Vsig4+ resident single-Kupffer cells improve hepatic inflammation and fibrosis in NASH.

BACKGROUND: The role of macrophages in the pathogenesis of nonalcoholic steatohepatitis (NASH) is complex and unclear. METHODS: Single-cell RNA sequencing was performed on nonparenchymal cells isolated from NASH and control mice. The expression of Vsig4+ macrophages was verified by qPCR, flow cytometry and immunohistochemistry. Primary hepatic macrophages were cocultured with primary hepatocytes or hepatic stellate cells (LX2) cells by Transwell to detect immunofluorescence and oil red O staining. RESULTS: Two main single macrophage subsets were identified that exhibited a significant change in cell percentage when NASH occurred: resident Kupffer cells (KCs; Cluster 2) and lipid-associated macrophages (LAMs; Cluster 13). Nearly 82% of resident single KCs in Cluster 2 specifically expressed Cd163, and an inhibited subgroup of Cd163+ resident single-KCs was suggested to be protective against NASH. Similar to Cd163, Vsig4 was both enriched in and specific to Cluster 2. The percentage of Vsig4+-KCs was significantly decreased in NASH in vivo and in vitro. Hepatocytes and hepatic stellate cells produced less lipid droplet accumulation, proinflammatory protein (TNF-α) and profibrotic protein (α-SMA) in response to coculture with Vsig4+-KCs than in those cocultured with lipotoxic KCs. CONCLUSIONS: A subgroup of Vsig4+ resident single-KCs was shown to improve hepatic inflammation and fibrosis in NASH.

CircRNA608-microRNA222-PINK1 axis regulates the mitophagy of hepatic stellate cells in NASH related fibrosis.

BACKGROUND: Increasing evidences have confirmed the relationship between mitophagy and nonalcoholic steatohepatitis (NASH). The exact mechanism of upstream circular RNAs (circRNAs) regulating PTEN-induced putative kinase 1 (PINK1) mediated mitophagy and its contribution to NASH-related liver fibrosis was explored in our study. METHODS: Primary hepatic stellate cells (PHSCs) from C57BL/6 mice transfected with small interfering RNAs against PINK1 (si-PINK1) and negative control (si-NC) were prepared to perform circRNA sequence. Differentially expressed circRNAs, bioinformatic analysis and predicting software were performed to select axis of circ608/miR-222/PINK1. The expressions of circ608/miR-222/PINK1 were verified by RT-qPCR. The mitochondrial function was evaluated by immunofluorescence staining of COX4 and LC3B. RESULTS: PINK1-mediated mitophagy was inhibited in NASH-related liver fibrosis mice. CircRNA sequence revealed there were 37 DE-circRNAs between si-PINK1 PHSCs and si-NC PHSCs. Bioinformatic analysis showed these DE-circRNAs were related to enriched signaling pathways (such as Wnt, Rap1, mTOR, Hippo) regulating liver fibrosis and mitophagy. Circ608 was significantly down-regulated in lipotoxic HSCs and in livers of NASH-related liver fibrosis mice. MiR222 was identified to be the target miRNA of circ608 and was negatively regulated by circ608 in lipotoxic HSCs. MiR222 also had a binding site with PINK1 and could negatively regulate PINK1. So, the axis of circ608-miR222-PINK1 was proved to participate in NASH-related liver fibrosis by regulating mitophagy. These results illustrated that circ608 might promote PINK1-mediated mitophagy though inhibiting miR222 in lipotoxic HSCs. CONCLUSION: Circ608 could promote PINK1-mediated mitophagy of HSCs though inhibiting miR222 in NASH-related liver fibrosis.

The oncogenic miR-27a/BTG2 axis promotes obesity-associated hepatocellular carcinoma by mediating mitochondrial dysfunction.

Obesity is closely related to the initiation and development of hepatocellular carcinoma (HCC). The regulatory mechanism of obesity-associated HCC remains unclear. HepG2 cells treated with palmitic acid (PA) and diethylnitrosamine (DEN)-induced HCC mice fed a high-fat diet (HFD) were established. The expression of miR-27a and B-cell translocation gene 2 (BTG2) mRNA and protein were detected via qPCR and western blotting. Prediction software and luciferase assays were employed to verify the miR-27a/BTG2 axis. The biological effects of HepG2 cells were evaluated with ORO staining, MTT assays, Transwell assays, Mito-Timer, and Mito-SOX staining. Significantly upregulated miR-27a and downregulated BTG2 mRNA and protein were observed in HepG2 cells and liver tissues of HCC mice. Overexpressing miR-27a (mi-miR-27a) markedly promoted cellular lipid accumulation, proliferation, and invasion, accompanied by aggravated mitochondrial dysfunction (increased fading and ROS products of mitochondria) in HepG2 cells. Additionally, these effects were further reinforced in HepG2 cells treated with mi-miR-27a and PA. BTG2 was identified as a direct target and was negatively regulated by miR-27a. Similarly, BTG2 knockdown (sh-BTG2) had effects identical to those of mi-miR-27a on HepG2 cells. Additionally, PA evidently enhanced these effects of sh-BTG2 in HepG2 cells. Moreover, BTG2 overexpression effectively reversed the effects of miR-27a, including lipotropic and oncogenic effects, and simultaneously promoted mitochondrial imbalance in HepG2 cells. Thus, obesity-associated miR-27a acts as an oncogene to promote lipid accumulation, proliferation, and invasion by negatively regulating BTG2-mediated mitochondrial dysfunction in HCC.

linc-SCRG1 accelerates liver fibrosis by decreasing RNA-binding protein tristetraprolin.

The biologic roles of long noncoding RNAs (lncRNAs) in liver fibrosis remained unknown. Through microarray analysis, linc-SCRG1 (a lncRNA with transcript length 3118 bp) was found up-regulated 13.62-fold in human cirrhotic tissues. Quantitative PCR verified that linc-SCRG1 increased along with liver fibrosis progression in human tissues and in activated LX2 cells induced by TGF-ß1. Knockdown of linc-SCRG1 significantly reversed the effects of TGF-ß1 on LX2, including inhibiting activation, promoting apoptosis, reducing proliferation, lessening invasion, and down-regulating genes [fibrosis-related mRNA: α-smooth muscle actin ( α-SMA), type I collagen, and B-cell lymphoma-2; invasion-related mRNA: matrix metallopeptidase-2 ( MMP-2), MMP-9, and MMP-13; inflammation-related mRNA: TNF-α, IL-6, and IL-10]. linc-SCRG1 had binding sites with tristetraprolin (TTP), a kind of RNA-binding protein, and specifically combined to TTP proteins. Overexpression of linc-SCRG1 would cause TTP mRNA unstably and proteins decreasing. TTP mRNA was proved having negative relevance with linc-SCRG1 and was gradually reduced during human liver fibrosis progression. Overexpressing TTP resulted in knockdown of lincSCRG1 and degraded downstream target genes ( MMP-2 and TNF-α) in activated LX2. Overexpressing TTP had the same effects as small interfering RNA-lincSCRG1 (si- lincSCRG1), whereas knockdown of TTP had reversal effects on si- lincSCRG1 in activated LX2. In summary, linc-SCRG1 reduced TTP and restricted its degradation of target genes TNF-α and MMP-2. Therefore, linc-SCRG1 had a repressing TTP-elicited inactivation effect on hepatic stellate cell (HSC) phenotypes. Inhibition of linc-SCRG1 may be a novel therapeutic approach to inactivate HSCs and extenuate human liver fibrosis.-Wu, J.-C., Luo, S.-Z., Liu, T., Lu, L.-G., Xu, M.-Y. linc-SCRG1 accelerates liver fibrosis by decreasing RNA-binding protein tristetraprolin.

Prediction of hepatic necroinflammatory activity in patients with chronic hepatitis B by a simple noninvasive model.

BACKGROUND: A model was constructed using clinical and serum variables to discriminate between chronic hepatitis B (CHB) patients with and without significant necroinflammatory activity (score 4-18 vs. score 0-3). METHODS: Consecutive CHB patients who underwent liver biopsy were divided into two sequential groups: a training group (n = 401) and a validation group (n = 401). Multivariate analysis identified alanine aminotransferase, γ-glutamyltransferase, prothrombin time and albumin as independent predictors of necroinflammatory activity. RESULTS: The area under the receiver operating characteristic curve was 0.826 for the training group and 0.847 for the validation group. Using a cut-off score of H ≤ 0.375, significant necroinflammatory activity (score 4-18) was excluded with high accuracy [78.2% negative predictive value (NPV), 72% positive predictive value (PPV), and 90.8% sensitivity] in 238 (59.4%) of 401 patients in the training group and with the same certainty (88.1% NPV, 61.2% PPV, and 95.1% sensitivity) among 204 (50.9%) of 401 patients in the validation group. Similarly, applying a cut-off score of H > 0.720, significant necroinflammatory activity was correctly identified with high accuracy (90.8% PPV, 57.7% NPV, and 92.0% specificity) in 150 (37.4%) of 401 patients in the training group and with the same certainty (91.8% PPV, 64.6% NPV, and 95.4% specificity) in 188 (46.9%) of 401 patients in the validation group. CONCLUSIONS: A predictive model based on easily accessible variables identified CHB patients with and without significant necroinflammatory activity with a high degree of accuracy. This model may decrease the need for liver biopsy for necroinflammatory activity grading in 72.1% of CHB patients.

Stat3 signaling activation crosslinking of TGF-ß1 in hepatic stellate cell exacerbates liver injury and fibrosis.

BACKGROUND/AIMS: The role of signal transducer and activator of transcription 3 (Stat3) in liver fibrosis is still controversial. Since hepatic stellate cells (HSCs) and transforming growth factor-ß1 (TGF-ß1) are central to the fibrogenesis, our goal was to clarify the mechanism of Stat3 crosslinking of TGF-ß1 signaling. METHODS: Stat3, TGF-ß1 mRNA and protein expressions were examined in liver tissues of chronic hepatitis B (CHB) patients and diethylinitrosamine (DEN)-induced rat fibrosis model. The effect of Stat3 activation or suppression on TGF-ß1 signaling in HSCs was tested in vitro and in vivo. RESULTS: Stat3 expression as well as TGF-ß1 was increased in CHB patients and DEN-induced fibrosis rat model. This was strongly correlated with increase in fibrosis staging. TGF-ß1, a mediator of fibrosis, was enhanced by Stat3, but suppressed by siRNA-mediated RNA knockdown of Stat3 (siStat3) or Janus kinase 2 inhibitor (AG490) both in vivo and in vitro. Stat3 crosslinking TGF-ß1 signaling plays an important role in HSC activation and increasing fibrosis related products. TGF-ß1 could not achieve profibrogenic cytokine and anti-apoptosis characteristics without Stat3 activation in HSCs. CONCLUSION: We provide a novel role of Stat3 cooperating TGF-ß1 in activation and anti-apoptotic effect of HSCs. Stat3 worsens liver fibrosis through the up-regulation of TGF-ß1 and fibrotic product expression.

Causal relationship between gut microbiota and risk of esophageal cancer: evidence from Mendelian randomization study.

BACKGROUND: The causative implications remain ambiguous. Consequently, this study aims to evaluate the putative causal relationship between gut microbiota and Esophageal cancer (EC). METHODS: The genome-wide association study (GWAS) pertaining to the microbiome, derived from the MiBioGen consortium-which consolidates 18,340 samples across 24 population-based cohorts-was utilized as the exposure dataset. Employing the GWAS summary statistics specific to EC patients sourced from the GWAS Catalog and leveraging the two-sample Mendelian randomization (MR) methodology, the principal analytical method applied was the inverse variance weighted (IVW) technique. Cochran's Q statistic was utilized to discern heterogeneity inherent in the data set. Subsequently, a reverse MR analysis was executed. RESULTS: Findings derived from the IVW technique elucidated that the Family Porphyromonadaceae (P = 0.048) and Genus Candidatus Soleaferrea (P = 0.048) function as deterrents against EC development. In contrast, the Genus Catenibacterium (P = 0.044), Genus Eubacterium coprostanoligenes group (P = 0.038), Genus Marvinbryantia (P = 0.049), Genus Ruminococcaceae UCG010 (P = 0.034), Genus Ruminococcus1 (P = 0.047), and Genus Sutterella (P = 0.012) emerged as prospective risk contributors for EC. To assess reverse causal effect, we used EC as the exposure and the gut microbiota as the outcome, and this analysis revealed associations between EC and seven different types of gut microbiota. The robustness of the MR findings was substantiated through comprehensive heterogeneity and pleiotropy evaluations. CONCLUSIONS: This research identified certain microbial taxa as either protective or detrimental elements for EC, potentially offering valuable biomarkers for asymptomatic diagnosis and prospective therapeutic interventions for EC.

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

AIM: To understand how liver sinusoidal endothelial cells (LSECs) respond to nonalcoholic steatohepatitis (NASH). METHODS: We 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. RESULTS: Three 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. CONCLUSIONS: Hence, 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.

Serum dihydroxyacetone kinase peptide m/z 520.3 as predictor of disease severity in patients with compensated chronic hepatitis B.

BACKGROUND AND AIM: Due to known limitations of liver biopsy, reliable non-invasive serum biomarkers for chronic liver diseases are needed. We performed serum peptidomics for such investigation in compensated chronic hepatitis B (CHB) patients. METHODS: Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed peptides in sera from 40 CHB patients (20 with S0G0-S1G1 and 20 with S3G3-S4G4). Ion pair quantification from differentially expressed peptides in a validation set of sera from 86 CHB patients was done with multiple reaction monitoring (MRM). RESULTS: 21 differentially represented peptide peaks were found through LC-MS/MS. Ion pairs generated from eleven of these peptides (m/z < 800) were quantified by MRM. Summed peak area ratios of 6 ion pairs from peptide m/z 520.3 (176.1, 353.7, 459.8, 503.3, 351.3, 593.1), which was identified as dihydroxyacetone kinase (DAK) fragment, decreased from mild to advanced stages of fibrosis or inflammation. Area Under Receiver Operating Characteristic Curves (AUROCs) of five ion models discriminating fibrosis degrees were 0.871 ~ 0.915 (S2-4 versus S0-1) and 0.804 ~ 0.924 (S3-4 versus S0-2). AUROCs discriminating inflammation grades were 0.840 ~ 0.902 (G2-4 versus G0-1) and 0.787 ~ 0.888 (G3-4 versus G0-2). The diagnostic power of these models provides improved sensitivity and specificity for predicting disease progression as compared to aspartate aminotransferase to platelet ratio index (APRI), FIB-4, Forn's index and serum DAK protein. CONCLUSIONS: The peptide fragment (m/z 520.3) of DAK is a promising biomarker to guide timing of antiviral treatment and to avoid liver biopsy in compensated CHB patients.

Prediction of significant fibrosis and cirrhosis in hepatitis B e-antigen negative patients with chronic hepatitis B using routine parameters.

AIM: As liver biopsy has considerable limitations in the assessment of liver fibrosis, non-invasive models have achieved great progress in the past. However, many tests consist of variables that are not readily available, and there are few data about patients with hepatitis B e-antigen (HBeAg) negative chronic hepatitis B (CHB). The aim of this study was to develop a model using routine data to predict liver fibrosis in HBeAg negative CHB patients. METHODS: We randomly divided 349 patients who underwent liver biopsy into training (n = 200) and validation (n = 149) sets. Multivariable logistic regression and receiver-operator curve (ROC) analyses were used to develop a model for predicting both significant fibrosis (stages 2-4) and cirrhosis (stage 4) in the training set. The model was validated in 149 patients in comparison to FIB-4, Forn's, S and aspartate aminotransferase-to-platelet ratio index indices using ROC. RESULTS: Multivariable logistic regression analysis showed that the parameters of the model for predicting both significant fibrosis and cirrhosis included sex, age, prothrombin time, platelet count, cholesterol and γ-glutamyltransferase. In the training set, the areas under the ROC (AUC) for predicting significant fibrosis and cirrhosis were 0.856 and 0.956, respectively. In the validation group, the AUC for predicting significant fibrosis and cirrhosis were 0.889 and 0.937, respectively. Using the best cut-off values, significant fibrosis and cirrhosis can be accurately predicted in 40.9% and 91.3% of patients, respectively. CONCLUSION: Our model can accurately predict both significant fibrosis and cirrhosis and may decrease the need of liver biopsy in a considerable proportion of patients with HBeAg negative CHB.

Global transcriptome analysis of Escherichia coli exposed to immobilized anthraquinone-2-sulfonate and azo dye under anaerobic conditions.

The immobilization of quinone compounds is regarded as a promising strategy to accelerate anaerobic decolorization of xenobiotic compounds azo dyes in the presence of quinone-reducing microorganisms. However, little is known about the basic response of these microorganisms to immobilized quinones in the presence of azo dyes. In the present study, whole-genome DNA microarrays were used to investigate a quinone-reducing bacterium Escherichia coli K-12 transcription response to immobilized anthraquinone-2-sulfonate (AQSim) reduction and azo dye acid red 18 (AR 18) decolorization. Transcriptome analysis showed that AQSim was more accessible for the cells of E. coli K-12 than AR 18. Despite there being some differences between AQSim and soluble AQS mediated decolorization of AR 18, AQSim reduction and AR 18 decolorization, more similarity could be observed in the four processes. Among over 60 % shared genes, several groups of genes exhibited high expression levels, including those genes encoding terminal reductases, menaquinone biosynthesis, formate dehydrogenases and outer membrane proteins. Especially, nrfABCD, frdBCD and dsmABC encoding terminal reductases were significantly upregulated. Further gene deletion experiments demonstrated that the above three groups of genes were involved in AQSim-mediated AR 18 decolorization. In addition, significant upregulation of stress response genes was observed, which indicated the adaptation of E. coli K-12 to AQSim and AR 18 exposures.

Deciphering the role and mechanism of nano zero-valent iron on medium chain fatty acids production from CO2 via chain elongation in microbial electrosynthesis.

The integrated system of microbial electrosynthesis (MES) coupled with chain elongation has been considered a promising platform for carboxylic acids production. However, this biotechnology is still in its infancy, and many limitations are needed to be transcended, such as low electron transfer efficiency between cathode and microbes. In this study, nano zero-valent iron (NZVI) was employed to improve carboxylic acid production in the integrated system, and the promotion mechanisms were revealed. Results suggested that the highest production concentrations of acetate, butyrate, and caproate were observed at 7.5 g/L optimized NZVI dosage, increasing the total yield and coulomb efficiency by 23.7 % and 40.3 % compared to the control. Mechanism studies indicated that the hydrogen and electron released by the anaerobic corrosion of NZVI could be used as additional reducing equivalents, thereby enhancing the electron transfer performance. Besides, NZVI was also proven to facilitate the formation of electroactive biofilms according to the results of biofilm characterization and total DNA. In functional microbes' respect, the moderate NZVI enriched the chain elongator in biofilm, like Clostridium_sensu-stricto_12, and upregulated the activities of key enzymes of homoacetogenesis and chain elongation metabolic pathways, like carbon-monoxide dehydrogenase and hydroxyacyl-CoA dehydratase. This study provided the evidence and revealed how NZVI assisted carboxylic acid production from CO2 via chain elongation in MES.

18α-glycyrrhetinic acid down-regulates expression of type I and III collagen via TGF-Β1/Smad signaling pathway in human and rat hepatic stellate cells.

OBJECTIVE: To investigate the effects of 18α-glycyrrhetinic acid (18α-GA) on the expression of type I and III collagen in human and rat hepatic stellate cells (HSC) and to explore the role of TGF-ß1/Smad signaling pathway involved. METHODS: Following 18α-GA treatment, the cell viability and cell growth were detected to determine the optimal concentration of 18α-GA. The expressions of TGF-ß1/Smad signaling-related genes including type I and III collagen in human and rat HSCs before and after 18α-GA treatment were measured by real time PCR. The expression of related proteins was verified by western blot assay. The phosphorylation level of Smad2 and Smad3 was detected by immunocytochemistry. The DNA binding activities of SP-1, AP-1 and NF-κB were measured by both EMSA and ArrayStar transcription factor activity assay. RESULTS: 18α-GA could decrease the mRNA and protein expression of Smad3, type I and III collagen, increase the Smad7 expression in human and rat HSCs (P<0.05), and reduce phosphorylation level of Smad3 at 24 h and 48 h after treatment. The DNA binding activities of transcription factors were suppressed by 18α-GA in human and rat HSCs at 24 h, and the activities reduced in a time dependent manner with the lowest activities at 48 h, especially for SP-1. CONCLUSION: 18α-GA could inhibit the mRNA and protein expression of type I and III collagen in human and rat HSCs, which may be attributed to down-regulation of Smad3, up-regulation of Smad7, and inhibition of DNA binding activities of SP-1, AP-1 and NF-κB.

18α-Glycyrrhizin induces apoptosis and suppresses activation of rat hepatic stellate cells.

BACKGROUND: To investigate the potential mechanisms underlying the protective effects of 18α Glycyrrhizin (GL) on rat hepatic stellate cells (HSCs) and hepatocytes in vivo and in vitro. MATERIAL/METHODS: Sprague-Dawley (SD) rats were randomly divided into 5 groups: normal control group, liver fibrosis group, high-dose 18α GL group (25 mg/ kg/d), intermediate-dose 18α GL group (12.5 mg/kg/d) and low-dose 18α GL group (6.25 mg/ kg/d). The rat liver fibrosis model was induced by carbon tetrachloride (CCl4). The expressions of alpha-smooth muscle actin (αSMA) and NF-kappaB were determined by real-time PCR and immunohistochemistry. RESULTS: 18αGL dose-dependently inhibited the CCl4-induced liver fibrosis. There were significant differences in the mRNA and protein expressions of αSMA between the fibrosis group and 18α-GL treatment groups, suggesting that 18α GL can suppress the proliferation and activation of HSCs. Few HSCs were apoptotic in the portal area and fibrous septum in the liver fibrosis group. However, the double-color staining of a-SMA and TUNEL showed that 18α-GL treatment groups increased HSC apoptosis. NF-kappaB was mainly found in the nucleus in the fibrosis group, while cytoplasmic expression of NF-kappaB was noted in the 18αGL groups. In the in vitro experiments, 18α GL promoted the proliferation of hepatocytes, but inhibited that of HSCs. HSCs were arrested in the G2/M phase following 18α GL treatment and were largely apoptotic. CONCLUSIONS: 18α-GL can suppress the activation of HSCs and induce the apoptosis of HSCs by blocking the translocation of NF-kappaB into the nucleus, which plays an important role in the protective effect of 18α-GL on liver fibrosis.

Serum N-glycomic markers in combination with panels improves the diagnosis of chronic hepatitis B.

AIM: The present study aimed to evaluate the changes in the serum N-glycome profiles in chronic hepatitis B (CHB) patients and to assess the role of N-glycome-derived markers in the noninvasive diagnosis of liver fibrosis. MATERIALS AND METHODS: After liver biopsy for pathological grading and staging, 128 CHB patients underwent serum N-glycomic analysis using DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) and sensitive markers were screened. RESULTS: Peaks 1, 2, 8 and 10 in the N-glycome profiles could, to some extents, distinguish liver fibrosis at different stages. In addition, the N-glycome-derived marker log(peak2/peak8) possessed the highest diagnostic accuracy. The areas under the receiver operating characteristic (AUROCs) curves of the log(peak2/peak8) were 0.675, 0.736 and 0.754 in the diagnosis of significant fibrosis, advanced fibrosis and early cirrhosis, respectively. In combination with some marker panels (SLFG, S index, Fibrometer, Hui, Forns, APRI and Hepascore), it showed the best diagnostic potency in distinguishing significant fibrosis (SLFG + log[peak2/peak8], AUROC = 0.813) from advanced fibrosis (SLFG + log[peak2/peak8], AUROC = 0.899) and a better diagnostic potency in the identification of early cirrhosis (S index + log[peak2/peak8], AUROC = 0.903, lower than Hui model [AUROC = 0.927]) in the validation cohort. CONCLUSIONS: N-glycomic changes are present in the serum of CHB patients with liver fibrosis, and N-glycan profiling is a noninvasive and effective tool to assess the liver fibrosis, especially in combination with serum marker panels.

[Prognostic value of the model for end-stage liver disease combined with serum sodium levels in patients with decompensated cirrhosis].

OBJECTIVE: To investigate the ability of the model for end-stage liver disease (MELD) score combined with serum sodium measurements to effectively evaluate the prognosis of patients with decompensated liver cirrhosis. METHODS: A total of 212 patients with decompensated cirrhosis were retrospectively analyzed. Each patient's MELD scores, and sodium-based MELD scores (MELD-Na, MELDNa, and MESO) were calculated at three-month intervals. The area under the receiver operating characteristic (ROC) curve (AUC) was used to compare the predictive abilities of the four scores for 3-, 6- and 12-month mortality. Kaplan-Meier survival curves were created using the best cut-off values for each score identified by the ROC. RESULTS: Among the 212 patients, 46 died within three months, 56 died within six months, and 87 died within 12 months. The MELD, MELD-Na, MELDNa and MESO scores were significantly different between patients who survived and those who died within three and 12 months (P less than 0.01). The AUCs for the four separate scores were all more than 0.8 at the 3- and 6-month time points; however, the AUCs of MELDNa (3-month: 0.846; 6-month: 0.869) and MESO (0.831; 0.850) were significantly better than those of MELD (0.812; 0.841) (P less than 0.05). At the 12-month time point, the AUCs of MELD, MELD-Na, MELDNa, and MESO were not significantly different (0.774, 0.775, 0.786, and 0.777, respectively). Survival curves showed that all the scores were able to clearly discriminate the patients who survived from those who died within 12 months (P=0.000). CONCLUSION: The MELD score and its sodium-based variants (MELD-Na, MELDNa, and MESO) can precisely predict mortality of patients with decompensated cirrhosis for short and intermediate periods. The MELDNa and MESO scores are superior for predicting 3- and 6-month survival.

Hepatocyte-derived exosomal miR-27a activateshepatic stellate cells through the inhibitionof PINK1-mediated mitophagy in MAFLD.

The role of exosome-mediated mitophagy in the crosstalk between hepatocytes (HCs) and hepatic stellate cells (HSCs) in metabolic-associated fatty liver disease (MAFLD) remains unknown. Serum exosomal miR-27a levels were markedly increased and positively correlated with liver fibrosis in MAFLD patients and mice. Exosomal miR-27a was released from lipotoxic HCs and specifically transmitted to recipient-activated HSCs. PINK1, the key target of miR-27a, primarily mediates mitophagy. Overexpression of miR-27a or knockdown of PINK1 or lipotoxic HC-exosomal miR-27a impaired mitochondria (inhibiting mitophagy, respiration, membrane potential, and transcription while promoting reactive oxygen species production) in activated HSCs and stimulated HSC-derived fibroblasts (promoting activation and proliferation while inhibiting autophagy). High exosomal miR-27a serum levels and a lack of hepatic PINK1-mediated mitophagy were directly related to liver fibrosis in MAFLD mice. Lipotoxic HC exosome transplantation aggravated the degree of PINK1-mediated mitophagy suppression, steatohepatitis, lipidosis, and fibrosis in the livers of MAFLD mice with cirrhosis. Both in vitro and in vivo, exosomes derived from miR-27a-knockdown HCs could not facilitate the abovementioned deteriorating effects. In conclusion, lipotoxic HC-exosomal miR-27a plays a pivotal role in inhibiting mitophagy and in promoting MAFLD-related liver fibrosis by negatively regulating PINK1 expression.

Linc-SCRG1 accelerates progression of hepatocellular carcinoma as a ceRNA of miR26a to derepress SKP2.

BACKGROUND: Increasing evidence has demonstrated that long noncoding RNAs (lncRNAs) have regulatory functions in hepatocellular carcinoma (HCC). The link between lincSCRG1 and HCC remains unclear. METHODS: To explore the lincSCRG1 regulation axis, bioinformatics, RIP and luciferase reporter assay were performed. The expressions of lincSCRG1-miR26a-SKP2 were detected in HCC tissues and cell lines through qPCR and western blot. The functions of HCC cells were investigated through in vitro assays (MTT, colony formation, transwell and flow cytometry) and the inner effect of lincSCRG1-miR26a in vivo was evaluated by xenografts and liver metatstatic nude mice models. RESULTS: LincSCRG1 was found to be strongly elevated in human HCC tissues and cell lines. MiR26a and S phase kinase-related protein 2 (SKP2) were predicted as the target miRNA for lincSCRG1 and the target gene for miR26a with direct binding sites, respectively. LincSCRG1 was verified as a competing endogenous RNA (ceRNA) via negative regulation of miR26a and derepression of SKP2 in HCC cells. Both overexpression of lincSCRG1 (ov-lincSCRG1) and inhibition of miR26a (in-miR26a) obviously stimulated cellular viability, colony formation, migration and proliferation of S phase cells and also significantly increased the protein levels of cyclinD1, CDK4, MMP2/3/9, Vimentin, and N-cadherin or inhibited the protein level of E-cadherin of HCC cells, while knockdown of lincSCRG1 (sh-lincSCRG1) and upregulation of miR26a (mi-miR26a) had the opposite effects on HCC cells. Cotransfection of in-miR26a or overexpression of SKP2 (ov-SKP2) with sh-lincSCRG1 could rescue the anticancer functions of sh-lincSCRG1, including suppressing proliferation and migration of HCC cells. Additionally, sh-lincSCRG1 could effectively inhibit the growth of subcutaneous xenograft tumours and lung metastasis, while the anticancer effect of sh-lincSCRG1 could be reversed by cotransfection of in-miR26a. CONCLUSIONS: LincSCRG1 acts as a ceRNA of miR26a to restrict its ability to derepress SKP2, thereby inducing the proliferation and migration of HCC cells in vitro and in vivo. Depletion of lincSCRG1 could be used as a potential therapeutic approach in HCC.

Lipotoxic hepatocyte-derived exosomal miR-1297 promotes hepatic stellate cell activation through the PTEN signaling pathway in metabolic-associated fatty liver disease.

BACKGROUND: Exosomes play an important role in metabolic-associated fatty liver disease (MAFLD), but the mechanism by which exosomes participate in MAFLD still remain unclear. AIM: To figure out the function of lipotoxic exosomal miR-1297 in MAFLD. METHODS: MicroRNA sequencing was used to detect differentially expressed miRNAs (DE-miR) in lipotoxic exosomes derived from primary hepatocytes. Bioinformatic tools were applied to analyze the target genes and pathways regulated by the DE-miRs. Quantitative real-time PCR (qPCR) was conducted for the verification of DE-miRs. qPCR, western blot, immunofluorescence staining and ethynyl-20-deoxyuridine assay were used to evaluate the function of lipotoxic exosomal miR-1297 on hepatic stellate cells (LX2 cells). A luciferase reporter experiment was performed to confirm the relationship of miR-1297 and its target gene PTEN. RESULTS: MicroRNA sequencing revealed that there were 61 exosomal DE-miRs (P < 0.05) with a fold-change > 2 from palmitic acid treated primary hepatocytes compared with the vehicle control group. miR-1297 was the most highly upregulated according to the microRNA sequencing. Bioinformatic tools showed a variety of target genes and pathways regulated by these DE-miRs were related to liver fibrosis. miR-1297 was overexpressed in exosomes derived from lipotoxic hepatocytes by qPCR. Fibrosis promoting genes (α-SMA, PCNA) were altered in LX2 cells after miR-1297 overexpression or miR-1297-rich lipotoxic exosome incubation via qPCR and western blot analysis. Immunofluorescence staining and ethynyl-20-deoxyuridine staining demonstrated that the activation and proliferation of LX2 cells were also promoted after the above treatment. PTEN was found to be the target gene of miR-1297 and knocking down PTEN contributed to the activation and proliferation of LX2 cells via modulating the PI3K/AKT signaling pathway. CONCLUSION: miR-1297 was overexpressed in exosomes derived from lipotoxic hepatocytes. The lipotoxic hepatocyte-derived exosomal miR-1297 could promote the activation and proliferation of hepatic stellate cells through the PTEN/PI3K/AKT signaling pathway, accelerating the progression of MAFLD.

Diagnostic Performance of FibroTouch Ultrasound Attenuation Parameter and Liver Stiffness Measurement in Assessing Hepatic Steatosis and Fibrosis in Patients With Nonalcoholic Fatty Liver Disease.

INTRODUCTION: To evaluate the diagnostic performance of ultrasound attenuation parameter (UAP) and liver stiffness measurement (LSM) by FibroTouch for diagnosis of hepatic steatosis and fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). METHODS: We recruited 237 patients undergoing FibroTouch and liver biopsy within 2 weeks. The pathological findings of liver biopsy were scored by Nonalcoholic Steatohepatitis Clinical Research Network, and the diagnostic accuracy of UAP for steatosis and LSM for fibrosis was evaluated by area under the receiver operating characteristic curve (AUROC). The impacts of histological parameters on UAP and LSM were analyzed, and diagnostic performance of FibroTouch UAP and LSM was compared with other noninvasive biomarkers. RESULTS: The success rate of FibroTouch examination was 96.51%. The AUROC of UAP for diagnosis of steatosis ≥S1, ≥S2, and S3 was 0.88, 0.93, and 0.88, and the cutoff values were 244, 269, and 296 dB/m, respectively. The AUROC of LSM for the diagnosis of fibrosis stages ≥F2, ≥F3, and F4 was 0.71, 0.71, and 0.77, and the cutoff values were 9.4, 9.4, and 11 kPa, respectively. Multiple regression analysis showed that LSM was positively correlated with degree of fibrosis and NAFLD activity score. UAP was positively correlated with liver steatosis. The diagnostic performance of UAP for steatosis was significantly superior to that of the hepatic steatosis index. DISCUSSION: FibroTouch has a low failure rate with moderate to high diagnostic performance for discriminating the steatosis degree and fibrosis stage and is suitable for clinical evaluation and monitoring of patients with NAFLD.