Recompensation in treatment-naïve HBV-related decompensated cirrhosis: a 5-year multi-center observational study comparing patients with ascites and bleeding.

BACKGROUND AND AIMS: Recompensation between patients with ascites and bleeding was unknown in treatment-naïve HBV-related decompensated cirrhosis. METHODS: In this retrospective multi-center study, treatment-naïve HBV-related decompensated patients were enrolled at first decompensating event of ascites and/or variceal bleeding. Further complications and clinical characteristics were collected using standard case report form every 6 months to year-5 of antiviral treatment. Recompensation was defined as maintaining free of decompensation for one year and achieving liver function within Child-Pugh A and/or MELD < 10. RESULTS: Totally, 170 (170/298, 57.0%) patients in ascites group of 298 (298/383, 77.8%) treatment-naïve decompensated patients and 33 (33/85, 38.8%) in bleeding group of 85 (85/383, 22.2%) patients, achieved recompensation. Ascites group had higher 5-year rate of recompensation than bleeding group (63.3% vs. 46.5%, p = 0.012), respectively. Patients achieving recompensation in ascites group maintained lower rate of second decompensation than these in bleeding group (at year-5: 26.7% vs. 43.3%, p = 0.032). Specifically, recompensated patients in ascites group had predominantly 5-year rate of further ascites (24.0%) and lower rate of further bleeding (6.0%), which differed from the pattern of these in bleeding group, with lower rate of further ascites (16.0%, p = 0.599) and significantly higher rate of further bleeding (33.9%, p < 0.001). Both patients had superior long-term prognosis (death/LT rate at year-5: 0.6% vs. 3.0%, p = 0.196). CONCLUSION: Ascites patients could achieve higher rate of recompensation through antiviral therapy than bleeding patients. Recompensated patients in ascites group had better prognosis in terms of preventing further bleeding.

Vaspin Prevents Tumor Necrosis Factor-α-Induced Apoptosis in Cardiomyocytes by Promoting Autophagy.

Visceral adipose tissue-derived serine protease inhibitor (Vaspin) is an adipocytokine that has been shown to exert anti-inflammatory effects and inhibits apoptosis under diabetic conditions. This study was designed to investigate the impact of vaspin on autophagy in tumor necrosis factor (TNF)-α-induced injury in cardiomyocytes and its cardioprotective effects in the pathogenesis of diabetic cardiomyopathy (DCM). H9C2 cells were treated with TNF-α with or without vaspin in vitro. Tumor necrosis factor-α treatment inhibited autophagy and promoted apoptosis in H9C2 cells after stimulating for 24 hours. Pretreatment with vaspin significantly mitigated apoptosis induced by TNF-α partly because of augment effects of vaspin on autophagy as demonstrated by a higher ratio of LC3-II/LC3-I, higher expression of Beclin-1, and increased autophagosomes formation. Furthermore, the AKT agonist IGF-1 significantly reversed the effect of vaspin on autophagy. In vivo DCM model was also developed by treating rats with streptozotocin followed by intraperitoneal injection with vaspin. In DCM rats, upregulation of vaspin reversed cardiac dysfunction, as identified by increased left ventricular ejection fractions and fractional shortening levels, a higher Em/Am ratio, and lower levels of TNF-α, lactate dehydrogenase, creatine kinase, and creatine kinase-myocardial isoenzyme. In conclusion, vaspin attenuated the TNF-α-induced apoptosis by promoting autophagy probably through inhibiting the PI3K/AKT/mTOR pathway and further ameliorated the cardiac dysfunction in DCM rats.

Autophagy attenuates endothelial-to-mesenchymal transition by promoting Snail degradation in human cardiac microvascular endothelial cells.

Endothelial-to-mesenchymal transition (EndMT) mainly exists in cardiovascular development and disease progression, and is well known to contribute to cardiac fibrosis. Recent studies indicated that autophagy also participates in the regulation of cardiac fibrosis. However, the precise role of autophagy in cardiac fibrosis and the underlying molecular mechanism remain unclear. The present study aimed to explore the role of autophagy in EndMT, reveal the underlying molecular mechanism, and seek new therapy for cardiac fibrosis. In the present study, we found that EndMT and autophagy were induced simultaneously by hypoxia in human cardiac microvascular endothelial cells (HCMECs). Rapamycin, an autophagy enhancer, attenuated EndMT with promoting angiogenesis, while 3-methyladenine (3-MA) and chloroquine (CQ), agents that inhibit autophagy, accelerated the progression accompanied by the decrease in counts of tube formation under hypoxia conditions. Interestingly, intervening autophagy by rapamycin, 3-MA, or CQ did not affect hypoxia-induced autocrine TGFß signaling, but changed the expression of Snail protein without alterations in the expression of Snail mRNA. Furthermore, the colocalization of LC3 and Snail indicated that autophagy might mediate Snail degradation under hypoxia conditions in HCMECs. Interaction of p62, the substrate of autophagy, with Snail by co-immunoprecipitation especially in hypoxia-incubated cells confirmed the hypothesis. In conclusion, autophagy serves as a cytoprotective mechanism against EndMT to promote angiogenesis by degrading Snail under hypoxia conditions, suggesting that autophagy targetted therapeutic strategies may be applicable for cardiac fibrosis by EndMT.

RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells.

Endothelial-mesenchymal transition (EndMT) is an essential mechanism in the cardiovascular system, for both cardiovascular development and cardiovascular diseases (CVDs). Recent studies indicate that runt-related transcription factor 3 (RUNX3) contributes to EndMT and endothelial cell dysfunction. However, the underlying molecular mechanism remains unknown. The present study was designed to investigate the role of RUNX3 in EndMT and endothelial cell function, and to elucidate the underlying molecular mechanism. Human cardiac microvascular endothelial cells (HCMECs) were incubated in strictly controlled hypoxic conditions (1% O2). HCMECs were cultured under normoxic conditions (21% O2), and then moved to a strictly controlled hypoxic environment (1% O2). Under this hypoxic condition, the cells were transfected with the lentiviral vector containing RUNX3 or an empty lentiviral vector for 8 h. After the cells were cultured under hypoxic conditions for 4 days, CD31 and α-smooth muscle actin colocalization were assessed by immunofluorescence microscopy. Transwell migration and tube formation assays were used to examine the migration and angiogenesis ability. RT-qPCR and western blotting were used to determine the expression of molecules involved in EndMT. Hypoxia induced the transition of HCMECs to mesenchymal cells and markedly promoted tube formation and cell migration. Transforming growth factor-ß (TGF-ß) and Notch signaling were activated during the hypoxia-induced EndMT of HCMECs. RUNX3 knockdown attenuated EndMT of HCMECs, promoted angiogenic phenotype, and reduced endothelial cell migration. In conclusion, our results showed that RUNX3 knockdown attenuated hypoxia-induced EndMT and reversed endothelial cell functions. RUNX3 is a common downstream target of TGF-ß and Notch signaling, and may be a novel therapeutic target for treating CVD mediated by EndMT.

[Knockdown of RUNX3 inhibits hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells].

Objective To investigate the effects of Runt-related transcription factor 3 (RUNX3) knockdown on hypoxia-induced endothelial-to-mesenchymal transition (EndoMT) of human cardiac microvascular endothelial cells (HCMECs), and elucidate the underlying molecular mechanism. Methods HCMECs were cultured in hypoxic conditions and infected with RUNX3-RNAi lentivirus to knock-down the expression of RUNX3. Reverse transcription PCR was performed to detect the mRNA expressions of RUNX3 and EndoMT related genes such as CD31, vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1); Western blotting was used to determine the protein expressions of RUNX3, CD31, α-SMA and another molecules involved in EndoMT; and immunofluorescence cytochemistry was applied to observe the colocalization of CD31 and α-SMA. Results Hypoxia induced the transition of HCMECs to mesenchymal cells. Hypoxia up-regulated the expression of TGF-ß2, Smad2/3, phosphorylation of Smad2/3 (p-Smad2/3), Notch-1, Hes1, and Hey1; knockdown of RUNX3 down-regulated the levels of Smad2/3, p-Smad2/3, Hes1, and Hey1 to different extents, and raised the levels of TGF-ß2 and Notch-1. Conclusion Knockdown of RUNX3 in HCMECs attenuates hypoxia-induced EndoMT via partially inhibiting TGF-ß and Notch signaling pathway.

[Effects of antihistamine treatment on immune function in rats with experimental hepatitis].

OBJECTIVE: To investigate the effects of antihistamine treatment on immune function in rats with experimental hepatitis. METHODS: Thirty Wistar rats were randomly allocated into three groups:experimental hepatitis group (EH group), antihistamine treatment group (AH group) and normal control group (NC group). Rats in the EH group received the subcutaneous injection of 40% carbon tetrachloride oil solution and were fed on diet with low-protein, low-choline, high-fat and high-alcohol,while rats in the AH group received antihistamine treatment(ketotifen + vitamin C) additionally.They were sacrificed after 4 weeks, and the levels of serum alanine aminotransferase(ALT), total bilirubin (TBil), histamine(HA), IFNgamma, IL-12, IL-4 and IL-10 were determined. The levels of IL-12 mRNA and IFN-gamma mRNA in liver tissue were determined via real-time reverse transcriptional polymerase chain reaction(RT-PCR). RESULTS: (1) Compared to the NC group, in the EH group, the levels of ALT, TBil, and circulating and intrahepatic HA were significantly increased(P less than 0.05); intrahepatic HA were significantly decreased(P less than 0.05) after antihistamine treatment. (2) Compared to the NC group, in the EH group, the levels of IL-4, IL-10 were significantly increased((0.504+/-0.202)ng/ml and (29.025+/-1.478) pg/ml vs (0.811+/-0.244)ng/ml and (33.72+/-4.293)pg/ml respectively, P less than 0.05), and the levels of IL-12 were decreased ((6.515+/-2.893)pg/ml vs (3.519+/-1.113)pg/ml, P less than 0.05); and after antihistamine treatment the levels of IL-4 and IL-10 were significantly decreased (were (0.423+/-0.168)ng/ml and (30.412+/-3.275)pg/ml, P less than 0.05), the levels of IL-12 were significantly increased (P less than 0.05), but the level of IFNgamma had no significance (P more than 0.05). The levels of intrahepatic IL-12 mRNA and IFNgamma mRNA had similar results. CONCLUSION: Antihistamine treatment may improve liver function and correct Th1/Th2 unbalance.

[Changes in mast cells and hepatic expression of c-kit and stem cell factor in the rat model of chronic hepatitis].

OBJECTIVE: To study the potential role of mast cells and the related molecular mechanism in chronic hepatitis (CH) using a rat model system. METHODS: Thirty Wistar rats (15 males, 15 females; weight range: 230-290 g) were randomly divided into the normal contrast (NC) group and experimental CH group. The CH group received subcutaneous injection of CCl4 and a diet high in cholesterol and alcohol content and low in protein and choline content. Throughout the 4-week modeling period, aseptic blood samples were taken to test plasma tryptase (TS) and hyaluronic acid (HA) levels. The rats were euthanized to assess the changes in liver mast cells by histology and morphology analyses and the changes in liver expression of c-kit and stem cell factor (SCF) proteins by immunohistochemistry and mRNAs by RT-PCR. RESULTS: Compared to the NC group, the CH group had higher plasma and liver concentration of HA (78.09 +/- 38.55 vs. 145.14 +/- 52.54 ng/ml, 51.58 +/- 20.45 vs. 106.59 +/- 43.15 ng/100 mg; t = 2.457 and 2.825 respectively, both P less than 0.05) and TS (0.416 +/- 0.143 vs 0.753 +/- 0.210 mg/ml; t = 4.165, P less than 0.05). The CH group also showed fatty degeneration and fibrosis with many degranulating and degranulated mast cells filled with purple granula located around the liver blood vessels and in fiber-intervals. The CH livers also showed a significantly higher number of mast cells (2.167 +/- 0.924 vs. NC: 10.92 +/- 1.575; t = 7.633, P less than 0.05) and stronger intensity of c-kit staining (2.783 +/- 0.577 vs. 12.86 +/- 3.126; t = 9.511, P less than 0.05) and SCF staining (3.383 +/- 1.583 vs. 15.58 +/- 6.431; t = 9.625, P less than 0.05). The expressions of c-kit and SCF were positively correlated with HA level (r = 0.478 and 0.556 respectively, both P less than 0.05). The c-kit and SCF mRNA expression levels were also significantly higher in the CH liver tissues. CONCLUSION: Mast cell degranulation and histamine release is significantly increased under conditions of chronic hepatitis, and the related mechanism may involve up-regulation of the membrane receptor c-kit and its ligand SCF.

[Effects of lipopolysaccharide on the maturation and secretion of human peripheral dendritic cells].

OBJECTIVE: To study the effects of Lipopolysaccharide (LPS) on the maturation and secretion of human peripheral dendritic cells (DCs). METHODS: DCs from healthy human peripheral monocytes (PBMCs) were induced in vitro with rhGM-CSF, rhIL-4, Flt3-L and TNFalpha. The subjects were divided into 3 groups: the long-term group stimulated with LPS 1 microg/ml at day 1, 4, 7, 9 post culture; the short-term group stimulated with LPS 1 microg/ml at day 7 and 8 post culture, and the DCs without LPS stimulation was control group. After 10 days of culture, the morphologic features of DCs were observed by light and electron microscopes, the phenotypic patterns were characterized by flow cytometry, the proliferation of T cell were evaluated with mixed leukocytes reaction (MLR) and the levels of IL-12 and IFNgamma produced by DCs were analyzed with ELISA. RESULTS: Compared with the short-term group, the expressions of HLA-DR (65.81%+/-10.96%), CD86 (48.81%+/-18.13%), CD80 (13.56%+/-5.48%), CD83 (11.52%+/-5.09%), the secretions of IFNgamma(15.60+/-5.83 pg/ml) and IL-12 (51.77+/-11.02 pg/ml) by the DCs in long-term group were decreased obviously (P is less than 0.05) and the proliferation of homogenic lymphocyte cells (1.548+/-0.365) stimulated by DCs was also impaired (P < 0.05). CONCLUSION: Long-term LPS stimulation can suppress the maturation and secretion of DCs, which might be the reason of poor immunity in the patients with intestinal endotoxemia.

[Construction of cDNA library of protozoan ciliates Euplotes octocarinatus].

UNLABELLED: The protein biosynthesis in cells is an open process cooperatively regulated by many protein factors and enzymes, which form a complicated protein network and signal transduction pathway for mRNA metabolism and protein translation. OBJECTIVE: To provide a platform for studying on the function of proteins involving in process of translation termination and the relationship among these proteins in ciliates. METHODS: We constructed the cDNA library of protozoan ciliates Euplotes octocarinatus strictly following the procedure of BD Matchmaker Library Construction & Screening kit from Clontech. RESULTS: We obtained a cDNA library of ciliates Euplotes, the titer of which was about 2.437 x 10(7) cfu/mL, which could suffice for functional gene screen. By using the class II polypeptide release factor (eRF3) as bait protein, we obtained some putative genes, including a partial cDNA putatively encoding RNA helicase. CONCLUSION: This library will provide a convenient platform for identifying the functional genes in ciliates.