time. Group (3) chicks treated with florfenicol and tocotrienol (170 mg/kg b.w.) for 7 successive days. Group (4) Chicks treated with florfenicol and selenium yeast (0.15mg /kg b.w., on feed) for 7 successive days. Group (5) Chicks treated with a combination of florfenicol, tocotrienol and selenium yeast. Chickens treated with florfenicol exhibited an increased level in hepatic malondialdehyde (MDA), as well asa decreasedlevel in hepatic superoxide dismutase (SOD) and reduced glutathione (GSH). Tocotrienol and selenium yeast decreased the MDA and increased SOD and GSH in hepatic tissue as well as return ALP, cholesterol, triglyceride and VLDL to their normal levels. Treated chicks with tocotrienol returned serum (ALT) to normal activity but serum total protein and albumin levels were increased. Theselenium yeast treated groups showed an increase of serum total globulin. Histopathologically, florfenicol treated group had focal hepatic leukocytic infiltration and focal coagulative necrosis of hepatocytes but chickens with the combination of tocotrienol and selenium yeast had activated Kupffer cells and revealed less evident necrotic changes in liver. In conclusion, tocotrienol and selenium yeast administrated alone or in combination highlightedimproved antioxidant effects and mitigated the lipid peroxidation in broilers’ liver treated with florfenicol. Thus, tocotrienol and selenium yeastcan improve the safety of using florfenicol in broiler chickens under experimental conditions.

Further Delineation of the Spectrum of XMEN Disease in Six Chinese Pediatric Patients

X-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect (XMEN) disease is a primary immunodeficiency caused by loss-of-function variants in the MAGT1 gene. Only two patients from one family have been diagnosed with XMEN in China. In this study, we retrospectively analyzed the genetic, clinical, and immunological characteristics of six pediatric patients in a Chinese cohort. Medical records were retrieved, immunological phenotypes were assessed, and infectious microbes in patients were detected. Six male patients (mean age, 6.3 years) from five unrelated families were genetically diagnosed as XMEN. Five patients presented with a major complaint of elevated liver enzymes, while one patient was referred for recurrent fever, cough and skin rash. Five patients developed EBV viremia, and one patient developed non-Hodgkin’s lymphoma. Histopathological findings from liver biopsy tissues showed variable hepatic steatosis, fibrosis, inflammatory infiltration, and glycogenosis. Immune phenotypes included CD4 T-cell lymphopenia, elevated B cells, inverted CD4/CD8 ratios, and elevated αβDNTs. No pathogenic microbes other than EBV were identified in these patients. This study reports the clinical and molecular features of Chinese patients with XMEN. For patients with transaminase elevation, chronic EBV infection and EBV-associated lymphoproliferative disease, the possibility of XMEN should be considered in addition to isolated liver diseases.

Loss of bile salt export pump aggravates lipopolysaccharide-induced liver injury in mice due to impaired hepatic endotoxin clearance.

Background and AimsLipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear.Approach and ResultsWild‐type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two‐photon microscopy, and the findings in Bsep KO mice were compared to common bile duct–ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS‐induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule‐dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice.ConclusionsAbsence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.

Hepatology Highlights.

Potential conflict of interest: Nothing to report. DR CELLS ROUND UP NEW TUBES Ryan Watkins1 and Sumera I. Ilyas2 Liver injury increases ductular reaction (DR) and contributes to the wound‐healing process, but how DR cells affect the local microenvironment is not fully understood. The slit guidance ligand 2 (SLIT2)/roundabout guidance receptor 1 (ROBO1) axis promotes angiogenesis in select solid tumors, but whether this signaling pathway induces intrahepatic angiogenesis in acute or chronic liver injury is unknown. Using murine liver injury models and human liver specimens, Coll et al. elucidate that SLIT2 secreted by DR cells binds ROBO1 on new endothelial cells and promotes neoangiogenesis in chronic liver injury. Administering mice a diet enriched in 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidin or a choline‐deficient, ethionine‐supplemented (CDE) diet results in DR expansion. This was paralleled by increased neoangiogenesis, which is diminished in mice heterozygous for ROBO1/2. Similar findings were demonstrated in a cohort of patients with alcohol‐associated liver disease (ARLD). Interestingly, SLIT2 serum levels correlated with disease severity in patients with alcohol‐associated hepatitis. In a separate cohort of patients with NAFLD or HBV infected and without cirrhosis, liver DR markers correlated with SLIT2‐ROBO1 signaling, indicating that this response is not specific to ARLD. Altogether, these findings suggest that intrahepatic angiogenesis mediated by DR cell SLIT2 expression in response to chronic liver injury is an important and clinically relevant process in liver repair. (Hepatology 2022;75:353‐69). THE CHOLESTATIC CONUNDRUM Daniel D. Penrice2 and Sumera I. Ilyas2 Patients with intestinal failure related to short bowel syndrome or other causes are often on prolonged parenteral nutrition (PN) and thus are at risk of developing PN‐associated cholestasis (PNAC). PNAC is characterized by persistent hyperbilirubinemia during PN administration. Although the development of PNAC is multifactorial, phytosterols present in the PN solution have been implicated in the process and shown to suppress hepatocyte farnesoid X receptor (FXR) signaling. FXR is activated by bile acids and plays a major regulatory role in the liver. El Kasmi et al. used a murine model of PNAC using the combination of dextran sodium sulfate–induced intestinal injury and PN infusion. In this model, the FXR agonist, GW4064, activated FXR signaling with restoration of bile acid transporter expression and normalization of the serum bile acid profile. Moreover, FXR agonism reduced hepatic macrophage infiltration with a consequent reduction in hepatic inflammation and cholestasis. These findings suggest that FXR agonists added to PN are a potential therapeutic strategy to decrease the incidence of PNAC. (Hepatology 2022;75:252‐66). CHECK THE TIME: IS CCA READY FOR IMMUNOTHERAPY? Caitlin B. Conboy3 and Sumera I. Ilyas2 Cancer initiation and progression are facilitated by an immunosuppressive tumor immune microenvironment (TIME), characterized by suppressive immune cells and immune checkpoint molecules. Immune checkpoint inhibitors (ICI) have transformed the treatment of many cancers, yet their efficacy in cholangiocarcinoma (CCA) is limited to date. Carapeto et al. sought to identify which immune checkpoints are salient in CCA and how their expression correlates with leukocyte infiltration, underlying genetic drivers, and patient survival. The investigators focused on a subset of patients with resected, fluke‐negative, and predominantly HBV/HCV‐negative intrahepatic CCA (N = 96). Using immunohistochemistry profiling, they identified that both immune cell (CD4+/CD8+ T cells, macrophages) and checkpoint molecule expression (lymphocyte activating 3 [LAG3], OX40, programmed cell death 1 [PD‐1], and T‐cell immunoglobulin and mucin‐domain containing 3) were decreased in the tumor center compared to the invasive margin. Only a small subset of patients had a “hot” TIME. In contrast, a signature of low T‐cell infiltration and high PD‐1 or LAG3 expression was associated with poor overall survival. Correlation of immune checkpoints and genetic mutations identified an association of BRCA1‐associated protein 1 loss‐of‐function mutation with increased expression of the immune checkpoint, B7H4. Tumors with a “cold” TIME have been inherently resistant to ICI therapy. However, broad profiling of the CCA TIME may facilitate development of, and predict response to, novel immunotherapy approaches. (Hepatology 2022;75:297‐308). ALCOHOL AND OBESITY ADDITIVELY INCREASE CIRRHOSIS RISK Gustavo Ayares4 and Juan Pablo Arab4 The relationship between alcohol and obesity on the risk of cirrhosis morbidity remains unclear given that some studies have demonstrated a reduction and others have demonstrated an additive or supra‐additive effect. To assess this interaction in a large population‐based study, Innes et al. used data from the UKB (United Kingdom Biobank) study and examined the relationship between body mass index (BMI) and alcohol intake on cirrhosis morbidity, which was defined by first‐time hospital admission for cirrhosis. Incidence of cirrhosis morbidity increased with alcohol intake and BMI independently. Excess cumulative incidence was highest for obese harmful drinkers (1.83%; 95% CI, 1.46–2.20), compared to harmful drinking alone (1.22%; 95% CI, 0.89–1.55), and obesity alone (0.4%; 95% CI, 0.34–0.46). These findings are consistent with previous studies that have demonstrated an additive effect of alcohol and BMI on the risk of liver disease. (Hepatology 2022;75:369‐78).

Lanzhang Granules Ameliorate Nonalcoholic Fatty Liver Disease by Regulating the PPARα Signaling Pathway

Background There is still a lack of effective therapeutic drugs for nonalcoholic fatty liver disease (NAFLD) to date. In this study, we applied mouse model experiments to clarify the effect of Chinese herbal medicine “Lanzhang Granules (LZG)” on NAFLD and further explore the potential mechanism to provide an alternative method for NAFLD treatment. Methods Male C57BL/6J mice were fed with a high-fat diet (HFD) for twenty-two weeks to induce the NAFLD model. LZG intervention was then performed by gavage daily for another eight weeks. At the end of the treatment, serum and liver tissues were collected. Serum biochemical indexes, insulin levels, and liver histopathology were measured to assess the effect of LZG on NAFLD. The liver tissues were then analyzed by RNA sequence for differentially expressed genes and signaling pathways. Results were further analyzed by Protein-Protein Interaction (PPI) networks between the LZG and model groups. The selected different genes and signaling pathways were further verified by RT-PCR and Western blot analysis. Moreover, alpha mouse liver 12 (AML12) cells with lipid accumulation induced by fatty acid were treated with LZG, Fenofibrate (PPARα agonist), or Gw6471 (PPARα antagonist) to confirm the potential pharmacological mechanism. Results LZG was found to downregulate liver weight, body weight, liver index, and serum levels of ALT, AST, and serum lipid in HFD-induced NAFLD mice. HE and Oil Red O staining showed the improvement of hepatic steatosis and inflammatory infiltration in the mice with LZG treatment. The homeostasis model assessment-insulin resistance (HOMA-IR) index indicated that LZG improved the insulin resistance of NAFLD mice. The RNA sequencing and PPI analysis confirmed the role of LZG in lipid metabolism regulation and identified the peroxisome proliferator-activated receptor alpha (PPARα) signaling pathway as one of the major underlying mechanisms. Western blot and RT-PCR results verified the regulatory effect of LZG on the PPARα pathway, including the upregulation of PPARα, acyl-coenzyme A oxidase 1 (ACOX1), and enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase (EHHADH) and the downregulation of TNFα. In vitro experiments showed the effect of LZG in improving lipid accumulation and cell viability in AML12 cells induced by fatty acids, which were alleviated by Gw6471 coincubation. Gw6471could also reverse the transcription of PPAR target genes ACOX1 and EHHADH, which were upregulated by LZG treatment. Conclusion LZG can improve NAFLD in mice or cell models. A major underlying mechanism may be the regulation of the PPARα signaling pathway to improve lipid metabolism and inhibit the inflammatory response. This study will help to promote the clinical application of LZG for the treatment of NAFLD.

NLRP6 Inflammasome Modulates Disease Progression in a Chronic-Plus-Binge Mouse Model of Alcoholic Liver Disease.

A considerable percentage of the population is affected by alcoholic liver disease (ALD). It is characterized by inflammatory signals from the liver and other organs, such as the intestine. The NLR family pyrin domain containing 6 (NLRP6) inflammasome complex is one of the most important inflammatory mediators. The aim of this study was to evaluate a novel mouse model for ALD characterized by 8-week chronic-plus-binge ethanol administration and to investigate the role of NLRP6 inflammasome for intestinal homeostasis and ALD progression using Nlrp6-/- mice. We showed that chronic-plus-binge ethanol administration triggers hepatic steatosis, injury, and neutrophil infiltration. Furthermore, we discovered significant changes of intestinal microbial communities, including increased relative abundances of bacteria within the phyla Bacteroidota and Campilobacterota, as well as reduced Firmicutes. In this ALD model, inhibiting NLRP6 signaling had no effect on liver steatosis or damage, but had a minor impact on intestinal homeostasis via affecting intestinal epithelium function and gut microbiota. Surprisingly, Nlrp6 loss resulted in significantly decreased hepatic immune cell infiltration. As a result, our novel mouse model encompasses several aspects of human ALD, such as intestinal dysbiosis. Interfering with NLRP6 inflammasome activity reduced hepatic immune cell recruitment, indicating a disease-aggravating role of NLRP6 during ALD.

TOTAL CHOLESTEROL / HIGH DENSITY LIPOPROTEIN CHOLESTEROL RATIO, TRIGLYCERIDES / HIGH DENSITY LIPOPROTEIN CHOLESTEROL WITH THE CONTROLLED ATTENUATION PARAMETER (CAP) IN NON-ALCOHOLIC FATTY LIVER

Non-Alcoholic Fatty Liver Disease (NAFLD) is a global public health problem. The ratio of Total Cholesterol (TC) to High Density Lipoprotein Cholesterol (HDL) and Ultrasensitive C-Reactive Protein have been a biomarker of cardiovascular risk. In a study of the Chinese population, the CT/HDL ratio was established as a predictor of metabolic syndrome and NAFLD >3.8 The optimal cut-off level to predict NAFLD according to the ratio triglycerides (TG)/HDL was established in women >0.9 and in men > 1.4, with sensitivity and specificity of 78 and 77.3% respectively. There are optimal cut-off values of CT/HDL and TG/HDL to predict NAFLD 4 . However, they have not been associated with the degree of hepatic fat infiltration using Transient Elastography (FibroScan®) study by CAP, with a sensitivity of 82% and specificity of 91%. Objective: To describe the relationship CT/HDL, TG/HDL with the controlled attenuation parameter in patients diagnosed with non-alcoholic fatty liver. Retrospective study in patients registered in the database of the Gastroenterology outpatient clinic of the Juárez Hospital in Mexico, with a diagnosis of NAFLD, from January 1, 2017 to April 30, 2019, who underwent Fibroscan. The data were obtained from the records of the outpatient clinic and clinical record, it was analyzed in the statistical program Jamovi 1.1.9, to obtain means, medians and percentages. Chi-square test was used for analysis of categorical variables and unidirectional analysis of variance (ANOVA) for continuous variables, establishing a p <0.05 as significant. In total 48 patients, middle age 49 years (20-76), female 38 (79.2%), with no previous history 29 (60.4%) metabolic syndrome 33 (68.8%) diabetic 19 (39.6%) obesity 27 (56.3%) The CAP for steatosis evaluation: S1, 4 (8.3%) S2, 20 (41.7%) and S3, 24 (50%), for degree of liver fibrosis in Fibroscan: F0, 33 (68.8%) and F1, 14 (29.2%) and by FIB4 scale (<1.30) with NAFLD scale indeterminate fibrosis. The median CT/HDL ratio is not higher with the degree of CAP per FibroScan, being S1, 3.9 (3.1- 4.1) S2, 3.8 (2.3- 6.8) S3, 4.5 (3.1-7.2), ( p 0.076). The higher the median TG/HDL ratio, the higher the degree of CAP per FibroScan, being S1, 2.3 (1.6- 2.7) S2, 3.8 (0.9- 7.9) S3, 4.6 (2-16.9) p value 0.008. It was associated that the higher the degree of CAP per Fibroscan, the higher the triglyceride levels above the normal upper value and the greater the association of presenting metabolic syndrome with a p value of 0.009 and 0.003 respectively. (see table) Higher levels of the TG/HDL ratio, elevated triglyceride levels, and the presence of metabolic syndrome increase the degree of CAP in this group of NAFLD patients. This study the largest population was female (79.2%) so it cannot be inferred that the TG/HDL ratio and degree of CAP is relevant for male sex, so more studies should be carried out and these variables determined. This study has as a limitation that it was carried out in a single hospital center so the cohort sample should be increased and carried out prospectively to really determine whether or not it has utility CT/ HDL relationship in NAFLD, since in this study a result was obtained without statistical significance. The CT/HDL ratio was not associated with the degree of CAP by Fibroscan. It was determined that the higher the median TG/HDL ratio, the higher the degree of CAP in Fibroscan. However, it is necessary to consider this study in patients with high body mass index lowers its sensitivity and specificity, so it should be performed with other advanced imaging modalities. The authors declare that there is no conflict of interest.

Correlation between liver lipidosis, body condition score variation, and hepatic analytes in dairy cows.

Liver lipidosis is a metabolic disorder mostly observed in high yielding dairy cattle, especially during the transition period. The aim of this study was to determine the correlation between hepatic lipid infiltration, biochemical indicators of liver function, and body condition score (BCS) variation in dairy cows. Fifty-one multiparous Holstein cows raised in a confined system were evaluated. Liver biopsies and blood samples were collected, and BCS was measured on days 3 and 28 postpartum. Lipid infiltration was determined by histologic examination. The plasma activity of aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase and concentration of beta-hydroxybutyrate, non-esterified fatty acids, albumin, total bilirubin, and cholesterol were determined. BCS was measured using objective (camera) and subjective (visual) methods. Mild lipid infiltration was found in 3.92% of cows sampled on day 3 and 5.88% on day 28. Bilirubin was significantly higher on day 3 than on day 28 postpartum, and cholesterol was significantly higher on day 28 than on day 3 in all cows. There was no difference in biochemical analytes between cows with and without lipidosis. On day 3, mean subjective BCS was 3.10 and objective BCS was 3.16, while on day 28, these scores were 2.91 and 2.99, respectively. The calculated liver function index (LFI) was found to be a more sensitive indicator of liver function than the hepatic analytes evaluated. No correlation between BCS variation and lipid infiltration was found. Cholesterol and bilirubin levels showed the most remarkable changes during the early postpartum period. LFI is a potential indicator of postpartum liver function.

The role of MLKL in Hepatic Ischemia-Reperfusion Injury of Alcoholic Steatotic Livers.

Alcohol-related liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the primary causes of chronic liver disease in western countries. Liver transplantation is currently one of the most efficient approaches to save patients with liver failure, which is often associated with hepatic ischemia-reperfusion (IR) injury. IR injury is exacerbated by hepatic steatosis, yet the mechanism remains elusive. Necroptosis is a form of regulated cell death mediated by receptor-interacting protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL) protein, which has been implicated in the pathogenesis of ALD and NAFLD. Though necroptosis plays an important role in IR injury of high fat diet - induced steatotic livers, the role of necroptosis in IR injury of ethanol - induced steototic livers has not been investigated. In the present study, we used chronic plus binge alcohol (Gao-binge) feeding followed by IR surgery to investigate IR liver injury with ethanol-associated steatosis. We found that the levels of key necroptotic proteins MLKL and RIP3 increased in alcohol-fed mouse livers. Moreover, we observed increased liver injury after IR in control diet-fed mice, which was further exacerbated by alcohol feeding based on serum alanine aminotransferase (ALT) levels and TUNEL staining of necrotic cells. Hepatic neutrophil infiltration also increased in alcohol-fed mice after IR surgery. However, deletion of Mlkl did not protect against IR liver injury in alcohol-fed mice compared with matched wild-type mice. In conclusion, alcoholic steatosis promotes IR injury, which seems to be independent of MLKL-mediated necroptosis.

Neutrophil Extracellular Traps Mediate Acute Liver Failure in Regulation of miR-223/Neutrophil Elastase Signaling in Mice.

Background & AimsMarked enhancement of neutrophil infiltration in the liver is a hallmark of acute liver failure (ALF), a severe life-threatening disease with varying etiologies. However, the mechanisms and pathophysiological role corresponding to hepatic neutrophil infiltration during ALF development remain poorly characterized.MethodsExperimental ALF was induced in 10-week-old male microRNA-223 (miR-223) knockout (KO) mice, neutrophil elastase (NE) KO mice, and wild-type controls by intraperitoneal injection of galactosamine hydrochloride and lipopolysaccharide. Age-matched mice were injected with phosphate-buffered saline and served as vehicle controls.ResultsMouse liver with ALF showed evident formation of neutrophil extracellular traps (NETs), which were enhanced markedly in miR-223 KO mice. The blockade of NETs by pharmacologic inhibitor GSK484 significantly attenuated neutrophil infiltration and massive necrosis in mouse liver with ALF. ALF-related hepatocellular damage and mortality in miR-223 KO mice were aggravated significantly and accompanied by potentiated neutrophil infiltration in the liver when compared with wild-type controls. Transcriptomic analyses showed that miR-223 deficiency in bone marrow predominantly caused the enrichment of pathways involved in neutrophil degranulation. Likewise, ALF-induced hepatic NE enrichment was potentiated in miR-223 KO mice. Genetic ablation of NE blunted the formation of NETs in parallel with significant attenuation of ALF in mice. Pharmaceutically, pretreatment with the NE inhibitor sivelestat protected mice against ALF.ConclusionsThe present study showed the miR-223/NE axis as a key modulator of NETs, thereby exacerbating oxidative stress and neutrophilic inflammation to potentiate hepatocellular damage and liver necrosis in ALF development, and offering potential targets against ALF.