Nonalcoholic Steatohepatitis Fibrosis Research Articles

Single-cell transcriptome reveals a novel mechanism of C-Kit+-liver sinusoidal endothelial cells in NASH

AimTo understand how liver sinusoidal endothelial cells (LSECs) respond to nonalcoholic steatohepatitis (NASH).MethodsWe profiled single-LSEC from livers of control and MCD-fed mice. The functions of C-Kit+-LSECs were determined using coculture and bone marrow transplantation (BMT) methods.ResultsThree special clusters of single-LSEC were differentiated. C-Kit+-LSECs of cluster 0, Msr1+-LSECs of cluster 1 and Bmp4+Selp+-VECs of cluster 2 were revealed, and these cells with diverse ectopic expressions of genes participated in regulation of endothelial, fibrosis and lipid metabolism in NASH. The number of C-Kit+-primary LSECs isolated from MCD mice was lower than control mice. Immunofluorescence co-staining of CD31 and C-KIT showed C-Kit+-LSECs located in hepatic sinusoid were also reduced in NASH patients and MCD mice, compared to AIH patients and control mice respectively. Interestingly, lipotoxic hepatocytes/HSCs cocultured with C-Kit+-LSECs or the livers of MCD mice receipting of C-Kit+-BMCs (bone marrow cells) showed less steatosis, inflammation and fibrosis, higher expression of prolipolytic FXR and PPAR-α, lower expression of TNF-α and α-SMA. Furthermore, coculturing or BMT of C-Kit+-endothelial derived cells could increase the levels of hepatic mitochondrial LC3B, decrease the degree of mitochondrial damage and ROS production through activating Pink1-mediated mitophagy pathway in NASH.ConclusionsHence, a novel transcriptomic view of LSECs was revealed to have heterogeneity and complexity in NASH. Importantly, a cluster of C-Kit+-LSECs was confirmed to recovery Pink1-related mitophagy and NASH progression.

Dynamics of cellular plasticity in non-alcoholic steatohepatitis (NASH)

Non-alcoholic steatohepatitis (NASH) is a pathogenic stage of the broader non-alcoholic fatty liver disease (NAFLD). Histological presentation of NASH includes hepatocyte ballooning, macrophage polarization, ductular reaction, and hepatic stellate cell (HSCs) activation. At a cellular level, a heterogenous population of cells such as hepatocytes, macrophages, cholangiocytes, and HSCs undergo dramatic intra-cellular changes in response to extracellular triggers, which are termed “cellular plasticity. This dynamic switch in the cellular structure and function of hepatic parenchymal and non-parenchymal cells and their crosstalk culminates in the perpetuation of inflammation and fibrosis in NASH. This review presents an overview of our current understanding of cellular plasticity in NASH and its molecular mechanisms, along with possible targeting to develop cell-specific NASH therapies.

Meta-analysis: Efficacy and safety of fibroblast growth factor 21 analogues for the treatment of non-alcoholic steatohepatitis and non-alcoholic steatohepatitis-related fibrosis.

Fibroblast growth factor 21 (FGF21) analogues have emerged as promising therapeutic targets for non-alcoholic steatohepatitis (NASH). However, the effects and safety of these analogues on NASH and NASH-related fibrosis remain unexplored. To estimate the efficacy and safety of FGF21 analogues for treating NASH and NASH-related fibrosis. PubMed, Embase, and the Cochrane Library were searched for relevant studies up to 11 October 2023. Primary outcomes were defined as the fibrosis improvement ≥1 stage without worsening of NASH and NASH resolution without worsening fibrosis. Secondary outcomes included biomarkers of fibrosis, liver injury, and metabolism. Treatment-related adverse events were also analysed. Nine studies, including 1054 patients with biopsy-proven NASH and stage F1-F4 fibrosis, were identified. Seven studies reported histological outcomes. The relative risk (RR) for obtaining fibrosis improvement ≥1 stage efficacy was 1.79 (95% CI 1.29-2.48, I2  = 37%, p < 0.001) with FGF21 analogues relative to placebo. Although no statistically significant difference was observed between FGF21 analogues in NASH resolution, sensitivity analyses and fragility index suggest that this result is unstable. The drugs improved hepatic fat fraction (HFF), along with other biomarkers of fibrosis, liver injury, and metabolism (MRE, LSM, Pro-C3, ELF, ALT, AST, TG, HDL-C, and LDL-C). Additionally, no significant difference in serious adverse event incidence rate was observed (RR = 1.26, 95% CI 0.82-1.94, I2  = 24%, p = 0.3). FGF21 analogues appear as promising agents for the treatment of NASH and NASH-related fibrosis, and they generally seem to be safe and well tolerated.

Antioxidative Hyaluronic Acid-Bilirubin Nanomedicine Targeting Activated Hepatic Stellate Cells for Anti-Hepatic-Fibrosis Therapy.

Liver fibrosis is a life-threatening and irreversible disease. The fibrosis process is largely driven by hepatic stellate cells (HSCs), which undergo transdifferentiation from an inactivated state to an activated one during persistent liver damage. This activated state is responsible for collagen deposition in liver tissue and is accompanied by increased CD44 expression on the surfaces of HSCs and amplified intracellular oxidative stress, which contributes to the fibrosis process. To address this problem, we have developed a strategy that combines CD44-targeting of activated HSCs with an antioxidative approach. We developed hyaluronic acid-bilirubin nanoparticles (HABNs), composed of endogenous bilirubin, an antioxidant and anti-inflammatory bile acid, and hyaluronic acid, an endogenous CD44-targeting glycosaminoglycan biopolymer. Our findings demonstrate that intravenously administered HABNs effectively targeted the liver, particularly activated HSCs, in fibrotic mice with choline-deficient l-amino acid-defined high-fat diet (CD-HFD)-induced nonalcoholic steatohepatitis (NASH). HABNs were able to inhibit HSC activation and proliferation and collagen production. Furthermore, in a murine CD-HFD-induced NASH fibrosis model, intravenously administered HABNs showed potent fibrotic modulation activity. Our study suggests that HABNs have the potential to serve as a targeted anti-hepatic-fibrosis therapy by modulating activated HSCs via CD44-targeting and antioxidant strategies. This strategy could also be applied to various ROS-related diseases in which CD44-overexpressing cells play a pivotal role.

Panaxydol extracted from Panax ginseng inhibits NLRP3 inflammasome activation to ameliorate NASH-induced liver injury

Activation of NOD-like receptor protein 3 (NLRP3) inflammasome exacerbates liver inflammation and fibrosis in nonalcoholic steatohepatitis (NASH), suggesting that development of inflammasome inhibitor can become leading candidate to ameliorate NASH. Panax ginseng (P. ginseng) contains numerous bioactive natural components to reduce inflammation. This study aims to identify inhibitory components of P. ginseng for NLRP3 inflammasome activation. We separated polar and non-polar fractions of P. ginseng and tested modulation of NLRP3 inflammasome, and then identified pure component for inflammasome inhibitor which ameliorates diet-induced NASH. Non-polar P. ginseng fractions obtained from ethyl acetate solvent attenuated IL-1β secretion and expression of active caspase-1. We revealed that panaxydol (PND) is pure component to inhibit NLRP3 inflammasome activation. PND blocked inflammasome cytokines release, pyroptotic cell death, caspase-1 activation and specking of inflammasome complex. Inhibitory effect of PND was specific to NLRP3-dependent pathway via potential interaction with ATP binding motif of NLRP3. Moreover, in vivo studies showed that PND plays beneficial roles to reduce tissue inflammations through disruption of NLRP3 inflammasome and to ameliorate the development of NASH. These results provide new insight of natural products, panaxydol, for NLRP3 inflammasome inhibitor and could offer potential therapeutic candidate for reliving NASH.

Mic19 depletion impairs endoplasmic reticulum-mitochondrial contacts and mitochondrial lipid metabolism and triggers liver disease

Endoplasmic reticulum (ER)-mitochondria contacts are critical for the regulation of lipid transport, synthesis, and metabolism. However, the molecular mechanism and physiological function of endoplasmic reticulum-mitochondrial contacts remain unclear. Here, we show that Mic19, a key subunit of MICOS (mitochondrial contact site and cristae organizing system) complex, regulates ER-mitochondria contacts by the EMC2-SLC25A46-Mic19 axis. Mic19 liver specific knockout (LKO) leads to the reduction of ER-mitochondrial contacts, mitochondrial lipid metabolism disorder, disorganization of mitochondrial cristae and mitochondrial unfolded protein stress response in mouse hepatocytes, impairing liver mitochondrial fatty acid β-oxidation and lipid metabolism, which may spontaneously trigger nonalcoholic steatohepatitis (NASH) and liver fibrosis in mice. Whereas, the re-expression of Mic19 in Mic19 LKO hepatocytes blocks the development of liver disease in mice. In addition, Mic19 overexpression suppresses MCD-induced fatty liver disease. Thus, our findings uncover the EMC2-SLC25A46-Mic19 axis as a pathway regulating ER-mitochondria contacts, and reveal that impairment of ER-mitochondria contacts may be a mechanism associated with the development of NASH and liver fibrosis.

High coffee consumption is not associated with less liver fibrosis in non-alcoholic steato-hepatitis: Results of the COCANASH study

BackgroundThe beneficial effect of coffee consumption on the progression of liver fibrosis in NASH is controversial. AimsTo compare coffee consumption in NASH patients with and without advanced fibrosis. MethodsCross-sectional observational study on 97 patients with NASH diagnosed by histology or the association of steatosis, metabolic syndrome, elastometry>6kPa, and exclusion of other liver diseases. Usual coffee and caffeine intake were assessed using a standardized questionnaire. Liver fibrosis was evaluated by elastometry (advanced fibrosis if ≥10kPa). ResultsAmong the 97 patients, 49 patients (51%) had non-advanced fibrosis (group 1) and 48 (49%) advanced fibrosis (group 2). The mean consumption of caffeine from coffee was 251mg±293 per day in group 1 and 257mg±286 per day in group 2 (P=0.92). In multivariable analysis, the odds ratio for the mean consumption of caffeine from coffee (100mg/d) between groups was 1.00 (1.00–1.00, P=0.92). There was no correlation between elastometry and the consumption of caffeine from coffee. ConclusionWe found no relationship between caffeine or coffee consumption and the degree of liver fibrosis in NASH. Our result does not support a beneficial effect of coffee consumption on the progression of fibrosis in NASH.

Vascular endothelial cell-specific overexpression of CNP did not improve liver fibrosis in HFFCD-induced NASH, but did improve renal lesions

Mice with endothelial-cell-specific overexpression of C-type natriuretic peptide (E-CNP Tg mice) were shown to be protected against hepatic fibrosis and inflammation induced by high fat diet (HFD) feeding, with improved insulin sensitivity and attenuated weight gain. A recently developed high-fat, high-fructose, high-cholesterol diet (HFFCD) is considered to be a superior model to HFD, owing to the resemblance to human non-alcoholic steatohepatitis (NASH). In this study, we therefore aimed to reveal whether these previous findings with E-CNP Tg mice on HFD can be observed in a newly developed NASH model.　Patients with NASH have been suggested to be at higher risk of developing chronic kidney disease, so we also assessed the kidney histology of these mice. After 8 months of HFFCD feeding, the livers of E-CNP Tg mice and controls showed progressive fibrosis, which resembled the features of human NASH. However, no significant differences were observed in NAFLD activity scores between E-CNP Tg mice and controls, although there was a tendency for improvement in E-CNP Tg mice. The reduced levels of GCB, a receptor for CNP, may have weakened the action of CNP in the current model. In the kidneys, HFFCD showed glomerular hypertrophy and tubular atrophy in the cortical region, which were suppressed in E-CNP Tg mice. The present study did not prove the therapeutic effect of CNP on NASH in the HFFCD model, but provided evidence of its potential beneficial effects on NASH-associated renal damage.

Semaglutide reduces tumor burden in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH-HCC with advanced fibrosis

Non-alcoholic steatohepatitis (NASH) is emerging as a major cause of hepatocellular carcinoma (HCC), however, it is not resolved if compounds in late-stage clinical development for NASH may have additional therapeutic benefits in NASH-driven HCC (NASH-HCC). Here, we profiled monotherapy with semaglutide (glucagon-like-receptor-1 receptor agonist) and lanifibranor (pan-peroxisome proliferator-activated receptor agonist) in a diet-induced obese (DIO) mouse model of NASH-HCC. Disease progression was characterized in male C57BL/6 J mice fed the GAN (Gubra Amylin NASH) diet high in fat, fructose and cholesterol for 12–72 weeks (n = 15 per group). Other GAN DIO-NASH-HCC mice fed the GAN diet for 54 weeks and with biopsy-confirmed NASH (NAFLD Activity Score ≥ 5) and advanced fibrosis (stage F3) received vehicle (n = 16), semaglutide (30 nmol/kg, s.c., n = 15), or lanifibranor (30 mg/kg, p.o., n = 15) once daily for 14 weeks. GAN DIO-NASH-HCC mice demonstrated progressive NASH, fibrosis and HCC burden. Tumors presented with histological and molecular signatures of poor prognostic HCC. Consistent with clinical trial outcomes in NASH patients, both lanifibranor and semaglutide improved NASH while only lanifibranor reduced fibrosis in GAN DIO-NASH-HCC mice. Notably, only semaglutide reduced tumor burden in GAN DIO-NASH-HCC mice. In conclusion, the GAN DIO-NASH-HCC mouse is a clinical translational model of NASH-HCC. Semaglutide improves both NASH and tumor burden in GAN DIO-NASH-HCC mice, highlighting the suitability of this preclinical model for profiling novel drug therapies targeting NASH-HCC.

BRP39 Regulates Neutrophil Recruitment in NLRP3 Inflammasome-Induced Liver Inflammation

Background and AimsBRP-39 (Chi3L1) and its human homolog YKL-40, is an established biomarker of liver fibrosis in nonalcoholic steatohepatitis (NASH) patients, but its role in NASH pathogenesis remains unclear. We have recently identified Chi3L1 as one of the top upregulated genes in mice with inducible gain-of-function NOD-like receptor protein 3 (NLRP3) activation that mimics several liver features of NASH. This study aimed to investigate the effects of BRP39 deficiency on NLRP3-induced liver inflammation using Tamoxifen-inducible Nlrp3 knock-in mice sufficient (Nlrp3A350V CRT) and deficient for BRP39 (Nlrp3A350V /BRP-/- CRT). MethodsUsing Tamoxifen-inducible Nlrp3A350V knock-in mice (Nlrp3A350V CRT) mice and Tamoxifen-inducible Nlrp3A350V knock-in BRP39 deficient mice (Nlrp3A350V BRP-/- CRT), we investigated the consequence of BRP39 deficiency influencing NLRP3 induced liver inflammation. ResultsOur results showed that BRP39 deficiency in NLRP3 induced inflammation improved body weight and liver weight. Moreover, liver inflammation, fibrosis and HSC activation were significantly reduced, corresponding to significantly decreased Ly6C+ infiltrating macrophages, CD68+ OPN+ hepatic LAMs (lipid associated macrophages), and activated Ly6G+ and H3Cit+ neutrophils accumulation in the liver. Further investigation revealed that circulatory neutrophils from NLRP3 induced BRP39 deficient mice have impaired chemotaxis and migration ability, and this was confirmed by RNA-bulk sequencing showing reduced immune activation, migration and signaling responses in neutrophils. ConclusionThese data showcase the importance of BRP39 in regulating the NLRP3 inflammasome during liver inflammation and fibrotic NASH by altering cellular activation, recruitment and infiltration during disease progression, and revealing BRP39 to be a potential therapeutic target for future treatment of inflammatory NASH and its associated diseases.

Case-control analysis of venous thromboembolism risk in non-alcoholic steatohepatitis diagnosed by transient elastography.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. Studies have shown a strong association between non-alcoholic steatohepatitis (NASH) cirrhosis and portal vein thrombosis. Specifically, there is paucity of data on the association of NASH and venous thromboembolism (VTE), with one such study predicting a 2.5-fold increased risk for VTE compared to other liver diseases in hospitalized patients. The mechanism is believed to be a hepatocellular injury, which causes a chronic inflammatory state leading to the unregulated activation of procoagulant factors. There has been no prior analysis of the degree of steatosis and fibrosis (measured using transient elastography, commonly known as FibroScan) in NASH and its association with VTE. To examine the association between the degree of hepatic steatosis and fibrosis, quantified by transient elastography, and the incidence of VTE in patients with NASH. In our case-control study, we included patients with a documented diagnosis of NASH. We excluded patients with inherited thrombophilia, hemoglobinopathy, malignancy, alcohol use disorder, autoimmune hepatitis, and primary biliary cirrhosis. The collected data included age, demographics, tobacco use, recreational drug use, medical history, and vibration controlled transient elastography scores. VTE-specific data included the location, type of anticoagulant, need for hospital stay, and history of VTE recurrence. Steatosis was categorized as S0-S1 (mild) and S2-S3 (moderate to severe) based on the controlled attenuation parameter score. Fibrosis was classified based on the kilopascal score and graded as F0-F1 (Metavir stage), F2, F3, and F4 (cirrhosis). χ2 and Mann-Whitney U tests were used for the qualitative and quantitative variable analyses, respectively. Furthermore, we performed a logistic regression using VTE as the dependent variable. A total of 415 patients were analyzed, and 386 met the inclusion criteria. 51 and 335 patients were included in the VTE and non-VTE groups, respectively. Patients with VTE had a mean age of 60.63 years compared to 55.22 years in the non-VTE group (P < 0.014). Patients with VTE had a higher body mass index (31.14 kg/m² vs 29.30 kg/m²) and a higher prevalence of diabetes mellitus (29.4% vs 13.1%). The history of NASH was significantly higher in the VTE group (45.1% vs 30.4%, P < 0.037). Furthermore, moderate-to-severe steatosis was significantly higher in the VTE group (66.7% vs 47.2%, P < 0.009). Similarly, the F2-F4 fibrosis grade had a prevalence of 58.8% in the VTE group compared to 38.5% in the non-VTE group (P < 0.006). On logistic regression, using VTE as a dependent variable, diabetes mellitus had an odds ratio (OR) =1.702 (P < 0.015), and F2-F4 fibrosis grade had an OR = 1.5 (P < 0.033). Our analysis shows that NASH is an independent risk factor for VTE, especially deep vein thrombosis. There was a statistically significant association between the incidence of VTE, moderate-to-severe steatosis, and fibrosis. All hospitalized patients should be considered for medical thromboprophylaxis, particularly those with NASH.

Seladelpar combined with complementary therapies improves fibrosis, inflammation, and liver injury in a mouse model of nonalcoholic steatohepatitis.

Seladelpar, a selective peroxisome proliferator-activated receptor δ (PPARδ) agonist, improves markers of hepatic injury in human liver diseases, but histological improvement of nonalcoholic steatohepatitis (NASH) and liver fibrosis has been challenging with any single agent. To discover how complementary agents could work with seladelpar to achieve optimal outcomes, this study evaluated a variety of therapeutics (alone and in combination) in a mouse model of NASH. Mice on a high-fat amylin liver NASH (AMLN) diet were treated for 12 wk with seladelpar, GLP-1-R (glucagon-like peptide-1 receptor) agonist liraglutide, apoptosis signal-regulating kinase 1 (ASK1) inhibitor selonsertib, farnesoid X receptor (FXR) agonist obeticholic acid, and with seladelpar in combination with liraglutide or selonsertib. Seladelpar treatment markedly improved plasma markers of liver function. Seladelpar alone or in combination resulted in stark reductions in liver fibrosis (hydroxyproline, new collagen synthesis rate, mRNA indices of fibrosis, and fibrosis staining) compared with vehicle and the other single agents. Robust reductions in liver steatosis were also observed. Seladelpar produced a reorganization of metabolic gene expression, particularly for those genes promoting peroxisomal and mitochondrial lipid oxidation. In summary, substantial improvements in NASH and NASH-induced fibrosis were observed with seladelpar alone and in combination with liraglutide in this model. Broad gene expression analysis suggests seladelpar should be effective in concert with diverse mechanisms of action.NEW & NOTEWORTHY NASH is a chronic, progressive, and increasingly problematic liver disease that has been resistant to treatment with individual therapeutics. In this study using a diet-induced mouse model of NASH, we found that the PPARδ agonist seladelpar reduced fibrosis and NASH pathology alone and in combinations with a GLP-1-R agonist (liraglutide) or an ASK1 inhibitor (selonsertib). Liver transcriptome analysis comparing each agent and coadministration suggests seladelpar should be effective in combination with a variety of therapeutics.

Hepatocyte-targeted siTAZ therapy lowers liver fibrosis in NASH diet-fed chimeric mice with hepatocyte-humanized livers

Nonalcoholic steatohepatitis (NASH) is emerging as the most common cause of liver disease. Although many studies in mouse NASH models have suggested therapies, translation to humans is poor, with no approved drugs for NASH. One explanation may lie in differences between mouse and human hepatocytes. We used NASH diet-fed chimeric mice reconstituted with human hepatocytes (hu-liver mice) to test a mechanism-based hepatocyte-targeted small interfering RNA (siRNA), GalNAc-siTaz, shown previously to block the progression to fibrotic NASH in mice. Following ablation of endogenous hepatocytes, male mice were reconstituted with human hepatocytes from a single donor with the rs738409-C/G PNPLA3 risk variant, resulting in ∼95% human hepatocyte reconstitution. The mice were then fed a high-fat choline-deficient l-amino acid-defined diet for 6weeks to induce NASH, followed by six weekly injections of GalNAc-siTAZ to silence hepatocyte-TAZ or control GalNAc-siRNA (GalNAc-control) while still on the NASH diet. GalNAc-siTAZ lowered human hepatic TAZ and IHH, a TAZ target that promotes NASH fibrosis. Most important, GalNAc-siTAZ decreased liver inflammation, hepatocellular injury, hepatic fibrosis, and profibrogenic mediator expression versus GalNAc-control, indicating that GalNAc-siTAZ decreased the progression of NASH in mice reconstituted with human hepatocytes. In conclusion, silencing TAZ in human hepatocytes suppresses liver fibrosis in a hu-liver model of NASH.

Dynamics of Liver Macrophage Subsets in a Novel Mouse Model of Non-Alcoholic Steatohepatitis Using C57BL/6 Mice.

Macrophages are critical for the development of non-alcoholic steatohepatitis (NASH). Our previous findings in TSNO mouse livers showed that an iHFC (high-fat/cholesterol/cholate) diet induced liver fibrosis similar to human NASH and led to the accumulation of distinct subsets of macrophage: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+/Ly6C- cells were associated with the promotion of advanced liver fibrosis in NASH. On the other hand, CD11c-/Ly6C+ cells exhibited an anti-inflammatory effect and were involved in tissue remodeling processes. This study aimed to elucidate whether an iHFC diet with reduced cholic acid (iHFC#2 diet) induces NASH in C57BL/6 mice and examine the macrophage subsets accumulating in the liver. Histological and quantitative real-time PCR analyses revealed that the iHFC#2 diet promoted inflammation and fibrosis indicative of NASH in the livers of C57BL/6 mice. Cell numbers of Kupffer cells decreased and recruited macrophages were accumulated in the livers of iHFC#2 diet-fed C57BL/6 mice. Notably, the iHFC#2 diet resulted in the accumulation of three macrophage subsets in the livers of C57BL/6 mice: CD11c+/Ly6C-, CD11c-/Ly6C+, and CD11c+/Ly6C+ cells. However, CD11c+/Ly6C+ cells were not distinct populations in the iHFC-fed TSNO mice. Thus, differences in cholic acid content and mouse strain affect the macrophage subsets that accumulate in the liver.

Hepatocyte CYR61 polarizes profibrotic macrophages to orchestrate NASH fibrosis.

Obesity is increasing worldwide and leads to a multitude of metabolic diseases, including cardiovascular disease, type 2 diabetes, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis (NASH). Cysteine-rich angiogenic inducer 61 (CYR61) is associated with the progression of NASH, but it has been described to have anti- and proinflammatory properties. We sought to examine the role of liver CYR61 in NASH progression. CYR61 liver-specific knockout mice on a NASH diet showed improved glucose tolerance, decreased liver inflammation, and reduced fibrosis. CYR61 polarized infiltrating monocytes promoting a proinflammatory/profibrotic phenotype through an IRAK4/SYK/NF-κB signaling cascade. In vitro, CYR61 activated a profibrotic program, including PDGFa/PDGFb expression in macrophages, in an IRAK4/SYK/NF-κB-dependent manner. Furthermore, targeted-antibody blockade reduced CYR61-driven signaling in macrophages in vitro and in vivo, reducing fibrotic development. This study demonstrates that CYR61 is a key driver of liver inflammation and fibrosis in NASH.

Immunohistochemical expression of NRF2 is correlated with the magnitude of inflammation and fibrosis in chronic liver disease.

The nuclear factor E2-related factor 2-Kelch-like Ech-associated protein (NRF2-KEAP1) pathway is a major cellular defense mechanism against oxidative stress. However, the role of NRF2-KEAP1 signaling in the development of chronic liver disease remains unclear. Clinical liver specimens from 50 hepatocellular carcinoma (HCC) developed from non-alcoholic steatohepatitis (NASH), 49 HCCs developed from chronic viral hepatitis C (CHc), and 48 liver metastases of colorectal cancer (CRC) from both tumorous and non-tumorous areas were collected during hepatic resection surgery. They were evaluated by immunohistochemical analyses of hematoxylin-eosin, Masson's trichrome, NRF2, and KEAP1, and compared with clinicopathological information. Hepatic inflammation and fibrosis were more severe in the low-intensity NRF2 group than in the high-intensity NRF2 group both between CRC and NASH (Low vs. High: inflammation; p = 0.003, fibrosis; p = 0.014), and between CRC and CHc (Low vs. High: inflammation; p = 0.031, fibrosis; p = 0.011), which could indicate that NRF2 expression in cytosol of hepatocytes was inversely correlated with liver inflammation and fibrosis in non-tumorous areas. The dense staining of NRF2 in the nuclei of non-tumor hepatocytes positively correlated with liver inflammation (CRC and NASH; R = 0.451, p < 0.001, CRC and CHc; R = 0.502, p < 0.001) and fibrosis (CRC and NASH; R = 0.566, p < 0.001, CRC and CHc; R = 0.548, p < 0.001) in both NASH and CHc, and was inversely correlated with hepatic spare ability features such as platelet count (R = -0.253, p = 0.002) and prothrombin time (R = -0.206, p = 0.012). However, KEAP1 expression was not correlated with NRF2 expression levels and nuclear staining intensity. Nuclear translocation of NRF2 was correlated with the magnitude of liver inflammation and fibrosis in chronic liver disease. These results suggest that NRF2 plays a protective role in the development of chronic liver diseases such as NASH and CHc.

Antimullerian Hormone, a Marker of Ovarian Reserve, Is Protective Against Presence and Severity of NASH in Premenopausal Women

Antimüllerian hormone (AMH) is a marker of ovarian reserve with emerging data linking lower levels to some metabolic and inflammatory diseases in women. Whether AMH levels influence nonalcoholic fatty liver disease (NAFLD) is unknown. Leveraging the NASH Clinical Research Network we determined the association of AMH levels within 6 months of liver biopsy with presence and severity of histologic measures of NAFLD in premenopausal women. Outcomes included presence of nonalcoholic steatohepatitis (NASH), presence and severity of fibrosis, and NAFLD Activity Score and its components. Logistic and ordinal logistic regression models were adjusted for age, race/ethnicity, homeostatic model assessment for insulin resistance, body mass index, dyslipidemia, polycystic ovary syndrome, estrogen-progestin use, and menstrual cyclicity. Median cohort age was 35 years; 73% were white and 24% Hispanic. Thirty-three percent had diabetes, 81% had obesity, and 95% had dyslipidemia. On biopsy 71% had NASH, 68% had any fibrosis, and 15% had advanced fibrosis. On adjusted analysis (n= 205), higher AMH quartiles were inversely associated with NAFLD histology including prevalent NASH (adjusted odds ratio [AOR], 0.64; 95% confidence interval [CI], 0.41-1.00), NAFLD Activity Score ≥5 (AOR, 0.52; 95% CI, 0.35-0.77), Mallory hyaline (AOR, 0.54; 95% CI, 0.35-0.82), and higher fibrosis stage (AOR, 0.70; 95% CI, 0.51-0.98). The protective effects of AMH were more pronounced among women without polycystic ovary syndrome (n= 164), including lower odds of NASH (AOR, 0.53; 95% CI, 0.32-0.90) and any NASH fibrosis (AOR, 0.54; 95% CI, 0.32-0.93). AMH may reflect a unique biomarker of NASH in premenopausal women and findings suggest a novel link between reproductive aging and histologic severity of NAFLD in women.

Differential effects of PGAM5 knockout on high fat high fructose diet and methionine choline-deficient diet induced non-alcoholic steatohepatitis (NASH) in mice

BackgroundPhosphoglycerate mutase 5 (PGAM5), a phosphatase involved in mitochondrial homeostasis, is reported to be closely related to the metabolic stress induced by high-fat diet (HFD) or cold. In this study, we aimed to investigate the effects of PGAM5 on hepatic steatosis, inflammation and fibrosis in nonalcoholic steatohepatitis (NASH).Methods and resultsWe generated PGAM5 global knockout (GKO) mice and their wildtype (WT) littermates using CRISPR/CAS9. The mice were fed with a high fat high fructose (HFHF) diet for 12 weeks or a methionine choline-deficient (MCD) diet (methionine choline supplemented (MCS) as control) for 6 weeks. Hepatic PGAM5 expression was up-regulated in humans with NASH and WT mice fed with HFHF and MCS, and reduced in WT mice fed with MCD diet. In HFHF-fed mice, GKO had reduced body weight, hepatic triglyceride (TG) content and serum transaminase along with decreased hepatic pro-inflammatory and pro-fibrotic responses compared with their WT control. GKO had increased expression of antioxidative gene glutathione peroxidase-6 (GPX6) and activation of mammalian target of rapamycin (mTOR). In mice fed with MCS diet, GKO significantly increased serum TNF-α and IL-6 and decreased hepatic GPX6 mRNA expression. There was no difference in hepatic steatosis, inflammation or fibrosis between GKO and WT mice fed with MCD diet. We investigated the role of PGAM5 deficiency in a variety of cell types. In differentiated THP-1 cells, PGAM5 silencing significantly increased pro-inflammatory cytokine secretion and decreased antioxidative proteins, including nuclear factor erythroid 2- related factors (NRF2), heme oxygenase-1 (HO-1) and GPX6 without affecting mTOR activity. In HepG2 cells with steatosis, PGAM5 knockdown reduced insulin sensitivity, increased mTOR phosphorylation and reduced the expression of NRF2, catalase (CAT), HO-1 and GPX6. Conversely, PGAM5 knockdown reduced TG accumulation, increased insulin sensitivity, and increased antioxidative genes in 3T3-L1 cells, despite the up-regulation in mTOR phosphorylation.ConclusionsPGAM5-KO relieved hepatic steatosis and inflammation in HFHF model, promoted inflammation in MCS-fed mice and had no effects on the MCD-fed model. The distinct effects may be owing to the different effects of PGAM5-KO on anti-oxidative pathways in energy-dependent, possible involves mTOR, and/or cell type-dependent manner. Our findings suggest that PGAM5 can be a potential therapeutic target for NASH.

Production, Exacerbating Effect, and EV-Mediated Transcription of Hepatic CCN2 in NASH: Implications for Diagnosis and Therapy of NASH Fibrosis.

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis, hepatocyte ballooning, and inflammation and may progress to include increasingly severe fibrosis, which portends more serious disease and is predictive of patient mortality. Diagnostic and therapeutic options for NASH fibrosis are limited, and the underlying fibrogenic pathways are under-explored. Cell communication network factor 2 (CCN2) is a well-characterized pro-fibrotic molecule, but its production in and contribution to NASH fibrosis requires further study. Hepatic CCN2 expression was significantly induced in NASH patients with F3-F4 fibrosis and was positively correlated with hepatic Col1A1, Col1A2, Col3A1, or αSMA expression. When wild-type (WT) or transgenic (TG) Swiss mice expressing enhanced green fluorescent protein (EGFP) under the control of the CCN2 promoter were fed up to 7 weeks with control or choline-deficient, amino-acid-defined diet with high (60%) fat (CDAA-HF), the resulting NASH-like hepatic pathology included a profound increase in CCN2 or EGFP immunoreactivity in activated hepatic stellate cells (HSC) and in fibroblasts and smooth muscle cells of the vasculature, with little or no induction of CCN2 in other liver cell types. In the context of CDAA-HF diet-induced NASH, Balb/c TG mice expressing human CCN2 under the control of the albumin promoter exhibited exacerbated deposition of interstitial hepatic collagen and activated HSC compared to WT mice. In vitro, palmitic acid-treated hepatocytes produced extracellular vesicles (EVs) that induced CCN2, Col1A1, and αSMA in HSC. Hepatic CCN2 may aid the assessment of NASH fibrosis severity and, together with pro-fibrogenic EVs, is a therapeutic target for reducing NASH fibrosis.

Weighted Gene Co-Expression Network Analysis of Immune Infiltration in Nonalcoholic Fatty Liver Disease.

Immune cell infiltration is an important component of nonalcoholic fatty liver disease (NAFLD) pathogenesis. This study aimed to explore novel genes associated with immune infiltration in the progression of NAFLD. CIBERSORT was used to evaluate the abundance of immune infiltration in the human NAFLD via a high-throughput sequencing dataset. Further weighted gene co-expression network analysis (WGCNA) was performed to search for the susceptibility gene module and hub genes associated with differential immune cells. The expression of hub genes in different liver non-parenchymal cell clusters and NAFLD-associated hepatocellular carcinoma (HCC) was also explored. Four hub genes (ITGBL1, SPINT1, COL1A2, and THBS2) were ultimately identified, which may be associated with immune infiltration, fibrosis progression, and activity score. The receiver operating characteristic curve (ROC) analysis suggested that these genes had good predictive value for NASH and advanced fibrosis. A single-cell analysis showed that COL1A2 was highly expressed in hepatic stellate cells (HSCs), especially in the later stage, while SPINT1 was highly expressed in cholangiocytes (Cho). In addition, ITGBL1, COL1A2, and THBS2 might be associated with transforming from nonalcoholic steatohepatitis (NASH) to HCC. Our findings identified several novel genes that might be related to immune infiltration in NAFLD. These genes may serve as potential markers for the assessment of immune infiltration as well as therapeutic targets for NAFLD. More studies are needed to elucidate the biological mechanism of these genes in the occurrence and development of NAFLD.