E3 ubiquitin ligase ring finger protein 5 protects against hepatic ischemia reperfusion injury by mediating phosphoglycerate mutase family member 5 ubiquitination.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion (HIR) injury, a common clinical complication of liver transplantation and resection, affects patient prognosis. Ring finger protein 5 (RNF5) is an E3 ubiquitin ligase that plays important roles in endoplasmic reticulum stress, unfolded protein reactions, and inflammatory responses; however, its role in HIR is unclear. APPROACH AND RESULTS: RNF5 expression was significantly down-regulated during HIR in mice and hepatocytes. Subsequently, RNF5 knockdown and overexpression of cell lines were subjected to hypoxia-reoxygenation challenge. Results showed that RNF5 knockdown significantly increased hepatocyte inflammation and apoptosis, whereas RNF5 overexpression had the opposite effect. Furthermore, hepatocyte-specific RNF5 knockout and transgenic mice were established and subjected to HIR, and RNF5 deficiency markedly aggravated liver damage and cell apoptosis and activated hepatic inflammatory responses, whereas hepatic RNF5 transgenic mice had the opposite effect compared with RNF5 knockout mice. Mechanistically, RNF5 interacted with phosphoglycerate mutase family member 5 (PGAM5) and mediated the degradation of PGAM5 through K48-linked ubiquitination, thereby inhibiting the activation of apoptosis-regulating kinase 1 (ASK1) and its downstream c-Jun N-terminal kinase (JNK)/p38. This eventually suppresses the inflammatory response and cell apoptosis in HIR. CONCLUSIONS: We revealed that RNF5 protected against HIR through its interaction with PGAM5 to inhibit the activation of ASK1 and the downstream JNK/p38 signaling cascade. Our findings indicate that the RNF5-PGAM5 axis may be a promising therapeutic target for HIR.

Regulator of G-protein signaling 14 protects the liver from ischemia-reperfusion injury by suppressing TGF-ß-activated kinase 1 activation.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion injury (IRI) is a common complication of hepatectomy and liver transplantation. However, the mechanisms underlying hepatic IRI have not been fully elucidated. Regulator of G-protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates the G-protein and mitogen-activated protein kinase (MAPK) signaling pathways. However, the role of RGS14 in hepatic IRI remains unclear. APPROACH AND RESULTS: We found that RGS14 expression increased in mice subjected to hepatic ischemia-reperfusion (IR) surgery and during hypoxia reoxygenation in hepatocytes. We constructed global RGS14 knockout (RGS14-KO) and hepatocyte-specific RGS14 transgenic (RGS14-TG) mice to establish 70% hepatic IRI models. Histological hematoxylin and eosin staining, levels of alanine aminotransferase and aspartate aminotransferase, expression of inflammatory factors, and apoptosis were used to assess liver damage and function in these models. We found that RGS14 deficiency significantly aggravated IR-induced liver injury and activated hepatic inflammatory responses and apoptosis in vivo and in vitro. Conversely, RGS14 overexpression exerted the opposite effect of the RGS14-deficient models. Phosphorylation of TGF-ß-activated kinase 1 (TAK1) and its downstream effectors c-Jun N-terminal kinase (JNK) and p38 increased in the liver tissues of RGS14-KO mice but was repressed in those of RGS14-TG mice. Furthermore, inhibition of TAK1 phosphorylation rescued the effect of RGS14 deficiency on JNK and p38 activation, thus blocking the inflammatory responses and apoptosis. CONCLUSIONS: RGS14 plays a protective role in hepatic IR by inhibiting activation of the TAK1-JNK/p38 signaling pathway. This may be a potential therapeutic strategy for reducing incidences of hepatic IRI in the future.

Integrating serum microRNAs and electronic health records improved the diagnosis of tuberculosis.

BACKGROUND: To verify the differential expression of miR-30c and miR-142-3p between tuberculosis patients and healthy controls and to investigate the performance of microRNA (miRNA) and subsequently models for the diagnosis of tuberculosis (TB). METHODS: We followed up 460 subjects suspected of TB, and finally enrolled 132 patients, including 60 TB patients, 24 non-TB disease controls (TB-DCs), and 48 healthy controls (HCs). The differential expression of miR-30c and miR-142-3p in serum samples of the TB patients, TB-DCs, and HCs were identified by reverse transcription-quantitative real-time PCR. Diagnostic models were developed by analyzing the characteristics of miRNA and electronic health records (EHRs). These models evaluated by the area under the curves (AUC) and calibration curves were presented as nomograms. RESULTS: There were differential expression of miR-30c and miR-142-3p between TB patients and HCs (p < 0.05). Individual miRNA has a limited diagnostic value for TB. However, diagnostic performance has been both significantly improved when we integrated miR-142-3p and ordinary EHRs to develop two models for the diagnosis of tuberculosis. The AUC of the model for distinguishing tuberculosis patients from healthy controls has increased from 0.75 (95% CI: 0.66-0.84) to 0.96 (95% CI: 0.92-0.99) and the model for distinguishing tuberculosis patients from non-TB disease controls has increased from 0.67 (95% CI: 0.55-0.79) to 0.94 (95% CI: 0.89-0.99). CONCLUSIONS: Integrating serum miR-142-3p and EHRs is a good strategy for improving TB diagnosis.

Interaction analysis of gene variants related to one-carbon metabolism with chronic hepatitis B infection in Chinese patients.

BACKGROUND: The risk of chronic hepatitis B (CHB) infection is influenced by aberrant DNA methylation and altered nucleotide synthesis and repair, possibly caused by polymorphic variants in one-carbon metabolism genes. In the present study, we investigated the relationship between polymorphisms belonging to the one-carbon metabolic pathway and CHB infection. METHODS: A case-control study using 230 CHB patients and 234 unrelated healthy controls was carried out to assess the genetic association of 24 single nucleotide polymorphisins (SNPs) determined by mass spectrometry. RESULTS: Three SNPs, comprising rs10717122 and rs2229717 in serine hydroxymethyltransferase1/2 (SHMT2) and rs585800 in betaine-homocysteine S-methyltransferase (BHMT), were associated with the risk of CHB. Patients with DEL allele, DEL.DEL and DEL.T genotypes of rs10717122 had a 1.40-, 2.00- and 1.83-fold increased risk for CHB, respectively. Cases inheriting TA genotype of rs585800 had a 2.19-fold risk for CHB infection. The T allele of rs2229717 was less represented in the CHB cases (odds ratio = 0.66, 95% confidence interval = 0.48-0.92). The T allele of rs2229717 was less in patients with a low hepatitis B virus-DNA level compared to the control group (odds ratio = 0.49, 95% confidence interval = 0.25-0.97) and TT genotype of rs2229717 had a significant correlation with hepatitis B surface antigen level (p = 0.0195). Further gene-gene interaction analysis showed that subjects carrying the rs10717122 DEL.DEL/DEL.T and rs585800 TT/TA genotypes had a 2.74-fold increased risk of CHB. CONCLUSIONS: The results of the present study suggest that rs10717122, rs585800 and rs2229717 and gene-gene interactions of rs10717122 and rs585800 affect the outcome of CHB infection, at the same time as indicating their usefulness as a predictive and diagnostic biomarker of CHB infection.

Effects of chitinase-3-like protein 1 on brain death-induced hepatocyte apoptosis via PAR2-JNK-caspase-3.

Hepatocyte apoptosis is a crucial factor affecting liver quality in brain-dead donors. The identification of key molecular proteins involved in brain-death (BD)-induced hepatocyte apoptosis may help determine an effective method for improving the quality of livers from brain-dead donors. In this study, we used in vivo and in vitro models to investigate the role of chitinase-3-like protein 1 (CHI3L1) in promoting liver cell apoptosis after BD. Chitin was used to inhibit CHI3L1 in a rat model of BD. Macrophage polarization of THP-1 cells and hypoxia/reoxygenation (H/R) of LO-2 cells were used to mimic BD-induced cell stress in liver. We found that CHI3L1 played a vital role in promoting liver cell apoptosis. Six hours after BD, CHI3L1 expression was significantly upregulated in liver macrophages and was associated with BD-induced M1 polarization of these cells. In liver cells cultured under H/R conditions, recombinant CHI3L1 activated the protease-activated receptor 2 (PAR2)/c-June N-terminal kinase (JNK) apoptotic pathway and aggravated apoptosis. Compared with the control group, chitin particles inhibited the expression of CHI3L1 in the liver of brain dead rats, thereby reducing activation of the hepatocyte surface receptor, PAR2, and its downstream JNK/caspase-3 signaling pathway, ultimately reducing hepatocyte apoptosis. In conclusion, our results indicate that CHI3L1 relies on a PAR2/JNK-mediated mechanism to promote BD-induced hepatocyte apoptosis.

Tripartite Motif-Containing 27 Attenuates Liver Ischemia/Reperfusion Injury by Suppressing Transforming Growth Factor ß-Activated Kinase 1 (TAK1) by TAK1 Binding Protein 2/3 Degradation.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion (I/R) injury, which mainly involves inflammatory responses and apoptosis, is a common cause of organ dysfunction in liver transplantation (LT). As a critical mediator of inflammation and apoptosis in various cell types, the role of tripartite motif-containing (TRIM) 27 in hepatic I/R injury remains worthy of study. APPROACH AND RESULTS: This study systemically evaluated the putative role of TRIM27/transforming growth factor ß-activated kinase 1 (TAK1)/JNK (c-Jun N-terminal kinase)/p38 signaling in hepatic I/R injury. TRIM27 expression was significantly down-regulated in liver tissue from LT patients, mice subjected to hepatic I/R surgery, and hepatocytes challenged by hypoxia/reoxygenation (H/R) treatment. Subsequently, using global Trim27 knockout mice (Trim27-KO mice) and hepatocyte-specific Trim27 transgenic mice (Trim27-HTG mice), TRIM27 functions to ameliorate liver damage, reduce the inflammatory response, and prevent cell apoptosis. In parallel in vitro studies, activating TRIM27 also prevented H/R-induced hepatocyte inflammation and apoptosis. Mechanistically, TRIM27 constitutively interacted with the critical components, TAK1 and TAK1 binding protein 2/3 (TAB2/3), and promoted the degradation of TAB2/3, leading to inactivation of TAK1 and the subsequent suppression of downstream JNK/p38 signaling. CONCLUSIONS: TRIM27 is a key regulator of hepatic I/R injury by mediating the degradation of TAB2/3 and suppression of downstream TAK1-JNK/p38 signaling. TRIM27 may be a promising approach to protect the liver against I/R-mediated hepatocellular damage in transplant recipients.

Synaptopodin-2 promotes hepatocellular carcinoma metastasis via calcineurin-induced nuclear-cytoplasmic translocation.

Hepatocellular carcinoma (HCC), a type of malignant liver tumor, has a grim prognosis. As a functional protein, synaptopodin-2 (SYNPO2) has been associated with malignancy; however, the expression profile and function of SYNPO2 in HCC remains unknown. Herein, we revealed that SYNPO2 was transcriptionally downregulated in HCC tissues from both The Cancer Genome Atlas cohort and our cohort, and was also decreased at the translational level as determined by western blotting and immunohistochemical staining. Furthermore, reduced SYNPO2 expression correlated significantly with short overall survival and recurrence free survival of HCC patients. Restoring SYNPO2 expression inhibited the proliferation and aggressiveness of hepatocarcinoma cells. Mechanistically, increasing the ratio of cytoplasmic SYNPO2 to nuclear SYNPO2 was positively associated with recurrence rate in HCC patients; calcineurin (CaN) activity positively correlated with cytoplasmic SYNPO2 levels in HCC tissues; and nuclear-cytoplasmic translocation of SYNPO2 was induced by CaN to facilitate metastasis of HCC through assembly of peripheral actin bundles. In short, our findings uncover a novel role of SYNPO2 in HCC metastasis via the CaN/SYNPO2/F-actin axis, and indicate that SYNPO2 may serve as a possible prognostic marker and novel therapeutic target.

Six-Transmembrane Epithelial Antigen of the Prostate 3 Deficiency in Hepatocytes Protects the Liver Against Ischemia-Reperfusion Injury by Suppressing Transforming Growth Factor-ß-Activated Kinase 1.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion (I/R) injury remains a major challenge affecting the morbidity and mortality of liver transplantation. Effective strategies to improve liver function after hepatic I/R injury are limited. Six-transmembrane epithelial antigen of the prostate 3 (Steap3), a key regulator of iron uptake, was reported to be involved in immunity and apoptotic processes in various cell types. However, the role of Steap3 in hepatic I/R-induced liver damage remains largely unclear. APPROACH AND RESULTS: In the present study, we found that Steap3 expression was significantly up-regulated in liver tissue from mice subjected to hepatic I/R surgery and primary hepatocytes challenged with hypoxia/reoxygenation insult. Subsequently, global Steap3 knockout (Steap3-KO) mice, hepatocyte-specific Steap3 transgenic (Steap3-HTG) mice, and their corresponding controls were subjected to partial hepatic warm I/R injury. Hepatic histology, the inflammatory response, and apoptosis were monitored to assess liver damage. The molecular mechanisms of Steap3 function were explored in vivo and in vitro. The results demonstrated that, compared with control mice, Steap3-KO mice exhibited alleviated liver damage after hepatic I/R injury, as shown by smaller necrotic areas, lower serum transaminase levels, decreased apoptosis rates, and reduced inflammatory cell infiltration, whereas Steap3-HTG mice had the opposite phenotype. Further molecular experiments showed that Steap3 deficiency could inhibit transforming growth factor-ß-activated kinase 1 (TAK1) activation and downstream c-Jun N-terminal kinase (JNK) and p38 signaling during hepatic I/R injury. CONCLUSIONS: Steap3 is a mediator of hepatic I/R injury that functions by regulating inflammatory responses as well as apoptosis through TAK1-dependent activation of the JNK/p38 pathways. Targeting hepatocytes, Steap3 may be a promising approach to protect the liver against I/R injury.

Biflavones from Ginkgo biloba as inhibitors of human thrombin.

Ginkgo Biloba leaf extract has been widely used for the prevention and treatment of thrombosis and cardiovascular disease in both eastern and western countries, but the bioactive constituents and the underlying mechanism of anti-thrombosis have not been fully characterized. The purpose of this study was to investigate the inhibitory effects of major constituents in Ginkgo biloba on human thrombin, a key serine protease regulating the blood coagulation cascade and the processes of thrombosis. To this end, a fluorescence-based biochemical assay was used to assay the inhibitory effects of sixteen major constituents from Ginkgo biloba on human thrombin. Among all tested natural compounds, four biflavones (ginkgetin, isoginkgetin, bilobetin and amentoflavone), and five flavonoids (luteolin, apigenin, quercetin, kaempferol and isorhamnetin) were found with thrombin inhibition activity, with the IC50 values ranging from 8.05 µM to 82.08 µM. Inhibition kinetic analyses demonstrated that four biflavones were mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki values ranging from 4.12 µM to 11.01 µM. Molecular docking method showed that the four biflavones could occupy the active cavity with strong interactions of salt bridges and hydrogen bonds. In addition, mass spectrometry-based lysine labeling reactivity assay suggested that the biflavones could bind on human thrombin at exosite I rather than exosite II. All these findings suggested that the biflavones in Ginkgo biloba were naturally occurring inhibitors of human thrombin, and these compounds could be used as lead compounds for the development of novel thrombin inhibitors with improved efficacy and high safety profiles.

Breviscapine ameliorates CCl4­induced liver injury in mice through inhibiting inflammatory apoptotic response and ROS generation.

Acute liver injury is characterized by fibrosis, inflammation and apoptosis, leading to liver failure, cirrhosis or cancer and affecting the clinical outcome in the long term. However, no effective therapeutic strategy is currently available. Breviscapine, a mixture of flavonoid glycosides, has been reported to have multiple biological functions. The present study aimed to investigate the effects of breviscapine on acute liver injury induced by CCl4 in mice. C57BL/6 mice were subjected to intraperitoneal injection with CCl4 for 8 weeks with or without breviscapine (15 or 30 mg/kg). Mice treated with CCl4 developed acute liver injury, as evidenced by histological analysis, Masson trichrome and Sirius Red staining, accompanied with elevated levels of alanine aminotransferase and aspartate aminotransferase. Furthermore, increases in pro­inflammatory cytokines, chemokines and apoptotic factors, including caspase­3 and poly(ADP ribose) polymerase­2 (PARP­2), were observed. Breviscapine treatment significantly and dose­dependently reduced collagen deposition and the fibrotic area. Inflammatory cytokines were downregulated by breviscapine through inactivating Toll­like receptor 4/nuclear factor-κB signaling pathways. In addition, co­administration of breviscapine with CCl4 decreased the apoptotic response by enhancing B­cell lymphoma-2 (Bcl­2) levels, while reducing Bcl­2­associated X protein, apoptotic protease activating factor 1, caspase­3 and PARP activity. Furthermore, CCl4­induced oxidative stress was blocked by breviscapine through improving anti­oxidants and impeding mitogen­activated protein kinase pathways. The present study highlighted that breviscapine exhibited liver­protective effects against acute hepatic injury induced by CCl4 via suppressing inflammation and apoptosis.

Oridonin synergistically enhances JQ1-triggered apoptosis in hepatocellular cancer cells through mitochondrial pathway.

Bromodomain and Extra-Terminal Domain (BET) inhibitors, such as JQ1 have emerged as novel drug candidates and are being enthusiastically pursued in clinical trials for the treatment of cancer. However, many solid cancers are resistance to BET inhibitors. To explore methods for improving the therapeutic potential of BET inhibitors, we investigated the combinational activity of JQ1 with Oridonin, a bioactive molecules derived from Traditional Chinese Medicine in hepatocellular carcinoma (HCC) cells. Our results showed that Oridonin synergistically enhanced the abilities of JQ1 to inhibit cell viability in HCC cells and, significantly augmented JQ1-triggered apoptosis in HCC cells and in HCC cancer stem-like cells. Moreover, Oridonin dose-dependently inhibited the expression of several anti-apoptotic proteins, such as Bcl-2, Mcl-1, and x-linked inhibitor of apoptosis (xIAP) in HCC cells. Cell fractionation and western blotting analysis showed that the enhancement of apoptosis by Oridonin was associated with cytochrome c release, activation of caspase-9, -3 and cleavage of PARP, indicating the activation of mitochondrial apoptosis pathway. Altogether, our findings demonstrate that Oridonin may be used to effectively enhance the sensitivity of BET inhibitors in HCC therapy via downregulation of the expression of multiple anti-apoptotic proteins.

Kruppel-like factor 6 suppresses growth and invasion of hepatocellular carcinoma cells in vitro and in vivo.

Kruppel-like factor 6 (KLF6) as a novel tumor suppressive gene participates in multiple biological behaviors and plays an important role in regulating tumor cell growth and invasion. However, the functions of KLF6 in hepatocellular carcinoma (HCC) remain poorly understood. The expression level of KLF6 was examined by immunohistochemical assay in human HCC tissues, and KLF6-overexpressed HCC cells (SMCC-7721 and HepG2) were used for evaluating cell proliferation and invasion by MTT and Transwell assays. A subcutaneous HCC tumor model was established for assessing tumor growth in vivo. Our results showed that the expression of KLF6 was significantly downregulated in HCC tissues compared with the adjacent non-cancerous tissues (50.0% vs. 72.0%, P = 0.034) and negatively associated with the lymph-vascular space invasion (LVSI) in HCC patients (P = 0.003). Furthermore, overexpression of KLF6 reduced cell proliferation and weakened the cell invasive potential followed with the decreased expression of PCNA and MMP-9 in HCC cells. The in vivo experiment indicated that KLF6 overexpression suppressed the xenograft tumor growth. Therefore, our findings show that KLF6 suppresses growth and invasion of HCC cells in vitro and in vivo, suggesting a tumor suppressive function in HCC and provides the potential therapeutic target for the treatment of HCC.

Withdrawal of immunosuppression in liver transplantation and the mechanism of tolerance.

BACKGROUND: Immunosuppression reagents have side effects and cause considerable long-term morbidity and mortality in patients after liver transplantation. Sufficient evidences showed that minimization or withdrawal of immunosuppression reagents does not deteriorate the recipient's immune response and physiological function and therefore, is feasible in some recipients of liver transplantation. However, the mechanisms are not clear. The present review was to update the current status of immunosuppression in liver transplantation and the mechanism of minimization or withdrawal of immunosuppression in liver recipients. DATA SOURCES: We searched articles in English on minimization or withdrawal of immunosuppression in liver transplantation in PubMed. We focused on the basic mechanisms of immune tolerance in liver transplantation. Studies on immunosuppression minimization or withdrawal protocols and biomarker in tolerant recipients were also analyzed. RESULTS: Minimization or withdrawal of immunosuppression can be achieved by the induction of immune tolerance, which may not be permanent and can be affected by various factors. However, accurately evaluating immune status post-transplant is a prerequisite to achieve individualized immunosuppression. Numerous mechanisms for immune tolerance have been found, including immunophenotypic shift of memory CD8+ T cells and CD4+ T cell subsets. Activation of the inflammasome through apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) in dendritic cells is associated with rejection after liver transplantation. CONCLUSIONS: Minimization or withdrawal of immunosuppression can be achieved by the induction of immune tolerance via different mechanisms. This process could be affected by immunophenotypic shift of memory CD8+ T cells and CD4+ T cell subsets, which may be correlated with activation of the inflammasome through ASC in dendritic cells.