Experimental And Clinical Research Of Propyl Gallate On Regulating Vascular Inflammatory Reaction And Platelet Activation

BACKGROUNDInteraction between inflammatory reaction and platelet activation plays a key role in the pathogenesis of acute ischemic myocardial events such as rupture of unstable coronary plaque and thus acute coronary syndrome. How to regulate vascular inflammatory reaction and platelet activation is one of the focus in the research domain of prevention and treatment of cardiovascular diseases. Propyl gallate(PrG) is alkyl ester of gallic acid which is an active ingredient of Radix Paeoniae Rubra, with actions of inhibiting platelet aggragation induced by arachidonic acid and anti-inflammation. This research contains three subunits, ie: in vitro studies, in vivo experiments and clinical study. We maily explores the effects of PrG on cyclooxygenase ( COX ) pathway, cell adhesion between vascular endothelial cell(VEC) and polymorphonuclear leukocyte (PMN), inflamma- tory factors, platelet activation and thrombosis, with the aims to provide new thoughts and methods in the research of herbal medicine ingredients for regulating vascular inflammatory reaction and platelet activation, and to provide theoretical evidence for the treatment of ischemic heart disease.PART I : In Vitro StudiesObjective: To investigate the effects of PrG on COX pathway as well as cellular adhesion between VEC and PMN. Methods: A screening model for COX inhibitors in vitro based on murine peritoneal macrophages was used. COX-1 activity was determined by the level of 6-keto-PGF1α in supernatants of cultured macrophages which were stimulated with Calcium ionophore A23187 for 1 hour, while COX-2 activity was determined by the level of PGE2 in supernatants of cultured macrophages which were stimulated with lipopolysaccharide (LPS) for 9 hours; Semi-quantative RT-PCR was used to determine the effect of PrG on mRNA expression of COX-1 and COX-2 in RAW murine macrophage cell line stimulated by LPS; Western blotting was used to determine determine the effect of PrG on protein expression of COX-1 and COX-2 in RAW cell stimulated by LPS. In addition , human VECinflammation model induced by TNF- a was also established . Rose bengal vital staining method was used to measure the adherence rate of PMN to VEC, while flow cytometry was used to determine the ' expression of E-selectin and ICAM-1 in VEC surface. Results: PrG did not affect 6-keto-PGFia synthesis at concentration of 1 * 10"5? 5 * 10'6 mol ■ L'1 ( P>0.05 ) , but enhanced 6-keto-PGFi 0 synthesis at concentration of 1 * 10"\ 5 * 10"7^ 1 * 10"7 mol ■ L"1 ( P<0.01 ) in vitro. It inhibited PGE2 synthesis at concentration of 1 * 10"\ 1 * 10"6 mol ■ L"1 ( P<0.05 ) . PrG ( 0.001mM-5mM ) could inhibit adherence of PMN to VEC at different degree. PrG (5-lmM) could inhibit VEC surface expression of E-selectin and ICAM-1 at concentrations of in a dose-dependent way. PrG at lower concentrations (lOOuM-luM) showed no effect on ICAM-1 expression, while showed a slight increasing trend in E-selectin (P>0.05). lOmM ASA had no obvious effect on positive rate of E-selectin and ICAM-1. Conclusion: Within the range of 1 * 10"5 to 5 * 10"8 mol ? L"\ PrG activated COX-1 at lower concentrations and inhibited COX-2 at higher concentrations in murine peritoneal macrophages. PrG had no effect on COX-1 > COX-2 mRNA expression, while PrG could inhibit COX-2 protein expression. In addition, high concentration of PrG (0.1-lmM) is an effective inhibitor of cellular adherence of PMN to HUVEC, which is related to its inhibitory effect on HUVEC surface expression of ICAM-1 and E-selectin. Its action concentration was lower than that of ASA.PART n : In Vivo Experiments HA: Objective: To investigate the effect of PrG on occlusion time( OT ) and coagulation/fibrinolysis system in experimental carotid artery thrombosis model of rats. Methods: SD rats were randomly divided into 4 groups: normal saline , heparin(1250IU/Kg,iv), PrG( 30mg/Kg, iv ) , PrG ( 60mg/Kg, iv ) . 30 min after iv of saline or corresponding drugs, carotid artery thrombus of rats were induced by continuous electric stimulation. The duration from initiation of electric stimulation to sudden descending of carotid temperature was recorded as OT. Contents of serum t-PA and PAI were determined by ELISA.Results: PrG(30, 60mg/Kg) could prolong OT ( P<0.05 ) , but the action was obviously weaker than that of heparin. Compared with normal saline group, PrG (30, 60mg/Kg) showed a uptrend in elevating the ratio of t-PA and PAL Conclusion: PrG had a slight antithrombotic effect and showed a trend in regulating t-PA /PAI imbalance. II B: Objective : To investigate the effect of PrG on serum inflammatory factors as well as protein expression of COX-2 and ICAM-1 in ischemic myocardium of acute myocardial infarction (AMI) rats. Methods: The proximal position of left anterior descending (LAD) coronary artery was ligated in wistar rats to produce AMI model. Rats with ST segment elvation in standard limb lead II of electrocardiogram were proved to have AMI. Rats were then randomly divided into 6 groups: normal control, sham control, model control(further divided into 3 subgroups : 24h, 3d, 7d after AMI), PrG(80mg ? kg'1 ? d'1 ip), PrG(40mg ? kg"1 ? d'1 ip), ASA (25mg ? kg"1 ■ d"1 ig). Test drugs were given in succession for 7 days. Radioimmunoassay (RIA) was used to determine serum content of IL-1 |3 and TNF- a . Immunohistochemistry was used to determine the level of COX-2 and ICAM-1 protein expression in myocardium. Results: Compared with sham control, TNF- a level of model group(7d) increased significantly, but not IL-1 P . COX-2 and ICAM-1 protein expression in ischemic myocardium of model group increased at 24 h, and peaked at the 7th day. Compared with model group, PrG (40mg ? kg"1 ? d"1) could decrease the serum level of TNF- a and inhibit COX-2 and ICAM-1 protein expression in ischemic myocardium . Conclusion: PrG (40mg ? kg" 1 ? d"1) could decrease the serum level of inflammatory factor of TNF-a and slightly inhibit COX-2 and ICAM-1 protein expression in ischemic myocardium of AMI rats.PARTffl: Clinical StudyObjective : To investigate the therapeutic effects of PrG in combination with mordern medcine standard medication on patients with non-ST segment elevation acute coronary syndrome and on serum inflammatory markers and platelet activation. Methods: 55 patientswith UA/NSTMI were randomly divided into PrG group(27 cases) and salvia composite for injection group(28 cases). On the basis of standard medication of mordern medcine, patients were treated with PrG or salvia consecutively for 14 days. Flow cytometry (FCM) was used to detect the positive rate and mean fluorescence density(MFI) of GP II b -III a and CD62P expression on platelet surface both before and after treatment. ELISA was used to detect the serum concentration of Hs-CRP of patients both before and after treatment. Results: The availability rates of angina and electrocardiogram between the two groups showed no significant difference. Serum level of Hs-CRP, GP II b -III a MFI and CD62P positive rate after treatment were significantly lower than that before treatment for each group(P<0.05). Compared with salvia group, PrG showed a trend in further lowering the level of Hs-CRP and GPU b -III a MFI, but there was no statistical difference between the 2 groups. Conclusion: In combination with standard medication of modern medcine, PrG and salvia showed no obvious difference in therapeutic effect and the effects on the level of Hs-CRP and platelet activation. Compared with salvia group, PrG seemed to show a superiority in lowering the level of Hs-CRP and GPU b -III a MFI.SUMMARYIn conclusion, on the basis of previous presumption that PrG could act on the link of COX, this research established two screening models for COX inhibitors in vitro based on murine peritoneal macrophages and human VEC respectively, and proved that PrG was a inhibitor of COX-2 activity and protein expression, but showed a contribution to COX-1 activity. By establishing VEC inflammatory model, we found that PrG ( >0.1mM ) is an effective inhibitor of adherence of PMN to VEC. With a lower effectory concentration than that of ASA, the action mechanism of PrG was likely to be related to its inhibitory effect on HUVEC surface adherence molecule expression such as ICAM-1 and E-Slectin at high concentrations. By establishing the experimental carotid artery thrombosis model and the AMI model of rats, we also found that PrG had a slight anti-thrombotic effect and could alleviate...