Serum ANGPTL4 and SIRT1 factor levels and the Carotid Atherosclerotic plaque stability relationship analysis.

This study was to investigate the relationship between the levels of Angiopoietin-Like Protein 4 (ANGPTL4) and Silent Mating-type Information Regulation 2 Homolog 1 (SIRT1) and the stability of carotid atherosclerotic plaque. For this purpose, 108 patients with coronary heart disease in our hospital from Jan 2021 to May 2022 were selected as the coronary heart disease (CHD) group and 80 patients with the healthy examination as the control group. Patients' serum levels of ANGPTL4 and SIRT1 were collected, and their stability of carotid atherosclerotic plaque was determined by carotid ultrasound. According to their stability results, patients were divided into three subgroups: No plaque, Stable plaque, and Unstable plaque. The serum ANGPTL4 and SIRT1 levels were analyzed in different groups, and the correlation between their serum levels and the stability of carotid atherosclerotic plaque was analyzed by rank correlation. Results showed that the CHD group's serum ANGPTL4 and SIRT1 levels were lower, with statistical significance (P<0.05); A statistically significant difference in serum ANGPTL4 and SIRT1 levels were observed among patients with No plaques, Stable plaques, and Unstable plaques (P<0.05); A negative correlation was observed between serum levels of ANGPTL4 and SIRT1 and the stability of carotid atherosclerotic plaque (r=-0.438, -0.717, P<0.001); Serum ANGPTL4 and SIRT1 can be used as the evaluation method of carotid atherosclerotic plaque stability. When ANGPTL4 ≤ 30.17mg/L and SIRT1 ≤ 6.91µg/L, patients were more likely to develop unstable plaques; When ANGPTL4 ≤ 30.40mg/L and SIRT1 ≤ 6.87µg/L, patients were more likely to develop plaques (instability and/or stability). In conclusion, the serum levels of ANGPTL4 and SIRT1 in patients with CHD decreased. ANGPTL4 and SIRT1 will participate in the formation and development of carotid plaque, which can be used as a serological evaluation index to evaluate the occurrence and carotid atherosclerotic plaque's stability.

Effects of Netrin-1 and NHE1 Participation on the Migration of Macrophages Driven by CCL2.

This experiment was designed to investigate the relationship between NHE1 gene expression differences between Netrin-1 and NHE1. For this purpose, the blank control, CCL2, CCL2 + Netrin-1 groups were constructed, and cell migration ability was detected by scratch tests and Transwell experiments; Commercial over-expressed NHE1 adenovirus vector (over-expressed NHE1 group), shRNA adenoviral vector silencing NHE1 (silencing NHE1 group) and negative control without carrying virus (negative control group) were subjected to RT-PCR test 24h after infection and pH recovery rate after acid loading was measured. The percentage of wound healing area and the number of cell migration of macrophages in the blank control group, CCL2 group, CCL2+Netrin-1 group, over-expressed NHE1 group, silencing NHE1 group and negative control group were compared. Results showed that in terms of migration ability, the percentage of wound healing area and migration in CCL2 increased (P <0.05), in CCL2 + Netrin-1 (P <0.05) and increased NHE1 mRNA (P <0.05), and not in NHE1 (P <0.05).pH response rate after acid load (NHE1 activity) showed that NHE1 activity was enhanced compared with the blank group, while NHE1 activity in silent NHE1 group decreased (P <0.05); from macrophage migration ability after overexpression/silencing, the percentage of macrophage wound healing area and cell migration increased/decreased compared with CCL2 group and Netrin-1 + CCL2 group (P <0.05). Then Upregulation of NHE1 can promote CCL2-driven macrophage RAW264.7 cell migration, and the downregulation of NHE1 can inhibit its cell migration; Netrin-1 can inhibit CCL2-driven RAW264.7 cell migration regardless of NHE1 regulation.

NCF2, MYO1F, S1PR4, and FCN1 as potential noninvasive diagnostic biomarkers in patients with obstructive coronary artery: A weighted gene co-expression network analysis.

This study aims to explore the predictive noninvasive biomarker for obstructive coronary artery disease (CAD). By using the data set GSE90074, weighted gene co-expression network analysis (WGCNA), and protein-protein interactive network, construction of differentially expressed genes in peripheral blood mononuclear cells was conducted to identify the most significant gene clusters associated with obstructive CAD. Univariate and multivariate stepwise logistic regression analyses and receiver operating characteristic analysis were used to predicate the diagnostic accuracy of biomarker candidates in the detection of obstructive CAD. Furthermore, functional prediction of candidate gene biomarkers was further confirmed in ST-segment elevation myocardial infarction (STEMI) patients or stable CAD patients by using the datasets of GSE62646 and GSE59867. We found that the blue module discriminated by WGCNA contained 13 hub-genes that could be independent risk factors for obstructive CAD (P < .05). Among these 13 hub-genes, a four-gene signature including neutrophil cytosol factor 2 (NCF2, P = .025), myosin-If (MYO1F, P = .001), sphingosine-1-phosphate receptor 4 (S1PR4, P = .015), and ficolin-1 (FCN1, P = .012) alone or combined with two risk factors (male sex and hyperlipidemia) may represent potential diagnostic biomarkers in obstructive CAD. Furthermore, the messenger RNA levels of NCF2, MYO1F, S1PR4, and FCN1 were higher in STEMI patients than that in stable CAD patients, although S1PR4 showed no statistical difference (P > .05). This four-gene signature could also act as a prognostic biomarker to discriminate STEMI patients from stable CAD patients. These findings suggest a four-gene signature (NCF2, MYO1F, S1PR4, and FCN1) alone or combined with two risk factors (male sex and hyperlipidemia) as a promising prognostic biomarker in the diagnosis of STEMI. Well-designed cohort studies should be implemented to warrant the diagnostic value of these genes in clinical purpose.

[Impact of inhaled anesthetics on the male reproductive system and its mechanisms: An update].

The safety of occupational exposure to inhaled anesthetics remains a concern among the medical staff in hospitals. Few reports are seen about the impact of inhaled anesthetics on the reproductive system, particularly that of males. Several clinical and basic studies on isoflurane and others suggest that inhaled anesthetics affect the reproductive system of rodents by decreasing the sperm count, inducing sperm morphological abnormality, reducing sperm motility, and changing the levels of reproductive hormones, the underlying mechanisms of which are mainly associated with the alteration of the hypothalamic-pituitary-gonadal axis and DNA damage and apoptosis of reproductive cells. This article reviews the main impacts of inhaled anesthetics on the male reproductive system and the possible mechanisms.

SGLT2i Alleviates Atherosclerosis by Inhibiting NHE1 Activation to Protect against Macrophage Senescence Induced by Angiotensin II.

BACKGROUND: Sodium-dependent glucose transporter (SGLT2) inhibitors (SGLT2i) have been found to have anti-atherosclerotic effects in clinical treatment. OBJECTIVES: The aim of this study was to explore whether angiotensin II (Ang II) induces changes in the expression of Na+/H+ exchanger of cytoplasmic membrane channel proteins (NHE1) and SGLT2 in macrophages and whether dapagliflozin (DAPA), an SGLT2i, protects against Ang II induced macrophage senescence by inhibiting NHE1 activation to alleviate Atherosclerosis (AS). METHODS: After intervention with DAPA plus gavage or feeding them a high-fat diet, the mice's aortas were dissected, and oil red O staining was performed. Cell proliferation and toxicity detection, western blot, immunofluorescence, and ß-galactosidase staining methods were adopted to detect cell activity, expressions of senescence-related genes, and number of senescent cells after different concentrations of Ang II or DAPA or plasmid NHE1 were treated with RAW264.7 cells. RESULTS: (1) The formation of AS plaques in ApoE -/- mice showed a downward trend under DAPA. (2) After the intervention of Ang II, the cell activity of RAW264.7 decreased, and the expression of senescent cells and related genes increased. (3) Under the Ang II condition, the expression of SGLT2 and NHE1 increased, and SGLT2, NHE1, and senescence-related genes decreased with the addition of DAPA. (4) The expression of NHE1, senescent cells and related genes decreased in RAW264.7 cells after DAPA treatment with plasmid NHE1 intervention. CONCLUSION: SGLT2i alleviates atherosclerosis by inhibiting NHE1 activation to protect against macrophage senescence induced by Ang II.

Downregulating NHE-1 decreases the apoptosis of hippocampal cells in epileptic model rats based on the NHE-1/calpain1 pathway.

Seizure is associated with pathological changes of hippocampus, but the mechanism by which hippocampal neuronal apoptosis promotes epilepsy is unclear. Our previous study showed that the expression of NHE-1 was increased in epileptic model rats. Therefore, this study further explores the effect of NHE-1 on hippocampal cells apoptosis and seizure in lithium chloride-pilocarpine epileptic model rats. First, we established a lithium chloride-pilocarpine induced epileptic rat model and detected the expression of NHE-1, calpain1 and apoptosis in the hippocampus. Then, we further down-regulated NHE-1 to observe the expression of calpain1 and apoptosis in the hippocampus, as well as its effect on seizures in rats. We found that the expression of NHE-1 and calpain1 and apoptosis in the hippocampus was significant increased in the model group. After down-regulating NHE-1, the expression of calpain1 was decreased, and hippocampal cell apoptosis was alleviated. In addition, down-regulation of NHE-1 reduced the frequency and duration of seizures in epileptic rats. Therefore, hippocampal NHE-1 overexpression is closely related to the development of neuronal apoptosis in a rat model of epilepsy, and downregulating NHE-1 expression can reduce cell apoptosis. Moreover, the NHE-1/calpain1 signaling pathway may be an important mechanism leading to hippocampal cell apoptosis.

Downregulation of NHE1 expression attenuates apoptosis of primary hippocampal neurons of an epilepsy model through the calpain-1 pathway.

OBJECTIVE: Na(+)/H(+) exchanger isoform 1 (NHE1), a membrane protein that regulates intracellular pH, is abundantly expressed in brain tissues. It is associated with pathophysiologies in several brain diseases. The present study aimed to investigate the effects of NHE1 on the apoptosis of primary neurons of an epilepsy model. METHODS: Primary hippocampal neurons were cultured in an Mg2+-free medium to establish an epilepsy cell model. Designed shNHE1 lentivirus was used to silence NHE1 level in primary neurons. Nonselective pharmacological inhibitor MDL-28170 (20 µmol/L) was used to inhibit calpain-1 protein in neurons treated with Mg2+-free medium. The expression levels of NHE1 and calpain-1, intracellular Ca2+ (Ca2+i) and H+ (H+i) levels, and the expression levels of apoptosis-related proteins Bcl-2 and Bax were detected in neurons. TUNEL staining was performed to determine apoptosis in different groups. RESULTS: NHE1 expression was increased in primary neurons treated with an Mg2+-free medium, and it was correlated with increased expression of calpain-1 and cell apoptosis. Neurons from the in vitro epilepsy model showed significantly decreased Bcl-2 protein expression and significantly increased Bax protein expression. In the presence of LV-shNHE1 and the calpain-1 inhibitor MDL-28170, the changes in the expression of apoptosis-related proteins Bcl-2 and Bax were blocked in the epileptic model, and the percentage of apoptotic neurons among neurons from the in vitro epilepsy model was significantly decreased. The increase in calpain-1 expression was suppressed by LV-shNHE1; however, the inhibition of calpain-1 did not affect NHE1 expression. CONCLUSION: These results demonstrate that NHE1 participates in the promotion of neuronal apoptosis of epilepsy model in vitro through the calpain-1 pathway. Downregulation of NHE1 expression could exert a neuroprotective effect on epilepsy.

Exploratory Study of Differentially Expressed Genes of Peripheral Blood Monocytes in Patients with Carotid Atherosclerosis.

BACKGROUND: The abundance of circulating monocytes is closely associated with the development of atherosclerosis in humans. OBJECTIVE: This study aimed to further research into diagnostic biomarkers and targeted treatment of carotid atherosclerosis (CAS). METHODS: We performed transcriptomics analysis through weighted gene co-expression network analysis (WGCNA) of monocytes from patients in public databases with and without CAS. Differentially expressed genes (DEGs) were screened by R package limma. Diagnostic molecules were derived by the least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE) algorithms. NetworkAnalyst, miRWalk, and StarBase databases assisted in the construction of diagnostic molecule regulatory networks. The DrugBank database predicted drugs targeting the diagnostic molecules. RT-PCR tested expression profiles. RESULTS: From 14,369 hub genes and 61 DEGs, six differentially expressed monocyte-related hub genes were significantly associated with immune cells, immune responses, monocytes, and lipid metabolism. LASSO and SVM-RFE yielded five genes for CAS prediction. RT-PCR of these genes showed HMGB1 was upregulated, and CCL3, CCL3L1, CCL4, and DUSP1 were down-regulated in CAS versus controls. Then, we constructed and visualized the regulatory networks of 9 transcription factors (TFs), which significantly related to 5 diagnostic molecules. About 11 miRNAs, 19 lncRNAs, and 39 edges centered on four diagnostic molecules (CCL3, CCL4, DUSP1, and HMGB1) were constructed and displayed. Eleven potential drugs were identified, including ibrutinib, CTI-01, roflumilast etc. Conclusion: A set of five biomarkers were identified for the diagnosis of CAS and for the study of potential therapeutic targets.

Suppression of NHE1 by small interfering RNA inhibits HIF-1α-induced angiogenesis in vitro via modulation of calpain activity.

Hypoxia-inducible factor-1 (HIF-1) orchestrates angiogenesis under hypoxic conditions mainly due to increased expression of such target genes as vascular endothelial growth factor (VEGF). Na+/H+exchanger-1 (NHE1), a potential HIF target gene product, plays a pivotal role in proliferation, survival, migration, adhesion and so on. However, it is unknown whether NHE1 is involved in HIF-1α-induced angiogenesis. This present study demonstrated that the expression of NHE1 was much higher in human umbilical vein endothelial cells (HUVECs) infected with adenovirus encoding HIF-1α (rAd-HIF) than with vacuum adenovirus (vAd). HIF-1α also increased the expression of VEGF, the expression and activity of calpains, and the intracellular pH. Moreover, small interfering RNA targeting NHE1 (NHE1 siRNA) dramatically decreased the expression of NHE1 and thus lowered the intracellular pH, and it also attenuated the protein expression of calpain-2 but not calpain-1, resulting in the lower calpain activity. Furthermore, HIF-1α enhanced the proliferation, migration and Matrigel tube formation, which were inhibited by NHE1 siRNA. Finally, the inhibitory effect of NHE1 siRNA was reversed by VEGF and the reversibility of the later was abrogated by the calpain inhibitor ALLM. In conclusion, the findings have revealed that NHE1 might participate in HIF-1-induced angiogenesis due, at least in part, to the alteration of the calpain activity, suggesting that NHE1 as well as calpains might represent a potential target of controlling angiogenesis in response to the hypoxic stress under various pathological conditions.

Bone marrow mesenchymal stromal cells with support of bispecific antibody and ultrasound-mediated microbubbles prevent myocardial fibrosis via the signal transducer and activators of transcription signaling pathway.

BACKGROUND AIMS: This study was initiated to investigate the efficacy of myocardial fibrosis intervention via signal transducer and activators of transcription (STAT) signaling using bone marrow (BM) mesenchymal stromal cells (MSC) with the aid of bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB). METHODS: BiAb (anti-CD29 × anti-myosin light chain antibody; AMLCA) was prepared and combined with isolated MSC from male mice and transfused into female mice with isoproterenol-induced myocardial fibrosis via the tail vein, followed by MB (MSC + BiAb + MB). This study included seven groups: MSC + BiAb + MB; MSC; BiAb; MB; MSC + BiAb; untreated; and control. Five weeks after treatment, expression levels of the sex-determining region of Y-chromosome (SRY), matrix metalloproteinases (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1 and vascular endothelial growth factor (VEGF) in myocardium were detected by fluorescent quantitative real-time polymerase chain reaction (qRT-PCR). Collagen distribution was observed using Sirius Red staining. The protein expression of signal transducer and activators of transcription (STAT)1 and STAT3 was detected by Western blot. RESULTS: The highest homing number of MSC was in the MSC + BiAb + MB group, second highest in the MSC + BiAb group, and lowest in MSC alone. Compared with the untreated group, MSC + BiAb + MB, MSC + BiAb and MSC groups had decreased levels of MMP-9, TIMP-1, STAT1 and collagen deposition, and increased levels of STAT3. Upregulated STAT3 and downregulated TIMP-1 were significantly different in MSC + BiAb + MB compared with MSC alone or MSC + BiAb. CONCLUSIONS: The homing rate and repairing efficacy of MSC improved with treatment utilizing a combination of BiAb and MB. MSC can improve MMP-TIMP expression in injured myocardium and interfere with myocardial fibrosis after homing, a mechanism that may be related to the STAT-mediated signaling pathway.

Heme oxygenase-1 (HO-1) alleviates vascular restenosis after balloon injury in a rabbit carotid artery model.

Percutaneous coronary intervention (PCI) is used commonly for coronary artery disease (CAD); however, restenosis is a proliferative response and frequent sequela to this treatment. Although the introduction of drug-eluting stents has convincingly reduced the incidence of vascular restenosis, restenosis remains a problem. The present study was designed to investigate the effects of the heme oxygenase-1 (HO-1) on restenosis formation after balloon injury in a rabbit carotid artery model. We found that involvement of the HO-1 in defensive restenosis formation was independent of the levels of blood lipid. Activation of HO-1 induced by chlorhematin treatment alleviated vascular restenosis after balloon injury in a rabbit carotid artery model, whereas inhibition of HO-1 by zinc protoporphyrin treatment exacerbated restenosis formation. Furthermore, overexpression of HO-1 inhibited nuclear factor kappa B subunit 1 (NF-кB) activity and decreased tumor necrosis factor-alpha (TNF-α) and endothelin 1 (ET-1) expression. In conclusion, our study provides preliminary data suggesting that HO-1 alleviates vascular restenosis after balloon injury in a rabbit carotid artery model by inhibiting NF-кB, TNF-α and ET-1 expression, indicating induction of HO-1 activation may be a feasible therapeutic target for treating vessels resistant to restenosis.

Aberrant T cell subsets and cytokines expression profile in systemic lupus erythematosus.

To assess T cell subsets and levels of chemokines and cytokines in patients with SLE and determine their relationships between disease activity and organ involvement. Blood samples from SLE patients (n = 24) and healthy controls (n = 36) were analyzed. Frequency of circulating follicular help T cells (Tfh), central memory T cells (Tcm), effector memory T cells (Tem), and naïve T cell subsets was enumerated and their surface markers expression of inducible T cell co-stimulator (ICOS) and programmed death 1(PD-1) protein was examined by flow cytometry. The disease state in SLE patients was evaluated using the SLE Disease Activity Index (SLEDAI). Concentrations of autoantibodies, serum C-reactive protein (CRP), the erythrocyte sedimentation rate (ESR), lgG, complement 3, complement 4, cytokines, and chemokines, such as IL-21, IL-17A, and IL-1ß, were measured. The frequencies of circulating Tfh and Tcm cell subsets were significantly lower than those in healthy controls. However, the percentages of circulating PD1+ICOS+Tfh, PD1+ICOS+Tcm, and PD1+ICOS+Tem of PBMCs from SLE patients were higher than those in healthy controls. Furthermore, increased levels of serum IL-1ß, IL-4, IL-6, MCP-1, IL-21, and IL-17A were detected in the patients with SLE compared to healthy controls. In addition, patients with immune thrombocytopenia displayed elevated proportions of serum IL-10, IL-17A, and IL-1ß. Aberrant T cell subsets and cytokines expression profile were observed in SLE patients. PD1+ICOS+Tem cell subset was clearly influenced by disease activity and serum IL-10, IL-17A, and IL-1ß were significantly increased in patients with immune thrombocytopenia. Therefore, PD1+ICOS+Tem cells might serve as an important tool for recognition and serum IL-10, IL-17A, and IL-1ß might be an effective monitor for SLE patients with immune thrombocytopenia.

[Overexpression of NHE1 suppresses ABCA1 protein expression via increasing calpain activity in RAW264.7 cells].

Objective To investigate the effect of over-expressed Na+/H+ exchanger 1 (NHE1) on the protein expression of adenosine three phosphate binding cassette transporter A1 (ABCA1) in RAW264.7 cells. Methods RAW264.7 cells were infected with the adenoviral vector encoding NHE1-EGFP (AdNHE1). The infected RAW264.7 cells were subjected to Western blot analysis for NHE1-EGFP fusion protein. The subcellular localization of NHE1-EGFP fusion protein was observed by confocal laser scanning microscopy. NHE1 activity was measured by the method of pH recovery in response to an acute acid pulse. Furthermore, Western blotting was performed to determine ABCA1 protein levels and calpain activity in NHE1-overexpressing RAW264.7 cells. The effect of calpain inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) on ABCA1 protein levels in the presence of TO-901317 was examined by Western blotting. Results NHE1-EGFP fusion protein was highly expressed and localized in cytoplasm and cell membrane of RAW264.7 cells infected with AdNHE1. NHE1-EGFP fusion protein reduced ABCA1 protein expression and increased calpain activity. The calpain inhibitor ALLN blocked the decrease of ABCA1 protein expression. Conclusion Overexpressed NHE1 suppresses the expression of ABCA1 protein via increasing the calpain activity in RAW264.7 cells.

Analysis of the correlation between non-high density lipoprotein cholesterol and coronary heart disease in elderly Chinese.

OBJECTIVE: In the aged population, serum cholesterol is directly correlated with coronary heart disease (CHD) risk. We aimed to investigate the correlation between non-high density lipoprotein cholesterol (non-HDL-C) level and CHD and coronary lesions in an aged population. METHODS: 1,272 cases of old patients who were more than 65 years old and accepted for coronary angiography were analyzed retrospectively. Based on the result of coronary angiography, the patients were divided into control group and CHD group. Further, 767 CHD patients were divided into subgroups according to the number of branches with pathological changes and Gensini score. Serum TC, HDL-C, LDL-C and TG were assayed to measure the level of non-HDL-C. The differences in non-HDL-C among groups were compared, and the correlation between non-HDL-C and coronary artery disease degree was also analyzed. RESULTS: The non-HDL-C level in the CHD group was significantly higher than that in the control group (p < 0.01). Further, the serum non-HDL-C showed an increasing tendency accompanied by the increase in branches with lesions. Compared with patients with single or double coronary artery branches lesions, the non-HDL-C in those with multiple lesions in branches was significantly increased (p < 0.01, p < 0.05). With the aggravation of coronary artery stenosis, the serum non-HDL-C level was gradually increased. The most significant increase was observed in the group with Gensini score of more than 40. There was a significant difference between Gensini score > 40 group and Gensini score of 20-40 group and Gensini score < 20 group, respectively (p < 0.05, p < 0.01). Meanwhile, there were significant positive correlations between the serum non-HDL-C level and coronary lesions area and severity (r = 0.147, p < 0.01; r = 0.152, p < 0.01). CONCLUSION: In conclusion, serum non-HDL-C was closely associated with development of CHD and coronary artery lesions severity.

Procollagen III N-terminal peptide predicts short-term prognosis and cardiac remodeling in coronary heart disease patients with metabolic syndrome.

INTRODUCTION: Many patients with coronary heart disease (CHD) also have metabolic syndrome (MS); however, little is known about the condition of cardiovascular remodeling in these patients. The objective of this study to explore the role of plasma procollagen III N-terminal peptide (PIIINP) in predicting the prognosis and cardiac remodeling in patients with CHD with MS. METHODS: One hundred eight patients were classified into high and low PIIINP groups according to the median value of plasma PIIINP. Cardiovascular examinations including echocardiogram, carotid color ultrasound examination, coronary angiography and the 6-minute walking test (6MWT) were performed before and after a 1-year follow-up. Readmission for cardiac and cerebrovascular events was assessed during the follow-up period. RESULTS: Plasma PIIINP level was significantly correlated with age, high-sensitivity C-reactive protein (hs-CRP) and body mass index in a multiple stepwise regression model. There was a positive correlation between the LnPIIINP and an increased left ventricular mass index in partial correlation analysis. The Cox proportional hazard model analysis indicated that the level of PIIINP, left ventricular ejection fraction and hs-CRP were independent predictors of readmission owing to cardiac and cerebrovascular events during the follow-up. A PIIINP value of 4.0 µg/L was the best threshold value for determining the need for readmission. CONCLUSIONS: PIIINP levels rise with increases in age, hs-CRP and body mass index in patients with CHD with MS, and a high level of PIIINP indicates recent deterioration of cardiac remodeling and exercise tolerance and a poor prognosis.