Mu-opioid receptor alleviated ferroptosis in hepatic ischemia-reperfusion injury via the HIF-1α/KCNQ1OT1 axis.

Ferroptosis is the ideal therapeutic target for hepatic ischemia and reperfusion injury (HIRI). The µ opioid receptor (MOR) is associated with ferroptosis in HIRI. We aimed to determine the ferroptosis-related therapeutic mechanism of MOR in HIRI. A model of HIRI was established in BALB/c mice. Primary hepatocytes isolated from mice were stimulated by hypoxia/reoxygenation (H/R). Changes in histopathology were determined by H&E staining. Alterations in ferroptosis were evaluated by malondialdehyde (MDA), iron, glutathione (GSH), ACSL4, GPX4, and mitochondrial morphology. ALT and AST were used to determine hepatic function. First, we found that hepatic ischemia/reperfusion (I/R) induced the destruction of hepatic tissue structure and dead hepatocytes and determined that ferroptosis occurred in vivo and in vitro. During HIRI, the expression levels of HIF-1α and KCNQ1OT1 were significantly upregulated. We demonstrated that sufentanil improved the damage in the liver and hepatocytes undergoing I/R. Importantly, sufentanil inhibited ferroptosis in HIRI. In addition, sufentanil downregulated the expression levels of HIF-1α and KCNQ1OT1 in HIRI. Increases in HIF-1α and KCNQ1OT1 reversed the role of sufentanil in ferroptosis and HIRI. Subsequently, we determined that HIF-1α could activate the transcription of KCNQ1OT1 by binding to its promoter. In addition, KCNQ1OT1 was demonstrated to enhance ACSL4 stability by interacting with SRSF1. Finally, we observed that KCNQ1OT1 downregulation protected hepatocytes from hepatic I/R and inhibited ferroptosis. KCNQ1OT1 upregulation aggravated ferroptosis and hepatic injury during I/R. However, decreases in ACSL4 and SRSF1 reversed the harmful role of KCNQ1OT1 upregulation in HIRI. MOR alleviated ferroptosis in HIRI via the HIF-1α/KCNQ1OT1 axis.

Silencing lncRNA KCNQ1OT1 reduced hepatic ischemia reperfusion injury-induced pyroptosis by regulating miR-142a-3p/HMGB1 axis.

BACKGROUND: Based on pre-existing evidence, KCNQ1OT1 has been pointed out to be closely related to myocardial and cerebral ischemia reperfusion injury diseases. Herein, the objective of our study is to probe into the potential function as well as the underlying mechanism of KCNQ1OT1 on hepatic ischemia reperfusion injury (HIRI). METHODS: Using C57BL/6 J mice and primary mouse hepatocytes were conducted to establish HIRI model in vivo and in vitro. Cell viability was examined using CCK-8 assay and EdU assay. Flow cytometric analysis was performed to evaluate the pyroptosis. Dual-luciferase reporter assay was employed to verify the interaction relationships. qRT-PCR and Western blot were adopted to analyze the mRNA and protein level. Histopathological alteration of liver tissue was evaluated by HE staining. Immunohistochemistry (IHC) was performed to measure NLRP3 and caspase 1. RESULTS: Our data revealed that KCNQ1OT1 expression was ascending in hepatic tissue of HIRI mouse. Moreover, deprivation of KCNQ1OT1 mitigated I/R-induced hepatic injury and pyroptosis in vivo. Further experiments demonstrated that silencing KCNQ1OT1 promoted proliferation and inhibited pyroptosis in hypoxia/reoxygenation (H/R)-induced primary mouse hepatocytes. Mechanistically, KCNQ1OT1 functioned as a competing endogenous RNA which sponged miR-142a-3p, therefore promoted HMGB1 expression to activate TLR4/NF-κB signaling pathway in HIRI. CONCLUSION: LncRNA KCNQ1OT1 elevated HMGB1 expression through binding to miR-142a-3p, thereby promoting pyroptosis in HIRI.

The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region.

Avian leukosis virus subgroup J (ALV-J) can cause neoplastic diseases in poultry and is still widely prevalent in China. Chicken telomerase reverse transcriptase (chTERT) is the core component of telomerase, which is closely related to the occurrence and development of tumors. Our previous studies showed that chTERT is overexpressed in ALV-J tumors, but the mechanism is still not completely clear. Therefore, this study aims to analyze the possible molecular mechanism of chTERT overexpression in ALV-J tumors from the perspective of DNA methylation and promoter mutation. Methylation sequencing of the chTERT amplicon showed that ALV-J replication promoted the methylation level of the chTERT promoter. And the methylation level of the chTERT promoter in ALV-J tumors was significantly higher than that in tumor-adjacent and normal tissues. Compared with the tumor-adjacent and normal tissues, the chTERT promoter in each ALV-J tumors tested had a mutation of -183 bp C > T, and 36.0% (9/25) of the tumors also had mutations of -184 bp T > C, -73 bp::GGCCC and -56 bp A > T in the chTERT promoter, which formed the binding sites for the transcription factors NFAT5, TFAP2A and ZEB1, respectively. The results of RT-qPCR and Western blotting showed that the occurrence of these mutations significantly increased the expression level of chTERT. In conclusion, this study demonstrated that the high expression of chTERT in ALV-J tumors is positively correlated with the level of hypermethylation and mutation in its promoter, which provides a new perspective for further research on the molecular mechanism of chTERT in ALV-J tumorigenesis.

Chicken telomerase reverse transcriptase promotes the tumorigenicity of avian leukosis virus subgroup J by regulating the Wnt/ß-catenin signaling pathway.

This research aimed to analyze the regulatory effect of chicken telomerase reverse transcriptase (chTERT) on the Wnt/ß-catenin signaling pathway and its effect on the tumorigenicity of avian leukosis virus subgroup J (ALV-J) through in vivo experiments. The chTERT eukaryotic expression plasmid and its recombinant lentivirus particles were constructed for in vivo transfection of chTERT to analyze the effect of chTERT continuously overexpressed in chickens on the tumorigenicity of ALV-J. During 156 days of the artificial ALV-J tumor-inducing process, 7 solid tumors developed in 3 chickens in the chTERT-overexpression group (n = 26*2) and no tumors developed in the control group (n = 26*2). Another 18 tumors induced by ALV-J were confirmed and collected from breeding poultry farms. And we confirmed that chTERT was significantly highly expressed in ALV-J tumors. The ELISA data suggested that the protein levels of ß-catenin and c-Myc in the chicken plasma of the chTERT-overexpressing group with ALV-J infected were consistently and significantly higher than those of the control group. Compared with that of the tumor-adjacent tissues, the activity of the Wnt/ß-catenin signaling pathway and expression of the c-Myc was significantly increased in ALV-J tumors. And the percentage of apoptosis in ALV-J tumors significantly lower than that in tumor-adjacent tissues. Immunohistochemistry, Western blot and RT-qPCR suggested that the replication level of ALV-J in tumors was significantly higher than that in tumor-adjacent tissues. This study suggests that chTERT plays a critical role in the tumorigenicity of ALV-J by enhancing the Wnt/ß-catenin signaling pathway, which will contribute to further elucidating the tumor-inducing mechanism of ALV-J.

Mutual regulation between chicken telomerase reverse transcriptase and the Wnt/ß-catenin signalling pathway inhibits apoptosis and promotes the replication of ALV-J in LMH cells.

This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/ß-catenin signalling pathway and its effects on cell growth and avian leukosis virus subgroup J (ALV-J) replication in LMH cells. First, LMH cells stably overexpressing the chTERT gene (LMH-chTERT cells) and corresponding control cells (LMH-NC cells) were successfully constructed with a lentiviral vector expression system. The results showed that chTERT upregulated the expression of ß-catenin, Cyclin D1, TCF4 and c-Myc. chTERT expression level and telomerase activity were increased when cells were treated with LiCl. When the cells were treated with ICG001 or IWP-2, the activity of the Wnt/ß-catenin signalling pathway was significantly inhibited, and chTERT expression and telomerase activity were also inhibited. However, when the ß-catenin gene was knocked down by small interfering RNA (siRNA), the changes in chTERT expression and telomerase activity were consistent with those in cells treated with ICG001 or IWP-2. These results indicated that chTERT and the Wnt/ß-catenin signalling pathway can be mutually regulated. Subsequently, we found that chTERT not only shortened the cell cycle to promote proliferation but also inhibited apoptosis by downregulating the expression of Caspase 3, Caspase 9 and BAX; upregulating BCL-2 and BCL-X expression; and promoting autophagy. Moreover, chTERT significantly enhanced the migration ability of LMH cells, upregulated the protein and mRNA expression of ALV-J and increased the virus titre. ALV-J replication promoted chTERT expression and telomerase activity.

Residues 140-142, 199-200, 222-223, and 262 in the Surface Glycoprotein of Subgroup A Avian Leukosis Virus Are the Key Sites Determining Tva Receptor Binding Affinity and Infectivity.

Subgroup A avian leukosis virus (ALV-A) invades cells through gp85-encoded surface glycoprotein (SU) via specifically recognizing the cellular receptor Tva. To identify the key residues of ALV-A SU that determine the Tva binding affinity and infectivity in DF-1 cells, a strategy of substituting corresponding residues of SU between ALV-A RSA and ALV-E ev-1 (using Tvb as the receptor) was adopted. A series of chimeric soluble gp85 proteins were expressed for co-immunoprecipitation (co-IP) analysis and blocking analysis of viral entry, and various recombinant viruses based on replication-competent avian retrovirus vectors containing Bryan polymerase (RCASBP) were constructed for transfection into DF-1 cells and measurement of the percentage of GFP-positive cells. The results revealed that the substitution of residues V138, W140, Y141, L142, S145, and L154 of host range region 1 (hr1), residues V199, G200, Q202, R222, and R223 of host range region 2 (hr2), and residue G262 of variable region 3 (vr3) reduced the viral infectivity and Tva binding affinity, which was similar to the effects of the -139S, -151N, -155PWVNPF, -201NFD, Δ214-215, and -266S mutations. Our study indicated that hr1 and hr2 contain the principal receptor interaction determinants, with new identified-vr3 also playing a key role in the receptor binding affinity of ALV-A.

Exogenous NAD(+) supplementation protects H9c2 cardiac myoblasts against hypoxia/reoxygenation injury via Sirt1-p53 pathway.

Nicotinamide adenine dinucleotide (NAD(+) ) not only transfers electrons in mitochondrial respiration, but also acts as an indispensable cosubstrate for Sirt1, the class III histone/nonhistone deacetylase. However, NAD(+) is depleted in myocardial ischemia/reperfusion (IR) injury. The objective of this study was to investigate the role of exogenous NAD(+) supplementation in hypoxia/reoxygenation (HR)-stressed H9c2 cardiac myoblasts. Firstly, the effects of distinct treating time points and doses of NAD(+) supplementation on the viability of HR-stressed H9c2 cells were detected. Secondly, intracellular NAD(+) levels in HR-stressed H9c2 cells at various extracellular NAD(+) concentrations were determined. Thirdly, the role of NAD(+) supplementation in HR-induced cell apoptosis and its relevance to Sirtuin 1-p53 pathway were investigated. Exogenous NAD(+) supplementation elevated intracellular NAD(+) level and reduced HR-induced cell death in both time- and concentration-dependent manners. It appeared that NAD(+) supplementation exerted the greatest protection when extracellular concentration ranged from 500 to 1000 µm and when NAD(+) was added immediately after reoxygenation began. NAD(+) replenishment restored Sirt1 activity, reduced the acetylation level of p53 (Lys373 & 382), and attenuated cell apoptosis in HR-stressed H9c2 cells, whereas inhibition of Sirt1 activity alleviated the effects of NAD(+) replenishment. These results indicated that exogenous NAD(+) supplementation attenuated HR-induced cell apoptosis, which was at least partly mediated by restoring Sirt1 activity and subsequently inhibiting p53 activity via deacetylating p53 at lysine 373 and 382.

Distinct effects of Nampt inhibition on mild and severe models of lipopolysaccharide-induced myocardial impairment.

The study aimed to investigate the variance of myocardial and serum Nampt levels and the role of Nampt inhibition by FK866 in relatively mild endotoxemia- and severe endotoxemia-induced myocardial injury. Different doses of LPS were injected intraperitoneally to establish relatively mild endotoxemia (4mg/kg) and severe endotoxemia (20mg/kg). FK866 (10mg/kg b.w.) was injected intraperitoneally at hour one after LPS injection. The hearts were isolated from rats at hour six after LPS treatment and mounted in a Langendorff setup to measure cardiac function. Myocardial expression of Nampt was determined with immunohistochemistry assay and western blot. Serum Nampt level and myocardial TNF-α level were determined with ELISA. The myocardial level of TNF-α mRNA was detected with RT-PCR. The degree of myocardial oxidative injury was reflected by measuring lipid peroxidation and GSH/GSSG ratio. The apoptosis of cardiomyocytes was determined with detecting caspase-3 activity and with TUNEL assay. Myocardial expression of Nampt was markedly increased in 4mg/kg LPS-induced endotoxemia but decreased in 20mg/kg LPS-induced endotoxemia. Serum Nampt level was consistently up-regulated in both severities of endotoxemia. Inhibition of Nampt by FK866 reduced myocardial inflammation, oxidative injury and apoptosis of cardiomyocytes and improved cardiac function in 4mg/kg LPS-induced endotoxemia. In 20mg/kg LPS-induced endotoxemia, FK866 reduced myocardial inflammation, exacerbated apoptosis of cardiomyocytes, and failed to attenuate myocardial oxidative injury and cardiac dysfunction. In conclusion, the variance of myocardial Nampt expression may be associated with severities of endotoxemia. Nampt may play complicated roles and consequently application of Nampt inhibition should be critically evaluated in endotoxemia-induced myocardial impairment.

[Effect of hydrogen inhalation on p38 MAPK activation in rats with lipopolysaccharide- induced acute lung injury].

OBJECTIVE: To investigate the effect of hydrogen inhalation on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the underlying molecular mechanisms. METHODS: Thirty-two male SD rats were randomly divided into 4 groups (n=8), namely the normal saline group (SA), saline with 2% hydrogen gas inhalation group (SH group), ALI group, and ALI with hydrogen inhalation group (LH group). In the two ALI groups, ALI was induced by intraperitoneal injection of 15 mg/kg LPS. Treatments with inhalation of 2% hydrogen gas for 6 h was administered after the injection of LPS or saline. The concentrations of tumor necrosis factor-α (TNF-α) in the lung tissue and serum were examined with ELISA. The expression of p38 MAPK in the lung tissue was detected by Western blotting.. RESULTS: Hydrogen inhalation decreased the expression of p-p38 MAPK in the lung tissue, and significantly reduced TNF-α content in the lung tissue and serum of rats with ALI. CONCLUSION: Hydrogen inhalation can decrease the expression of TNF-α in the lung tissue and serum, and this effect may be related with reduced p38 MAPK expression and inhibition of p38 MAPK activation.