HMGB1-induced autophagy facilitates hepatic stellate cells activation: a new pathway in liver fibrosis.

High-mobility group box-1 (HMGB1) plays a context-dependent role in autophagy, which is required for hepatic stellate cells (HSCs) activation. However, the significance of HMGB1-induced HSCs autophagy in liver fibrosis has not been elucidated. Here, we first documented an enrichment of peripheral and intrahepatic HMGB1 signal in hepatitis B virus (HBV)-related liver fibrosis progression, and presented a direct evidence of anatomic proximity of HMGB1 with a-SMA (a marker for HSCs activation) in cirrhotic liver specimens. Then, we demonstrated the autophagy-inducing effects by serum-sourced HMGB1 in both primary murine HSCs and human HSCs cell line (LX-2), reflected by increased number of autophagic vacuoles (AVs) under the transmission electron microscope (TEM) and up-regulated protein expression of lipidated microtubule-associated light chain 3 (LC3-II) (a marker for autophagosome) in Western blot analysis. Intriguingly, there is a possible translocation of endogenous HMGB1 from the nucleus to cytoplasm to extracellular space, during exogenous HMGB1-induced HSCs autophagy. Meanwhile, the dose- and time-dependent effects by recombinant HMGB1 (rHMGB1) in enhancing LX-2 autophagy and fibrogenesis have been revealed with activated extracellular regulated protein kinase (ERK)/c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) and restrained mammalian target of rapamycin (mTOR)/STAT3 signaling pathways. Additionally, the ERK or JNK inhibitor could not only inhibit rHMGB1-induced autophagy and fibrogenesis in LX-2 cells, but also restore the suppressed mTOR and STAT3 pathways. Furthermore, using LC3-siRNA transfected LX-2, we found HMGB1-induced fibrogenesis is dependent on its autophagy-inducing effects. Finally, we elucidated the involvement of extracellular HMGB1-receptor for advenced glycation end product (RAGE) axis and endogenous HMGB1 in exogenous HMGB1-induced effects. Our findings could open new perspectives in developing an antifibrotic therapy by targetting the HSCs autophagy.

Effect of Additional Clostridium butyricum on the Intestinal Flora of Chronic Hepatitis B Patients Treated with Entecavir.

INTRODUCTION: To explore the influence of intestinal flora on the occurrence, development and antiviral therapy of chronic hepatitis B (CHB), 16S rDNA amplification sequencing was performed to investigate the intestinal flora in CHB patients treated with entecavir (ETV) and Clostridium butyricum (CB). METHODS: CHB patients were divided into the ETV group (treatment with ETV alone) and ETV + CB group (treatment with ETV and CB). After 8-week treatment, feces samples were collected and processed for 16S rDNA amplicon sequencing; blood samples were collected for the biochemical, immunologic and virologic evaluations, which were compared between groups. RESULTS: ETV treatment for 8 weeks significantly decreased the serum levels of alanine aminotransferase (ALT), interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α) and HBV DNA compared to those before treatment, but there were no marked differences between the ETV group and ETV + CB group. The intestinal flora changed significantly in the CHB patients after ETV + CB treatment: there were marked differences in 13 unique species before treatment and 4 unique species after ETV + CB treatment; at the phylum level, the top five bacteria with significant difference between patients before treatment and ETV + CB patients were Firmicutes, Actinobacteria, Cyanobacteria, Euryarchaeota and Synergistetes. There were significant differences in 25 unique species in the ETV group and 4 unique species in the ETV + CB group; at the phylum level, the top five bacteria with significant difference between ETV patients and ETV + CB patients were Actinobacteria, Fusobacteria, Proteobacteria, Saccharibacteria and Synergistetes. CONCLUSION: ETV treatment improves the serum biochemical, immunologic and virologic variables, but additional CB fails to further improve these variables. Of note, additional CB affects the intestinal flora in the CHB patients treated with ETV.

Nomogram for predicting the risk of gastroesophageal varices after primary endoscopic prophylaxis for variceal hemorrhage in patients with cirrhosis.

BACKGROUND AND AIMS: The aim of this study was to identify predictors of non-high-risk gastroesophageal varices and evaluate the probability of the residual high-risk varices in cirrhosis patients after the primary endoscopic treatment. PATIENTS AND METHODS: Medical records of the patients with cirrhosis admitted for primary endoscopic prophylaxis gastroesophageal varices hemorrhage were retrospectively analyzed. The patients were divided into high-risk varices and non-high-risk varices groups according to the endoscopy. A nomogram was developed based on the results of multivariate Cox analyses. Accuracy of this model was validated by the concordance index (Harrell's c-index) and calibration curve. RESULTS: Altogether 117 patients were enrolled between March 2014 and April 2018. The multivariate Cox analyses identified spleen length <140 mm [odds ratio (OR) = 2.715; P = 0.037), small or medium size of esophageal varices (OR = 4.412; P = 0.017), unaccompanied with gastric varices (OR = 7.025; P = 0.003) and frequency of endoscopic variceal ligation ≥one time per 4 months (OR = 3.834; P = 0.034) as independent factors of non-high-risk varices. All significant predictors were incorporated into a nomogram to predict the residual high-risk varices, which showed a notable accuracy with the concordance index (0.833). CONCLUSION: The nomogram-based prediction of residual high-risk varices can be used for risk stratification in cirrhosis patients with gastroesophageal varices.

Significance of MR/OPN/HMGB1 axis in NAFLD-associated hepatic fibrogenesis.

AIMS: The activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis, however non-alcoholic fatty liver disease (NAFLD) associated liver fibrogenesis have been poorly understood. We aimed to determine the significance of mineralocorticoid receptor (MR)/osteopontin (OPN)/high-mobility group box-1 (HMGB1) axis in this setting. MAIN METHODS: Liver specimens were collected from NAFLD patients and murine NAFLD models established with 12-week high fat diet (HFD) for analysis of both upstream signals of MR and intrahepatic MR/OPN/HMGB1 axis. The in vitro cell model of NAFLD-associated liver fibrogenesis was established by treating LX-2 (a cell line of human HSCs) with free fatty acids (FFA). The effects of MR signaling were evaluated using with ALD (MR activator) or eplerenone (Ep, MR antagonist). Moreover, the in vitro loss- and gain- of function approaches were applied to confirm the upstream and downstream relationships of mediators contained in the intracellular MR/OPN/HMGB1 axis of LX-2. KEY FINDINGS: In NAFLD condition, both human and mouse liver tissue samples demonstrated a significant up-regulation of MR/OPN/HMGB1 axis simultaneously with enhanced expression of pro-fibrogenic markers, including ACTA2, TIMP1, TGFB1 and COL1A1. Besides, enhanced production of serum aldosterone (ALD) was also observed in mouse NAFLD models. Moreover, the in vitro data demonstrated MR play an essential role in FFA-induced HSCs fibrogenesis. Meanwhile, MR acts as the upstream effector mediator of OPN and shares downstream HMGB1 with OPN. SIGNIFICANCE: The MR/OPN/HMGB1 axis could be therapeutically targeted to treat NAFLD associated hepatic fibrogenesis.

The in vitro effects of phospholipase D1-mTOR axis in liver fibrogenesis.

AIMS: The activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis progression. Phospholipase D (PLD) enzymes participate in multiple cellular activities. However, whether and how PLD regulates HSCs activation remain elusive. MAIN METHODS: The expression of intrahepatic PLD1 and PLD2 was determined in CCl4-induced mouse liver fibrosis models by western blot and immunohistochemistry. Cell model of liver fibrogenesis was constructed using rat HSCs line (HSC-T6) treated with recombinant transforming growth factor ß1 (TGFß1). Fibrogenesis was evaluated on the aspects of proliferation, expression of pro-fibrogenic markers and migration. The effects mediated by PLD1-mTOR axis on TGFß1-induced fibrogenesis were evaluated using HSC-T6 treated with small-molecular PLD1 inhibitors, PLD1-SiRNA, rapamycin (mTOR inhibitor) and MHY1485 (mTOR activator). KEY FINDINGS: Significant increase of PLD1, not PLD2 was documented in CCl4-induced cirrhotic compared to normal liver tissues. Suppression of PLD1 activities by PLD inhibitors or down-regulation of PLD1 expression in HSC-T6 could significantly restrain TGFß1-induced fibrogenesis, as reflected by decreased cell proliferation and reduced expression of pro-fibrogenic markers. Besides, either PLD1 inhibitor or PLD1-SiRNA significantly inhibited mTOR activity of HSC-T6. Moreover, PLD1 inhibitors not only exhibited similar effects with rapamycin in TGFß1-induced fibrogenesis, but also blunted MHY1485 enhanced cell proliferation of HSC-T6. SIGNIFICANCE: The PLD1-mTOR axis of HSCs could be therapeutically targeted in advanced liver fibrosis.

[Effects of bone morphogenic proteins-7 on production of collagen from hepatocytes and hepatic stellate cells during liver fibrosis].

OBJECTIVE: To study the effects of bone morphogenic proteins-7 (BMP-7) on the production of collagen from hepatocytes and hepatic stellate cells (HSC) during liver fibrosis, and the possible mechanism thereof. METHODS: Ten SD rats underwent repeated peritoneal injection of pig serum twice a week for 8 weeks so as to establish liver fibrosis models. Another 5 rats were injected intraperitoneally with normal saline as control group. Then the rats were sacrificed with their livers taken out. Primary hepatocytes and HSC were isolated from the fibrotic livers and cultured. BMP-7 at the concentrations of 10, 50, or 100 mg/L was added into the culture media for 24 and 96 hours respectively. ELISA was used to detect the levels of collagen of type I, III, and IV. BMP-7 at the concentration of 50 mg/L was added into the culture media for 96 h, then immunofluorescence staining was conducted on the cells to examine the levels of TbetaR-I and TbetaR-II, TGF-beta1 receptors on the cell surface, and the Smad2/3 level. Real-time RT-PCR was used to detect the TGF-beta1 mRNA expression in the cells. RESULTS: The levels of collagen of type I and III in the HSC treated by BMP-7 were all significantly lower than those in the control group especially 50 mg/L BMP-7 decreased the expression of collagen I by 40% +/- 6% (P < 0.01) and decreased the collagen III expression by 60% +/- 8% (P < 0.01), however, the levels of type IV collagen in the hepatocytes treated by BMP-7 were not significantly different from those of the control group. The levels of collagen of type I, III, and IV in the hepatocytes treated by BMP-7 were not significantly different from those of the control group. The impact of 50 mg/L BNP-7 on the HSC was stronger than that of the 10 mg/L BMP-7, however, not significantly different from that of the 100 mg/L BMP-7. There were not significant differences in the TbetaR-I and TbetaR-II levels in the HSC culture fluid treated by 50 mg/L BMP-7. 96 h after the treatment of 50 mg/L BMP-7 the Smad2/3 immunofluorescent strength in the HSC and hepatocytes decreased remarkably, and the TGF-beta1 level in the HSC decreased to the level 88% as high as that before treatment. CONCLUSION: BMP-7 inhibits the production of type I, and III collagen in activated HSC with the possible mechanism of inhibition of phosphorylated Smad2/3 in HSCs and down-regulation of the expression of TGF-beta1.

Migration of hepatic stellate cells in fibrotic microenvironment of diseased liver model.

BACKGROUND: In liver fibrosis, alterations within the space of Disse microenvironment facilitate the progression of chronic liver disease. The normal basement membrane-like matrix in the space of Disse converts to a matrix rich in fibril-forming collagens during the fibrosis. This study aimed to investigate the impact of alterations in the space of Disse microenvironment on the migration of hepatic stellate cells (HSCs) in the process of liver fibrosis, and to explore the novel mechanism of liver fibrosis from the viewpoint of cell migration. METHODS: A modified in vitro Boyden chamber system was employed to partially mimic the in vitro microenvironment of the Disse space in normal liver and in fibrosis. The effects of fibrogenetic growth factors on the migration of HSCs in simulated liver fibrosis were assessed by cell migration and cell proliferation experiments. RESULTS: Enhanced platelet-derived growth factor (PDGF)-BB, transforming growth factor-beta1 (TGF-beta1) and/or epithelial growth factor (EGF) in liver fibrosis resulted in an increase in migratory capacity of activated HSCs. The enhanced migration of HSCs induced by PDGF-BB was proliferation-independent. The elevation of basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) during liver fibrosis had no effect on the migration of HSCs. CONCLUSIONS: The study provides valuable insights into the role of the space of Disse microenvironment in regulating the migratory behavior of HSCs. TGF-beta1, PDGF-BB and EGF, which increase in liver fibrosis, induce the migration of activated HSCs. However, bFGF and VEGF have no effect although they also increase during liver fibrosis.

[Mechanism of hepatic stellate cell migration during liver fibrosis].

OBJECTIVE: To study the mechanism of migration of hepatic stellate cells (HSCs) within the space of Disse microenvironment during liver fibrosis, and to explore the novel pathogenesis of liver fibrosis from the view of cell migration. METHODS: Human HSCs of the line LX2-HSC were cultured. A modified in vitro Boyden chamber system was used to partially mimic the microenvironment of Disse space of normal basement membrane like matrix or that in fibrosis. HSCs were put in the upper chamber, and transforming growth factor (TGF)-beta1, plate-derived growth factor (PDGF)-BB, epidermal growth factor (EGF), vascular epithelial growth factor (VEGF), bfibroblast growth factor (bFGF), and collagen type I or type IV were put into the lower chamber. Four hours later cell migration assay was conducted. HSCs were put into 6-well plate and then added with TGF-beta1, PDGF-BB, EGF, VEGF, bFGF, and collagen type I or type IV, zymography was used to examine the activity of matrix metalloproteinases 2 and 9. SDS-gel immunoblotting was used too. RESULTS: Stimulation of HSCs with PDGF-BB, TGF-beta1, and/or EGF resulted in an increase in their migratory capacity and up-regulated MMP-2 activity. And the increase of MMP-2 could enhance Migration of HSC by 4.9-fold. The migration of HSCs (3.2-fold) was induced by type I collagen and inhibited by type IV collagen (1.2-fold). Migration induced by PDGF-BB, TGF-beta1, and collagen I could be inhibited by alpha1- and/or alpha2-integrin blocking antibodies. CONCLUSION: In liver fibrosis, alterations within the space of Disse microenvironment facilitate the migration of HSCs; the mechanism is associated with up-regulation of MMP-2 and with mediation of alpha1beta1 and alpha2beta1 integrins. Extracellular matrix by it self shows feedback actions to migration of HSCs.

Hepatopulmonary syndrome: the role of intra-abdominal hypertension and a novel mouse model.

OBJECTIVE: Hepatopulmonary syndrome (HPS) is considered as a triad of chronic liver disease, pulmonary vascular ectasia and severe hypoxemia. The study aims to investigate the pathological mechanism of intra-abdominal pressure (IAP) in HPS and establish a novel mouse model. METHODS: Fifty male ICR mice were randomly divided into experimental and control group, receiving subcutaneous injection of carbon tetrachloride and water, respectively. Mice in experimental group were then divided into 4 sub-groups with the intraperitoneal injection of different volume of albumin to form different IAP (0, 5, 10 and 20 cmH2O). All the mice were then sacrificed 24 hours later and blood gas analysis was conducted. In addition, liver and lung histopathology was also examined. RESULTS: Blood gas analysis in different IAP suggested the respiratory alkalosis. Arterial partial pressure of oxygen significantly decreased in the IAP=10 cmH2O (68.13 ± 3.56, P<0.01) and 20 cmH2O (66.00 ± 3.78, P<0.01). Alveolar-arterial oxygen pressure difference increased markedly in the IAP=10 cmH2O (54.60 ± 6.80, P<0.001) and 20 cmH2O (57.04 ± 5.60, P<0.001). According to lung histopathology, macrophages were found to accumulate in the alveolar spaces and the widened alveolar walls were detected. In addition, there was visible blood stasis in the alveolar walls and numerous red blood cells extravasated into air space in the IAP=10 and 20 cmH2O. CONCLUSIONS: Our study suggested that intra-abdominal hypertension was a significant pathological mechanism of HPS. Meanwhile, we have established a novel mouse model that will now be optimized with further investigation of the mechanism and therapeutic targets of HPS.

Hepatorenal syndrome: insights into the mechanisms of intra-abdominal hypertension.

OBJECTIVE: Hepatorenal syndrome is one of the serious complications of cirrhosis and closely associated with the increasing intra-abdominal pressure (IAP). The study aims to explore the potential mechanism of intra-abdominal hypertension in the development of hepatorenal syndrome in mouse models. METHODS: Eighty male mice were randomly divided into model group (subcutaneous injection of carbon tetrachloride) and control group (subcutaneous injection of olive oil). After 12 weeks, parts of the mice were sacrificed and liver histopathology was detected. Then, albumin (30 g/L) and normal saline were separately injected into the peritoneal cavity of mice to induce the different IAP levels (0, 5, 10 and 20cmH2O). Blood urea nitrogen, serum creatinine and renal histopathology were examined 24 hours later. RESULTS: Blood urea nitrogen and serum creatinine levels were statistically significant high in the group of IAP= 10 and 20cmH2O as compared with the IAP= 0cmH2O. From results of renal histopathology, the constrictive renal tubular lumen and inflammatory infiltration in the interstitial were observed in groups of IAP= 5 and 10cmH2O. Besides, the formed casts and hyperemia in the renal interstitial could be detected in group of IAP= 20cmH2O. The cellular swelling and edema of renal tubular epithelial cells were found in model group simultaneously. CONCLUSIONS: Our study suggested that intra-abdominal hypertension was a significant pathological mechanism and a potential independent risk factor of hepatorenal syndrome.

Inhibitory effect of bone morphogenetic protein-7 on hepatic fibrosis in rats.

AIM: Hepatic cirrhosis is a serious clinical problem caused by the accumulation of extracellular matrix, which can ultimately progress into hepatic failure. Transforming growth factor-beta1 (TGF-ß1) plays a pivotal role in extracellular matrix production. Bone morphogenetic protein-7 (BMP-7), as a member of the TGF-ß1 superfamily, has been well proved to be capable of reversing renal fibrosis in mice. In this study, we aim to investigate the potential effect of BMP-7 on hepatic fibrosis in rats. METHODS: Sprague-Dawley rats were randomly divided into five groups. In the hepatic fibrosis model group (n=8), rats was treated with porcine serum at 0.5 ml each time, twice a week. In the negative control group (n=10), rats were intraperitoneally injected with equal amount and frequency saline. Rats were injected with BMP-7 (100 µg/kg weight) before porcine serum intraperitoneal injection in the preventive group (n=9). For the early (n=10) and late (n=8) treatment group, rats were received with BMP-7 (100 µg/kg weight) every other day since the second and fourth week respectively after porcine serum injection. After eight weeks, the degree of liver fibrosis in rats was evaluated and the expression of TGF-ß1 in liver tissues was detected by Western blot and immunohistochemistry. RESULTS: The grade of hepatic fibrosis was significant attenuated by BMP-7 prevention and treatment compared with the rats in negative control group (P<0.05). In addition, the expression of TGF-ß1 greatly decreased in the BMP-7 preventive and treatment groups detected by both Western blot and immunohistochemistry. CONCLUSIONS: BMP-7 can attenuate and even prevent the level of hepatic fibrosis in rats through inhibiting the expression of TGF-ß1 in the liver fibrotic tissues. Therefore, it may be a potential clinical drug for the prevention and treatment of hepatic fibrosis.