Abstract:Aim To explore whether inhibiting P53 and increasing the expression of glucose transporter 4 (GLUT4) in cardiomyocytes can improve glucose metabolism disorder and reduce apoptosis induced by high glucose (HG) combined with ischemia-hypoxia(IH). Methods The cardiomyocyte HG＋IH model was induced in vitro. The experimental groups were the control group, HG group, IH group, HG＋IH group, HG＋IH＋P53 inhibitor group (HG＋IH＋Pifithrin-α group), HG＋IH＋P53 inhibitor＋GLUT4 inhibitor group (HG＋IH＋Pifithrin-α＋Fasentin group). Cell viability was detected by the CCK-8 method, lactate dehydrogenase (LDH), glycolytic key enzyme activity, and ATP content were detected by the kit, protein expression of P53, GLUT4, Bax/Bcl-2 and Caspase-3 were detected by Western blot, and cardiomyocyte apoptosis was detected by flow cytometry. Results ①In the HG+IH cardiomyocyte model in vitro, compared with the control group, the expression of P53 increased by 75%, the expression of GLUT4 decreased by 16%, the content of ATP decreased by 51%, the cell viability decreased by 45%, the LDH activity increased by 3.6 times, the expression of Caspase-3 and Bax/Bcl-2 increased by 54% and 77% respectively, and the apoptosis rate increased (all P＜0.05). ②After inhibiting the expression of P53 in cardiomyocytes, compared with the HG＋IH group, the expression of GLUT4 in cardiomyocytes increased by 34%, the content of ATP increased by 60%, the cell viability increased by 50%, the LDH activity decreased by 13%, the expression of Caspase-3 and Bax/Bcl-2 decreased by 31% and 53% respectively, and the apoptosis rate decreased in HG＋IH＋Pifithrin-α group (all P＜0.05). ③After inhibiting GLUT4, compared with the HG＋IH＋Pifithrin-α group, the expression of GLUT4 in cardiomyocytes decreased by 22%, the content of ATP decreased by 39%, the cell viability decreased by 25%, the LDH activity increased by 21%, the expression of Caspase-3 and Bax/Bcl-2 increased by 43% and 89% respectively, and the apoptosis rate increased (all P＜0.05). Conclusions In the cardiomyocyte model of high glucose combined with ischemia-hypoxia, inhibiting P53 can increase the expression of GLUT4, improve the glucose metabolism disorder of cardiomyocytes induced by high glucose combined with ischemia-hypoxia, and reduce apoptosis.

Abstract:Aim To investigate the level of serum tp53-induced glycolysis and apoptotic and regulator (TIGAR) mRNA in patients with acute ischemic stroke and its relationship with prognosis. Methods 247 patients with acute ischemic stroke were selected as the study object. The age, sex, body mass index, hypertension, hyperlipidemia, diabetes, coronary heart disease, smoking, stroke distribution, stroke onset time, toast etiology, severity, stroke focus number, hemorrhage transformation, homocysteine (Hcy), C-reactive protein (CRP), D-Dimer (D-D), hemoglobin A1c (HbA1c), angiopoietin-1 (Ang-1) of patients were collected. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the relative expression of serum TIGAR mRNA, and its relationship with prognosis was analyzed. Results The incidence of poor prognosis in 247 patients with acute ischemic stroke was 47.77% within 90 days. The relative expression of TIGAR mRNA in the prognosis group was significantly higher than that in the poor prognosis group, and the difference was statistically significant (P＜0.05). The area under the curve AUC, sensitivity and specificity of TIGAR mRNA in evaluating the prognosis of patients with acute ischemic stroke were 0.6,8.14% and 80.62%, respectively.Logistic multivariate regression analysis showed that the onset time of stroke, TOAST etiology classification, severity, hemorrhagic transformation and TIGAR mRNA were closely related to the prognosis of patients with acute ischemic stroke. Conclusion The higher the relative expression of TIGAR mRNA in serum, the better the prognosis of patients with acute ischemic stroke. Detection of relative expression of TIGAR mRNA is helpful to assess the prognosis of patients.

Abstract:Aim To investigate the correlation between the p53 gene codon 72 polymorphism and hyperlipidemia. Methods Blood samples were collected from 202 people in hyperlipidemia group（both the level of total cholesterol and low-density lipoproteins increased） and 194 people in control group. PCR-RFLP and PCR method was used to detect the genotypes of p53 codon 72 polymorphism. Results The genotype of the p53 gene codon 72 was ArgArg/ArgPro/ProPro, and the frequencies of genotype in hyperlipidemia group were 39.1 %, 45.0% and 15.8%, respectively.The frequencies of genotype in control group were 28.4%, 55.7% and 16.0%, and the difference of distribution of genotype was not significant（P>0.05）. The frequencies of genotype of women in two groups were 42.8%, 42.1%, 15.1% and 27.3%, 58.0%, 14.7%, respectively, and the difference was statistically significant（P0.012）. The genotype distribution of men in two groups was not significantly different. The differences of allele frequency distributions in the two groups and in men or in woman were not significant（P>0.05）. Conclusion There was a correlation between the genotype distribution of p53 gene codon 72 and hyperlipidemia. The risk of hyperlipidemia was increased in women who carried the Arg/Arg genotype. The genotype Arg/Arg may be a risk factor of hyperlipidemia.

Abstract:Aim To explore the effect of hyperhomocysteinemia （HHcy） on the changes of myocardial enzyme spectrum and its correlation with P53 gene. Methods 36 male ApoE―/― mice were randomly divided into 3 groups （n＝12 for each group）: AopE-/- control group, hyperhomocysteinemia group and intervention group. They were respectively fed with nomal diet, normal diet enriched 1.7% methionine and nomal diet plus 1.7% methionine 0.006% folate and 0.000 4% vitamin B12 for 14 weeks. 12 healthy 5-week-old male C57BL/6J mice were taken as normal control group. The levels of serum homocysteine （Hcy） and myocardial enzyme levels in the four groups were measured by automatic biochemical analyzer. The content of serum oxidized low density lipoprotein （ox-LDL） was detected by enzymelinked immunosorbent assay （ELISA） the protein and mRNA expressions of P53 in the heart were detected by immunohis tochemical assay and real time polymerase chain reaction （RT-PCR）. Results The content of Hcy was significantly higher in ApoE―/― control group, HHcy group and folate treatment group than normal control group, a remarkable increase was observed in the HHcy mice （P<0.01）, the ox-LDL level showed a parallel increase with the Hcy, the difference was significant （P<0.05）. The contents of creatine kinase-MB （CK-MB）, creatine kinase （CK）, hydroxy butyrate dehydrogenase （HBDH） and lactate dehydrogenase （LDH） were increased significantly in HHcy group while folate and vitamin B12 decreased serum CK-MB, CK and HBDH levels compared with HHcy group. The results of RT-PCR and immunohistochemical assay exposed an up-expressed P53 mRNA and protein in HHcy group. The changes of P53 mRNA and serum CK levels had positive correlation （r＝0.706 1, P<0.01）. Conclusions Hyperhomocysteinemia could result in the changes of myocardial enzyme spectrum, the homocysteine-induced oxidative stress might be its major pathogenic mechanism. The up-expressed P53 might also be involved in its pathogenesis. Replenishment of folate and vitamin B12 could significantly antagonize the detrimental effects of exogenous Hcy.

Abstract:Aim To explore the effect of the angiotensin Ⅱ type 1 (AT 1) receptor antagonist irbesartan on cardiomyocytes apoptosis in spontaneous hypertensire rats (SHR),and gain insight into the regulation of cardiac apoptosis. Methods Twenty 13 weeks old SHR were randomly devide into two groups: SHR positive control group,irbesartan treating group [50 mg/(kg·d)],ten normotensive Wistar-Kyoto rats were acted as normal control group. Monitoring blood pressure of rats periodically, After 15 weeks, putting all rats to death, measuring heart weight, then we investigated the changes of cardiomyocytes, apoptosis using in situ TDT-mediated dUTP nick end labeling (TUNEL). The expression of Bcl-2, Bax and P53 was assessed by immunolistochemical detection. Results Compared with WKY, untreated SHR exhibited increased apoptosis (1.59±0.38 vs 0.33±0.11, p<0.01) increased Bax (1.76±0.31 vs 0.59±0.11,p<0.01) and similar Bcl-2(0.88±0.26 vs 0.82±0.19, p>0.05), The Bcl-2/Bax ratio was lower in untreated SHR than in WKY (0.53±0.17 vs 1.41±0.34, p<0.01) . The chronic administration of irbesartan was associated with the the normalization of apoptosis (0.56±0.17 vs 1.59±0.38, p<0.01), Bax expression (0.84±0.23 vs 1.76±0.31, p<0.01) and the Bcl-2/Bax ratio (1.12±0.35 vs 0.53±0.17, p<0.01). No changes in the expression of Bcl-2 were observed in these rats after treatment(0.92±0.28 vs 0.88±0.26,p>0.05). Conclussion Chronic blockade of AT1 receptors prevents Bax overexpression and normalizes apoptosis in the left ventricular of SHR independenty of its hemoclynamic effect. AT1 blocker may prevented apoptosis by acting through a receptor mechanism involving the AT1 receptor, and may participate in the stimulation of Bax protein,which in turn renders cardiomyocytes more susceptible to apoptosis.

Abstract:Aim To investigate expression of ubiquitin conjugating enzyme (Ubc E2) and p53 in the apoptosis of human monocyte cell line U937 induced by oxidized low density lipoprotein (ox LDL). Methods U937 cells were incubated with ox LDL. The apoptotic cells were determined by DNA fragment analysis and flow cytometric analysis. The level of p53 and ubiquitin conjugating enzyme mRNA were quantified by reverse transcription polymerase chain reaction (RT PCR). The protein contents of ubiquitin conjugating enzyme, P53 and apolipoprotein B (apo B) were analyzed by immunofluoresence staining and flow cytometric method. Results The results showed that the increase of the degree of U937 cell apoptosis was concentration dependent. Ox LDL could down regulate the gene/protein expression of ubiquitin conjugating enzyme and up regulate the gene/protein expression of P53, meanwhile, the apo B accumulated in U937 cells. Conclusion The results implicated that ox LDL induced U937 cell apoptosis by accumulation of P53 and apo B through down regulating ubiquitin conjugating enzyme. Cellular defense system, either dependent or independent on the ubiquitin system, was weakened by the ox LDL induced toxicity.

Abstract:Aim To better understand the inhibition of growthand induction of apoptosis in smooth muscle cells(VSMC) by nitric oxide (NO) and the mechanism inthese processes.Methods Cultured rat VSMC at passage 5 to 8 wereused for experiment. After reaching 60 % ～70 % con-fluence, VSMCs were preincubated for 24 h (at least)with serum free Dubeccos modified Eagles medium(DMEM ) to become quiescent before the experiments.Then SNAP (0. 4 mol/L) was added to the medium.The cells were fixed with 3% paraformaldehyde after 8h. Expression of Bcl-2, Bax, P53, and Fas in VSMCwere examined by fluorescein immunocytochemistrymethods through ACAS 570.Results Expression of Bcl-2 was decreased by NO.But expression of Bax , P53 , and Fas were increased byNO.Conclusion Bcl-2, Bax, P53, and Fas may take partin the effects of NO on inhibition of growth and induc-tion of apoptosis in VSMCs.

Abstract:Aim To study the expression of P21 gene in vascu-lar smooth muscle cells(VSMC)induced by introduc-tion of wild-type P53 gene and explore the mechanism of regulation for cell cycle progression by wild-type P53 gene.Methods A P53 gene recombinant adenovirus vec-tor,AdCMVP53.was transfected into the cultured SMC derived from human umbilical artery.The gene expression of P21 was quantified by reverse transcrip-tion-polymerase chain reaction(RT-PCR).The con-tent of P21 was detected by immunochemical staining.Flow cytometry was used for cell cycle analysis.Results The level of P21 mRNA and protein was increased in VSMC transfected with AdCMVP53.Overexpression of wild-type P53 could arrest the SMC at G0/G1 phase of the cell cycle.Conclusion Wild-type P53 gene could induce the expression of P21 gene,leading to repression of cell cycle progression.

Abstract:Aim To observe the induction of apoptosis in vascular smooth muscle cell (SAC) by the introduction of wild-type P53 gene and investigate the mechanism of inhibition for SMC proliferation by wild-type P53 gene.Methods A replication--deficient adenovirus vector encoding a wild-type P53,AdCMV P53,was constructed and trans feeted into the cultured rabbit aortic SMC.The cell cycle of SMC was analysed with flow cytometry. The 3H-thymidine incorporation assay was used to measure DNA synthesis in SAC. The apoptotic cells was determined by termmed deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and agarose gel electrophoresis.Results Introduction of wild-type P53 gene into SMC can arrest 77% of AdCMV P53--infected cells at GO/GI phases of the cell cycle, leading to inhibition of DNA synthesis. Overexpression of wild-type P53 induced apoptosis of cells infected with AdCMV P53 as detected by TUNEL and flow cytometry. Electrophoresis of genomic DNA showed internucleosomal fragments of DNA isolated from the AdCMV P53--infected SMC.Conclusion Wild-type P53 gene suppress SMC pro liferation by induction of apoptosis in SMC.

Abstract:DNA was extracted from aldehydride fixed human atherosclerotic plaques. By the method of PCR, P53 gene fragments were amplified. Cloning these fragments into SK vector and sequencing them, we found that p53 gene T~(272) changed into C~(272). Immunohistochemistry showed that in plaques,the mutatant protein expressed. These results indicated that p53 gene mutation may be related with cell proliferation of atherosclerosis.