Abstract:Aim To investigate the levels of krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase 3 (NOS3) in the serum of patients with acute cerebral infarction (ACI) of large-artery atherosclerosis (LAA) type, and to analyze their value in the diagnosis and disease assessment of LAA type ACI. Methods A total of 150 patients with LAA type ACI were divided into mild group (n＝36), moderate group (n＝48), and severe group (n＝66) based on their condition. Additionally, a control group (n＝150) was selected from health exminers during the same period. The levels of serum KLF2 and NOS3 in each group were compared; receiver operator characteristic(ROC) curve was applied to analyze the diagnostic value of serum KLF2 and NOS3 levels for LAA type ACI and the predictive value for the occurrence of severe LAA type ACI, respectively. Results The serum KLF2 and NOS3 levels were significantly lower in LAA type ACI group than those in control group (P＜0.05). The serum KLF2 and NOS3 levels in the mild, moderate and severe groups were significantly decreased in turn(P＜0.05). The area under the curve (AUC) of the combined diagnosis of serum KLF2 and NOS3 for LAA type ACI was 0.858, with a sensitivity of 73.33% and a specificity of 86.00%, which was superior to the individual diagnosis of KLF2 and NOS3 (Zcombined detection-KLF2＝3.796, Zcombined detection-NOS3＝4.689, all P＜0.001). The AUC of combined prediction of serum KLF2 and NOS3 for the occurrence of severe LAA type ACI was 0.878, with a sensitivity of 77.27% and a specificity of 90.48%, which was superior to the independent prediction of KLF2 and NOS3 (Zcombined detection-KLF2＝2.401, P＝0.016; Zcombined detection-NOS3＝3.070, P＝0.002). Conclusions The serum levels of KLF2 and NOS3 in patients with LAA type ACI were significantly reduced and negatively correlated with the severity of the disease. The combination of the two has high evaluation efficacy in the diagnosis and disease prediction of LAA type ACI.

Abstract:Aim To observe the effect of TSB2 inhibiting the combination of heat shock protein 90 (HSP90) and endothelial nitric oxide synthase (eNOS) on the formation of atherosclerosis. Methods Human umbilical vein endothelial cells (HUVEC) were treated with TSB2 and the combination between HSP90 and eNOS was detected by co-immunoprecipitation. C57BL/6 mice and low density lipoprotein receptor knockout (LDLR－/－) mice were fed with normal diet (ND) or high fat diet (HFD) for 12 weeks while injected with phosphate buffered saline (PBS) or TSB2 intraperitoneally. The mice were divided into four groups:C57BL/6＋ ND＋PBS group, LDLR－/－＋ND＋PBS group, LDLR－/－＋HFD＋PBS group, LDLR－/－＋HFD ＋TSB2 group. Then the aorta was isolated. The combination between HSP90 and eNOS in aorta was measured. The atherosclerotic plaque in aorta and aortic sinus were determined. The production of nitric oxide (NO) and superoxide anion (O2·－) were also detected. At the same time, L-monomethyl-arginine (L-NMMA), a competitive substrate of L-arginine, was used to determine the production of NO, and L-nitroarginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, was used to determine the production of O2·－. Results Compared with control group, the combination between HSP90 and eNOS was decreased by 41.06% (P＜0.05) in cultured HUVEC treated with TBS2. Compared with LDLR－/－＋HFD＋PBS group,the combination between HSP90 and eNOS in the mouse aortas was decreased by 40.95% (P＜0.05) in LDLR－/－ ＋HFD＋TSB2 group, and the production of O2·－ was decreased by 63.73% (P＜0.05) (L-NAME significantly inhibited the production of O2·－ in LDLR－/－＋HFD＋PBS group), while the production of NO had no significant change in the mouse aortic endothelial cells (L-NMMA inhibited NO production in all groups), and the formation of atherosclerotic lesions in aortas and aortic sinus were significantly decreased by 59.39% and 68.86% (P＜0.05) respectively in LDLR－/－＋HFD＋TSB2 group. Conclusion TSB2 can reduce the O2·－ production of uncoupled eNOS in vascular endothelial cells by inhibiting the combination of HSP90 and eNOS in aortic endothelial cells, and finally inhibits the formation of atherosclerosis.

Abstract:Stress refers to the adaptive changes and reconstruction of the homeostasis of the body in order to meet the corresponding needs when the body feels the strong stimulation of various factors. Oxidative stress is a stress response involving reactive oxygen species (ROS) as the main effector. Through oxidation, it participates in the adaptation and repair response of tissues and cells. ROS can be used as the second messenger of cell signal transduction in this process. Free radicals involved in oxidative stress include ROS and reactive nitrogen species (RNS). The stress response involving RNS can also be called nitrative stress, which is specifically manifested as increased expression of nitric oxide synthase (NOS), and increased expression of nitric oxide (NO), which ultimately leads to activity nitrogen levels increase, and the increase in reactive nitrogen can nitrate proteins. Stress is manifested as the adaptive response of cells and the repair of tissues. Fibrotic repair is a kind of incomplete repair of tissues in the late stage of stress response. It is the main repair method of tissues containing permanent cells. It is also the adaptive response of tissues to external stimuli in the late stage of inflammatory response, which is manifested as fibrosis of tissues or organs. This review summarized the recent progress on the relationship between protein nitration and fibrosis.

Abstract:Aim To study the preventive effect and mechanism of ligustrazine on doxorubicin-induced cardiotoxicity in mice. Methods The mouse model of cardiotoxicity induced by doxorubicin was established. The mice were randomly divided into three groups:sham group, doxorubicin group (doxorubicin 15 mg/kg), ligustrazine treated group (ligustrazine 60 mg/(kg·d)＋doxorubicin 15 mg/kg), echocardiography was performed to detect cardiac function of doxorubicin and ligustrazine intervention groups in mice. The sections were stained with HE staining or Massons trichrome staining for histological and collagen analysis, apoptosis of cardiomyocyte were analyzed by TUNEL staining, Western blot was used to analyze the expression of Akt, endothelial nitric oxide synthase (eNOS), the phosphorylation of Akt and eNOS.Results Compared with doxorubicin group, the fractional shortening (FS) and the ventricular ejection fraction (EF) in the ligustrazine treated group were significantly higher (P＜0.01). The ligustrazine treated group significantly decreased apoptosis of cardiomyocyte in mice as compared to the doxorubicin group (P＜0.01), the expression of p-Akt and p-eNOS in the ligustrazine treated group were stronger than those in the the doxorubicin group (P＜0.01). Meanwhile, the ligustrazine treated group significantly inhibited cleaved Caspase-3 as compared to the doxorubicin group. Conclusion Ligustrazine protects doxorubicin-induced myocardial injury by activating Akt/eNOS signaling pathway and inhibiting cardiomyocyte apoptosis, and it may be an underlying mechanism by which ligustrazine can prevent doxorubicin-induced cardiotoxicity.

Abstract:Aim To investigate the relationship between carotid stenosis and endothelial progenitor cells (EPC) and the regulation of insulin-like growth factor-1 (IGF-1) on EPC and its mechanism. Methods 35 patients with cerebral infarction and carotid stenosis were enrolled into the carotid stenosis group. At the same time, 11 healthy control subjects were selected as control group. According to the results of cerebral angiography, carotid stenosis group was divided into 3 subgroups: mild stenosis group, middle stenosis group, severe stenosis group. The serum IGF-1 concentration of the study subjects was determined. Mononuclear cells were isolated by density gradient centrifugation and cultured with endothelium growth medium 2 (EGM2), and the double staining method was used to identify EPC. The experiment was divided into 4 groups:untreated group, IGF-1 group, IGF-1＋NG-nitro-L-arginine methyl ester (L-NAME) group and L-NAME group. Cells were cultured with EGM2 for 2 to 3 weeks, and the proliferation and adhesion function of EPC were determined in each group. Results The more severe the degree of carotid stenosis, the lower the number of EPC colony forming (P＜0.05), the lower the serum IGF-1 concentration, and the proliferation and adhesion ability of EPC decreased with the increase of the degree of stenosis. Function experiments showed that EPC function in IGF-1 group was significantly higher than that in untreated group (P＜0.01), IGF-1＋L-NAME group had no obvious difference, and EPC function in L-NAME group was lower than that in untreated group. The endothelial nitric oxide synthase (eNOS) in IGF-1 group was significantly higher than that in untreated group, there was no significant difference in IGF-1＋L-NAME group, and eNOS in L-NAME group was lower than that in untreated group. Conclusion EPC may have a protective effect on carotid stenosis, and IGF-1 may enhance the function of EPC by affecting the synthesis of eNOS.

Abstract:Aim To investigate the relationship between intron 4a/b gene polymorphisms of endothelial nitric oxide synthase (eNOS) gene and coronary artery ectasia (CAE). Methods The research was performed by case-control study. The CAE group included 30 patients with coronary artery ectasia on coronary angiogram. The control group contained 41 patients with normal coronary artery. The study collected the data of patientsgender, age, smoking history, drinking history, ect. Polymorphism chain reaction (PCR) technique was used to identify the eNOS intron 4a/b gene polymorphisms. Results The lesion involved single coronary artery in most cases was 46.7%(14 cases), the lesions involved in two and three vessels were 26.7%(8 cases)and 26.7%(8 cases), respectively. The right coronary artery (RCA) was the most frequently involved vessel (44.4%), the left anterior descending artery (LAD) and the left circumflex artery (LCX) were 31.5% and 24.1%, respectively. The frequencies of eNOS gene phenotypes in CAE group and control group for “aa”, “ab”, “bb” were 13.3%, 33.3%, 53.3% and 4.9%, 17.1%, 78%(P＞0.05), respectively. The presence of “a” type allele of eNOS gene in CAE group and control group were 30% and 13.4%(P<0.05), respectively. Logistic regression analysis showed that “a” type allele of eNOS gene was an independent risk factor for CAE (P<0.05, OR =3.327, 95% CI=1.083 ~ 10.226). Conclusion The “a” type allele of eNOS gene may be an independent risk factor for the occurrence of CAE.

Abstract:Aim To explore the effect of sitagliptin on the expression of endothelin-1(ET-1) and endothelial nitric oxide synthase(eNOS) in human aortic endothelial cells(HAEC) and its underlying mechanism in high glucose environment. Methods HAEC were cultured in high glucose environment(25 mmol/L), and treated with different concentrations of sitagliptin(0, 5, 10 and 20 μmol/L, respectively). The mRNA and protein expressions of eNOS, ET-1, iNOS and phosphate nuclear factor-kappa B p65(p-NF-κB p65) were measured. The measurements for eNOS, ET-1, iNOS, NF-κB p65 on mRNA and protein levels in HAEC were evaluated after incubation with tumor necrosis factor-α(TNF-α) and sitagliptin. Results Compared with normal medium(glucose concentrations for 7 mmol/L), both the mRNA and the protein expression of eNOS in HAEC significantly decreased in high glucose medium, while those of ET-1, iNOS and p-NF-κB p65 protein significantly increased(P<0.05). Compared with 0 μmol/L sitagliptin, 20 μmol/L sitagliptin significantly increased mRNA and protein expressions of eNOS, while decreased those of ET-1, iNOS and p-NF-κB p65 protein(P<0.05). Compared with sitagliptin alone treated HAEC, both the mRNA and the protein expressions of eNOS significantly decreased in HAEC treated with TNF-α and sitagliptin, while those of ET-1, iNOS and p-NF-κB p65 protein expressions significantly increased(P<0.05). Conclusions Sitagliptin enhances eNOS, represses ET-1, iNOS expressions at the level of transcription and translation through inhibiting NF-κB p65 phosphorylation in HAEC in high glucose environment. This may contribute to the improvement of endothelial function and prevention of subsequent atherogenesis.

Abstract:Aim To explore the association between 4A4B insert/deletion polymorphism of endothelial nitric oxide synthase(eNOS) and coronary heart disease. Methods Genotypes of 4A4B locus were detected in 842 coronary heart disease patients and 842 age and gender matched healthy individuals using polymerase chain reaction and Gel electrophoresis and the association between them was analyzed. Results The frequency distributions of patients with smoking, diabetes, hypertension, obesity and lipid metabolic abnormity were significantly higher than those in controls. Genotype distributions in controls were in consistence with Hardy-Weinberg inheritance model. Allele 4A(10.2% vs. 6.9%, OR＝2.03, 95%CI＝1.39~2.44), genotype AA(3.0% vs. 0.9%, OR＝2.08, 95%CI＝1.45~2.67) and AA＋AB(17.4% vs. 13.1%, OR＝1.58, 95%CI＝1.08~1.94) of the locus were significantly associated with conorany heart disease. Stratifiying analysis results showed that genoytpe AA and 4A allele were significantly associated with the disease in each subgroup excluding non-drinking subgroup, respectively, and the disease risk of individuals carring AA genotype and 4A allele were 2.34, 2.59, 3.13 fold and 2.55, 2.77, 3.10 fold than those with BB genoytpe or 4B allele in diabetes, hepertension, and fact subgroups, respectively. Conclusions 4A allele and AA, AA＋AB genotypes of 4A4B locus within eNOS gene might be genetic susceptible factors for coronary heart disease, and these genetic factor of the locus could interact with status of smorking, drinking, diabetes, hypertension and fat to further increase susceptibility to the disease.

Abstract:Aim To investigate the effect of maternal perinatal high-salt diet on dimethylarginine dimethylamino-hydrolase 2 (DDAH2)/asymmetric dimethylarginine (ADMA)/endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway of the mesenteric artery in the male offspring rats. Methods The rats were divided into 2 groups:normal-salt diet (NSD) group and high-salt diet (HSD) group, and fed respectively with normal-salt diet (1%NaCl) and high-salt diet (8%NaCl) during perinatal period. After delivery, the male offspring rats were fed with the same diet for 16 weeks. Blood pressure, mesenteric artery endothelial-dependent diastolic function, NO content, eNOS activity and ADMA content in plasma and mesenteric artery, DDAH2 activity and protein expression of DDAH1 and DDAH2 in mesenteric artery were detected by various methods. Results The systolic blood pressure (SBP) in HSD group was significantly higher than that in NSD group (P<0.01). The endothelium-dependent tension of mesenteric artery in HSD group was lower than that in NSD group (P<0.01). After incubation with ADMA, the blood vessel tension was significantly decreased in NSD group, while no significant change was found in HSD group. Compared with the NSD group, NO content was decreased (P<0.05), eNOS activity was decreased (P<0.01), ADMA content was increased (P<0.05) in plasma in HSD group, and NO content was decreased (P<0.01), eNOS activity was decreased (P<0.01), ADMA content was increased (P<0.05) on mesenteric artery in HSD group. In HSD group, DDAH2 activity and protein expression were decreased (P<0.01), but DDAH1 protein expression was not changed significantly. In HSD group, correlation analysis of mesenteric arterial indexes showed that eNOS activity was positively correlated with NO content, ADMA content was negatively correlated with eNOS activity, DDAH2 activity and DDAH2 protein expression were negatively correlated with ADMA content. Conclusion The high-salt diet in the maternal perinatal period results in the increase of SBP and the endothelial-dependent diastolic dysfunction on mesenteric arteries in male offspring rats, which are related to the decrease of DDAH2 activity and the disorder of DDAH2/ADMA/eNOS/NO pathway in mesenteric arteries.

Abstract:Aim To investigate the effect of metformin on angiotensin Ⅱ （AngⅡ） -induced proliferation of adult rat cardiac fibroblasts （CF） and its underlying mechanism. Methods The adult rat CF was isolated by a combination of trypsin and collagenase Ⅱ digestion.The cell proliferation was induced by AngⅡ （100 nmol/L） stimulation,and the CF was treated with metformin in different concentrations （10,50,and 200 μmol/L）.The proliferation of CF was evaluated by MTT assay,and the DNA synthesis was detected by EdU incorporation.The expression of endothelial nitric oxide synthase （eNOS） and phosphorylated endothelial nitric oxide synthase （p-eNOS） were detected by Western blot.The level of NO in CF culture supernatant fluids was measured by nitrate reductase method. Results Stimulation with AngⅡ for 48 h induced the proliferation of adult rat CF,and this effect was inhibited by pretreatment of CF with metformin in a concentration-dependent manner.Metformin significantly increased the phosphorylation level of eNOS in CF as well as the level of NO in cell culture supernatant fluids in a concentration-dependent manner.In addition,pretreatment of CF with eNOS inhibitor L-NAME markedly attenuated the inhibitory effect of metformin on AngⅡ-induced cell proliferation. Conclusion Metformin inhibits AngⅡ-induced proliferation of adult rat CF,and this effect may be associated with the activation of the eNOS/NO pathway.