Study On Mechanism Of Endothelial Cell Senescence Associated With Vascular Aging And Intervention Of Yiqi Huoxue Medicine

Vascular aging is a well-documented risk factor for cardiovascular disease. At present, China has entered the aged society, and the aged population in China will reach400million in2037. Therefore, the researches on vascular aging have been the impelling social requirement with aged society coming rapidly. Endothelial cells are the critical component of the vasculature, their undergoing senescence can have a significant impact on vascular integrity and function and on the progression of vascular disease. Studies have shown that, endothelial cells senescence is one of the important pathological changes in vascular aging; thus, endothelial cell has been the key area of vascular aging researches. We have previously demonstrated that endothelial cells senescence can be induced by AngⅡ through up-regulating ATI and NADPH p22phox contributing to increasing oxidative stress, and the oxidative stress may play an important role in vascular aging. The Yiqi Huoxue medicine can evidently decrease the intensity of AngⅡ signal, moreover, the role of low-dose medicine intervention is more effective.Cell proliferation inhibition is the important feature of aged cell. The degradation of the structure and function of tissues and organs in the aged is closely related to its reduction of the number of cells, and cell proliferation is the key mechanism to maintain the number of cells. The cell proliferation is regulated by extracellular signals and intracellular signaling cascades, which induce the cell cycle arrest. Studies suggested that, the expression of cyclin-dependent kinase inhibitory factor (CDI) could inhibit cell proliferation, which may play a role in endothelial cells senescence.The peroxisome proliferator activated receptor (PPAR)γ is a nuclear hormone receptor and a well-known target of antidiabetic drugs. PPARγ signaling has important antiatherogenic properties and is associated with decreased oxidative stress and increased NO formation, which are the most important component affecting the process of ECs senescence.Clinical study indicated that, the structure and function of vessels changed with vascular aging, and the incidence of many cardiovascular diseases increased rapidly. The stiffening of vascular associated with aging results in various adverse hemodynamic consequences, for example, elevated systolic BP, diastolic dysfunction and heart failure. Pulse wave velocity (PWV) is the major non-invasive method of assessing arterial stiffness. Increased arterial stiffness associated with vascular aging results in increased speed of the pulse wave in the artery. In foreign studies, PWV was an independent predictor of cardiovascular death and events in acute myocardium infarction patients.Therefore, the study aims to clarify the mechanism of vascular aging and the intervention of Chinese medicine from endothelial cell senescence further, and explore the clinical significance and TCM syndrome characteristics of vascular aging through investigating STEMI patients, based on the preliminary work. First, from the view of cell proliferation regulation, explore the intrinsic mechanism of ECs senescence induced by Ang Ⅱ and vascular aging and the intervention of Yiqi Huoxue medicine. Second, as PPARy a target, investigate the compensatory protective effect on ECs senescence and its changes, and reveal the possible mechanism of Yiqi Huoxue medicine intervention. Again, as PWV the detection index of vascular aging, analyze the relation between vascular aging and short-term prognosis of STEMI patients, and inspect the perspective on TCM tongue diagnosis of vascular aging. This can provide new theories, new perspectives and new targets for researches, prevention and treatment of vascular aging, but also provide new ideas for assessing the prognosis of cardiovascular disease from the angle of vascular aging.The research includes three parts:the basic experimental research, the clinical research and the literature review.Basic experimental research:including the following four sections.Section1:The characteristics changes with vascular endothelial cell senescence induced by Ang Ⅱ.Objective To explore the cytological features changes of aged vascular endothelial cells induced by angiotensin Ⅱ(AngⅡ). Methods Human umbilical vein endothelial cells(HUVECs) were obtained from the umbilical cord of healthy mothers and cultured in vitro until fourth passage. ECs were intervened by AngⅡ (stimulated with AngⅡ10-6mol/L for48h). The cytological features changes of aged cells were observed by morphological observation, β-gal staining, NO or eNOS production, CCK-8and cell cycle analysis. Results ECs appeared morphological changes induced by Ang Ⅱ. The positive cell number of β-gal staining was significantly higher in AngⅡ group than control group (p<0.01). AngⅡ down-regulated the expression of NO and eNOS (p<0.01). The number of surviving cells was also greatly reduced in AngⅡ group (p<0.01). The cell cycle was arrested at G0/G1phase (p<0.01). And the senescence level in48h is more severe than that in24h through above indexes (p<0.05). Conclusion The key cytological features changes of vascular endothelial cell senescence include cell proliferation inhibition and dysfunction.Section2:The intervention of Yiqi Huoxue medicine on cytological features of aged ECs.Objective To test and verify the effect of Yiqi Huoxue medicine on cytological features of aged ECs. Methods The aged ECs induced by AngⅡ (10-6mol/L) were intervened by Yiqi Huoxue medicine (including ginseng, panax and chuanxiong)(20mg/L) and Telmisartan (10-6mol/L), and divided into4groups:the control group, AngⅡ group, Chinese medicine group and Telmisartan group. MTT, β-gal staining, NO or eNOS production, CCK-8and cell cycle analysis were conducted. Results At24hours and48hours after intervention, we found Yiqi Huoxue medicine can reduce the positive cell number of β-gal staining, up-regulate the expression of NO and eNOS, increase the surviving cells and improve the cell cycle arrest (p<0.05). Moreover, the effect of Yiqi Huoxue medicine is more prominent at24hours after induction by AngⅡ than that of Telmisartan (p<0.05), while Telmisartan is more effective at48hours compared to Chinese medicine (p<0.05). Conclusion Yiqi Huoxue medicine can delay ECs senescence induced by Ang Ⅱ, and improve cell proliferation inhibition and dysfunction partly. In addition, Chinese medicine may play an important role in early senescence. Section3:Study on gene expression of p53, p16in aging ECs induced by Ang Ⅱ and intervention of Yiqi Huoxue medicine.Objective To study the p53, p16gene expression associated with ECs senescence induced by Ang Ⅱ and the effect of Yiqi Huoxue medicine. Methods Aged ECs were divided into4groups:the control group, Ang Ⅱgroup (10-6mol/L), Chinese medicine group (CM20mg/L+Ang Ⅱ10-6mol/L) and Telmisartan group (Telmisartan, AngⅡ10-6mol/L).The expressions of p53, p16were detected by immunocytochemical staining. And RT-PCR and western-blot analysis were used to identify the expression of p53and p16at12h,24h,36h,48h after intervention in each group. Results In AngⅡ group, p53, p16mRNA and protein expression increased markedly (p<0.05), but p53expression appeared earlier than p16. Simultaneously, Chinese medicine decreased evidently p53, p16expression compared to those in Ang Ⅱ group (p<0.05), and it was more effective than Telmisartan at24h (p<0.05). Conclusion The proliferation obstacle with ECs senescence might be associated with increasing the expression level of p53and p16. Chinese medicine improves ECs aging, especially in the early.Section4:Study on expression of PPARy in aging ECs induced by Ang Ⅱ and possible mechanisms of Chinese medicine intervention.Objective To study the PPARy gene expression associated with ECs senescence induced by Ang Ⅱ and the mechanisms of Chinese medicine intervention. Methods Aged ECs were divided into6groups:the control group, Ang Ⅱ group(10-6mol/L), Chinese medicine group (CM20mg/L+Ang Ⅱ10-6mol/L), Telmisartan group (Telmisartan, Ang Ⅱ10-6mol/L), AngⅡ+blocker group (Ang Ⅱ10-6mol/L+GW966210-5mol/L) and Chinese medicine+blocker group(CM20mg/L+AngⅡ10-6mol/L+GW966210-5mol/L). RT-PCR and western-blot analysis were used to identify the expression of PPARy, p53and p16at12h,24h,36h,48h after intervention. Analyze the molecular mechanisms of ECs senescence and Chinese medicine intervention. Results Ang Ⅱ up-regulated the expression of PPARγ mRNA of ECs with time dependency mode, but its protein expression decreased after24hours. Chinese medicine increased significantly the PPARymRNA and protein expression at every time point (p<0.05). At24h, the effect of Chinese medicine is more significant than Telmisartan (p<0.05); while at36and48h, Telmisartan exceeded Chinese medicine (p<0.05). After blocking PPARy signaling, the p53, p16expressions in Ang Ⅱ+blocker group were evidently higher compared to those in Ang Ⅱ group (p<0.05). Accordingly, the expression of p53, p16in Chinese medicine+blocker group was higher than that in Chinese medicine group (p<0.05), but lower than that in Ang Ⅱ group (p<0.05). Conclusion Activating PPARy signaling may play an important role in delaying ECs senescence, and improve the proliferation inhibition of aged cells through down-regulating p53and p16. But, its protection function was waning, because of protein translation obstacle. Yiqi Huoxue medicine can increase the PPARy expression, partly through which, Chinese medicine delay ECs senescence.Clinical research:including the following two sections.Section1:The association between different levels of vascular aging and short-term prognosis of ST-segment elevation acute myocardial infarction patients.Objective To evaluate the association of the PWV level and the short term mortality and combined end point events in patients with ST-segment elevation acute myocardial infarction (STEMI). Methods This is an observational analysis of282Chinese STEMI patients from the Department of Cardiology in Wangjing Hospital of China Academy of Traditional Chinese Medicine and the First Affiliated Hospital of Tsinghua University from2005to2008. The conventional treatment was used for the selected patients, including reperfusion and pharmacological therapy. The PWV was measured prospectively at hospital admission, within30days after hospitalization, respectively. The patients were grouped by the PWV level:the control group (PWV<1400cm/s), the mild increasing group (PWV:1400-1900cm/s), the moderate increasing group (PWV:1901-2400cm/s) and the severe increasing group (PWV>2400cm/s). The incidence of all-cause mortality and combined end point events of death, recurrence ischemia, re-infarction, cardiogenic shock, stroke within30days were analyzed in the four groups. Results Compared with the control group,30-day mortality and combined end point events increased as PWV increased. There was not significant difference in mortality with various PWV groups(p>0.05), while there was significant difference in combined end point events with every groups (p<0.001). Multivariate logistic regression analysis showed that, the moderate and severe increasing of PWV had independent associations with30-day mortality and combined end point events. The severe increasing group had an increased mortality (p=0.021, OR:9.242) and combined end point events (p=0.014, OR:5.932), respectively. Conclusion PWV>1900cm/s significantly increased30-day mortality and combined end point events. Vascular aging is an important independent risk factor of short-term prognosis in STEMI patients.Section2:The association between different levels of vascular aging and purple tongue in STEMI patients.Objective To study the relationship of purple tongue and the PWV level in STEMI patients. Methods Based on the same patients database of section1, two methods were used to assess purple tongue. The tongue was measured in TY-SX3recognition and analysis system, and was diagnosed by Chinese medicine experts for7days after pathogenesis. The tongue of all patients were collected and judged from admission to the6th day after hospitalization, once a day, total7days. The incidence of1-day purple tongue,3-day purple tongue and7-day purple tongue were analyzed in patients of various PWV level groups. Results The incidence of3-day and7-day purple tongue significantly increased in PWV moderate and severe increasing groups. Multivariate logistic regression analysis showed that, the PWV moderate and severe increasing groups had an increased incidence of7-day purple tongue respectively. The severe increasing group had an increased7-day purple tongue (p<0.01, OR:1.497). Conclusion PWV>1900cm/s significantly increased7-day purple tongue. The continuous occurrence of purple tongue may be one of the TCM syndrome characteristics of vascular aging in STEMI patients. Literature review:including the research progress and TCM understanding of vascular aging, contribution of PPARy, p53and p16pathways to regulation of vascular endothelial cells senescence.In summary, the study has shown that Ang Ⅱ can induce vascular endothelial cells senescence. The results suggested that, the key cytological features changes of vascular endothelial cell senescence include cell proliferation obstacle and dysfunction. Ang Ⅱ up-regulated the expressions of p53, p16mRNA and protein of ECs with time dependency mode, and leaded to the cell proliferation inhibition. On the contrary, PPARy signaling has important anti-senescence properties and affects the process of aging in ECs. But its protection function was waning in the late. Yiqi Huoxue medicine delays EC senescence mainly by the regulation of these genes expression. Our study suggests that, the proliferation inhibition of aged ECs may be relevant to the up-regulated expressions of p16and p53. The down-regulated expressions of PPARy in the late may be related to the protein translation obstacle. The treatment with Chinese medicine delayed ECs senescence by activation of PPARy signaling partly. Especially, it indicated the important role of prevention in the early during aging. In addition, the clinical research demonstrated that vascular aging can contribute to cardiovascular diseases, and purple tongue may be one of the TCM syndrome characteristics of vascular aging. In short, the innovations of this study were shown that, the precise mechanism of vascular aging and the targets of Chinese medicine intervention were explored from regulation of cell proliferation and anti-senescence role; the time-effectiveness law of Chinese medicine was further clarified by dynamic observation; the association between different levels of vascular aging and short-term prognosis or purple tongue in STEMI patients was expressed by multivariate logistic regression analysis.