Z-nucleic acid sensor ZBP1 in sterile inflammation.

Z-DNA binding protein 1 (ZBP1), a cytosolic nucleic acid sensor for Z-form nucleic acids (Z-NA), can detect both exogenous and endogenous nucleic acids. Upon sensing of self Z-NA or exposure to diverse noxious stimuli, ZBP1 regulates inflammation by activating nuclear factor kappa B and interferon regulating factor 3 signaling pathways. In addition, ZBP1 promotes the assembly of ZBP1 PANoptosome, which initiates caspase 3-mediated apoptosis, mixed lineage kinase domain like pseudokinase (MLKL)-mediated necroptosis, and gasdermin D (GSDMD)-mediated pyroptosis (PANoptosis), leading to the release of various damage-associated molecular patterns. Thereby, ZBP1 is implicated in the development and progression of diverse sterile inflammatory diseases. This review outlines the expression, structure, and function of ZBP1, along with its dual roles in controlling inflammation and cell death to orchestrate innate immunity in sterile inflammation, especially autoimmune diseases, and cancers. ZBP1 has emerged as an attractive therapeutic target for various sterile inflammatory diseases.

HSF1 functions as a key defender against palmitic acid-induced ferroptosis in cardiomyocytes.

Palmitic acid (PA)-induced myocardial injury is considered a critical contributor to the development of obesity and type 2 diabetes mellitus (T2DM)-related cardiomyopathy. However, the underlying mechanism has not been fully understood. Here, we demonstrated that PA induced the cell death of H9c2 cardiomyoblasts in a dose- and time-dependent manner, while different ferroptosis inhibitors significantly abrogated the cell death of H9c2 cardiomyoblasts and primary neonatal rat cardiomyocytes exposed to PA. Mechanistically, PA decreased the protein expression levels of both heat shock factor 1 (HSF1) and glutathione peroxidase 4 (GPX4) in a dose- and time-dependent manner, which were restored by different ferroptosis inhibitors. Overexpression of HSF1 not only alleviated PA-induced cell death and lipid peroxidation but also improved disturbed iron homeostasis by regulating the transcription of iron metabolism-related genes (e.g., Fth1, Tfrc, Slc40a1). Additionally, PA-blocked GPX4 protein expression was evidently restored by HSF1 overexpression. Inhibition of endoplasmic reticulum (ER) stress rather than autophagy contributed to HSF1-mediated GPX4 expression. Moreover, GPX4 overexpression protected against PA-induced ferroptosis, whereas knockdown of GPX4 reversed the anti-ferroptotic effect of HSF1. Consistent with the in vitro findings, PA-challenged Hsf1-/- mice exhibited more serious ferroptosis, increased Slc40a1 and Fth1 mRNA expression, decreased GPX4 and TFRC expression and enhanced ER stress in the heart compared with Hsf1+/+ mice. Altogether, HSF1 may function as a key defender against PA-induced ferroptosis in cardiomyocytes by maintaining cellular iron homeostasis and GPX4 expression.

Integrated study of miR-215 promoting breast cancer cell apoptosis by targeting RAD54B.

BACKGROUND: MicroRNAs (miRNAs) are widely distributed in cells and participate in the regulation of the pathophysiological process of many diseases. As an important part of non-coding RNA, miRNAs regulate a variety of molecules and signal pathways in tumour cells. However, the evidence for regulatory mechanisms of specific miRNAs in tumour cells is still lacking. METHODS: In this study, we used transcriptomics analysis and integrated a variety of public databases to screen miRNAs that have key regulatory effects on breast cancer (BC). In addition, we used in vitro and in vivo studies and combined clinical samples to verify its regulatory mechanism. RESULTS: We found that among the specific miRNAs, miR-215-5p is a key regulator in BC. Compared with normal adjacent tissues, miR-215-5p has a lower expression level in BC tissues. Patients with high expression levels of miR-215-5p have a longer survival time. miR-215-5p can specifically target the 3'UTR region of RAD54B mRNA and down-regulate the expression of RAD54B, thereby inhibiting the proliferation of BC cells and promoting the apoptosis of BC cells. CONCLUSIONS: Finally, we found that miR-215-5p can be used as an important biomarker for BC. We have clarified its function and revealed its mechanism of targeting RAD54B mRNA for the first time. This may provide important clues to reveal the deeper molecular regulation mechanism of BC.

Metformin protects against ischaemic myocardial injury by alleviating autophagy-ROS-NLRP3-mediated inflammatory response in macrophages.

Myocardial ischaemia is usually accompanied by inflammatory response which plays a critical role in the myocardial healing and scar formation, while persistent inflammatory response contributes greatly to the myocardial remodeling and consequent heart failure. Metformin (Met), a widely used hypoglycemic drug, has increasingly been shown to exert remarkable cardioprotective effect on ischaemic myocardial injury such as acute myocardial infarction (AMI). However, the underlying mechanisms are still far from being fully understood. In this study, a mouse model of AMI was established through ligating the left anterior descending coronary artery (LAD), 100 mg/kg Met was given immediately after operation once daily for 3 days. It was demonstrated that Met effectively improved the cardiac haemodynamics (LVSP, LVEDP, +dp/dt, -dp/dt), diminished the infarct size, alleviated the disarrangement of myocardial cells and reduced the infiltration of inflammatory cells (macrophages, neutrophils and lymphocytes) in the heart of AMI mice. Mechanistically, Met decreased the expression of NLRP3 and enhanced the accumulation of LC3 puncta in F4/80-positive macrophages in the heart of AMI mice. Single cell suspension of cardiac macrophages was prepared from AMI mice and exhibited increased NLRP3 mRNA and protein expression. In contrast, Met decreased the expression of NLRP3 and p62, whereas increased the ratio of LC3II/LC3I. Additionally, both conditioned medium from H9c2 cardiomyocytes exposed to hydrogen peroxide (H9c2-H2O2-CM) and combination of mtDNA and ATP (mtDNA-ATP) increased the expression of NLRP3 and cleaved caspase-1 (p10) as well as intracellular ROS production in RAW264.7 macrophages, which were abrogated by Met treatment. Strikingly, chloroquine (CQ), 3-methyladenine (3-MA) and knockdown of autophagy-related gene (Atg5) abrogated the inhibitory effects of Met on H9c2-H2O2-CM and mtDNA-ATP-induced NLRP3 expression, release of IL-1ß and IL-18 as well as ROS production in RAW264.7 macrophages. Collectively, these findings suggest that Met protects against ischaemic myocardial injury through alleviating autophagy-ROS-NLRP3 axis-mediated inflammatory response in macrophages.

Systematic prediction of key genes for ovarian cancer by co-expression network analysis.

Ovarian cancer (OC) is the most lethal gynaecological malignancy, characterized by high recurrence and mortality. However, the mechanisms of its pathogenesis remain largely unknown, hindering the investigation of the functional roles. This study sought to identify key hub genes that may serve as biomarkers correlated with prognosis. Here, we conduct an integrated analysis using the weighted gene co-expression network analysis (WGCNA) to explore the clinically significant gene sets and identify candidate hub genes associated with OC clinical phenotypes. The gene expression profiles were obtained from the MERAV database. Validations of candidate hub genes were performed with RNASeqV2 data and the corresponding clinical information available from The Cancer Genome Atlas (TCGA) database. In addition, we examined the candidate genes in ovarian cancer cells. Totally, 19 modules were identified and 26 hub genes were extracted from the most significant module (R2  = .53) in clinical stages. Through the validation of TCGA data, we found that five hub genes (COL1A1, DCN, LUM, POSTN and THBS2) predicted poor prognosis. Receiver operating characteristic (ROC) curves demonstrated that these five genes exhibited diagnostic efficiency for early-stage and advanced-stage cancer. The protein expression of these five genes in tumour tissues was significantly higher than that in normal tissues. Besides, the expression of COL1A1 was associated with the TAX resistance of tumours and could be affected by the autophagy level in OC cell line. In conclusion, our findings identified five genes could serve as biomarkers related to the prognosis of OC and may be helpful for revealing pathogenic mechanism and developing further research.

Nucleolin promotes Ang II-induced phenotypic transformation of vascular smooth muscle cells by regulating EGF and PDGF-BB.

RNA-binding properties of nucleolin play a fundamental role in regulating cell growth and proliferation. We have previously shown that nucleolin plays an important regulatory role in the phenotypic transformation of vascular smooth muscle cells (VSMCs) induced by angiotensin II (Ang II). In the present study, we aimed to investigate the molecular mechanism of nucleolin-mediated phenotypic transformation of VSMCs induced by Ang II. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) inhibitors were used to observe the effect of Ang II on phenotypic transformation of VSMCs. The regulatory role of nucleolin in the phenotypic transformation of VSMCs was identified by nucleolin gene mutation, gene overexpression and RNA interference technology. Moreover, we elucidated the molecular mechanism underlying the regulatory effect of nucleolin on phenotypic transformation of VSMCs. EGF and PDGF-BB played an important role in the phenotypic transformation of VSMCs induced by Ang II. Nucleolin exerted a positive regulatory effect on the expression and secretion of EGF and PDGF-BB. In addition, nucleolin could bind to the 5' untranslated region (UTR) of EGF and PDGF-BB mRNA, and such binding up-regulated the stability and expression of EGF and PDGF-BB mRNA, promoting Ang II-induced phenotypic transformation of VSMCs.

VEGF-A promotes angiogenesis after acute myocardial infarction through increasing ROS production and enhancing ER stress-mediated autophagy.

Proangiogenesis is generally regarded as an effective approach for treating ischemic heart disease. Vascular endothelial growth factor (VEGF)-A is a strong and essential proangiogenic factor. Reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy are implicated in the process of angiogenesis. This study is designed to clarify the regulatory mechanisms underlying VEGF-A, ROS, ER stress, autophagy, and angiogenesis in acute myocardial infarction (AMI). A mouse model of AMI was successfully established by occluding the left anterior descending coronary artery. Compared with the sham-operated mice, the microvessel density, VEGF-A content, ROS production, expression of vascular endothelial cadherin, positive expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/Bip), and LC3 puncta in CD31-positive endothelial cells of the ischemic myocardium were overtly elevated. Moreover, VEGF-A exposure predominantly increased the expression of beclin-1, autophagy-related gene (ATG) 4, ATG5, inositol-requiring enzyme-1 (IRE-1), GRP78/Bip, and LC3-II/LC3-I as well as ROS production in the human umbilical vein endothelial cells (HUVECs) in a dose and time-dependent manner. Both beclin-1 small interfering RNA and 3-methyladenine treatment predominantly mitigated VEGF-A-induced tube formation and migration of HUVECs, but they failed to elicit any notable effect on VEGF-A-increased expression of GRP78/Bip. Tauroursodeoxycholic acid not only obviously abolished VEGF-A-induced increase of IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I, but also negated VEGF-A-induced tube formation and migration of HUVECs. Furthermore, N-acetyl- l-cysteine markedly abrogated VEGF-A-increased ROS production, IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I in the HUVECs. Taken together, our data demonstrated that increased spontaneous production of VEGF-A may induce angiogenesis after AMI through initiating ROS-ER stress-autophagy axis in the vascular endothelial cells.

Heat-shock protein B1 upholds the cytoplasm reduced state to inhibit activation of the Hippo pathway in H9c2 cells.

Heat-shock protein B1 (HSPB1) is a multifunctional protein that protects against oxidative stress; however, its function in antioxidant pathways remains largely unknown. Here, we sought to determine the roles of HSPB1 in H9c2 cells subjected to oxidative stress. Using nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis, we found that increased HSPB1 expression promoted the reduced states of glutathione reductase (GR), peroxiredoxin 1 (Prx1), and thioredoxin 1, whereas knockdown of HSPB1 attenuated these responses following oxidative stress. Increased HSPB1 expression promoted the activation of GR and thioredoxin reductase. Conversely, knockdown of HSPB1 attenuated these responses following oxidative stress. Importantly, overexpression of HSPB1 promoted the complex formation between HSPB1 and oxidized Prx1, leading to dephosphorylation of STE-mammalian STE20-like kinase 1 (MST1) in H9c2 cells exposed to H2 O 2 , whereas downregulation of HSPB1 induced the opposite results. Mechanistically, HSPB1 regulated the Hippo pathway by enhancing the dephosphorylation of MST1, resulting in reduced phosphorylation of LATS1 and Yes-associated protein (YAP). Moreover, HSPB1 regulated YAP-dependent gene expression. Thus, HSPB1 promoted the reduced state of endogenous antioxidant pathways following oxidative stress in H9c2 cells and improved the redox state of the cytoplasm via modulation of the Hippo signaling pathway.

[Research advance in chondrogenic differentiation of adipose-derived stem cells].

Articular cartilage lesions due to injury or other pathology are often difficult to heal, and the outcomes of the clinical treatment widely used today are far from satisfaction. Adipose-derived stem cells (ADSCs) are multipotent stem cells from adipose tissue. Tissue engineering based on the ability of ADSCs to differentiate into chondrocytes provides a new idea for the repair and regeneration of articular cartilage defects. The method for inducing the differentiation of ADSCs into chondrocytes in vitro who have been quite well established, which mainly include the use of growth factors and scaffolds to mimic the in vivo microenvironment, thereby promoting the differentiation of ADSCs into chondrocytes.

Nucleolin mediated pro-angiogenic role of Hydroxysafflor Yellow A in ischaemic cardiac dysfunction: Post-transcriptional regulation of VEGF-A and MMP-9.

Hydroxysafflor Yellow A (HSYA), a most representative ingredient of Carthamus tinctorius L., had long been used in treating ischaemic cardiovascular diseases in China and exhibited prominently anticoagulant and pro-angiogenic activities, but the underlying mechanisms remained largely unknown. This study aimed to further elucidate the pro-angiogenic effect and mechanism of HSYA on ischaemic cardiac dysfunction. A C57 mouse model of acute myocardial infarction (AMI) was firstly established, and 25 mg/kg HSYA was intraperitoneally injected immediately after operation and given once, respectively, each morning and evening for 2 weeks. It was found that HSYA significantly improved ischaemia-induced cardiac haemodynamics, enhanced the survival rate, alleviated the myocardial injury and increased the expressions of CD31, vascular endothelial growth factor-A (VEGF-A) and nucleolin in the ischaemic myocardium. In addition, HSYA promoted the migration and tube formation of human umbilical vein endothelial cells (HUVECs), enhanced the expressions of nucleolin, VEGF-A and matrix metalloproteinase-9 (MMP-9) in a dose- and time-dependent manner. However, down-regulation of nucleolin expression sharply abrogated the effect mentioned above of HSYA. Further protein-RNA coimmunoprecipitation and immunoprecipitation-RT-PCR assay showed that nucleolin binded to VEGF-A and MMP-9 mRNA and overexpression of nucleolin up-regulated the mRNA expressions of VEGF-A and MMP-9 in the HUVECs through enhancing the stability of VEGF-A and MMP-9 mRNA. Furthermore, HSYA increased the mRNA expressions of VEGF-A and MMP-9 in the extract of antinucleolin antibody-precipitated protein from the heart of AMI mice. Our data revealed that nucleolin mediated the pro-angiogenic effect of HSYA through post-transcriptional regulation of VEGF-A and MMP-9 expression, which contributed to the protective effect of HSYA on ischaemic cardiac dysfunction.

A minimally invasive approach to induce myocardial infarction in mice without thoracotomy.

Acute myocardial infarction (MI) is a leading cause of morbidity and mortality in the world. Traditional method to induce MI by left coronary artery (LCA) ligation is typically performed by an invasive approach that requires ventilation and thoracotomy, causing serious injuries in animals undergoing this surgery. We attempted to develop a minimally invasive method (MIM) to induce MI in mice. Under the guide of ultrasound, LCA ligation was performed in mice without ventilation and chest-opening. Compared to sham mice, MIM induced MI in mice as determined by triphenyltetrazolium chloride staining and Masson staining. Mice with MIM surgery revealed the reductions of LVEF, LVFS, E/A and ascending aorta (AAO) blood flow, and the elevations of S-T segment and serum cTn-I levels at 24 post-operative hours. The effects of MI induced by MIM were comparable to the effects of MI produced by traditional method in mice. Importantly, MIM increased the survival rates and caused less inflammation after the surgery of LCA ligation, compared to the surgery of traditional method. Further, MIM induced angiogenesis and apoptosis in ischaemic hearts from mice at postoperative 28 days as similarly as traditional method did. Finally, the MIM model was able to develop into the myocardial ischaemia/reperfusion model by using a balloon catheter with minor modifications. The MI model is able to be efficiently induced by a minimally invasive approach in mice without ventilation and chest-opening. This new model is potentially to be used in studying ischaemia-related heart diseases.

MiR-15b-5p Regulates Collateral Artery Formation by Targeting AKT3 (Protein Kinase B-3).

OBJECTIVE: To identify circulating microRNAs that are differentially expressed in severe coronary heart disease with well or poorly developed collateral arteries and to investigate their mechanisms of action in vivo and in vitro. APPROACH AND RESULTS: In our study, we identified a circulating microRNA, miR-15b-5p, with low expression that, nevertheless, characterized patients with sufficient coronary collateral artery function. Moreover, in murine hindlimb ischemia model, in situ hybridization identified that miR-15b-5p was specifically expressed in vascular endothelial cells of adductors in sham group and was remarkably downregulated after femoral artery ligation. Overexpressed miR-15b-5p significantly inhibited arteriogenesis and angiogenesis in mice. In vitro, both under basal and vascular endothelial growth factor stimulation, loss-of-function or gain-of-function studies suggested that miR-15b-5p significantly promoted or depressed the migration and proliferation of endothelial cells. We identified AKT3 (protein kinase B-3) as a direct target of miR-15b-5p. Interestingly, AKT3 deficiency by injection with Chol-AKT3-siRNA obviously suppressed arteriogenesis and the recovery of blood perfusion after femoral ligation in mice. CONCLUSIONS: These results indicate that circulating miR-15b-5p is a suitable biomarker for discriminating between patients with well-developed or poorly developed collaterals. Moreover, miR-15b-5p is a key regulator of arteriogenesis and angiogenesis, which may represent a potential therapeutic target for ischemic disease.

Intraintestinal administration of ulinastatin protects against sepsis by relieving intestinal damage.

BACKGROUND: Intravenous administration of ulinastatin (UTI), a broad spectral protease inhibitor, has been used on an experimental basis with severe sepsis patients in Asia. However, the effects of intraintestinal administration of UTI on intestinal and multiple organ damage in sepsis have not been reported. MATERIALS AND METHODS: In this study, we established a sepsis model in rats using cecal ligation and puncture and compared the effects of intraintestinal administration of UTI through an artificial fistula of duodenum and intraperitoneal administration of UTI on the pathophysiological changes of sepsis. RESULTS: It was found that intraintestinal administration of UTI (1) significantly improved the survival of septic rats, (2) significantly reduced the serum levels of tumor necrosis factor-α, interleukin-1ß, interleukin-6 as well as intestinal injury biomarkers diamine oxidase, D-lactic acid, and fluorescein isothiocyanate-dextran 4, and (3) significantly reduced intestinal microscopic and ultrastructural damage of septic rats. In addition, the protective effects of intraintestinal administration of UTI were significantly better than those of intraperitoneal administration of UTI. CONCLUSIONS: Overall, the present study for the first time revealed that intraintestinal administration of protease inhibitor UTI could reduce systemic inflammatory responses and multiple organ dysfunction in rats with sepsis by inhibiting autodigestion of intestinal wall due to proteases and provided new research ideas and experimental evidences for treatment of sepsis by intraintestinal administration of UTI.

Asymmetric dimethylarginine triggers macrophage apoptosis via the endoplasmic reticulum stress pathway.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is emerging as a key contributing factor in atherogenesis, a process in turn known to involve macrophage apoptosis. The aim of this study was to determine the effect of ADMA on macrophage apoptosis, with specific reference to the endoplasmic reticulum (ER) stress pathway. Macrophage apoptosis was evaluated by Annexin V- Propidium iodide (PI) and Hoechst 33258 staining assays. Levels of the ER stress marker glucose regulated protein 78 (GRP78) were characterized by western blot. Levels of the proapoptotic C/EBP-homologous protein (CHOP) were evaluated by western blot and reverse transcription polymerase chain reaction (RT-PCR), and caspase-4 activity was measured using a colorimetric protease assay kit. We observed ADMA dose- and time-dependent increases in macrophage levels of GRP78. Similar ADMA dose- and time-dependent increases were detected in intracellular caspase-4 activity and macrophage apoptosis, all of which were sensitive to treatment with siRNAs for protein kinase RNA-like ER kinase and inositol-requiring protein-1 (IRE1), the ADMA antagonist L-arginine, as well as inhibitors of eukaryotic translation initiation factor-2 (salubrinal), IRE1 (irestatin 9389), and c-Jun N-terminal kinase (SP600125). Our results indicate that ADMA triggers macrophage apoptosis via the ER stress pathway.

Sinomenine hydrochloride protects against polymicrobial sepsis via autophagy.

Sepsis, a systemic inflammatory response to infection, is the major cause of death in intensive care units (ICUs). The mortality rate of sepsis remains high even though the treatment and understanding of sepsis both continue to improve. Sinomenine (SIN) is a natural alkaloid extracted from Chinese medicinal plant Sinomenium acutum, and its hydrochloride salt (Sinomenine hydrochloride, SIN-HCl) is widely used to treat rheumatoid arthritis (RA). However, its role in sepsis remains unclear. In the present study, we investigated the role of SIN-HCl in sepsis induced by cecal ligation and puncture (CLP) in BALB/c mice and the corresponding mechanism. SIN-HCl treatment improved the survival of BALB/c mice that were subjected to CLP and reduced multiple organ dysfunction and the release of systemic inflammatory mediators. Autophagy activities were examined using Western blotting. The results showed that CLP-induced autophagy was elevated, and SIN-HCl treatment further strengthened the autophagy activity. Autophagy blocker 3-methyladenine (3-MA) was used to investigate the mechanism of SIN-HCl in vitro. Autophagy activities were determined by examining the autophagosome formation, which was shown as microtubule-associated protein light chain 3 (LC3) puncta with green immunofluorescence. SIN-HCl reduced lipopolysaccharide (LPS)-induced inflammatory cytokine release and increased autophagy in peritoneal macrophages (PM). 3-MA significantly decreased autophagosome formation induced by LPS and SIN-HCl. The decrease of inflammatory cytokines caused by SIN-HCl was partially aggravated by 3-MA treatment. Taken together, our results indicated that SIN-HCl could improve survival, reduce organ damage, and attenuate the release of inflammatory cytokines induced by CLP, at least in part through regulating autophagy activities.

[Protective effect of heart-fatty acid binding protein on lipopolysaccharide-induced cardiomyocyte damage].

OBJECTIVE: To observe the protective effect of heart-fatty acid binding protein (H-FABP) on lipopolysaccharide (LPS)-induced cardiomyocyte damage. METHODS: The cardiomyocytes were isolated and cultured from 1-3 days old neonatal rats. The specific siRNA or plasmid of H-FABP were transfected into cells to alter H-FABP expression, which was evaluated by Western blot and quantitative-PCR. LPS-induced cardiomyocyte damage and inflammation were estimated by detecting the contents of lactate dehydrogenase(LDH), TNF-α, and IL-1ß as well as cell viability. RESULTS: LPS treatment induced inflammation and cell damage indicated by a decrease in cell viability and an increase in LDH, TNF-α and IL-1ß in the medium. When H-FABP was downregulated by siRNA transfection, the LPS-induced inflammation and cell damage were augmented. In contrast, when H-FABP was overexpressed by pcDNA3.1-H-FABP transfection, the LPS-induced inflammation and cell damage were suppressed. CONCLUSION: H-FABP protects cardiomyocytes from LPS-induced inflammation and cell injury.

Mipu1 protects H9c2 myogenic cells from hydrogen peroxide-induced apoptosis through inhibition of the expression of the death receptor Fas.

Mipu1 (myocardial ischemic preconditioning upregulated protein 1), a novel rat gene recently identified in our lab, was expressed abundantly and predominantly in the brain and heart and upregulated in myocardium during myocardial ischemia/reperfusion in rats. In our previous study we found that Mipu1 was an evolutionarily conserved zinc finger-containing transcription factor. However, whether Mipu1 confers myocardial protection remains unknown. In this study, H9c2 myogenic cells were treated with hydrogen peroxide (H2O2) to simulate oxidative stress during myocardial ischemia-reperfusion injury. The expression of Mipu1 at mRNA and protein levels was detected by RT-PCR and Western blotting analysis. To study the effect of Mipu1 on apoptosis and expression of Fas induced by H2O2, full-length Mipu1 cDNA and Mipu1-RNAi plasmids were transiently transfected into H9c2 myogenic cells, and flow cytometry was used to quantitate the percentage of apoptotic cells. The expression of Fas was analyzed by Western blotting assay. The DNA binding and transcription activities of Mipu1 to the Fas promoter were detected by chromatin immunoprecipitation and luciferase reporter assays. The results showed that exposure of H9c2 myogenic cells to H2O2 resulted in a dose- and time-dependent increase in Mipu1 mRNA and protein levels; Mipu1 over-expression inhibited H2O2-induced apoptosis and upregulation of Fas induced by H2O2 in H9c2 myogenic cells; and knockdown of Mipu1 by RNAi promoted apoptosis and upregulation of Fas induced by H2O2. The chromatin immunoprecipition and reporter assays showed the DNA binding and transcription suppressor activities of Mipu1 to Fas promoter region. These results indicate that Mipu1 protected H9c2 myogenic cells from H2O2-induced apoptosis through inhibiting the expression of Fas.

Upregulation of PTPN1 aggravates endotoxemia-induced cardiac dysfunction through inhibiting mitophagy.

OBJECTIVES: To investigate the role of protein tyrosine phosphatase non-receptor type 1 (PTPN1) in mitophagy during sepsis and its underlying mechanisms and determine the therapeutic potential of PTPN1 inhibitors in endotoxemia-induced cardiac dysfunction. METHODS: A mouse model of endotoxemia was established by administering an intraperitoneal injection of lipopolysaccharide (LPS). The therapeutic effect of targeting PTPN1 was evaluated using its inhibitor Claramine (CLA). Mitochondrial structure and function as well as the expression of mitophagy-related proteins were evaluated. Rat H9c2 cardiomyocytes were exposed to mouse RAW264.7 macrophage-derived conditioned medium. Cryptotanshinone, a specific p-STAT3 (Y705) inhibitor, was used to confirm the role of STAT3 in PTPN1-mediated mitophagy following LPS exposure. Electrophoretic mobility shift and dual luciferase reporter assays were performed to discern the mechanisms by which STAT3 regulated the expression of PINK1 and PRKN. RESULTS: CLA alleviated LPS-induced myocardial damage, cardiac dysfunction, and mitochondrial injury and dysfunction in the mouse heart. PTPN1 upregulation exacerbated LPS-induced mitochondrial injury and dysfunction in H9c2 cardiomyocytes, but inhibited LPS-induced mitophagy. LPS promoted the interaction between PTPN1 and STAT3 and reduced STAT3 phosphorylation at Tyr705 (Y705), which was required to inhibit mitophagy by PTPN1. Upon LPS stimulation, PTPN1 negatively regulated the transcription of PINK1 and PRKN through dephosphorylation of STAT3 at Y705. STAT3 regulated the transcription of PINK1 and PRKN by binding to STAT3-responsive elements in their promoters. CONCLUSION: PTPN1 upregulation aggravates endotoxemia-induced cardiac dysfunction by impeding mitophagy through dephosphorylation of STAT3 at Y705 and negative regulation of PINK1 and PRKN transcription.

NINJ1: A Novel Sepsis Severity and Mortality Biomarker.

BACKGROUND: Multiple cell death modalities are implicated in sepsis pathobiology. However, the clinical relevance of NINJ1, a key mediator of plasma membrane rupture during lytic cell death, in sepsis progression and outcomes has remained poorly explored. METHODS: Circulating NINJ1 levels were measured in 116 septic ICU patients, 16 non-septic ICU controls, and 16 healthy controls. Comparative analysis of serum NINJ1 across these groups was performed. Correlations between NINJ1 and clinical disease severity scores (SOFA, APACHE II) as well as laboratory parameters were examined in the sepsis cohort. Furthermore, we assessed the prognostic performance of NINJ1 for predicting 28-day mortality in septic patients using receiver operating characteristic (ROC) analyses. RESULTS: Circulating NINJ1 levels were elevated in septic patients and positively correlated with sepsis severity scores. NINJ1 also showed positive correlations with liver injury markers (AST/ALT) and coagulation parameters (D-dimer, APTT, PT, TT) in sepsis. Further analysis using the ISTH overt DIC scoring system revealed an association between NINJ1 and sepsis-induced coagulopathy.ROC analysis demonstrated NINJ1 outperformed traditional inflammatory biomarkers PCT and CRP in predicting 28-day sepsis mortality, although its prognostic accuracy was lower than SOFA and APACHE II scores. Combining NINJ1 with SOFA improved mortality prediction from an AUC of 0.6843 to 0.773. CONCLUSIONS: Circulating NINJ1 serves as a novel sepsis biomarker indicative of disease severity, coagulopathy and mortality risk, and its integration with SOFA and APACHE II scores substantially enhances prognostic risk stratification. These findings highlight the prospective clinical utility of NINJ1 for sepsis prognostication and monitoring, warranting further validation studies to facilitate implementation.

MCP-1-induced protein promotes endothelial-like and angiogenic properties in human bone marrow monocytic cells.

Monocytic cells enhance neovascularization by releasing proangiogenic mediators and/or by transdifferentiating into endothelial-like cells. However, the mechanisms that govern this transdifferentiation process are largely unknown. Recently, monocyte chemotactic protein-1 (MCP-1)-induced protein (MCPIP) has been identified as a novel CCCH-type zinc-finger protein expressed primarily in monocytic cells. Here, we analyzed whether MCPIP might exert angiogenic effects by promoting differentiation of monocytic cells into endothelial cell (EC)-like phenotype. The expression of MCPIP increased during MCP-1-induced transdifferentiation in human bone marrow mononuclear cells (BMNCs). Knockdown of MCPIP with small interfering RNA (siRNA) abolished MCP-1-induced expression of EC markers Flk-1 and Tie-2 in human BMNCs. BMNCs transfected with MCPIP expression vector displayed EC-like morphology accompanied by downregulation of monocytic markers CD14 and CD11b, upregulation of EC markers Flk-1 and Tie-2, induction of cadherin (cdh)-12 and -19, activation of endoplasmic reticulum (ER) stress, and autophagy. Knockdown of cdh-12 or cdh-19 markedly inhibited MCPIP-induced enhancement of cell attachment and EC-marker expression. Inhibition of ER stress by tauroursodeoxycholate abolished MCPIP-induced expression of EC markers. Inhibition of autophagy by knockdown of Beclin-1 with siRNA or by an autophagy inhibitor 3'-methyladenine inhibited MCPIP-induced expression of EC markers. Expression of MCPIP in BMNCs enhanced uptake of acetylated low-density lipoprotein (acLDL), formation of EC-colony, incorporation of cells into capillary-like structure on Matrigel, and exhibited increased neovascularization in the ischemic hindlimb in mice. These results demonstrate that MCPIP may be an important regulator of inflammatory angiogenesis and provide novel mechanistic insights into the link between MCP-1 and cardiovascular diseases.