Rui-Nan Zhang

Controlled attenuation parameter for non-invasive assessment of hepatic steatosis in Chinese patients

AIM To evaluate the performance of a novel non-invasive controlled attenuation parameter (CAP) to assess liver steatosis. METHODS This was a multi-center prospective cohort study. Consecutive patients (aged ≥ 18 years) who had undergone percutaneous liver biopsy and CAP measurement were recruited from three Chinese liver centers. Steatosis was categorized as S0: < 5%; S1: 5%-33%; S2: 34%-66%; or S3: ≥ 67%, according to the nonalcoholic fatty liver disease (NAFLD) activity score. The FibroScan(®) 502 equipped with the M probe (Echosens, Paris, France) was used to capture both CAP and liver stiffness measurement values simultaneously. Receiver operating characteristic curves were plotted, and the areas under the curves were calculated to determine the diagnostic efficacy. The accuracy of the CAP values at the optimal thresholds was defined by maximizing the sum of sensitivity and specificity (maximum Youden index). RESULTS A total of 152 patients were recruited, including 52 (34.2%) patients with NAFLD and 100 (65.8%) with chronic hepatitis B (CHB) virus infection. After adjustment, the steatosis grade (OR = 37.12; 95%CI: 21.63-52.60, P < 0.001) and body mass index (BMI, OR = 6.20; 95%CI: 2.92-9.48, P < 0.001) were found independently associated with CAP by multivariate linear regression analysis. CAP was not influenced by inflammation, fibrosis or aetiology. The median CAP values and interquartile ranges among patients with S0, S1, S2 and S3 steatosis were 211 (181-240) dB/m, 270 (253-305) dB/m, 330 (302-360) dB/m, and 346 (313-363) dB/m, respectively. The cut-offs for the CAP values in all patients with steatosis ≥ 5%, ≥ 34% and ≥ 67% were 253 dB/m, 285 dB/m and 310 dB/m, respectively. The areas under the curves were 0.92, 0.92 and 0.88 for steatosis ≥ 5%, ≥ 34% and ≥ 67%, respectively. No significant differences were found in the CAP values between the NAFLD group and the CHB group in each steatosis grade. CONCLUSION CAP appears to be a promising tool for the non-invasive detection and quantification of hepatic steatosis, but is limited by BMI.

Impact of skin capsular distance on the performance of controlled attenuation parameter in patients with chronic liver disease

Controlled attenuation parameter (CAP) is a non‐invasive method for evaluating hepatic steatosis. However, larger skin capsular distance (SCD) can affect the accuracy. The aim of this study was to investigate the impact of SCD on the diagnostic performance of CAP and liver stiffness measurement (LSM).

Disease-specific miR-34a as diagnostic marker of non-alcoholic steatohepatitis in a Chinese population

AIM To assess disease-specific circulating microRNAs (miRNAs) in non-alcoholic steatohepatitis (NASH) patients. METHODS A total of 111 biopsy-proven non-alcoholic fatty liver disease (NAFLD) or chronic hepatitis B (CHB) patients and healthy controls from mainland China were enrolled to measure their serum levels of miR-122, -125b, -146b, -16, -21, -192, -27b and -34a. The correlations between serum miRNAs and histological features of NAFLD were determined. The diagnostic value of miRNA in NASH and significant fibrosis was analyzed and compared with that of cytokeratin-18 (CK-18), fibrosis-4 (FIB-4), and aspartate aminotransferase to platelet ratio index (APRI), respectively. RESULTS Circulating miR-122, -16, -192 and -34a showed differential expression levels between NAFLD and CHB patients, and miR-34a had an approximately 2-fold increase in NAFLD samples compared with that of CHB samples (P < 0.01). Serum miR-122, -192 and -34a levels were correlated with steatosis (R = 0.302, 0.323 and 0.470, respectively, P < 0.05) and inflammatory activity (R = 0.445, 0.447 and 0.517, respectively, P < 0.01); only serum miR-16 levels were associated with fibrosis (R = 0.350, P < 0.05) in patients with NAFLD. The diagnostic value of miR-34a for NASH (area under the receiver operating characteristic, 0.811, 95%CI: 0.670-0.953) was superior to that of alanine aminotransferase, CK-18, FIB-4 and APRI in NAFLD, but miR-16 showed a limited performance in the diagnosis of significant fibrosis in NASH. CONCLUSION Circulating miR-34a may serve as a disease-specific noninvasive biomarker for the diagnosis of NASH.

Sodium butyrate attenuates high-fat diet-induced steatohepatitis in mice by improving gut microbiota and gastrointestinal barrier.

AIM To investigate whether gut microbiota metabolite sodium butyrate (NaB) is an effective substance for attenuating non-alcoholic fatty liver disease (NAFLD) and the internal mechanisms. METHODS Male C57BL/6J mice were divided into three groups, normal control were fed standard chow and model group were fed a high-fat diet (HFD) for 16 wk, the intervention group were fed HFD for 16 wk and treated with NaB for 8 wk. Gut microbiota from each group were detected at baseline and at 16 wk, liver histology were evaluated and gastrointestinal barrier indicator such as zonula occluden-1 (ZO-1) were detected by immunohistochemistry and realtime-PCR, further serum or liver endotoxin were determined by ELISA and inflammation- or metabolism-associated genes were quantified by real-time PCR. RESULTS NaB corrected the HFD-induced gut microbiota imbalance in mice, while it considerably elevated the abundances of the beneficial bacteria Christensenellaceae, Blautia and Lactobacillus. These bacteria can produce butyric acid in what seems like a virtuous circle. And butyrate restored HFD induced intestinal mucosa damage, increased the expression of ZO-1 in small intestine, further decreased the levels of gut endotoxin in serum and liver compared with HF group. Endotoxin-associated genes such as TLR4 and Myd88, pro-inflammation genes such as MCP-1, TNF-α, IL-1, IL-2, IL-6 and IFN-γ in liver or epididymal fat were obviously downregulated after NaB intervention. Liver inflammation and fat accumulation were ameliorated, the levels of TG and cholesterol in liver were decreased after NaB intervention, NAS score was significantly decreased, metabolic indices such as FBG and HOMA-IR and liver function indicators ALT and AST were improved compared with HF group. CONCLUSION NaB may restore the dysbiosis of gut microbiota to attenuate steatohepatitis, which is suggested to be a potential gut microbiota modulator and therapeutic substance for NAFLD.

Linked PNPLA3 polymorphisms confer susceptibility to nonalcoholic steatohepatitis and decreased viral load in chronic hepatitis B

AIM To investigate the association of PNPLA3 polymorphisms with concurrent chronic hepatitis B (CHB) and nonalcoholic fatty liver disease (NAFLD). METHODS A cohort of Han patients with biopsy-proven CHB, with or without NAFLD (CHB group, n = 51; CHB + NAFLD group, n = 57), and normal controls (normal group, n = 47) were recruited from Northern (Tianjin), Central (Shanghai), and Southern (Zhangzhou) China. Their PNPLA3 polymorphisms were genotyped by gene sequencing. The association between PNPLA3 polymorphisms and susceptibility to NAFLD, and clinical characteristics of NAFLD were evaluated on the basis of physical indices, liver function tests, glycolipid metabolism, and histopathologic scoring. The association of PNPLA3 polymorphisms and hepatitis B virus (HBV) load was determined by the serum level of HBV DNA. RESULTS After adjusting for age, sex, and body mass index, we found that four linked single nucleotide polymorphisms (SNPs) of PNPLA3, including the rs738409 G allele (CHB + NAFLD group vs CHB group: odds ratio [OR] = 2.77, 95% confidence interval [CI]: 1.18-6.54; P = 0.02), rs3747206 T allele (CHB + NAFLD group vs CHB group: OR = 2.77, 95%CI: 1.18-6.54; P = 0.02), rs4823173 A allele (CHB + NAFLD group vs CHB group: OR = 2.73, 95%CI: 1.16-6.44; P = 0.02), and rs2072906 G allele (CHB + NAFLD group vs CHB group: OR = 3.05, 95%CI: 1.28-7.26; P = 0.01), conferred high risk to NAFLD in CHB patients. In patients with both CHB and NAFLD, these genotypes of PNPLA3 polymorphisms were associated with increased susceptibility to nonalcoholic steatohepatitis (NASH) (NAFLD activity score ≥ 3; P = 0.01-0.03) and liver fibrosis (> 1 Metavir grading; P = 0.01-0.04). As compared to those with C/C and C/G at rs738409, C/C and C/T at rs3747206, G/G and G/A at rs4823173, and A/A and A/G at rs2072906, patients in the CHB + NAFLD group with G/G at rs738409, T/T at rs3747206, A/A at rs4823173, and G/G at rs2072906 showed significantly lower serum levels of HBV DNA (P < 0.01-0.05). CONCLUSION Four linked SNPs of PNPLA3 (rs738409, rs3747206, rs4823173, and rs2072906) are correlated with susceptibility to NAFLD, NASH, liver fibrosis, and HBV dynamics in CHB patients.

Saturated Fatty Acid Inhibits Viral Replication in Chronic Hepatitis B Virus Infection With Nonalcoholic Fatty Liver Disease by Toll-Like Receptor 4-Mediated Innate Immune Response

Background: Chronic Hepatitis B (CHB) infection is common in patients with Non-Alcoholic Fatty Liver Disease (NAFLD). The replication level of Hepatitis B Virus (HBV) was inversely correlated with hepatic steatosis. Toll-Like Receptor (TLR) 4-mediated innate immunity plays a pivotal role in the occurrence of NAFLD and controls HBV replication. Objectives: This study aimed to investigate whether the TLR4-mediated innate immunity stimulates the pathogenesis of CHB in patients with NAFLD and to determine whether TLR4 plays a role in inhibiting HBV replication. Materials and Methods: The HBV transgenic mice were randomized into the HBV and HBV/NAFLD groups. HepG2.2.15 cells were treated with different concentrations (0 – 200 μM) of Stearic Acid (SA) to induce steatosis. The total RNA of the liver tissue was extracted for Real-Time Polymerase Chain Reaction (RT-PCR) detection, and immunohistochemistry or western blot was conducted for further validation. The Enzyme-Linked Immunosorbent Assay (ELISA) analysis was applied to evaluate the production of Interleukin 6 (IL-6), Tumor necrosis factor α (TNF-α) and Interferon β (IFN-β). Moreover, viral dynamics were analyzed using HBV DNA and HBV-related antigens (HBsAg and HBeAg). Results: Non-alcoholic fatty liver disease was induced in HBV-transgenic mice fed with High Fat Diet (HFD) for 8 - 24 weeks. Oil red-O staining positive droplets and the content of Triglyceride (TG) were increased in HepG2.2.15 cells treated with SA. TLR4, Myeloid differentiation factor 88 (MyD88), IL-6 and TNF-α expression levels were significantly higher in the HBV/NAFLD group and the steatotic HepG2.2.15 cells than those in their respective controls. Compared to the HBV group, significant reductions in serum levels of HBsAg, HBeAg, and HBV DNA titers occurred in the HBV/NAFLD group at 24 weeks, but the IFN-β level was remarkably increased. Similar data were also obtained from the steatoric HepG2.2.15 cells. Conclusions: Saturated Fatty Acids (SFAs) served as a potential ligand for TLR4 and activated TLR4 signaling pathway, which might be involved in the pathogenesis. Thus, SFAs can accelerate the mechanism of inhibiting HBV replication in CHB with NAFLD.

miR-192-5p regulates lipid synthesis in non-alcoholic fatty liver disease through SCD-1

AIM To evaluate the levels of miR-192-5p in non-alcoholic fatty liver disease (NAFLD) models and demonstrate the role of miR-192-5p in lipid accumulation. METHODS Thirty Sprague Dawley rats were randomly divided into three groups, which were given a standard diet, a high-fat diet (HFD), and an HFD with injection of liraglutide. At the end of 16 weeks, hepatic miR-192-5p and stearoyl-CoA desaturase 1 (SCD-1) levels were measured. MiR-192-5p mimic and inhibitor and SCD-1 siRNA were transfected into Huh7 cells exposed to palmitic acid (PA). Lipid accumulation was evaluated by oil red O staining and triglyceride assays. Direct interaction was validated by dual-luciferase reporter gene assays. RESULTS The HFD rats showed a 0.46-fold decrease and a 3.5-fold increase in hepatic miR-192-5p and SCD-1 protein levels compared with controls, respectively, which could be reversed after disease remission by liraglutide injection (P < 0.01). The Huh7 cells exposed to PA also showed down-regulation and up-regulation of miR-192-5p and SCD-1 protein levels, respectively (P < 0.01). Transfection with miR-192-5p mimic and inhibitor in Huh7 cells induced dramatic repression and promotion of SCD-1 protein levels, respectively (P < 0.01). Luciferase activity was suppressed and enhanced by miR-192-5p mimic and inhibitor, respectively, in wild-type SCD-1 (P < 0.01) but not in mutant SCD-1. MiR-192-5p overexpression reduced lipid accumulation significantly in PA-treated Huh7 cells, and SCD-1 siRNA transfection abrogated the lipid deposition aggravated by miR-192-5p inhibitor (P < 0.01). CONCLUSION This study demonstrates that miR-192-5p has a negative regulatory role in lipid synthesis, which is mediated through its direct regulation of SCD-1.

PNPLA3 rs1010023 Predisposes Chronic Hepatitis B to Hepatic Steatosis but Improves Insulin Resistance and Glucose Metabolism

PNPLA3 polymorphisms serve as the genetic basis of hepatic steatosis in normal population and lead to dysregulated glucose metabolism. Whether it underlies the hepatic steatosis and glucose homeostasis in chronic hepatitis B patients remains uncertain. Here, we investigated the PNPLA3 polymorphisms in biopsy-proven chronic hepatitis B patients with (CHB+HS group, n = 52) or without hepatic steatosis (CHB group, n = 47) and non-CHB subjects with (HS group, n = 37) or without hepatic steatosis (normal group, n = 45). When compared to the TT genotype, C-allele at PNPLA3 rs1010023 (CC and TC genotypes) conferred higher risk to hepatic steatosis in chronic hepatitis B patients (odds ratio (OR) = 1.768, 95% confidence interval (CI): 1.027–3.105; P = 0.045) independent of age, gender, and body mass index. In contrast to their role in hepatic steatosis, CC and TC genotypes of PNPLA3 rs1010023 were correlated to significant improvement of homeostasis model assessment index (HOMA-IR) as compared to TT genotype in the CHB+HS group. Downregulated fasting blood glucose also characterized the CHB+HS patients with C-allele at PNPLA3 rs1010023 (CC/TC versus TT: 4.81 ± 0.92 mmol/L versus 5.86 ± 2.11 mmol/L, P = 0.02). These findings suggest that PNPLA3 rs1010023 may predispose chronic hepatitis B patients to hepatic steatosis but protects them from glucose dysregulation by attenuating insulin resistance.

PNPLA3 rs139051 is associated with phospholipid metabolite profile and hepatic inflammation in nonalcoholic fatty liver disease

AIM To investigate the effect of PNPLA3 polymorphisms on serum lipidomics and pathological characteristics in nonalcoholic fatty liver disease (NAFLD). METHODS Thirty-four biopsy-proven NAFLD patients from Northern, Central, and Southern China were subjected to stratification by genotyping their single nucleotide polymorphisms (SNPs) in PNPLA3. Ultra performance liquid chromatographytandem mass spectrometry was then employed to characterize the effects of PNPLA3 SNPs on serum lipidomics. In succession, correlation analysis revealed the association of PNPLA3-related lipid profile and hepatic pathological characteristics on a basis of steatosis, activity, and fibrosis assessment. The variant-based scoring of hepatocyte steatosis, ballooning, lobular inflammation, and liver fibrosis was finally performed so as to uncover the actions of lipidomics-affecting PNPLA3 SNPs in NAFLD-specific pathological alterations. RESULTS PNPLA3 SNPs (rs139051, rs738408, rs738409, rs 2072906, rs2294918, rs2294919, and rs4823173) demonstrated extensive association with the serum lipidomics, especially phospholipid metabolites [lysophosphatidylcholine (LPC), lysophosphatidylcholine plasmalogen (LPCO), lysophosphatdylethanolamine (LPE), phosphatidylcholine (PC), choline plasmalogen (PCO), phosphatidylethanolamine (PE), ethanolamine plasmalogen (PEO)], of NAFLD patients. PNPLA3 rs139051 (A/A genotype) and rs2294918 (G/G genotype) dominated the up-regulatory effect on phospholipids of LPCs (LPC 17:0, LPC 18:0, LPC 20:0, LPC 20:1, LPC 20:2) and LPCOs (LPC O-16:1, LPC O-18:1). Moreover, subjects with high-level LPCs/LPCOs were predisposed to low-grade lobular inflammation of NAFLD (rho: -0.407 to -0.585, P < 0.05-0.001). The significant correlation of PNPLA3 rs139051 and inflammation grading [A/A vs A/G + G/G: 0.50 (0.00, 1.75) vs 1.50 (1.00, 2.00), P < 0.05] further demonstrated its pathological role based on the modulation of phospholipid metabolite profile. CONCLUSION The A/A genotype at PNPLA3 rs139051 exerts an up-regulatory effect on serum phospholipids of LPCs and LPCOs, which are associated with low-grade lobular inflammation of NAFLD.