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.

Discovery of novel arylamide derivatives containing piperazine moiety as inhibitors of tubulin polymerisation with potent liver cancer inhibitory activity.

In this work, a series of novel arylamide derivatives containing piperazine moiety were designed and synthesised as tubulin polymerisation inhibitors. Among 25 target compounds, compound 16f (MY-1121) exhibited low nanomolar IC50 values ranging from 0.089 to 0.238 µM against nine human cancer cells. Its inhibitory effects on liver cancer cells were particularly evident with IC50 values of 89.42 and 91.62 nM for SMMC-7721 and HuH-7 cells, respectively. Further mechanism studies demonstrated that compound 16f (MY-1121) could bind to the colchicine binding site of ß-tubulin and directly act on ß-tubulin, thus inhibiting tubulin polymerisation. Additionally, compound 16f (MY-1121) could inhibit colony forming ability, cause morphological changes, block cell cycle arrest at the G2 phase, induce cell apoptosis, and regulate the expression of cell cycle and cell apoptosis related proteins in liver cancer cells. Overall, the promising bioactivities of compound 16f (MY-1121) make the novel arylamide derivatives have the value for further development as tubulin polymerisation inhibitors with potent anticancer activities.

[Progress in antitumor activity of diterpenoid alkaloids in plants of Aconitum].

Small-molecule compounds with rich sources have diverse structures and activities. The active ingredients in traditional Chinese medicine(TCM) provide new sources for the discovery of new antitumor drugs. Aconitum plants as Chinese medicinal plants have the effects of dispelling wind, removing dampness, warming meridian, and relieving pain. They are mainly used to treat inflammation, pain, rheumatism, and tumors, improve heart function, and dilate blood vessels in clinical practice. Diterpenoid alkaloids are the main active components of Aconitum plants, including C20-, C19-, C18-diterpenoid alkaloids and bis-diterpenoid alkaloids. Stu-dies have demonstrated that diterpenoid alkaloids can effectively treat lung cancer, liver cancer, breast cancer, colon cancer and other cancers. Diterpenoid alkaloids are considered as the most promising natural compounds against cancers. In this review, we summarized the chemical structures and antitumor activities of C20-, C19-, C18-diterpenoid alkaloids and bis-diterpenoid alkaloids extracted from plants of Aconitum, aiming to provide reference for further development of diterpenoid alkaloids from Aconitum as antitumor drugs.

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.

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.

Perilipin2 inhibits the replication of hepatitis B virus deoxyribonucleic acid by regulating autophagy under high-fat conditions.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is often associated with increased lipid deposition in hepatocytes. However, when combined with non-alcoholic fatty liver disease or hyperlipidemia, it tends to have a lower HBV deoxyribonucleic acid (DNA) load. The relationship between lipid metabolism and HBV DNA replication and its underlying mechanisms are not well understood. AIM: To investigate the relationship between lipid metabolism and HBV DNA replication and its underlying mechanisms. METHODS: 1603 HBsAg-seropositive patients were included in the study. We first explored the relationship between patients' lipid levels, hepatic steatosis, and HBV DNA load. Also, we constructed an HBV infection combined with a hepatic steatosis cell model in vitro by fatty acid stimulation of HepG2.2.15 cells to validate the effect of lipid metabolism on HBV DNA replication in vitro. By knocking down and overexpressing Plin2, we observed whether Plin2 regulates autophagy and HBV replication. By inhibiting both Plin2 and cellular autophagy under high lipid stimulation, we examined whether the Plin2-autophagy pathway regulates HBV replication. RESULTS: The results revealed that serum triglyceride levels, high-density lipoprotein levels, and hepatic steatosis ratio were significantly lower in the HBV-DNA high load group. Logistic regression analysis indicated that hepatic steatosis and serum triglyceride levels were negatively correlated with HBV-DNA load. Stratified analysis by HBeAg showed significant negative correlations between HBV-DNA load and hepatic steatosis ratio in both HBeAg-positive and HBeAg-negative groups. An in vitro cell model was developed by stimulating HepG2.2.15 cells with palmitic acid and oleic acid to study the relationship between HBV-DNA load and lipid metabolism. The results of the in vitro experiments suggested that fatty acid treatment increased lipid droplet deposition and decreased the expression of cell supernatant HBsAg, HBeAg, and HBV DNA load. Western blot and polymerase chain reaction analysis showed that fatty acid stimulation significantly induced Plin2 protein expression and inhibited the expression of hepatocyte autophagy proteins. Inhibition of Plin2 protein expression under fatty acid stimulation reversed the reduction in HBsAg and HBeAg expression and HBV DNA load induced by fatty acid stimulation and the inhibition of cellular autophagy. Knocking down Plin2 and blocking autophagy with 3-methyladenine (3-MA) inhibited HBV DNA replication. CONCLUSION: In conclusion, lipid metabolism is a significant factor affecting HBV load in patients with HBV infection. The in vitro experiments established that fatty acid stimulation inhibits HBV replication via the Plin2-autophagy pathway.

Expression of the Vitreoscilla hemoglobin gene in Nannochloropsis oceanica regulates intracellular oxygen balance under high-light.

Nannochloropsis oceanica is widely used as a model photosynthetic chassis to produce fatty acids and carotenoid pigments. However, intense light typically causes excessive generation of reactive oxygen species (ROS) and photorespiration in microalgal cells, which results in decreased cell growth rate and unsaturated fatty acid content. In this study, the Vitreoscilla hemoglobin gene (vgb) was introduced into N. oceanica cells and expressed by using the light-harvesting complex promoter and its signal peptide. Compared with wild type (WT), the growth rate of transformants increased by 7.4%-18.5%, and the eicosapentaenoic acid content in an optimal transformant increased by 21.0%. Correspondingly, the intracellular ROS levels decreased by 56.9%-70.0%, and the catalase content in transformants was about 1.8 times that of WT. The photorespiration level of transformants was reduced by the measurement and calculation of the dissolved oxygen concentration under the condition of light-dark transition. The expression level of the key genes related to the photorespiration pathway in transformants was more than 80% lower than that in WT. These results indicated that Vitreoscilla hemoglobin could improve microalgal growth by reducing ROS damage and modulating photorespiration under stress conditions.

Inhibitory effect of melatonin on lung oxidative stress induced by respiratory syncytial virus infection in mice.

Previous research has shown that antioxidant (butylated hydroxyanisole) treatment ameliorates respiratory syncytial virus (RSV)-induced disease and lung inflammation. Melatonin has been reported to exhibit a wide varieties of biological effects, including antioxidant and anti-inflammation, and has no evident toxicity and side effect. But it is not known whether melatonin would modify RSV-induced lung disease and oxidative stress. The present study was to establish the involvement of oxidative stress in the pathogenesis of RSV-induced lung inflammation, and to investigate the protective effect of administration of melatonin in mice with RSV-induced oxidative pulmonary injury for 4 days. Malondialdehyde (MDA), an end product of lipid peroxidation, and glutathione (GSH) and superoxide dismutase (SOD) and nitric oxide (NO) levels were evaluated in lung tissue homogenates by spectrophotometry. Hydroxyl radical (.-OH), one of the indicators of free radical formation, was also detected in lung homogenates by Fenton reaction. Tumor necrosis factor-a (TNF-a) concentrations in mouse serum were measured with ELISA assay. The results demonstrated that the mice intranasally inoculated with RSV resulted in oxidative stress changes by increasing NO, MDA and .-OH levels, and decreasing GSH and SOD activities, whereas administration of melatonin significantly reversed all these effects. Furthermore, melatonin inhibited production of proinflammatory cytokines such as TNF-a in serum of RSV-infected mice. These results suggest that melatonin ameliorates RSV-induced lung inflammatory injury in mice via inhibition of oxidative stress and proinflammatory cytokine production and may be as a novel therapeutic agent in virus-induced pulmonary infection.

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.

[Spatial Distribution of Stable Isotope from the Lakes in Typical Temperate Glacier Region].

We focused mainly on the spatial variation and influencing factors of hydrogen and oxygen stable isotopes between water samples collected at the surface and different depths in the Lashi Lake in August, 2014. Hydrological supply characteristics of the lake in typical temperate glacier region were discussed. The results showed that the values of δ¹8O and δD in the Lashi Lake ranged from -12.98 per thousand to -8.16 per thousand with the mean of -9.75 per thousand and from -99.42 per thousand to -73.78 per thousand with the mean of -82.23 per thousand, respectively. There was a reversed spatial variation between δ¹8O and d. Relatively low values of δ¹8O with high values of d were found at the edge of the lake where the rivers drained into. Meanwhile, the values of d in the vertical profile varied little with depth, suggesting that the waters mixed sufficiently in the vertical direction. The d values increased at first and then decreased from east to west at different layers, but both increase and decrease exhibited different velocities, which were related to the river distribution, the locality of the lake and environmental conditions etc. River water and atmospheric precipitation were the main recharge sources of the Lashi Lake, and the melt-water of snow and ice might also be the supply resource. The δ¹8O values of lake water in glacier region decreased along the elevation (except for Lashi Lake), generally, this phenomenon was called "altitude effect". Moreover, high isotopic values of the lake water from non-glacier region were due to the evaporation effect.

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.

[An investigation on correlation of severity of brain injury with the expression of activin A and C-reactive protein].

OBJECTIVE: To determine the dynamic change in serum levels of activin A (ACTA) and C-reaction protein (CRP) in patients with brain injury, and to investigate its significance. METHODS: A prospective study was conducted. A total of 57 adult patients with brain injury occurring within 24 hours admitted to intensive care unit (ICU) of the First Affiliated Hospital of Zhengzhou University from August 2012 to June 2013 were enrolled. The patients were allocated into three groups according to their Glasgow coma scale (GCS) as follows: minor brain injury (GCS 13-15, n=17), moderate brain injury (GCS 9-12, n=18), heavy brain injury (GCS 3-8, n=22). The clinical and related laboratory data (reflecting the function of liver, kidney, lung, blood coagulability etc.) were recorded after ICU admission. At the same time, venous samples were collected on the day 1, 2, 3, 5, 7 after ICU admission for determination of ACTA with enzyme linked immunosorbent assay (ELISA) and CRP with fluorescence immunoassay technology. The correlation between ACTA and CRP was analyzed by linear correlation. The receiver operating characteristic (ROC) curve was plotted to analyze the accuracy of ACTA and CRP as a prognostic indicator in brain injury. Fifteen healthy persons were enrolled as the control group. RESULTS: The serum levels of ACTA and CRP in patients with minor, moderate and heavy brain injury were significantly higher than those in healthy control group [ACTA (µg/L): 23.96±3.55, 42.06±5.67, 52.32±4.46 vs. 13.66±2.45, all P<0.01; CRP (mg/L): 14.12±2.45, 23.05±2.85, 30.93±2.35 vs. 3.42±2.25, all P<0.01]. As the patients' condition worsening, levels of ACTA and CRP tended to elevate (all P<0.01). Levels of ACTA and CRP in minor, moderate and heavy brain injury groups were increased after ICU admission. On day 3, levels of serum ACTA and CRP reached the peak values [ACTA (µg/L):30.62±2.54, 51.35±2.55, 60.52±2.55; CRP (mg/L): 18.62±2.64, 30.35±2.25, 37.52±2.55], and then they lowered gradually. In minor and moderate brain injury groups, the levels of ACTA and CRP were slowly descending, and on day 7, they maintained at a lower level [ACTA (µg/L): 13.68±2.54, 37.74±2.55; CRP (mg/L): 6.68±2.44, 19.74±2.55]. On the contrary, the levels of ACTA and CRP in heavy brain injury group persistently maintained at a high level on day 7 [ACTA: (42.32±2.54) µg/L, CRP: (33.32±2.56) mg/L]. There were significant differences in ACTA and CRP among different degrees of brain injury groups (all P<0.01). There was a positive correlation between ACTA and CRP (r=0.958, P=0.007). ROC curve analysis showed that the sensitivity for brain injury prediction was 93.3% for ACTA with specificity 95.0%, area under ROC curve(AUC) 0.843, and the sensitivity for CRP was 89.1% with specificity 68.2%, AUC 0.723. CONCLUSIONS: Serum levels of ACTA and CRP in patients with brain injury are strongly correlated with the severity of the injury. Furthermore, ACTA is more sensitive than CRP in detecting early brain injury. Therefore, ACTA is a specific factor for detecting brain injury.