Mitofusin-2 gene polymorphisms and metabolic dysfunction associated fatty liver disease: a case-control study in a Chinese population.

OBJECTIVES: Mitofusion-2 (Mfn2) may have a role in mitochondrial oxidative stress and insulin resistance that can promote the development of metabolic dysfunction associated fatty liver disease (MAFLD). This retrospective and case control study aimed to explore the relationships between common Mfn2 single nucleotide polymorphisms (SNPs) and MAFLD in a northern Han Chinese population. METHODS: Six Mfn2 SNPs (rs2336384, rs873458, rs873457, rs4846085, rs2878677, and rs2236057) were genotyped using the ligase detection reaction in 466 MAFLD patients and 423 healthy controls. Genotype and allele frequencies were calculated, along with haplotype analysis and pairwise linkage disequilibrium. RESULTS: The genotype distribution of rs2336384, rs2878677, and rs2236057 among the MAFLD patients showed a significantly different pattern from that of healthy controls. The data showed that an increased risk of MAFLD was significantly correlated with patients carrying the GG genotype of rs2336384, CC genotype of rs873457, TT genotype of rs4846085, TT genotype of rs2878677, and the AA genotype of rs2236057. Moreover, The GGCTTA haplotype was found to be adversely linked with MAFLD by haplotype analysis. CONCLUSION: The current findings suggest a strong link between certain Mfn2 gene polymorphisms and MAFLD.

Plasma YAP1 predicts esophageal varices and the risk of variceal bleeding in liver cirrhosis.

Aim: To explore the predictive value of plasma YAP1 for esophageal varices (EV) and high-risk EV (HRV) in patients with liver cirrhosis. Materials & methods: A total of 208 patients with liver cirrhosis were enrolled and categorized into four groups. Correlation analysis, logistic regression analysis and receiver operating characteristic curve analysis were performed to evaluate the diagnostic performance of plasma YAP1 for EV and HRV. Results: Plasma YAP1 levels were significantly elevated with the occurrence and progression of EV in cirrhotic patients. The multivariate logistic regression analysis revealed that plasma YAP1 is an independent predictor for EV and HRV. For predicting EV and HRV, the YAP1 cut-off values of 5.43 and 6.98 ng/ml yielded the area under the receiver operating characteristic curves of 0.944 and 0.955, respectively. Conclusion: Plasma YAP1 is a potential novel noninvasive biomarker for predicting EV and HRV in patients with liver cirrhosis.

Hepatic Macrophage activation and the LPS pathway in patients with different degrees of severity and histopathological patterns of drug induced liver injury.

BACKGROUND: Inflammatory activation of hepatic macrophages plays a primary role in drug-induced liver injury (DILI). However, the exact mechanism underlying DILI remains unclear. METHODS: A total of 328 DILI patients and 80 healthy individuals were prospectively enrolled in this study. The DILI patients were categorized into subgroups based on either disease severity or histopathological patterns. Plasma soluble CD163 (sCD163) and hepatic CD163 were examined to determine hepatic macrophage activation, and CD8, CD20, and MUM-1 were assessed to determine cellular immunity using immunohistochemistry. The lipopolysaccharide (LPS) pathway proteins [e.g. LPS, soluble CD14 (sCD14), and LPS-binding protein (LBP)] were measured using enzyme-linked immunosorbent assay. RESULTS: Plasma sCD163 levels were nine-fold higher in DILI patients than in healthy controls at the baseline, but significantly decreased at the 4-week follow-up visit after treatment. The numbers of hepatic macrophages, B cells, and plasma cells were significantly higher in the liver tissues from DILI patients than those from healthy controls. Furthermore, the baseline levels of LPS pathway proteins in the DILI patients were significantly higher than those in the controls. Notably, these proteins significantly decreased at the 4-week follow-up visit but remained significantly higher than the levels for the controls. CONCLUSIONS: Hepatic inflammation in DILI involves the activation of hepatic macrophages and cellular immunity, in which the LPS pathway likely plays a role, at least in part. As such, this study has improved our understanding of the pathological mechanisms for DILI and may facilitate the development of better treatments for patients with DILI.

Clusterin contributes to hepatitis C virus-related hepatocellular carcinoma by regulating autophagy.

AIMS: To explore the potential regulatory mechanism of differentially expressed mRNAs in Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). MAIN METHODS: Patients with HCV-related HCC and age- and gender-matched healthy subjects were enrolled. Differentially expressed mRNAs in the plasma were detected by digital gene expression (DGE) profile analysis. HepG2 and SMMC7721 cells stably transfected with HCV-core protein and the control plasmid were established. And small interfering RNA (siRNA) was used to knockdown the target gene in HCV core-expressing HCC cell lines. mRNA expression was determined by qRT-PCR. Protein expression was measured by Western blot and immunohistochemistry staining. KEY FINDINGS: DGE profile data showed aberrant mRNA expression contributed to the progression of HCV-HCC, and clusterin (CLU), which was significantly highly expressed, was chosen as a candidate gene. Further evidence showed CLU was highly expressed in tumor tissues of HCV-HCC patients and HCV core-expressing HCC cell lines, accompanied with enhanced autophagy and upregulation of pro-autophagy genes. And knockdown of CLU in HCC cell lines suppressed cell autophagy, which was indicated by decreased expression of autophagy marker light chain 3B (LC3B) ІІ/І ratio, and downregulated pro-autophagy genes like Beclin1, autophagy-related protein 7 (Atg7) and Lamp2. On the other hand, anti-autophagy genes or regulators, including p62 and phosphorylated mammalian target of rapamycin (p-mTOR), were notably upregulated. SIGNIFICANCE: CLU could promote the progression of HCV-related HCC by regulating autophagy, which might be a potential therapeutic target of HCV-HCC.

Heme oxygenase-1 alleviated non-alcoholic fatty liver disease via suppressing ROS-dependent endoplasmic reticulum stress.

AIMS: The endoplasmic reticulum (ER) stress response plays a crucial role in the development of nonalcoholic steatohepatitis (NASH). Heme oxygenase-1 (HO-1) exerts beneficial effects against oxidative injury in NASH. This study is aimed to clarify whether HO-1 is an effective therapeutic strategy for NASH via regulation of ER stress. METHODS: The C57BL/6J mice were fed with methionine-choline deficient (MCD) for 4 weeks and high fat-high carbohydrate-high cholesterol (HFD) diet for 16 weeks, with hemin or zinc protoporphyrin IX (ZnPP-IX), respectively. The LO-2 cells were cultured in palmitic medium, with transfected pEX-HO-1 or sh-HO-1 plasmid for 24 h. Meanwhile, thirty NASH patients and 15 health controls were enrolled. The ER ultrastructure was observed by transmission electron microscopy (TEM) and confocal microscopy. The expressions of mRNAs and proteins of HO-1, ER stress related genes were detected by real time PCR, western blot and immunohistochemical staining, respectively. RESULTS: The swelled and broken rough endoplasmic reticulums were observed in MCD and HFD fed mice. The reactive hepatic expression of HO-1 was related with the increased ROS production and ER stress, companied with upregulation of GRP78, p-IRE1, PERK, ATF6. Through hemin administration, hepatocyte apoptosis was suppressed companied down-regulation of CHOP, caspase12 and up-regulation of BCL2. Conserved results were exhibited in ZnPP-IX administrated mice and HO-1 silent cells. Consistent results were observed in the NASH Patients. CONCLUSIONS: HO-1 could serve as a protective factor in the progression of nutritional steatohepatitis by suppresses hepatocyte excessive ER stress and might be a potential target for therapy of nonalcoholic steatohepatitis.

Yiqi Huoxue Recipe Improves Liver Regeneration in Rats after Partial Hepatectomy via JNK Pathway.

The liver is the only visceral organ that exhibits a remarkable capability of regenerating in response to partial hepatectomy (PH) or chemical injury. Improving liver regeneration (LR) ability is the basis for the favourable treatment outcome of patients after PH, which can serve as a potential indicator for postoperative survival. The present study aimed to investigate the protective effects of Yiqi Huoxue recipe (YQHX) on LR after PH in rats and further elucidate its underlying mechanism. A two-thirds PH rat model was used in this study. Wistar rats were randomly divided into four groups: sham-operated, PH, YQHX + PH, and Fuzheng Huayu decoction (FZHY) + PH groups. All rats were sacrificed under anesthesia at 24 and 72 h after surgery. The rates of LR were calculated, and the expression levels of cyclin D1 and c-jun were determined by immunohistochemical staining. The protein levels of p-JNK1/2, JNK1/2, p-c-jun, c-jun, Bax, and Bcl-2 were detected by Western blotting, while the mRNA levels of JNK1, JNK2, c-jun, Bax, and Bcl-2 were examined by real-time polymerase chain reaction (RT-PCR). At the corresponding time points, YQHX and FZHY administration dramatically induced the protein levels of p-JNK1/2 compared to the PH group (p < 0.05), while FZHY + PH group showed prominently increase in p-JNK1/2 protein levels compared to the YQHX + PH group (p < 0.05). A similar trend was observed for the expression levels of p-c-jun. Compared to the PH group, YQHX and FZHY markedly reduced the mRNA and protein expression levels of Bax at 24 h after PH, while those in the FZHY + PH group decreased more obviously (p < 0.05). Besides, in comparison with the PH group, YQHX and FZHY administration predominantly upregulated the mRNA and protein expression levels of Bcl-2 at 24 and 72 h after PH (p < 0.05). In conclusion, YQHX improves LR in rats after PH by inhibiting hepatocyte apoptosis via the JNK signaling pathway.

Study on the long-term curative effect of repeat percutaneous balloon mitral valvuloplasty in patients with mitral restenosis.

To study the long-term curative effect of repeat percutaneous balloon mitral valvuloplasty (PBMV) in patients with mitral restenosis.In our study, mitral restenosis developed in 39 patients after PBMV. Repeat PBMV was performed according to the improved Inoue method. All patients were followed up.Of 39 patients, 36 were successfully treated with repeat PBMV (achievement ratio, 92.3%). Immediately after repeat PBMV, clinical symptoms and left atrial mean pressure (LAP), pulmonary artery systolic pressure (PASP), mitral valve gradient (MVG), and mitral valve orifice area (MVA) improved significantly (24.50 ±â€Š6.54 mmHg vs 9.66 ±â€Š4.21 mmHg for LAP, 1.05 ±â€Š0.19 cm vs 2.23 ±â€Š0.22 cm for MVA, 17.03 ±â€Š4.52 mmHg vs 7.79 ±â€Š4.07 mmHg for MVG, 58.12 ±â€Š12.68 mmHg vs 31.45 ±â€Š10.02 mmHg for PASP; P <.05). Meanwhile, left atrial end-diastolic dimension (LAD) was altered slightly (4.71 ±â€Š0.75 vs 4.07 ±â€Š0.69, P >.05). The 36 patients were followed up for 69 ±â€Š23 (12-146) months. After long-term follow-up immediately after repeat PBMV, the results did not show a significant change (2.23 ±â€Š-0.22 cm vs 2.02 ±â€Š-0.21 cm for MVA, 7.79 ±â€Š-4.07 mmHg vs 9.15 ±â€Š-4.11 mmHg for MVG; P >.05) and were approximated to those shortly after repeat PBMV (2.23 ±â€Š0.22 cm vs 2.02 ±â€Š0.21 cm for MVA, 7.79 ±â€Š4.07 mmHg vs 9.15 ±â€Š4.11 mmHg for MVG; P > 0.05). LAD did not change significantly (4.13 ±â€Š0.71 cm vs. 4.07 ±â€Š0.69 cm; P >.05). The long-term follow-up results showed that cardiac function and quality of life were significantly improved in most patients.It would be safe for patients with mitral restenosis to undergo repeat PBMV. Appropriate cases should be selected, and treatment should be performed cautiously. Short- and long-term curative effects would be satisfactory. We suggested that repeat PBMV be the first choice for patients with mitral restenosis after first PBMV.

Role of LncRNA-activated by transforming growth factor beta in the progression of hepatitis C virus-related liver fibrosis.

Long non-coding RNA (LncRNA)-activated by transforming growth factor-beta (LncRNA-ATB) is a key regulator of transforming growth factor-beta (TGF-ß) signaling pathway, and is positively correlated with the development of liver cirrhosis and vascular invasion of hepatocellular carcinoma (HCC). However, the role of LncRNA-ATB in hepatitis C virus (HCV)-related liver fibrosis remains largely unknown. In the present study, we confirmed a high expression level of LncRNA-ATB in the liver tissues and plasma samples of patients with HCV-related hepatic fibrosis, and the plasma level of LncRNA-ATB was significantly correlated with liver fibrosis stages. Furthermore, increased expression level of LncRNA-ATB was also present in activated hepatic stellate cells (HSCs), and knockdown of LncRNA-ATB inhibited the expression of alpha-smooth muscle actin (α-SMA) and alpha-1 type I collagen (Col1A1). LncRNA-ATB was found to share the common miRNA responsive element of miR-425-5p with TGF-ß type II receptor (TGF-ßRII) and SMAD2. Ectopic expression of LncRNA-ATB in HSCs could upregulate the protein expression of TGF-ßRII and SMAD2 by inhibiting the endogenous miR-425-5p. Moreover, overexpression of miR-425-5p could partly abrogate the expression of TGF-ßRII and SMAD2 induced by LncRNA-ATB. Hence, we conclude that LncRNA-ATB promotes HCV-induced liver fibrogenesis by activating HSCs and increasing collagen I production through competitively binding to miR-425-5p. LncRNA-ATB may be a novel diagnostic biomarker and a potential therapeutic target for HCV-related hepatic fibrosis.

miR-1273g-3p modulates activation and apoptosis of hepatic stellate cells by directly targeting PTEN in HCV-related liver fibrosis.

MicroRNA (miRNA) play a pivotal role in the development of liver fibrosis. However, the functions of miRNA in hepatitis C virus (HCV)-related liver fibrosis remain unclear. In this study, we systematically analyzed the microarray data of the serum miRNA in patients with HCV-induced hepatic fibrosis. Among 41 dysregulated miRNA, miR-1273g-3p was the most significantly upregulated miRNA and correlated with the stage of liver fibrosis. Overexpression of miR-1273g-3p could inhibit translation of PTEN, increase the expression of α-SMA, Col1A1, and reduce apoptosis in HSCs. Hence, we conclude that miR-1273g-3p might affect the activation and apoptosis of HSCs by directly targeting PTEN in HCV-related liver fibrosis.

Gamma-glutamyl transpeptidase to platelet ratio index is a good noninvasive biomarker for predicting liver fibrosis in Chinese chronic hepatitis B patients.

Objective To evaluate whether gamma-glutamyl transpeptidase to platelet ratio index (GPRI) can diagnose the extent of liver fibrosis in Chinese patients with chronic hepatitis B (CHB) infection. Methods This prospective observational study used liver biopsy results as the gold standard to evaluate the ability of GPRI to predict hepatic fibrosis compared with two other markers, the aspartate aminotransferase (AST) to platelet ratio index (APRI) and fibrosis-4 score (FIB-4). The clinical and demographic factors that affected GPRI, independent of liver fibrosis, were assessed using multivariate linear regression analyses. Results This study enrolled 312 patients with CHB. GPRI had a significantly positive correlation with liver fibrosis stage and the correlation coefficient was higher than that for APRI and FIB-4. The areas under the receiver operating curves for GPRI for significant fibrosis, bridging fibrosis, and cirrhosis were 0.728, 0.836, and 0.842, respectively. Of the three indices, GPRI had the highest diagnostic accuracy for bridging fibrosis and cirrhosis. Age, elevated AST and elevated total bilirubin levels were independent determinants of increased GPRI. Conclusion GPRI was a more reliable laboratory marker than APRI and FIB-4 for predicting the stage of liver fibrosis in Chinese patients with CHB.

Stabilization and immune response of HBsAg encapsulated within poly(lactic-co-glycolic acid) microspheres using HSA as a stabilizer.

The aim of this study was to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres containing hepatitis B virus surface antigen (HBsAg) using human serum albumin (HSA) as a stabilizer. Lyophilization and emulsification of HBsAg solution with dichloromethane caused a considerable loss of HBsAg antigenicity. Thus, the effects of HSA and trehalose on HBsAg recovery during lyophilization and emulsification were investigated. Adding HSA to HBsAg solutions significantly improved antigen recovery to >90% during lyophilization and emulsification. The effects of co-encapsulated HSA on the characteristics of the PLGA microspheres and stability of HBsAg released from the microspheres were also investigated. The in vitro release test showed that HBsAg was released from the PLGA microspheres continuously over seventy days. A large amount of released HBsAg was inactive without co-encapsulation of HSA. On the contrary, with HSA co-encapsulation, the released HBsAg retained approximately 90% of its antigenicity. The single injection of the HBsAg-HSA-loaded PLGA microspheres in rats resulted in higher anti-HBsAg IgG and Th1 cytokine levels than the single injection of the HBsAg-loaded microspheres or two injections of the conventional aluminum-adjuvanted HBsAg vaccine. Based on these findings, the HBsAg-HSA-loaded PLGA microspheres could be an effective carrier for HBsAg and form a promising depot system.

[Clinical study on FibroTouch and multi-parameter model for diagnosis of hepatic fibrosis in patients with chronic liver disease].

OBJECTIVE: To explore the clinical application and related factors of FibroTouch in the diagnosis of liver fibrosis in patients with chronic liver disease through comparison of the specificity and sensitivity of FibroTouch and multi-parameter models, and to identify whether FibroTouch is a more accurate and safe method in diagnosis of liver fibrosis and evaluation of the therapeutic effect. METHODS: A total of 190 patients with chronic liver disease were performed liver biopsy and underwent liver stiffness measurement (LSM) using FibroTouch in department of Traditional and Western Medical Hepatology, Third Hospital of Hebei Medical University from January 2014 to February 2015. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were tested by enzymic method with automatic biochemistry analyzer. Blood platelet counts were detected by automatic blood cell analyzer. AST-to-PLT ratio index (APRI) and fibrosis index based on the 4 factor (FIB-4) were calculated. The diagnostic values of FibroTouch, APRI and FIB-4 for liver fibrosis degree were calculated and compared by receiver operating characteristic (ROC) curves. The related factors of LSM were analyzed by Spearman analysis. RESULTS: There was significant correlation between LSM and histological fibrosis (r=0.804, P=0.000). The area under ROC curve of LSM for S(≥2, S≥3 and S=4 was 0.894, 0.938 and 0.961, respectively, which was significantly higher than APRI (0.678, 0.698 and 0.658) and FIB-4 (0.765, 0.785 and 0.775). On Spearman analysis, LSM was positively correlated with age, ALT, AST, TBIL ((≥2×ULN) and the grade of liver inflammation (r=0.309, 0.558, 0.504, 0.492 and 0.532, respectively) but negatively with PLT (r=-0.444), (all P<0.05). CONCLUSIONS: LSM is a convenient and reliable approach for diagnosis of liver fibrosis in patients with chronic liver disease. The sensitivity and specificity of Fibrotouch in diagnosis of hepatic fibrosis is superior to APRI and FIB-4, and age, high level ofALT, AST and TBIL (≥2×ULN) were independent predictors of LSM inaccuracy.

Magnetic polycarbonate microspheres for tumor-targeted delivery of tumor necrosis factor.

OBJECTIVE: The specific expression of transferrin receptor can represent a diagnostic tool or therapeutic target in solid tumors expressing this antigen. Herein, the human transferrin receptor monoclonal antibody (T9) was investigated as a tumor-targeting group for active targeted-drug delivery systems. MATERIALS AND METHODS: A tumor-targeted conjugate T9-TNF was synthesized by the attachment of both human transferrin receptor monoclonal antibody (T9) as a tumor-targeting group and human tumor necrosis factor-α (TNF) as an anti-cancer drug to two terminated hydroxyl groups of poly(ethylene glycol). Subsequently, a solvent evaporation technique was adopted to produce anti-cancer magnetic polymer microspheres T9-TNF-PC-M containing T9-TNF and Fe3O4 magnetic ultrafine powders (M) using poly(trimethylene carbonate-co-5,5-dimethyl trimethylene carbonate) (PC, P(TMC-co-DTC)) as a polymeric carrier. RESULTS AND DISCUSSION: These magnetic polycarbonate microspheres possessed a steady TNF release rate in phosphate buffer saline solution, strong magnetic responsiveness and high T9-TNF loading capacity. In vitro cytotoxicity assays demonstrated the microspheres T9-TNF-PC-M and conjugate T9-TNF were strongly inhibitory to the human hepatic carcinoma (Bel-7204) cells. In vivo site-specific therapy in nude mice with human hepatic carcinoma indicated that the microspheres T9-TNF-PC-M and conjugate T9-TNF possessed markedly higher anti-tumor activity against Bel-7204 in mice than that of TNF. CONCLUSIONS: These results indicated that the magnetic polycarbonate microspheres were suitable as the potential-targeted treatment for hepatic carcinoma therapeutics.

Pharmacokinetic and pharmacodynamic profiles of recombinant human erythropoietin-loaded poly(lactic-co-glycolic acid) microspheres in rats.

AIM: To characterize the pharmacokinetic and pharmacodynamic profiles of the recombinant human erythropoietin (rhEPO)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres in rats. METHODS: The rhEPO-loaded microspheres were prepared using a solid-in-oil-in-water emulsion method. Pharmacokinetics and pharmacodynamics of the rhEPO-loaded microspheres were evaluated in male Sprague-Dawley rats. The serum rhEPO level was determined with ELISA. The level of anti-rhEPO antibody in the serum was measured to assess the immunogenicity of rhEPO released from the microspheres. RESULTS: rhEPO was almost completely released from the PLGA microspheres in vitro, following zero-order release kinetics over approximately 30 d. After intramuscular injection (10,000 or 30,000 IU rhEPO/kg) in the rats, the serum rhEPO concentration reached maximum levels on d 1, then decreased gradually and was maintained at nearly steady levels for approximately 4 weeks. Furthermore, the release of rhEPO from the PLGA microspheres was found to be controlled mainly by a dissolution/diffusion mechanism. A good linear correlation (R(2)=0.98) was obtained between the in vitro and in vivo release data. A single intramuscular injection of the rhEPO-loaded PLGA microspheres (10,000 or 30,000 IU rhEPO/kg) in the rats resulted in elevated hemoglobin and red blood cell concentrations for more than 28 d. Moreover, the immunogenicity of rhEPO released from the PLGA microspheres was comparable with that of the unencapsulated rhEPO. CONCLUSION: The results prove the feasibility of using the PLGA-based microspheres to deliver rhEPO for approximately 1 month.

Sustained release of low molecular weight heparin from PLGA microspheres prepared by a solid-in-oil-in-water emulsion method.

Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres for the sustained release of low molecular weight heparin (LMWH) were prepared by a soild-in-oil-in-water (s/o/w) emulsion method. Prior to encapsulation, the LMWH micro-particles were fabricated by a modified freezing-induced phase separation method. The micro-particles were subsequently encapsulated into PLGA microspheres. Process optimization revealed that the NaCl concentration in the outer phase of s/o/w emulsion played a critical role in determining the properties of the microspheres. When the NaCl concentration increased from 0% to 5%, the encapsulation efficiency significantly increased from 51.5% to 76.8%. The initial burst release also decreased from 37.3% to 12.4%. In vitro release tests showed that LMWH released from PLGA microspheres in a sustained manner for about 14 days. Single injection of LMWH-loaded PLGA microspheres into rabbits resulted in an elevation of an anti-factor Xa activity for about 6 days. Furthermore, the integrity of the encapsulated LMWH was preserved during encapsulation process.