| Lysophosphatidic Acid(LPA),as a cardiovascular active substances,plays an important role in cardiovascular system for its close correlation with arteriosclerosis,myocardial infarction and hypertension.Clinical researches revealed serum LPA level had direct relation with acute myocardial infarction. For acute myocardial infarction patients,8 hours after onset,the serum LPA level elevated 1 time;and after 48-72 hours,it reached 6 times as much; furthermore,even after 7 days,it is also higher than normal level.LPA benefits the proliferation,migration and dedifferentiation of vascular smooth muscle cells(VSMCs) and cause vessel stenosis.Currently,the mechanism of vessel restenosis is still not clear.Based on the existing basic and clinical researches,we think the mechanism refers to excessive intimal hyperplasia,vessel elastic recoil and thrombus formation in the lesion and negative vascular remodeling,with which excessive intimal hyperplasia is the biggest reason and VSMCs mainly account for intimal hyperplasia.For this reason,inhibition of VSMCs proliferation is critical for prevention of restenosis.Herbal compound xuefuzhuyu capsule is a new preparation of traditional formula with promoting blood circulation herbs and xuesaitong capsule based on panax notoginseng saponins(PNS) from chinese herb panax notoginseng. As representative traditional formula,the two drugs have been widely used in intervention of myocardial infarction and achieved great performance in clinical practice.However,few reports have been found in chinese herbs how to act on prevention of VSMCs proliferation and acting signal transduction mechanism. In this study,we observed the roles of formula in normal and LPA simulated VSMCs proliferation in myocardial infarction,and discussed the potential signal transduction mechanism for providing grounds and seeking effective targets to clinical practice of the formula.This study is divided into two parts:review and experimental research. 1.Review:This part includes three reviews:advances in the relationship between LPA and cardiovascular system,and restenosis mechanism as well as intervention of chinese herbs or effective ingredients on restenosis.2.Experimental research:This part includes 5 trails.2.1 The preparation of intervention serumGeneral chinese herbal serum pharmacological administration scheme was adopted,involving administration twice per day,and lasted 3days,1 hour after the final administration,blood was extracted and serum was collected with centrifugal analysis.The dosage was as 10 times as equivalent dose in order to remedy the serum dilution.It was appropriate when serum intervention concentration up to 10%measured by MTT in culture medium.2.2Primary Culture and identification of rat VSMCsTissue-piece was adopted to primary culture of SD rat VSMCs.The identification refers to observe cells morphological characteristics with inverted phase contrast microscope and identify with a-actin immunocytochemical staining.Results:cells emerged from the edge of tissue mass after 5-7 days of inoculation in culture flask,presented evident peak-valley curve.After a-actin immunocytochemical staining,over 90%cell showed positive,it described as cytoplasm brown-yellow and nucleus no changes.It also indicated that cells contained smooth muscle actin.2.3The role of the formula with prompting blood circulation in vascular VSMCs proliferationCells proliferation in each group was detected by MTT.Drug serum with diversified concentrations were performed to interfere cultured VSMCs in vitro, Internal environment of myocardial infarction simulated by LPA was also added. Moreover,PD98059,and PTX,H7,and PMA was used to interfere the individual signal transduction pathway.The impact of xuefuzhuyu and xuesaitong capsules on VSMCs proliferation of normal and LPA induced,as well as the changes after signal transduction intervention inhibitor added,and potential acting signal transduction pathway were discussed in the study.The VSMCs were divided into twelve groups:the blank serum group;the xuefuzhuyu(XFZY) capsules-containing serum group;the xuesetong(XST) capsules-containing serum group;the LPA+blank serum group;the LPA+XFZY capsules-containing serum group;the LPA+XST capsules-containing serum group;the PD98059+LPA+XSTcapsules-containing serum group;the PTX+LPA +XSTcapsules-containing serum group;the PMA+LPA+XSTcapsules-containing serum group;the H7+LPA+XSTcapsules-containing serum group;theLPA+captopril-containing serum group;the PD98059+LPA+XFZYcapsules-containing serum group.After serum intervention for 24hours,MTT was performed to detect cell activity.Results:In comparison with the blank serum group,the optical density(OD) of the XFZY capsules-containing serum group and the LPA+blank serum group was increased(P＜0.05,P＜0.01),the XST capsules-containing serum group has no difference with it;The OD of XFZY capsules-containing serum and XST capsules-containing serum added LPA increased smaller than the blank serum added LPA(P＜0.05),The OD of the LPA+blank serum group,the LPA+XFZYcapsules-containing serum group and the LPA+captopril-containing serum group has no difference,while the LPA+XST capsules-containing serum group is decreased than them(P＜0.05); Compar to the OD of the LPA+XST capsules-containing serum group,the ODs of the PD98059+LPA+XST capsules-containing serum group,the PTX+LPA+XST capsules-containing serum group and the H7+LPA+XST capsules-containing serum group were lower(P＜0.05),while the OD of the PMA+LPA+XST capsules-containing serum group was higher(P＜0.05),the OD of the PD98059+LPA+XFZYcapsules-containing serum group is decreased than the LPA+XFZYcapsules-containing serum group.2.4The role of the formula with prompting cell cycle in vascular smooth muscle cellscell cycle distribution was analyzed with flow cytometry in accordance with S phase fraction(SPF).As the randomized method mentioned above,after serum intervention for 24hours,cells were collected and stained by PI.Phase S in cell life circle was determined.Results:In comparison with the blank serum group,the S phase fraction(SPF) of the LPA+blank serum group and the XFZY capsules-containing serum group was increased(P＜0.05),the SPF of the the XST capsules-containing serum group has no difference with it;The SPF of XFZY capsules-containing serum and XST capsules-containing serum added LPA increased smaller than the blank serum added LPA(P＜0.05), Compar to the SPF of the LPA+blank serum group,the SPF of the LPA+XFZYcapsules-containing serum group was increased(P＜0.05),the SPF of the LPA+XST capsules-containing serum group and the LPA+captopril-containing serum group was decreased(P＜0.05);Comparison with the LPA+blank serum group,the SPFs of the PD98059+LPA+XST capsules-containing serum group,the PTX+LPA+XST capsules-containing serum group and the H7+LPA+XST capsules-containing serum group were decreased(P＜0.05),the SPF of the PMA+LPA+XST capsules-containing serum group was increased(P＜0.05),the SPF of the PD98059+LPA+XFZYcapsules-containing serum group is lower than the LPA+XFZYcapsules-containing serum group.2.5The role of the formula with prompting blood circulation in ERK1/2 activityWestern Bloting was performed to detect the expression of ERK and p-ERK., the relative level of p-ERK was used to reflect the activation level of ERK1/2. The divided groups are the same as experiment2.3,cells were collected after 24h invented by serums.Same method as 2.3,intervention for 24 hours,protein was extracted from the cells collected.Subsequently,through gelelectrophoresis and incubation of first and second antibody,ERK1/2 activity was calculated according to p-ERK,ERK1/2 expression.Results:The ERK1/2 activation of the XFZY capsules-containing serum group,the XST capsules-containing serum group and the LPA+blank serum group was increased than the blank serum group(P＜0.05);The ERK1/2 activation of XFZY capsules-containing serum and XST capsules-containing serum added LPA was obviously inhibited than the blank serum added LPA(P＜0.05),In comparison with the LPA+blank serum group The ERK1/2 activation of the LPA+XFZYcapsules-containing serum group,the LPA+XSTcapsules-containing serum group and the LPA+captopril-containing serum group was decreased(P＜0.05);Compar to the LPA+blank serum group,the PD98059+LPA+XST capsules-containing serum group,the PTX+LPA+XST capsules-containing serum group and the H7+LPA+XST capsules-containing serum group inhibit the ERK1/2 activity. The ERK1/2 activation of the PD98059+LPA+XFZYcapsules-containing serum group also decreased than the LPA+XFZYcapsules-containing serum group.In Conclusion,Elevation of LPA level after myocardial infarction onset did quicken the process of VSMC proliferation and cause restenosis.XST capsules showed no effect on natural VSMC,while XFZY capsules accelerated VSMC proliferation.Both of the formula mentioned above have good inhibition on VSMC proliferation induced by LPA.Restenosis could be prevented via the inhibition of the formula with promoting blood circulation herbs in ERK1/2 over activation induced by LPA and in transformation of VSMC to phase S.The signal transduction mechanism of VSMC proliferation post myocardial infarction maybe involve activation of protein kinase C by PKC-MEK1/2-ERK1/2 via G protein coupling receptors on membrane surface and phosphoinositide system,and then MAPK(ERK1/2) might be activated via Ras-Raf pathway and initiate the expressions of growth related genes. |
PDF Full Text Request