Abstract:Material and energy homeostasis is essential for maintaining cell survival and regular functions, and the loss of material and energy homeostasis may result in a variety of diseases. Recently, the relationship between cellular metabolism and function has become a hotspot in the study of various metabolic diseases, cancer and autoimmune diseases. This review focuses on the roles and regulatory mechanisms of T lymphocytes energy metabolism in its activation, differentiation and physiological function. It also discusses the significance of type M2 pyruvate kinase, which is the key enzyme in glycolysis, for the treatment of atherosclerosis.

Abstract:Aim To investigate the effect and mechanism of paeonol on the secretion of exosomes in THP-1 cells induced by lipopolysaccharide (LPS). Methods THP-1 cell culture supernatant exosomes were extracted by ultracentrifugation; the morphology of exosomes was observed by transmission electron microscope (TEM), and the marker proteins Alix, TSG101, CD9 and CD63 were detected by Western blot, and the dynamic light scattering (DLS) was used to detect exosome particle size. CCK-8 was used to detect the effect of different concentrations (0,0, 1,0.1,0.01 and 0.001 mg/L) of LPS on the survival rate of THP-1 cells to determine the optimal concentration; CCK-8 was used to detect the survival rate of THP-1 cells in different concentrations of paeonol (0,0, 0,0 and 15 μmol/L) medium for different time (2,4 and 48 h), to determine the optimal concentration and time; the amount of exosome protein was detected by BCA; the levels of N-SMase2 and p38 MAPK proteins in the cells were detected by Western blot. Results The results showed that the microvesicle structure obtained by ultracentrifugation was exosome. When the dose of LPS was 1 mg/L, it was suitable; when paeonol was administered at 60 μmol/L, the cell survival rate was higher at 24 h. The exosome secretion of the LPS group was significantly increased compared with the blank group, and the N-SMase2 protein level was increased, and the effect was similar to that of DNR. The exosome secretion of the paeonol group was significantly reduced compared with the LPS group, and the expression of N-SMase2 protein was decreased, and the effect was similar to that of GW4869. LPS promoted phosphorylation of p38 MAPK, and paeonol and SB203580 inhibited p38 MAPK phosphorylation.Paeonol inhibited the secretion of exosome of THP-1 cells after LPS stimulation. Conclusion Paeonol inhibits the secretion of THP-1 cell exosome, which is related to the inhibition of p38 MAPK/N-SMase2 pathway.

Abstract:Aim To construct lentiviral Hoxa3 vector, observe its transfection efficiency to human umbilical vein endothelial cells (HUVEC), study its effect on cell migration and angiogenesis, and explore the mechanism of Hoxa3 promoting angiogenesis. Methods Human Hoxa3 gene was obtained from the polymerase chain reaction library by gene synthesis, after being inserted into the bone plasmid, the lentiviral Hoxa3 vector was obtained by transfecting three plasmids into 293T cells, and the titer of lentiviral Hoxa3 vector was determined. Transfection of lentiviral Hoxa3 vector to HUVEC was performed to obtain the maximum transfective efficiency. HUVECs were divided into the control group and lentiviral Hoxa3 transfection group. HUVECs migration and tubule formation experiments were carried out to observe the effects of Hoxa3 on HUVEC migration and tubule formation. Western blot was used to detect the changes of urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-14 (MMP-14) protein expression in HUVECs transfected with lentiviral Hoxa3 vector. Results The lentiviral Hoxa3 vector was successfully constructed, and the titer of the virus was 8×10¹¹ TU/L. The transfection efficiency of 30 MOI lentiviral vector to HUVEC was over 99%. Western blot results showed that Hoxa3 could be effectively expressed in HUVEC after transfection of lentiviral Hoxa3 vector into HUVEC. Compared with the control group, transfection of HUVEC with lentiviral Hoxa3 vector significantly enhanced the migration and tubule formation of HUVEC, and significantly increased the expressions of uPAR and MMP-14 proteins (P＜0.05). Conclusion Successfully constructed lentiviral Hoxa3 vector can promote migration and tubule formation of HUVEC, and its mechanism may be related to its up-regulation of uPAR and MMP-14 proteins in HUVEC.

Abstract:Aim To investigate the effect of recombinant human vascular endothelial growth factor-dextran-polylactic acid-glycolic acid (VEGF-dextran-PLGA) sustained-release microspheres on promoting angiogenesis in ischemic lower limbs of rats. Methods VEGF-dextran-PLGA microspheres were prepared and their morphology was observed by scanning electron microscopy. The drug release properties were determined by ELISA in vitro. The fibrin gel loaded with VEGF-dextran-PLGA microspheres was injected into the ischemic lower limb muscle of rats, and histological and immunohistochemical staining were performed 1 month later to evaluate the angiogenesis. Results VEGF-dextran-PLGA microspheres released VEGF in vitro for more than 4 weeks. They could significantly increase the density of capillaries and α-smooth muscle actin-positive blood vessels at the injection site, and promote the mobilization of CD34, C-kit and VEGFR-2 positive cells. Conclusion Intramuscular injection of fibrin gel loaded with VEGF-dextran-PLGA microspheres is an ideal therapeutic angiogenesis method.

Abstract:Aim To investigate the expression and function of transmembrane protein 16A (TMEM16A) after acute myocardial infarction (AMI) in the left ventricular fibroblasts of minipig. Methods The AMI models were induced by ferric chloride (FeCl₃)-induced thrombosis in the left anterior descending coronary artery (LAD). Four hours after AMI, the infarction models were evaluated by enzymology and echocardiography. And, twenty-four hours after AMI, the TMEM16A mRNA level was measured by quantitative real-time PCR (qRT-PCR) in minipig ventricular fibroblasts; the changes of current intensity were determined by patch-clamp. Results Compared with the sham groups, the concentration of cardiac troponin I (cTnI), creatine kinase isoenzyme (CK-MB) and myoglobin (MYO) were significantly increased in the AMI model groups, and the left ventricular ejection fraction (LVEF) had a significant decrease ((53.4±1.9)%, P<0.05). The gene expression level of TMEM16A was dramatically upregulated, and the current intensity was also significantly enhanced (P<0.05). The amplitude of current was (1.58±0.67) pA/pF, (3.69±1.26) pA/pF, (7.60±2.14) pA/pF, (12.94±2.38) pA/pF, (22.19±2.61) pA/pF at stimulation voltages of +20, +40, +60, +80 and +100 mV, respectively. Conclusions These results suggested that TMEM16A was expressed in ventricular fibroblast cells of minipig. Moreover, the TMEM16A expression level was up-regulated after AMI; and calcium-actived chloride current (ICl,Ca) was enhanced by influencing calcium-activated chloride channels (CaCC).

Abstract:Aim To investigate the effects of C1q/TNF-related protein 1 (CTRP1) on the expression of adhesion molecules and adhesion function of human umbilical vein endothelial cells(HUVEC) and related mechanisms.Methods HUVEC were cultured and treated with different doses of recombinant CTRP1(0,0,1 000 μg/L). After 24 hours of stimulation, the gene expression and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were detected by Real-time fluorescence quantitative PCR and Western blot respectively. Fluorescence labeled THP-1 monocytes were incubated with HUVEC which were pre-treated with recombinant CTRP1(0,0,1 000 μg/L)or TNF-α(10 μg/L)for 24 h. The number of adhesion monocytes was counted by fluorescent microscope. In in vivo adhesion experiment, recombinant CTRP1 (3 or 10 μg), positive control factor TNF-α (3 μg), or negative control protein bovine serum albumin (BSA) was injected intraperitoneally for 2 h. Real-time monitoring microscopy was used to examine the rolling and adhesion of monocytes to the wall of mesenteric arteries. HUVEC were incubated with recombinant CTRP1(1 000 μg/L) and the expression levels of phospho-p38MAPK,phospho-ERK,phospho-JNK and phospho-p65 were detected by Western blot. Simultaneously, HUVEC pre-treated with SB203580 (the p38MAPK special inhibitor) were incubated with recombinant CTRP1(1 000 μg/L). The expression of adhesion molecules were detected by Western blot. Results After 24 h stimulation with recombinant CTRP1, protein and mRNA levels of adhesion molecules(VCAM-1 and ICAM-1) were significantly increased in a concentration-dependent manner in HUVEC(P＜0.05). CTRP1 dose-dependently induced the adhesion of THP-1 cells to HUVEC in vitro and interactions of leukocytes to the wall of mesenteric arteries in vivo. CTRP1 activated the p38 mitogenprotein kinase(MAPK) pathway with concurrent phosphorylation of the p65 in HUVECs. Consistently administration of p38 inhibitor SB203580,virtually abolished CTRP1-induced expression of VCAM-1 and ICAM-1. Conclusion CTRP1 can induce the expression of VCAM-1 and ICAM-1 in HUVEC and promote the adhesion function of HUVEC through the activation of p38 MAPK signaling pathway.

Abstract:Aim To investigate the predictive value of mean platelet volume/lymphocyte ratio (MPVLR) in patients with acute ST-segment elevation myocardial infarction (STEMI) after primary percutaneous coronary intervention (pPCI). Methods From January 2015 to December 2017, a total of 304 patients who underwent pPCI, admitted within 12 hours from symptom onset, were enrolled and divided into two groups based on no-reflow. The platelet/lymphocyte ratio (PLR) and the MPVLR were calculated by collecting baseline data of patients enrolled and preoperative blood parameters. Logistic regression was used to analyze the risk factors. Results In univariate analysis, the lymphocyte count in the no-reflow group was significantly lower than that in the normal-reflow group (P＜0.05); Mean platelet volume, PLR, MPVLR, high-sensitive C-reactive protein(hs-CRP), tirofiban in operation, pain-to-balloon time, and average stent length in the no-reflow group were significantly higher than those in the normal-reflow group (P＜0.05). In multivariate analysis, PLR, MPVLR, and pain-to-balloon time were independent predictors of no-reflow after pPCI in patients with acute STEMI. The area under the ROC curves for PLR and MPVLR were 0.766 and 0.795, respectively. The sensitivity was 73.8% and 88.7%, and the specificity was 69.1% and 64.1%, respectively. Conclusions MPVLR can effectively predict the occurrence of no-reflow after pPCI in patients with acute STEMI. As a calculated value of common indicators from blood routine examination, MPVLR is easy to get and deserved to be generalized.

Abstract:Aim To investigate the correlation between carotid atherosclerotic plaque and bone mineral density (BMD) in patients with type 2 diabetes mellitus (T2DM). Methods Analysing the clinical data of 720 patients with T2DM, the selected subjects were divided into male group and female group, and each group was further divided into normal bone mass group and osteopenia group, carotid artery plaque group and without carotid artery plaque group. Results In the male osteopenia group (77 cases), there were 68 cases of atheromatous plaque (88.31%). Compared with the normal bone mass group (164 cases), the incidence of atherosclerotic plaque in the osteopenia group was significantly increased (P<0.05), and the bone mineral density (BMD) in the plaque formation group was significantly decreased (P<0.05). In the female osteopenia group (255 cases), 191 (74.90%) had atherosclerotic plaques. Compared with normal bone mass group (224 cases), the incidence of atherosclerotic plaques in osteopenia group was significantly increased (P<0.05), and BMD in plaque formation group was significantly decreased (P<0.05). Logistic regression analysis showed that the influence factors of male bone mass reduction included age (OR=1.059, P=0.002), BMI (OR=0.853, P=0.004), FBG (OR=1.138, P=0.044), presence of carotid artery plaque (OR=2.514, P=0.035); the influencing factors of female bone mass reduction included age (OR=1.117, P=0.000), menopause age (OR=0.946, P=0.031), BMI (OR=0.910, P=0.003). Conclusions Carotid atherosclerotic plaque is closely related to bone loss in T2DM patients, which often occurs together in women as a result of the existence of common risk factors. However, in male patients, the formation of carotid atherosclerotic plaques is a risk factor for osteoporosis. Therefore, the development of carotid atherosclerosis is often accompanied by changes in bone mass, which is prone to osteoporosis.

Abstract:Aim To investigate the relationship between Rho/ROCK signal pathway and hemorheology in patients with cerebral arteriosclerosis (CAS). Methods Blood samples were collected from 80 CAS patients and 80 healthy persons. The biochemical parameters, the levels of Ras homolog gene family member A (RhoA) and Rho-associated kinase 2 (ROCK2), blood cell-specific volume, whole blood apparent viscosity (high shear and low shear) and plasma viscosity were measured. Hemorheological indexes, RhoA and ROCK2 levels were compared between the two groups.The correlations between RhoA, ROCK2 levels and hemorheological parameters were analyzed in CAS patients. ResultsThe smoking rate, body mass index, fasting plasma glucose, 2-hour postprandial plasma glucose, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglyceride and serum homocysteine in CAS group were higher than those in control group (P＜0.05). The blood cell-specific volume, whole blood apparent viscosity (high shear and low shear), plasma viscosity, ROCK2 and RhoA levels in CAS group were higher than those in control group (P＜0.05). Pearson correlation analysis and multiple linear regression analysis showed that serum ROCK2 level was positively correlated with blood cell-specific volume, whole blood apparent viscosity (high shear and low shear) and plasma viscosity; RhoA level was positively correlated with whole blood apparent viscosity (high shear and low shear). Conclusion Rho/ROCK signal pathway is closely related to abnormal hemorheology in CAS patients, and it may be involved in the occurrence and development of CAS.

Abstract:Aim To investigate the clinical significance and predictive value of serum carbohydrate antigen 125 (CA125) and brain natriuretic peptide (BNP) in patients with acute myocardial infarction (AMI) and heart failure (HF) occurred during hospitalization. Methods AMI patients admitted to cardiovascular department of Zhengzhou People's Hospital were selected from January to December in 2017. Serum CA125, BNP and color Doppler echocardiography were examined. Cardiac function was assessed within 1 week after coronary artery infusion therapy. The correlation among serum CA125, BNP levels and the indexes of color echocardiography were analyzed. The levels of CA125, BNP and left ventricular ejection fraction (LVEF) in different NYHA cardiac function classification groups of HF patients were compared.Results A total of 124 AMI patients was enrolled, of whom 41 (33.1%) had met the diagnostic criteria for HF. Serum CA125 and BNP levels were negatively correlated with LVEF (r＝－0.227, P＝0.011; r＝－0.569, P＜0.001), and CA125 was positively correlated with BNP (r＝0.270, P＝0.002). The area under curve (AUC) of CA125 predicting HF occurrence after AMI was 0.772 (95%CI 0.679-0.864, P＜0.01), and AUC of BNP was 0.938 (95%CI 0.888-0.988, P＜0.01).Among different NYHA classification groups in 41 patients with HF after AMI, with the increase of NYHA classification, serum levels of CA125 and BNP increased, and LVEF decreased. The differences of CA125, BNP and LVEF among groups were statistically significant (P＜0.001). Conclusion Serum levels of CA125 and BNP have predictive value for the occurrence of HF after AMI, and are correlated with the clinical severity of HF.

Abstract:Aim To evaluate the diagnostic value of gemstone CT iodine-water based images for acute cerebral infarction after interventional therapy. Methods 31 patients with acute cerebral infarction were found to have abnormal intracranial high density on the immediate gemstone CT scan QC images after the interventional therapy. Iodine-water based images were used to reconstruct and analyze the images. Simultaneously, the iodine based value and water based value of high density were measured, and the images were compared with the results of 24 h ordinary CT review. Results By using iodine-water based images, 17 cases were diagnosed as iodine contrast agent exudation, 14 cases were diagnosed as cerebral hemorrhage transformation, which were identical with the results of 24 h ordinary CT reexamination (Kappa=1,P＜0.01). The immediate postoperative iodine based value:iodine contrast agent exudation(32.09±5.36) g/L, cerebral hemorrhage transformation(6.86±2.26) g/L, there was statistical significant difference (t=53.291, P＜0.01). The immediate postoperative water based value:the iodine contrast agent exudation(1 027.93±8.29) g/L, the cerebral hemorrhage transformation(1 069.68±7.18) g/L, there was statistical significant difference (t =－8.897, P＜0.01). Conclusion Gemstone CT iodine-water based images can accurately diagnose intracranial iodine contrast agent exudation and cerebral hemorrhage transformation after interventional therapy for acute cerebral infarction, which is worthy of clinical promotion.

Abstract:Aim To investigate the effects of butylphthalide combined with edaravone on carotid intima-media thickness (IMT) and neurological function in elderly patients with acute cerebral infarction. Methods From January 2014 to March 6,2 elderly patients with acute cerebral infarction in the Department of Neurology in our hospital were randomly divided into edaravone treatment group and butylphthalide combined edaravone treatment group (combined treatment group), 46 cases in each group. Edaravone treatment group was treated with edaravone, and combined treatment group was treated with edaravone and butylphthalide for 90 days. The therapeutic effects of the two groups were observed, carotid IMT and plaque area were evaluated, and neurological function was evaluated by NIHSS score, modified Rankin scale (mRS) score and Barthel index. At the same time, the occurrence of adverse reactions in the two groups was observed. Results The total effective rate of combined treatment group was significantly higher than that of edaravone treatment group (P＜0.05). After treatment, carotid IMT and plaque area in the two groups were significantly lower than those before treatment (P＜0.05), and significantly lower in the combined treatment group than those in the edaravone treatment group (P＜0.05), the NIHSS score and mRS score in the combined treatment group were lower than those in the edaravone treatment group (P＜0.05), and the Barthel index was significantly higher than that in the edaravone treatment group (P＜0.05), there was no significant difference in the incidence of adverse reactions between the two groups (P＞0.05). Conclusion Butylphthalide combined with edaravone can significantly reduce carotid IMT in elderly patients with acute cerebral infarction and improve their nerve function, the clinical effect is remarkable and the safety is high.

Abstract:Aim To generate mutant mouse with ALK7 gene inserted by LoxP sequences by CRISPR/Cas9, to further develop temperospatial specific ALK7 deleted mouse models via cross bred with the tissue-specific Cre mouse, providing a basis for functional study of ALK7 in special time and tissue. Methods The CRISPR/Cas9 technology was used to edit ALK7. Two sgRNAs were designed to direct Cas9 endonuclease cleavage in intron 3-4 and intron 6-7. The targeting vector with LoxP-ALK7-LoxP sequence was designed. sgRNA, Cas9 mRNA and targeting vector were co-injected into zygotes, which were transferred to pseudopregnant mice. The pups were genotyped by PCR and Southern blott. Real-time PCR and Western blot were performed for analysis of the expression of ALK7. Results The ALK7LoxP/LoxP mouse was generated by CRISPR/Cas9, and did not show influence on the expression of ALK7. Conclusion The CRISPR/Cas9 technology can successfully generate the ALK7LoxP/LoxP mouse by inserting LoxP sequence into ALK7 gene, which is a basis for the creation of tissue-specific ALK7 deleted mouse models.

Abstract:Atherosclerosis (As) is a chronic inflammatory disease triggered by various risk factors and is the main pathological basis of cardiovascular and cerebrovascular diseases. In recent years, the incidence of As has been increasing, but its pathogenesis remains unknown. Therefore, it is of great significance to explore the pathogenesis of As for early detection and treatment of the disease. As an important mediator of intercellular communication, exosomes plays an important role in the process of As. Micro RNA (miRNA) is involved in the formation of As as a key signal transduction and molecular regulation pathway. Therefore, this article reviews the regulatory role of exosomes inclusion miRNA in the formation and development of As.

Abstract:Triglyceride (TG) is predominantly present in triglyceride-rich lipoproteins (TRLs). Dysregulation of TRL metabolism is closely associated with the occurrence and development of atherosclerosis. Lipoprotein lipase (LPL), a glycoprotein secreted by parenchyma cells, can cleave TG within the TRLs to produce free fatty acid. A variety of factors are involved in TRL metabolism and atherosclerosis progression by regulating LPL expression and activity. Thus, clarifying the role TRL metabolism and its regulatory mechanisms could have significant implications for the prevention and treatment of cardiovascular disease.

Abstract:Atherosclerosis(As) is the main pathological basis of ischemic cardiovascular and cerebrovascular diseases such as coronary heart disease and cerebral infarction. Its etiology is complicated and the pathogenesis has not been fully clarified. In recent years, more and more studies revealed that sphingomyelin signaling pathway can affect the occurrence and development of atherosclerosis by regulating lipid metabolism, inflammation and vascular endothelial function. This paper reviews the relationship between sphingomyelin, ceramide and sphingosine-1-phosphate, the key molecules of sphingomyelin signaling pathway, and atherosclerosis in order to provide new ideas for the prevention and treatment of As.