Gastroesophageal reflux disease and atrial fibrillation: a bidirectional Mendelian randomization study.

Background: In observational studies, gastroesophageal reflux disease (GERD) is linked to atrial fibrillation (AF). It is uncertain whether the relationship is due to GERD-induced AF or GERD caused by AF, or confusion with factors related to GERD and AF such as obesity and sleep-disordered breathing. We applied bidirectional Mendelian randomization (MR), in which genetic variations are used as instrumental variables to resolve confounding and reverse causation issues, to determine the causal effect between GERD and AF. Methods: Using summary data from the GERD and AF genome-wide association study (GWAS), a bidirectional MR was performed to estimate the causative impact of GERD on AF risk and AF on GERD risk. The GWAS of GERD meta-analysis comprised 78707 cases and 288734 controls. GWAS summary data for AF, including 45766 AF patients and 191924 controls, were used to genetically predicted AF. The inverse variance weighted (IVW) method was the major MR approach used. MR-PRESSO was implemented to detect heterogeneity and correct the effect of outliers. Weighted median and MR-Egger regression were applied to test heterogeneity and pleiotropy. Results: The genetic instruments of GERD related to increasing the risk of AF, with an OR of 1.339 (95% CI: 1.242-1.444, p < 0.001). However, after removing the outlier 8 SNPs, genetically predicted AF was not associated with an elevated risk of GERD (p = 0.351). Conclusions: Our result suggested that GERD had a causal effect on AF. However, no evidence was identified that AF elevated the risk of GERD.

The Renoprotective and Anti-Inflammatory Effects of Human Urine-Derived Stem Cells on Acute Kidney Injury Animals.

BACKGROUND: Acute Kidney Injury (AKI) is defined as a sudden loss of kidney function, which is often caused by drugs, toxins, and infections. The large spectrum of AKI implies diverse pathophysiological mechanisms. In many cases, AKI can be lethal, and kidney replacement therapy is frequently needed. However, current treatments are not satisfying. Developing novel therapies for AKI is essential. Adult stem cells possess regenerative ability and play an important role in medical research and disease treatment. METHODS: In this study, we isolated and characterized a distinct human urine-derived stem cell, which expressed both proximal tubular cell and mesenchymal stem cell genes as well as certain unique genes. RESULTS: It was found that these cells exhibited robust protective effects on tubular cells and anti- inflammatory effects on macrophages in vitro. In an ischemia-reperfusion-induced acute kidney injury NOD-SCID mouse model, transplantation of USCs significantly protected the kidney morphology and functions in vivo. CONCLUSION: In summary, our results highlighted the effectiveness of USCs in protecting from PTC injury and impeding macrophage polarization, as well as the secretion of pro-inflammatory interleukins, suggesting the potential of USCs as a novel cell therapy in AKI.

Causal relationship between particulate matter 2.5 (PM2.5), PM2.5 absorbance, and COVID-19 risk: A two-sample Mendelian randomisation study.

Background: Several observational studies reported on the association between particulate matter ≤2.5µm (PM2.5) and its absorbance with coronavirus (COVID-19), but none use Mendelian randomisation (MR). To strengthen the knowledge on causality, we examined the association of PM2.5 and its absorbance with COVID-19 risk using MR. Methods: We selected genome-wide association study (GWAS) integration data from the UK Biobank and IEU Open GWAS Project for two-sample MR analysis. We used inverse variance weighted (IVW) and its multiple random effects and fixed effects alternatives to generally predict the association of PM2.5 and its absorbance with COVID-19, and six methods (MR Egger, weighted median, simple mode, weighted mode, maximum-likelihood and MR-PRESSO) as complementary analyses. Results: MR results suggested that PM2.5 absorbance was associated with COVID-19 infection (odds ratio (OR) = 2.64; 95% confidence interval (CI) = 1.32-5.27, P = 0.006), hospitalisation (OR = 3.52; 95% CI = 1.05-11.75, P = 0.041) and severe respiratory symptoms (OR = 28.74; 95% CI = 4.00-206.32, P = 0.001) in IVW methods. We observed no association between PM2.5 and COVID-19. Conclusions: We found a potential causal association of PM2.5 absorbance with COVID-19 infection, hospitalisation, and severe respiratory symptoms using MR analysis. Prevention and control of air pollution could help delay and halt the negative progression of COVID-19.

A national cross-sectional analysis of dietary copper intake and abdominal aortic calcification in the US adults: NHANES 2013-2014.

BACKGROUND AND AIMS: Copper is an essential dietary element with a crucial role in physiological regulation. However, the relationship between dietary copper intake and abdominal aortic calcification (AAC) remains uncertain. METHODS AND RESULTS: This study encompassed a cohort of 2535 adults aged over 40 years, derived from the National Health and Nutrition Examination Survey 2013-2014. Dietary copper intake from both food sources and supplements was assessed through two 24-h dietary recall interviews. AAC was measured by dual-energy X-ray absorptiometry and classified into 3 groups using the Kauppila score system. Multivariable logistic regression models were constructed to evaluate the association between dietary copper intake and AAC. Among the participants, a total of 771 individuals (30.4%) were diagnosed with AAC, of which 239 (9.4%) exhibited severe AAC. Higher dietary copper intake was significantly associated with a lower incidence of severe AAC. Specifically, for each 1 mg/day increase in dietary copper intake, the incidence of severe AAC decreased by 38% (odds ratios [OR] 0.62, 95% confidence intervals [CI] 0.39-0.98) after adjustment for relevant covariates. Moreover, individuals in the third tertile of copper intake had a 37% lower incidence of AAC compared to those in the first tertile [OR 0.63, 95% CI (0.43-0.95)]. However, no significant associations were found between supplemental copper intake or serum copper levels and AAC. CONCLUSIONS: This study demonstrates that lower dietary copper intake, rather than serum copper levels or supplement copper intake, is significantly associated with a higher prevalence of AAC in adults ≥40 years old in the United States.

Changes of plasma Rap1A levels in patients with in-stent restenosis after percutaneous coronary intervention and the underlying mechanisms. / ç»çš®å† çŠ¶åŠ¨è„‰ä»‹å ¥æ²»ç–—æœ¯åŽæ”¯æž¶å† å†ç‹­çª„æ‚£è€ è¡€æµ†Rap1Aæ°´å¹³å˜åŒ–åŠå ¶æœºåˆ¶ï¼ˆè‹±æ–‡ï¼‰.

OBJECTIVES: Percutaneous coronary intervention (PCI) is one of the most important treatments for coronary artery disease (CAD). However, in-stent restenosis (ISR) after PCI is a serious complication without effective measures for prevention and treatment. This study aims to investigate the Ras-related protein 1A (Rap1A) level in ISR patients and in the tumor necrosis factor-α (TNF-α)-induced inflammatory injury model of human umbilical vein endothelial cells (HUVECs), to explore the role of Rap1A in regulating TNF-α-induced inflammation in HUVECs and to provide a new potential target for ISR prevention and treatment. METHODS: A total of 60 CAD patients, who underwent PCI between December 2020 and July 2022 from the Department of Cardiovascular Medicine of Xiangya Hospital, Central South University, and re-examined coronary angiography (CAG) 1 year after the operation, were included. After admission, 27 patients were diagnosed with ISR and 33 patients were diagnosed with non-in-stent restenosis (non-ISR) according to the CAG. Clinical data were collected, and the plasma Rap1A level was determined by enzyme linked immunosorbent assay (ELISA). In cell experiments, an inflammatory injury model was established with TNF-α treatment (10 ng/mL, 24 h) in HUVECs. The mRNA and protein expression levels of Rap1A, interlukin-6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) were measured by real-time reverse transcription PCR and Western blotting. Small interfering RNA (siRNA) was used to explore the role of Rap1A in regulating TNF-α-induced inflammation in HUVECs. RESULTS: Compared with the non-ISR patients, a higher proportion of ISR patients had a history of smoking (P=0.005) and diabetes (P=0.028), and higher levels of glycosylated hemoglobin (HbA1c) (P=0.012), low-density lipoprotein cholesterol (LDL-c) (P=0.014), and hypersensitive C-reactive protein (hs-CRP) (P=0.027). The remaining projects did not show significant differences (all P>0.05). The plasma level of Rap1A in the ISR group was significantly higher than that in the non-ISR group [942.14 (873.28 to 1 133.81) µg/mL vs 886.93 (812.61 to 930.98) µg/mL; P=0.004]. Diabetes, LDL-c, and Rap1A were risk factors for ISR by univariate logistic regression analysis (all P<0.05). The mRNA and protein expression levels of inflammatory factors IL-6 and VCAM-1 were increased in HUVECs after 10 ng/mL TNF-α treatment for 24 h compared with the control group (all P<0.05), while the mRNA and protein levels of Rap1A were increased (both P<0.05). After inhibition of Rap1A in HUVECs, the mRNA and protein expression levels of IL-6 and VCAM-1 were significantly decreased (all P<0.05). CONCLUSIONS: The plasma Rap1A level was significantly elevated in patients with ISR, suggesting that Rap1A may be a potential biomarker for predicting ISR. In the TNF-α- induced HUVECs inflammatory injury model, the expression level of Rap1A was increased. The level of TNF-α-induced endothelial cell inflammation was decreased after inhibition of Rap1A expression, suggesting that Rap1A may be a potential target for the treatment of endothelial cell inflammation in ISR.

Identification of PFKFB2 as a key gene for the transition from acute to old myocardial infarction in peripheral blood.

Objective: This study aims to analyze the gene expression profile of peripheral blood in different stages of myocardial infarction (MI) by transcriptome sequencing, and to study the gene expression characteristics of peripheral blood after MI. Methods: Differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) were used to identify genes and modules associated with old myocardial infarction (OMI). Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation were applied to analyze the potential functions of genes. Hub genes were identified by Random Forest Classifier. CIBERSORT was used to provide an estimate of the abundance of 22 immune cells in peripheral blood. Quantitative polymerase chain reaction (qPCR) was used to detect gene expression levels in clinical samples. The cellular components (CC) of peripheral blood were counted by an automatic hematology analyzer. Results: Through differential gene analysis and co-expression network analysis, 11 candidate genes were obtained. A random forest classifier identified 10 hub genes. Immune cell distribution of peripheral blood was found that T cell CD4 memory resting, NK cells resting, Dendritic cells activated, Mast cells resting, Monocytes and Neutrophils were correlated with OMI. Spearman correlation analysis found that PFKFB2 is related to the above immune cells. Low expression of PFKFB2 in peripheral blood of OMI was detected in clinical samples, and the relationship between PFKFB2 and peripheral blood immune cell counts was analyzed, which showed monocytes were associated with PFKFB2 in our study. Conclusion: PFKFB2 was low expressed in OMI, and related to the distribution of immune cells. PFKFB2 may play a key role in reflecting the transition from AMI to OMI, and predicting the distribution of immune cells, which provided a new perspective for improving myocardial fibrosis and adverse remodeling.

Levels of plasma Quaking and cyclooxygenase-2 predict in-stent restenosis in patients with coronary artery disease after percutaneous coronary intervention. / 血浆Quakingå’ŒçŽ¯æ°§åˆé ¶-2æ°´å¹³é¢„æµ‹å† å¿ƒç— æ‚£è€ è¡Œç»çš®å† çŠ¶åŠ¨è„‰ä»‹å ¥æœ¯åŽæ”¯æž¶å† å†ç‹­çª„.

OBJECTIVES: Percutaneous coronary intervention (PCI) is one of the important methods for the treatment of coronary artery disease (CAD). In-sent restenosis (ISR) after PCI for patients suffered from CAD is considered to be an essential factor affecting long-term outcomes and prognosis of this disease. This study aims to investigate the correlation between plasma Quaking (QKI) and cyclooxygenase-2 (COX-2) levels and ISR in patients with CAD. METHODS: A total of 218 consecutive CAD patients who underwent coronary angiography and coronary arterial stenting from September 2019 to September 2020 in the Department of Cardiology of Xiangya Hospital of Central South University were enrolled in this study, and 35 matched individuals from the physical examination center were served as a control group. After admission, clinical data of these 2 groups were collected. Plasma QKI and COX-2 levels were measured by enzyme linked immunosorbent assay (ELISA). Follow-up angiography was performed 12 months after PCI. CAD patients were divided into a NISR group (n=160) and an ISR group (n=58) according to the occurrence of ISR based on the coronary angiography. The clinical data, coronary angiography, and stent features between the NISR group and the ISR group were compared, and multivariate logistic regression was used to explore the factors influencing ISR. The occurrence of major adverse cardiovascular events (MACE) 1 year after operation was recorded. Fifty-eight patients with ISR were divided into an MACE group (n=24) and a non-MACE group (n=34), classified according to the occurrence of MACE, and the plasma levels of QKI and COX-2 were compared between the 2 groups. Receiver operating characteristic (ROC) curves were utilized to analyze the diagnostic value of plamsa levels of QKI and COX-2 for ISR and MACE occurrences in patients after PCI. RESULTS: Compared with control group, plasma levels of QKI and COX-2 in the CAD group decreased significantly (all P<0.001). Compared with the NISR group, the plasma levels of QKI and COX-2 also decreased obviously in the ISR group (all P<0.001), while the levels of high sensitivity C-reactive protein (hs-CRP) and glycosylated hemoglobin (HbAlc) significantly increased (all P<0.001). The level of COX-2 was negatively correlated with hs-CRP (r=-0.385, P=0.003). Multivariate logistic regression analysis showed that high level of plasma QKI and COX-2 were protective factors for in-stent restenosis after PCI, while hs-CRP was a risk factor. ROC curve analysis showed that the sensitivity and specificity of plasma QKI for evaluating the predictive value of ISR were 77.5% and 66.5%, respectively, and the sensitivity and specificity of plasma COX-2 for evaluating the predictive value of ISR were 80.0% and 70.7%, respectively. The sensitivity and specificity of plasma QKI combined with COX-2 for evaluating the predictive value of ISR were 81.3% and 74.1%, respectively. The sensitivity and specificity of plasma QKI for evaluating the prognosis of ISR were 75.0% and 64.7%, respectively. The sensitivity and specificity of plasma COX-2 for evaluating the prognosis of ISR were 75.0% and 70.6%, respectively. The sensitivity and specificity of plasma QKI combined with COX-2 for prognostic evaluation of ISR were 81.7% and 79.4%, respectively. The sensitivity and specificity of plasma COX-2 combined with QKI for evaluating ISR and MACE occurrences in patients after PCI were better than those of COX-2 or QKI alone (P<0.001). CONCLUSIONS: High level of plasma QKI and COX-2 might be a protective factor for ISR, which can also predict ISR patient's prognosis.

Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing.

In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.

Correlation of plasma TSG-6 with cardiac function, myocardial fibrosis, and prognosis in dilated cardiomyopathy patients with heart failure. / 血浆TSG-6æ°´å¹³ä¸Žæ‰©å¼ åž‹å¿ƒè‚Œç— å¿ƒåŠ›è¡°ç«­æ‚£è€ å¿ƒåŠŸèƒ½ã€å¿ƒè‚Œçº¤ç»´åŒ–åŠé¢„åŽçš„ç›¸å ³æ€§.

OBJECTIVES: Tumor necrosis factor α stimulated gene 6 (TSG-6) protein is an inflammation-inducing protein. In recent years, TSG-6 protein has been found to play an anti-inflammatory and anti-fibrosis role in a variety of disease models. The level of TSG-6 protein in circulating blood is considered to be a biological indicator for the evaluation of acute coronary syndrome, severe infection, and other diseases, and it is closely related to the prognosis. The clinical correlation between TSG-6 protein and dilated cardiomyopathy (DCM) patients with heart failure has not been reported. This study aims to investigate the changes of plasma TSG-6 protein levels in cardiomyopathy patients with heart failure and its correlation with cardiac function, myocardial fibrosis, and prognosis. METHODS: Based on the prospective studies, a number of 90 DCM patients with heart failure were selected as a DCM heart failure group from Dec.1, 2019 to Sept.1, 2020. Thirty-nine healthy people were served as a control group. Plasma TSG-6, Collagen Ⅰ, Collagen III, and α-smooth muscle actin (α-SMA) were measured with ELISA test. Echocardiography was used to evaluate the structure and function of the heart. DCM patients with heart failure were followed up for 3 months. The patients were assigned into 2 groups according to whether they had major adverse cardiovascular events (MACE). The general clinical data, plasma TSG-6, Collagen Ⅰ, Collagen III, and α-SMA protein levels were compared between the control group and the DCM heart failure group. At the same time, the correlation between plasma TSG-6 protein level and cardiac function grade, myocardial fibrosis or prognosis of patients in the DCM heart failure group was analyzed. RESULTS: Compared with the control group, the heart rate, TSG-6, Collagen Ⅰ, Collage III, α-SMA, hemoglobin, atrial natriuretic peptide (NT-proBNP), hypersensitive C-reactive protein, aspartate aminotransferase, serum creatinine, lactate dehydrogenase, and left ventricular end diastolic diameter (LVEDD) increased significantly (all P<0.001). High-density lipoprotein, left ventricular short axis shortening rate (LVFS), and left ventricular ejection fraction (LVEF) decreased significantly in the DCM heart failure group (all P<0.001). Plasma levels of TSG-6 were positively correlated with NT-proBNP, Collagen Ⅰ, Collagen III, α-SMA, and LVEDD (all P<0.001), while they were negatively correlated with LVFS and LVEF (all P<0.001). With the increase of NYHA heart function classification, plasma levels of TSG-6, Collagen Ⅰ, Collagen III, and α-SMA increased significantly (all P<0.001). The increases in plasma levels of NT-proBNP and TSG-6 was associated with poor prognosis in DCM patients with heart failure (all P<0.05). The sensitivity and specificity of plasma NT-proBNP for evaluating the prognosis of DCM heart failure were 76.2% and 68.1%, respectively. The sensitivity and specificity of plasma TSG-6 for evaluating the prognosis of DCM heart failure were 95.2% and 66.7%, respectively. The sensitivity and specificity of plasma TSG-6 combined with NT-proBNP for prognostic evaluation of DCM heart failure were 85.7% and 81.2%, respectively. The specificity of plasma TSG-6 combined with NT-proBNP for the prognosis of heart failure was better than that of NT-proBNP or TSG-6 alone (P<0.001). CONCLUSIONS: The plasma levels TSG-6 in DCM patients with heart failure increase significantly, and the plasma levels TSG-6 could be used as a new predictor for cardiac function, myocardial fibrosis, and prognosis.

Transcriptomic Changes in Mouse Bone Marrow-Derived Macrophages Exposed to Neuropeptide FF.

Neuropeptide FF (NPFF) is a neuropeptide that regulates various biological activities. Currently, the regulation of NPFF on the immune system is an emerging field. However, the influence of NPFF on the transcriptome of primary macrophages has not been fully elucidated. In this study, the effect of NPFF on the transcriptome of mouse bone marrow-derived macrophages (BMDMs) was explored by RNA sequencing, bioinformatics, and molecular simulation. BMDMs were treated with 1 nM NPFF for 18 h, followed by RNA sequencing. Differentially expressed genes (DEGs) were obtained, followed by GO, KEGG, and PPI analysis. A total of eight qPCR-validated DEGs were selected as hub genes. Subsequently, the three-dimensional (3-D) structures of the eight hub proteins were constructed by Modeller and Rosetta. Next, the molecular dynamics (MD)-optimized 3-D structure of hub protein was acquired with Gromacs. Finally, the binding modes between NPFF and hub proteins were studied by Rosetta. A total of 2655 DEGs were obtained (up-regulated 1442 vs. down-regulated 1213), and enrichment analysis showed that NPFF extensively regulates multiple functional pathways mediated by BMDMs. Moreover, the 3-D structure of the hub protein was obtained after MD-optimization. Finally, the docking modes of NPFF-hub proteins were predicted. Besides, NPFFR2 was expressed on the cell membrane of BMDMs, and NPFF 1 nM significantly activated NPFFR2 protein expression. In summary, instead of significantly inhibiting the expression of the immune-related gene transcriptome of RAW 264.7 cells, NPFF simultaneously up-regulated and down-regulated the gene expression profile of a large number of BMDMs, hinting that NPFF may profoundly affect a variety of cellular processes dominated by BMDMs. Our work provides transcriptomics clues for exploring the influence of NPFF on the physiological functions of BMDMs.

Prolactin-Releasing Peptide Differentially Regulates Gene Transcriptomic Profiles in Mouse Bone Marrow-Derived Macrophages.

Prolactin-releasing Peptide (PrRP) is a neuropeptide whose receptor is GPR10. Recently, the regulatory role of PrRP in the neuroendocrine field has attracted increasing attention. However, the influence of PrRP on macrophages, the critical housekeeper in the neuroendocrine field, has not yet been fully elucidated. Here, we investigated the effect of PrRP on the transcriptome of mouse bone marrow-derived macrophages (BMDMs) with RNA sequencing, bioinformatics, and molecular simulation. BMDMs were exposed to PrRP (18 h) and were subjected to RNA sequencing. Differentially expressed genes (DEGs) were acquired, followed by GO, KEGG, and PPI analysis. Eight qPCR-validated DEGs were chosen as hub genes. Next, the three-dimensional structures of the proteins encoded by these hub genes were modeled by Rosetta and Modeller, followed by molecular dynamics simulation by the Gromacs program. Finally, the binding modes between PrRP and hub proteins were investigated with the Rosetta program. PrRP showed no noticeable effect on the morphology of macrophages. A total of 410 DEGs were acquired, and PrRP regulated multiple BMDM-mediated functional pathways. Besides, the possible docking modes between PrRP and hub proteins were investigated. Moreover, GPR10 was expressed on the cell membrane of BMDMs, which increased after PrRP exposure. Collectively, PrRP significantly changed the transcriptome profile of BMDMs, implying that PrRP may be involved in various physiological activities mastered by macrophages.

The Emerging Role of Macrophages in Immune System Dysfunction under Real and Simulated Microgravity Conditions.

In the process of exploring space, the astronaut's body undergoes a series of physiological changes. At the level of cellular behavior, microgravity causes significant alterations, including bone loss, muscle atrophy, and cardiovascular deconditioning. At the level of gene expression, microgravity changes the expression of cytokines in many physiological processes, such as cell immunity, proliferation, and differentiation. At the level of signaling pathways, the mitogen-activated protein kinase (MAPK) signaling pathway participates in microgravity-induced immune malfunction. However, the mechanisms of these changes have not been fully elucidated. Recent studies suggest that the malfunction of macrophages is an important breakthrough for immune disorders in microgravity. As the first line of immune defense, macrophages play an essential role in maintaining homeostasis. They activate specific immune responses and participate in large numbers of physiological activities by presenting antigen and secreting cytokines. The purpose of this review is to summarize recent advances on the dysfunction of macrophages arisen from microgravity and to discuss the mechanisms of these abnormal responses. Hopefully, our work will contribute not only to the future exploration on the immune system in space, but also to the development of preventive and therapeutic drugs against the physiological consequences of spaceflight.

Cellular processes involved in RAW 264.7 macrophages exposed to NPFF: A transcriptional study.

Neuropeptide FF (NPFF) is a neuropeptide that modulates various physiological processes. The regulatory role of NPFF in the immune and inflammatory response is currently being revealed. However, the effect of NPFF at the transcriptome level in macrophages has not been fully elucidated. Here, the impact of NPFF on gene expression at the transcriptome level of RAW 264.7 cells was investigated by RNA-seq. RAW 264.7 macrophages were treated with NPFF (1 nM) for 18 h, followed by RNA-seq examination. Differentially expressed genes (DEGs) were acquired, followed by GO, KEGG, and PPI analysis. A total of eight qPCR-verified DEGs were obtained. Next, three-dimensional models of the eight hub proteins were constructed by using homology modeling with Modeller (9v23). Finally, molecular dynamics simulation (300 ns) was performed with GROMACS 2018.2 to investigate the structural characteristics of these hub proteins. NPFF had no detectable effect on the morphology of RAW264.7 cells. A total of 211 DEGs were acquired, and an enrichment study demonstrated that the immune response-related pathway was significantly inhibited by NPFF. Moreover, the molecular dynamics optimized-protein models of the hub proteins were obtained. Collectively, NPFF inhibited the expression of immune-related genes in RAW 264.7 cells at the transcriptome level, which suggested a negative relationship between NPFF and this set of immune-related genes in RAW 264.7 macrophages. Therefore, our data may provide direct evidence of the role of NPFF in peripheral or central inflammatory diseases.

An Emerging Target in the Battle against Osteoarthritis: Macrophage Polarization.

Osteoarthritis (OA) is one of the most prevalent chronic joint diseases worldwide, which causes a series of problems, such as joint pain, muscle atrophy, and joint deformities. Benefiting from some advances in the clinical treatment of OA, the quality of life of OA patients has been improved. However, the clinical need for more effective treatments for OA is still very urgent. Increasing findings show that macrophages are a critical breakthrough in OA therapy. Stimulated by different factors, macrophages are differentiated into two phenotypes: the pro-inflammatory M1 type and anti-inflammatory M2 type. In this study, various therapeutic reagents for macrophage-dependent OA treatment are summarized, including physical stimuli, chemical compounds, and biological molecules. Subsequently, the mechanisms of action of various approaches to modulating macrophages are discussed, and the signaling pathways underlying these treatments are interpreted. The NF-κB signaling pathway plays a vital role in the occurrence and development of macrophage-mediated OA, as NF-κB signaling pathway agonists promote the occurrence of OA, whereas NF-κB inhibitors ameliorate OA. Besides, several signaling pathways are also involved in the process of OA, including the JNK, Akt, MAPK, STAT6, Wnt/ß-catenin, and mTOR pathways. In summary, macrophage polarization is a critical node in regulating the inflammatory response of OA. Reagents targeting the polarization of macrophages can effectively inhibit inflammation in the joints, which finally relieves OA symptoms. Our work lays the foundation for the development of macrophage-targeted therapeutic molecules and helps to elucidate the role of macrophages in OA.

Changes in plasma levels of RIPK1, RIPK3, and MLKL in patients with coronary atherosclerotic heart disease and its clinical predictive value. / å† çŠ¶åŠ¨è„‰ç²¥æ ·ç¡¬åŒ–æ€§å¿ƒè„ç— æ‚£è€ è¡€æµ†RIPK1，RIPK3及MLKLæ°´å¹³å˜åŒ–åŠå ¶ä¸´åºŠé¢„æµ‹ä»·å€¼.

OBJECTIVES: Coronary atherosclerotic heart disease (CHD) is caused by coronary atherosclerosis, which leads to stenosis and even occlusion of the lumen, resulting in myocardial ischemia, and necrosis subsequently. Its prevalence has been high for a long time. The prevention and treatment of CHD are important. The study aimed to investigate the role of plasma levels of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like protein (MLKL) in patients with CHD and its clinical predictive value. METHODS: A total of 190 patients with CHD who were diagnosed by coronary angiography and 70 healthy subjects in cardiovascular department from September 2015 to May 2017 were enrolled in this study. Patients with CHD were assigned into 4 groups: Patients with stable angina pectoris (SAP, n=46), patients with unstable angina pectoris (UAP, n=56), patients with non-ST-segment elevation myocardial infarction (NSTEMI, n=42), and patients with ST-segment elevation myocardial infarction (STEMI, n=46). Patients with CHD were assigned into a single-vessel lesion group, a double-vessel lesion group, and a multi-vessel lesion group according to the results of coronary angiography, and the severity of coronary artery stenosis was determined by Gensini score. Plasma levels of RIPK1, RIPK3, and MLKL were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The plasma levels of RIPK1, RIPK3, and MLKL in patients with CHD were significantly higher than those in the controls (P<0.05). The plasma levels of RIPK1, RIPK3, and MLKL in the UAP group were significantly higher than those in the SAP group (P<0.05). The plasma levels of RIPK1, RIPK3, and MLKL in NSTEMI and STEMI group were significantly higher than those in the UAP group (P<0.05). There was no significant difference between the NSTEMI group and STEMI group (P>0.05). The plasma levels of RIPK1, RIPK3 and MLKL were significantly increased with numbers of coronary artery lesions (P<0.05), which were positively correlated with Gensini scores. The multivariate logistic regression analysis showed that plasma levels of RIPK1, RIPK3, and MLKL were independent risk factors for severe coronary artery stenosis.The average period of follow-up was 24 months after hospital discharge. The patients were divided into 2 groups according to whether they had major adverse cardiovascular events (MACE). Compared with patient without MACE, patient with MACE had higher levels of RIPK1, RIPK3, and MLKL (P<0.05). Receiver operator characteristic (ROC) curve analysis showed that the area under curve of RIPK1 was 0.72 (P<0.001), the area under curve of RIPK3 was 0.83 (P<0.001), and the area under curve of MLKL was 0.75 (P<0.001). CONCLUSIONS: Plasma levels of RIPK1, RIPK3, and MLKL are closely related to CHD, and they have predictive value for the prognosis evaluation for patients with CHD.

Protective effect of dimethyl fumarate on oxidative damage and signaling in cardiomyocytes.

Myocardial ischemia/reperfusion (I/R) injury contributes to the pathogenesis of numerous diseases. Based on its antioxidant and anti­inflammatory effects, dimethyl fumarate (DMF) has been reported to exert protective effects against I/R. However, to the best of our knowledge, its potential role as a myocardial protective agent in heart disease has received little attention. Previous studies have suggested that DMF may exert its protective effects by activating nuclear factor erythroid 2­related factor 2 (Nrf2); however, the exact underlying mechanisms remain to be elucidated. The aim of the present study was to investigate the protective role of DMF in myocardial I/R injury, and to determine the role of Nrf2 in mediating the activity of DMF. H9c2 cells were incubated with DMF (20 µM) for 24 h before establishing the I/R model, and were then subjected to myocardial ischemia for 6 h, followed by reperfusion. Cell viability, lactate dehydrogenase levels, anti­oxidant enzyme expression levels and anti­apoptotic effects were evaluated, and AKT/Nrf2 pathway­associated mechanisms were investigated. The results of the present study indicated that DMF may reduce myocardial I/R injury in a Nrf2­dependent manner. DMF significantly improved cellular viability, suppressed the expression of apoptotic markers, decreased the production of reactive oxygen species and increased the expression of Nrf2­regulated antioxidative genes. Notably, these beneficial DMF­mediated effects were not observed in the control or I/R groups. In conclusion, the results of the present study suggested that DMF may exert protective effects against a myocardial I/R model, and further validated Nrf2 modulation as a primary mode of action. Thus suggesting that DMF may be a potential therapeutic agent for AKT/Nrf2 pathway activation in myocardial, and potentially systemic, diseases.

Selection of a Full Agonist Combinatorial Antibody that Rescues Leptin Deficiency In Vivo.

Growth factor deficiency in adulthood constitutes a distinct clinical syndrome with significant morbidities including abnormal body composition, reduced energy, affective disturbances, dyslipidemia, and increased cardiovascular risk. Protein replacement therapies using recombinant proteins or enzymes represent the only approved treatment. Combinatorial antibodies have shown great promise as a new class of therapeutic molecules because they act as "mechanism-based antibodies" with both agonist and antagonist activities. Using leptin, a key hormone in energy metabolism, as an example, a function-guided approach is developed to select combinatorial antibodies with high potency and full agonist activity that substitute natural growth factors in vivo. The identified antibody shows identical biochemical properties and cellular profiles as leptin, and rescues leptin-deficiency in ob/ob mice. Remarkably, the antibody activates leptin receptors that are otherwise nonfunctional because of mutations (L372A and A409E). Combinatorial antibodies have significant advantages over recombinant proteins for chronical usage in terms of immunological tolerance and biological stability.

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice.

BACKGROUND: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. METHODS: We employed the antibody to treat Cx30A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca2+ imaging and ATP release assay in vitro. FINDINGS: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. INTERPRETATION: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function.

Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a "leaky" character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies.

Somatic MIWI2 Hinders Direct Lineage Reprogramming From Fibroblast to Hepatocyte.

Remodeling of the gene regulatory network in cells is believed to be a prerequisite for their lineage reprogramming. However, its key regulatory factors are not yet elucidated. In this article, we investigate the role of PIWI proteins and provide evidence that one of them, MIWI2, is elicited during transdifferentiation of fibroblasts into hepatocyte-like cells. In coincidence with the peak expression of MIWI2, we identified the appearance of a unique intermediate epigenetic state characterized by a specific Piwi-interacting RNA (piRNA) profile consisting of 219 novel sequences. Knockout of MIWI2 greatly improved the formation of the induced hepatocytes, whereas overexpression of exogenous MIWI2 completely abolished the stimulated effect. A bioinformatics analysis of piRNA interaction network, followed by experimental validation, revealed the Notch signaling pathway as one of the immediate effectors of MIWI2. Altogether, our results show for the first time that temporal expression of MIWI2 contributes negatively to cell plasticity not only in germline, but also in developed cells, such as mouse fibroblasts. Stem Cells 2019;37:803-812.