Abstract:Aim To observe the effects of receptor interacting protein 2 (RIP2) on macrophage inflammatory activation and polarity transformation, and to explore the mechanism of RIP2 in macrophage phagocytosis of oxidized low density lipoprotein (ox-LDL). Methods THP-1 derived macrophages were treated with different doses (0,5 and 50 mg/L) of ox-LDL for 24 hours, and treated with 50 mg/L ox-LDL for 8,6 and 24 hours. Real-time quantitative PCR and Western blot were used to detect the expression of RIP2 mRNA and protein in THP-1 derived macrophages, and ELISA was used to detect the secretion of tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). Three pairs of RIP2 siRNA were designed, transfecting them into cells using hiperfict transfection reagent, real-time quantitative PCR and Western blot were used to detect the expression of RIP2 mRNA and protein in THP-1 derived macrophages after transfection, in order to screen for the optimal siRNA transfection concentration and the most effective pair of siRNA.After transfection with the most effective RIP2 siRNA, cells were treated with 50 mg/L ox-LDL for 24 hours, ELISA was used to detect the secretion of TNF-α, MCP-1, interleukin-10 (IL-10) and interleukin-12 (IL-12), real-time quantitative PCR was used to detect the expression of inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), flow cytometry was used to detect the expression of cell surface antigens CD86, CD80 and CD163. Results Ox-LDL induced the expression of RIP2 in macrophages in a dose-dependent and time-dependent manner. With the increase of ox-LDL treatment dose and time, the expression of RIP2 mRNA and protein increased. Among them, the expression of RIP2 protein in the 50 mg/L group was 7.6 times of the control group, and the expression of RIP2 protein in the 24 h group was 17.9 times of the control group (P＜0.001). The ELISA results showed that with the increase of ox-LDL treatment dose and time, the secretion of TNF-α and MCP-1 increased (P＜0.05). After transfection of RIP2 siRNA into cells, ELISA results showed that the secretion of TNF-α, MCP-1 and IL-10 in the ox-LDL group was 2.4 times, 2.9 times and 1.8 times of the control group (P＜0.01), and the secretion of IL-12 decreased by 34.2% compared to the control group (P＜0.01); the secretion of TNF-α, MCP-1 and IL-10 in the siRNA+ox-LDL group decreased by 37.4%, 45.3% and 27.4%, respectively, compared to the ox-LDL group (P＜0.01), while the secretion of IL-12 increased by 31.4% (P＜0.05). The results of flow cytometry and real-time quantitative PCR showed that the expression of CD86, CD80 and iNOS mRNA in the ox-LDL group was 14.2 times, 33.8 times and 4.5 times of those of the control group, respectively, while the expression of CD163 and Arg-1 mRNA decreased by 33.4% and 43.0%, respectively, compared with the control group (P＜0.05); the expression of CD86, CD80 and iNOS mRNA in the siRNA+ox-LDL group decreased by 27.6%, 29.3% and 34.3%, respectively, compared to the ox-LDL group, while the expression of CD163 and Arg-1 mRNA increased by 30.3% and 38.6%, respectively (P＜0.05). Conclusion RIP2 expression in macrophages can be induced by ox-LDL in a dose-dependent and time-dependent manner. RIP2 gene silencing can inhibit ox-LDL induced M1 macrophage transformation.

Abstract:Diabetes mellitus type 2 (T2DM) is a common chronic metabolic disease, compared to non-diabetics, T2DM patients have a higher risk of heart disease, and their risk of vascular diseases is twice that of non-diabetic individuals. Multiple clinical trials have confirmed that glucagon-like peptide-1 receptor agonists (GLP-1RA), in addition to their function of lowering blood glucose, can also decrease the risk of cardiovascular events in T2DM patients. This article reviews the mechanisms of GLP-1RA-mediated cardioprotection. It provides a comprehensive review of the recent progress in GLP-1RA-mediated cardiac protective mechanisms, elaborating on the protective effects of GLP-1RA on specific heart cell and further discussing its impact on heart failure (HF), provide assistance for clinical treatment of diabetic cardiomyopathy.

Abstract:Aim To explore the predictive value of serum D-dimer and the soluble receptor for advanced glycation end products (sRAGE) levels in the short-term poor prognosis of elderly patients with coronary heart disease after percutaneous coronary intervention (PCI). Methods The clinical data of 316 elderly patients with coronary heart disease first diagnosed in Huanggang Central Hospital from April 2019 to June 2020 were collected. According to whether the patients had major adverse cardiovascular events (MACE) during the follow-up period, they were divided into MACE group (n＝52) and non MACE group (n＝264). The independent influencing factors of postoperative MACE were analyzed by univariate analysis and multivariate Logistic regression, the nomogram prediction model was established and verified according to the independent influencing factors of patient prognosis. The threshold effect of D-dimer and sRAGE levels was determined by curve fitting and threshold effect analysis, and the influence of D-dimer and sRAGE levels on MACE was evaluated by Kaplan-Meier curve. Results During the one-year postoperative period, 52(16.46%) of the 316 elderly patients with coronary heart disease who were included experienced MACE. Body mass index (BMI), proportion of hypertension, proportion of diabetes, GRACE score, number of stents, apoliporotein (Apo) B, ApoB/ApoA, low density lipoprotein cholesterol (LDLC), liporotein (a) [Lp(a)] and D-dimer levels of patients in the MACE group were higher than those in the non MACE group, and sRAGE levels were lower than those in the non MACE group, with statistically significant differences(P＜0.05). Multivariate Logistic regression analysis showed that high GRACE score, high Lp(a) and high D-dimer levels were independent risk factors for MACE in elderly patients with coronary heart disease after PCI treatment, and high sRAGE level was protective factor (P＜0.05). Curve fitting found that the probability of MACE increased with the increase of D-dimer level and the decrease of sRAGE level. The Kaplan-Meier curve shows that the incidence of MACE in patients with higher D-dimer levels is significantly higher than that in patients with low D-dimer levels (P＜0.001), and the incidence of MACE in patients with lower sRAGE levels is significantly higher than patients with higher sRAGE levels (P＜0.001). The nomogram model was constructed based on independent prognostic factors, and its consistency index was 0.796 (95%CI:0.723～0.834), ROC curve AUC was 0.851 (95%CI:0.806～0.892), which has a good degree of discrimination. Conclusion High level of D-dimer and low level of sRAGE are important risk factors for MACE after PCI in elderly patients with coronary heart disease, and have a high predictive value for short-term adverse prognosis after PCI.

Abstract:Aim To explore the effect of succinate/G protein coupled receptor 91 (GPR91) on mitochondria in vascular endothelial cells and its regulatory mechanisms. Methods Transmission electron microscopy, Western blot and fluorescence microscopy were used to observe the effects of succinate analogues diethyl succinate (DS), GPR91 agonist and inhibitor on the mitochondrial morphology, cristae, cristate homeostasis related proteins reactive oxygen species (ROS) content, Ca2＋ concentration, mitochondrial membrane potential, the expression of dihydroorotate dehydrogenase (DHODH) and oxidized coenzyme Q10 (CoQ10). Fluorescence probes were used to observe the effect of DHODH inhibitor and CoQ10 on ROS level and Ca2＋concentration of endothelial cells. Results After DS treatment, the mitochondria showed pyknosis and mitochondrial volume significantly decreased, electron density of the mitochondrial membrane increased, and the number of cristae decreased in endothelial cells; the expression of cristae homeostasis related proteins MIC60 decreased by 23%, while cellular ROS level and Ca2＋ concentration increased; mitochondrial membrane potential decreased (P＜0.05 or P＜0.01). After GPR91 agonist treatment, the expression of cristae homeostasis related proteins MIC60 decreased by 31%, meanwhile, cellular ROS level increased by 27% and Ca2＋ concentration increased by 36%; mitochondrial membrane potential decreased (P＜0.05 or P＜0.01). After GPR91 inhibitor treatment, the expression of cristae homeostasis related proteins MIC60 increased by 22% and ATP5I increased by 40%; the levels of ROS decreased by 41% and Ca2＋ concentration decreased by 67%; and the mitochondrial membrane potential was restored to normal (P＜0.05 or P＜0.01). After DS treatment, the expression of DHODH decreased by 43% and the level of oxidized CoQ10 increased by 120% (P＜0.05 or P＜0.01). After GPR91 agonist treatment, the expression of DHODH decreased by 22% and the level of oxidized CoQ10 increased by 36% (P＜0.05 or P＜0.01). After GPR91 inhibitor treatment, the expression of DHODH increased by 40% and the level of oxidized CoQ10 decreased by 39% (P＜0.01). After DHODH inhibitor treatment, the ROS level increased by 20% and Ca2＋ concentration increased by 28%, and mitochondrial membrane potential reduced at same time (P＜0.05 or P＜0.01). Exogenous oxidized CoQ10 inhibited ROS production by 30% and decreased Ca2＋ concentration by 20% (P＜0.05 or P＜0.01). Conclusion Succinate/GPR91 promotes mitochondrial damage in endothelial cells, and its mechanism may relate to down-regulating the expression of DHODH and inhibiting the reduction of CoQ10 by affecting the mitochondrial cristae homeostasis.

Abstract:Aim To investigate the inhibition role and mechanism of adipose derived mesenchymal stem cell (ADMSC) exosomes (Exo) on adverse ventricular remodeling after myocardial infarction (MI). Methods The changes of autophagy and inflammasomes phenotype of cardiac fibroblasts after H2O2 treatment were observed. MI rats were injected with an equal volume of normal saline, adipose derived mesenchymal stem cell exosomes (MSC-Exo) or fibroblast exosomes (MEF-Exo) via a tail vein. The expression of autophagy related 16 like protein 1 (ATG16L1), autophagy related protein 7 (ATG7) and NOD-like receptor protein 3 (NLRP3), inflammatory response, the degree of myocardial fibrosis, and the cardiac function were observed in different groups. Results After treatment with H2O2 on cardiac fibroblasts, the expressions of ATG16L1 and ATG7 were significantly decreased (P＜0.001), NLRP3 was significantly increased (P＜0.001), and the levels of inflammatory cytokines interleukin-1β (IL-1β) and IL-18 were significantly elevated (P＜0.001). After MI rats were intervened with MSC-Exo, the expressions of autophagy related proteins ATG16L1 and ATG7 were significantly up-regulated (P＜0.001), NLRP3 was significantly down-regulated (P＜0.001), serum IL-1β and IL-18 levels were significantly decreased (P＜0.001), fibrosis-related proteins collagen Ⅰ and Ⅲ were significantly reduced (P＜0.001), myocardial fibrosis was significantly relieved (P＜0.001), and cardiac function was significantly improved (P＜0.001). Conclusion Adipose derived MSC-Exo play a role in inhibiting adverse ventricular remodeling after MI by regulating the balance of autophagy and NLRP3 inflammasomes.

Abstract:Aim To investigate the levels of a disintegrin and metalloprotease 10 (ADAM-10) and soluble urokinase plasminogen activator receptor (suPAR) in the serum of hypertension patients with atherosclerosis and their relationship with the severity of disease. Methods From February 2021 to February 3,5 hypertension patients with atherosclerosis who visited our hospital were collected as the study group, and 76 healthy people were collected as control group. The patients in the study group were grouped into mild group (n＝40), moderate group (n＝42) and severe group (n＝43) according to the degree of atherosclerosis, after 60 days of treatment, the patients were grouped into a good prognosis group (n＝74) and a poor prognosis group (n＝51) based on their prognosis. Enzyme linked immunosorbent assay (ELISA) was applied to measure serum ADAM-10 and suPAR levels; Clinical data of patients with different prognosis were compared; Pearson method was applied to analyze the relationship between serum ADAM-10, suPAR levels and carotid intima-media thickness (IMT) in patients with hypertension and atherosclerosis; Multivariate Logistic regression was applied to analyze the influencing factors of poor prognosis in patients with hypertension and atherosclerosis; Receiver operating characteristic (ROC) curve was applied to evaluate the predictive value of ADAM-10 and suPAR alone and jointly for poor prognosis of hypertension with atherosclerosis. Results The levels of serum ADAM-10 and suPAR were significantly higher in the study group than those in control group (P＜0.05); The serum ADAM-10 and suPAR levels in moderate group and severe group were obviously higher than those in mild group (P＜0.05), while the serum ADAM-10 and suPAR levels were obviously higher in severe group than those in moderate group (P＜0.05); The serum ADAM-10 and suPAR levels, left and right IMT, systolic blood pressure, and diastolic blood pressure of patients with poor prognosis were significantly higher than those of patients with good prognosis (P＜0.05); Pearson correlation analysis showed that serum ADAM-10 and suPAR levels in patients with hypertension and atherosclerosis were positively correlated with systolic blood pressure, diastolic blood pressure and IMT (P＜0.001); Logistic regression analysis showed that the increased levels of systolic blood pressure, diastolic blood pressure, ADAM-10, suPAR and IMT were risk factors for poor prognosis of hypertension patients with atherosclerosis (P＜0.05); ROC curve analysis showed that the area under the curve (AUC) of serum ADAM-10 and suPAR levels alone and jointly predicting poor prognosis of hypertension with atherosclerosis was 0.9,0.830 and 0.900, respectively, the combination of the two was superior to their individual predictions (Zcombination-ADAM-10＝2.766, P＝0.006; Zcombination-suPAR＝2.602, P＝0.009).Conclusion The levels of serum ADAM-10 and suPAR in patients with hypertension and atherosclerosis are significantly increased, and are positively correlated with the severity of atherosclerosis. Both of them have a high predictive value for evaluating the prognosis of patients with hypertension and atherosclerosis.

Abstract:Diabetes cardiomyopathy (DCM) is a cardiovascular disease with structural damage and dysfunction of myocardium under the condition of glucose and lipid metabolism disorder in diabetes. The main pathological characteristics are ventricular hypertrophy, myocardial remodeling and cardiomyocyte death, which induce heart failure. DCM has become one of the main causes of death in patients with diabetes. As a pro-inflammatory programmed cell death pathway, pyroptosis mediated by inflammasomes is considered an important driver of chronic inflammation due to its excessive activation. Recently, it was found that the over activation of pyroptosis promoted the course of DCM and became the hub connecting the disorder of glucose and lipid metabolism in diabetes and myocardial injury. With the discovery of more and more roles of Traditional Chinese Medicine (TCM) in regulating pyroptosis, TCM affects the occurrence and development of DCM by regulating pyroptosis or protein expression, which has become a research hotspot of myocardial protection strategies in diabetes. This article reviews the mechanism of pyroptosis in DCM and the research progress of TCM monomers and compound prescriptions in regulating the process of pyroptosis in DCM, in order to provide new ideas and targets for clinical research and treatment of DCM.

Abstract:Aim To investigate the role of myeloid angiotensin type 1 receptor (Mye AT1R) in vascular insulin resistance and vascular injury in deoxycorticosterone acetate (DOCA)/salt-sensitive hypertensive mice. Methods C57BL/6J mice (wild type, WT) and Mye AT1R－/－ mice were randomly divided into WT group, DOCA/salt-sensitive hypertension group (DOCA group), Mye AT1R－/－group and Mye AT1R－/－/DOCA group, 8 in each group. DOCA/salt-sensitive hypertension was induced by left nephrectomy and DOCA sustained-release tablet implantation. Systolic blood pressure (SBP) was measured by tail cuff method. HE staining was used to observe aortic wall thickness, immunofluorescence was used to detect F4/80 (monocyte/macrophage marker) of aorta, RT-PCR and Western blot were used for mRNA and protein expressions of AT1R, proinflammatory factors and insulin signaling molecules. Acetylcholine or insulin-induced endothelium-dependent vasodilation was determined by isolated vascular perfusion system. Results Compared with WT group, in DOCA group, systolic blood pressure increased by 37%, aortic wall thickness increased by 57%, acetylcholine or insulin-induced endothelium-dependent vasodilation decreased by 32% and 36% respectively (P＜0.05), the number of F4/80 positive cells increased by 195%, the protein expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and phosphorylated c-Jun N-terminal kinase (p-JNK) were significantly increased by 42%, 45% and 32% respectively, the protein expression of p-Akt and p-Enos decreased by 36% in the aorta of DOCA mice (P＜0.05). Specific knockout of myeloid AT1R, aortic thickness decreased by 14%, the number of F4/80 positive cells decreased by 44%, acetylcholine or insulin-induced endothelium-dependent vasodilation improved by 21% and 17% respectively, the protein expression of MCP-1, TNF-α and p-JNK decreased by 52%, 41% and 17% respectively, damaged insulin protein PI3K/Akt/eNOS signaling pathway was reversed, the protein expression of p-Akt and p-eNOS increased by 48% and 42% respectively (P＜0.05) without significant reduction in systolic blood pressure. Conclusion Knockout of Mye AT1R can reduce vascular insulin resistance and vascular injury caused by salt-sensitive hypertension, and its mechanism may be related to inhibition of vascular inflammation caused by macrophage infiltration in vascular wall.

Abstract:Aim To observe the effect of cardiac glucocorticoid receptor activation on cardiac remodeling and cardiac function after myocardial infarction (MI) in mice and its possible mechanism. Methods 28 male C57BL/6J mice were randomly divided into sham group, dexamethasone group, MI group and MI＋dexamethasone group, 7 mice in each group. The MI group ligated the left anterior descending coronary artery in mice to create an MI model; the sham group and the dexamethasone group only wound the thread without ligation, and injected normal saline or dexamethasone (20 mg/kg) by intraperitoneal injection 24 h before the operation; the MI＋dexamethasone group was injected with dexamethasone (20 mg/kg) by intraperitoneal injection 24 h before creating MI model. On the 7th day after surgery, small animal ultrasound was used to detect cardiac function indicators, heart tissue slices were taken and immunohistochemical staining was used to observe the activation level of glucocorticoid receptors, Masson staining was used to observe the infarct area, immunohistochemical staining was used to observe the levels of inflammatory factor IL-6 and chemokine CCL-5. Results Compared with the MI group, in the MI+dexamethasone group the left ventricular end-diastolic diameter reduced by 6.85%, the left ventricular end-systolic diameter reduced by 6.89%, the left ventricular end-diastolic volume reduced by 16.11%, the left ventricular end-systolic volume reduced by 17.18% (all P＜0.05), the ejection fraction increased by 10.63%, and the fractional shortening rate increased by 13.11% (all P＜0.05), indicating that dexamethasone pretreatment improved cardiac function in mice after MI; the level of phosphorylated glucocorticoid receptor increased 1.57 times; after MI, the infarct area decreased by 18.4% (P＜0.05); the expression level of inflammatory factor IL-6 decreased by 71.4%, and the expression level of chemokine CCL-5 decreased by 65% (all P＜0.05). Conclusion Activation of cardiac glucocorticoid receptors has an antagonistic effect on myocardial cell damage in mice after MI, which can improve myocardial remodeling, this effect may be related to its anti-inflammatory response.

Abstract:Aim To explore the role of circular RNA spindle and kinetochore-associated protein 3 (circ-SKA3) in regulating Toll-like receptor 4 (TLR4) axis in atherosclerosis (As) through miR-1303. Methods Carotid artery plaque, diseased vascular tissue, normal tissue adjacent to plaque and venous blood were collected from 30 patients with As treated by carotid endarterectomy from April 2019 to April 2022. Another 30 normal venous blood samples were collected. Microarray analysis, quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization (FISH) were used to detect the expression and localization of circ-SKA3 in plaque tissue of As patients, control samples, plasma exosome, human umbilical vein endothelial cell (HUVEC) and aorta of As model mice. The relationship among circ-SKA3, miR-1303 and TLR4 was verified by bioinformatics, double luciferase reporter gene detection and RNA immunoprecipitation. The proliferation, migration and angiogenesis of HUVEC were detected by CCK-8, scratch, Transwell and angiogenesis experiments. TLR4 axis-related protein expression was detected by Western blotting. Pathological changes of aorta in As model mice was observed by Oil red O staining, HE staining and Masson staining. TLR4 expression in aorta of As model mice was detected by immunohistochemistry and immunofluorescence. Results The expression levels of circ-SKA3 and TLR4 in plaque tissue, plasma exosome, oxidized low density lipoprotein (ox-LDL) treated HUVEC and circ-SKA3, TLR4 in aortic of As model mice were up-regulated (P＜0.05), while the expression level of miR-1303 was down-regulated (P＜0.05). Functional analysis showed that circ-SKA3 promoted HUVEC damage in vitro and As progress in vivo. Mechanism analysis showed that circ-SKA3 could promote TLR4 expression by adsorbing miR-1303 by sponge. Inhibition of circ-SKA3/miR-1303/TLR4 axis can inhibit the formation of As lesions. Conclusions circ-SKA3 is overexpressed in carotid plaque, plasma exosome, ox-LDL-treated HUVEC and As model mouse aorta in As patients. circ-SKA3/miR-1303/TLR4 axis can promote the development of As model in vivo and in vitro.