Study On Anti - Thrombosis And Bleeding Risk Of Traditional Chinese Medicine Combined With Anti - Drug

Balancing anti-thrombus and reducing the risk of hemorrhage play key roles in the management of cardio-cerebral diseases. In Traditional Chinese Medicine, Tonifying Qi and activating blood circulation treatments are commonly employed in treating cardiac and cerebral thrombotic diseases. Within the Eleventh Five-year Plan period, we conducted a multi-center random controlled study which provided the evidence to support that Tonifying Qi and activating blood circulation treatments combined with routine western medicine treatments can reduce the incidence of Acute Coronary Syndrome (ACS) and cardiovascular events (sudden death, non-fatal myocardial infarction and non-fatal brain stoke) after percutaneous coronary intervention therapies, while not raising the risk of hemorrhage. However, there are currently very few reports and mechanism discussions about whether combination therapy of Tonifying Qi and activating blood circulation TCM prescription with dual antiplatelet therapy (DAPT) (aspirin and clopidogrel) can further strengthen the anti-thrombosis effects, and at the same time reduce the risk of hemorrhage.Clinical researchObjective:To investigate the efficacy and safety of combination therapy of Tonyfing Qi and activating blood TCM and DAPT on patients with Qi-deficiency and blood-stasis coronary heart disease (CHD)Method:Treatment group included 50 patients of Qi-deficiency and blood-stasis with coronary heart disease and treated with TCM of Tonifying Qi and activating blood circulation in addition to DAPT therapy. Control group consists of patients diagnosed Qi-deficient and blood-stasis coronary heart disease in Anzhen Hospital from 2010 to 2012. There is no significant difference between baseline characteristics (demographic characteristics, heart rate, blood pressure, cardiovascular and cerebrovascular disease history, history of gastrointestinal disease, cardiovascular and cerebrovascular disease family history, smoking history, drinking history) in the two groups(P>0.05). Platelet aggregation rate, angina pectoris attack score, Qi deficiency and blood stasis symptom score and blood stasis score were evaluated upon each patient before the treatment, at the 4th week and the third month after the treatment. The patients were examined for gastrointestinal adverse reaction cases and the integral changes at third month and the 6th month post-treatment.Results:(1) In contrast to pre-medication conditions, platelet aggregation rate, angina pectoris, Qi deficiency and blood stasis syndrome score, blood stasis syndrome scores significantly decreased (P<0.05, P<0.01) in treatment group at 4 weeks and 3 months. After the 6th month treatment, gastrointestinal adverse reaction cases and the integral changes were increased (P<0.01); (2) In contrast to the control group, platelet aggregation rate, angina pectoris, Qi deficiency and blood stasis syndrome, blood stasis syndrome scores in treatment group were significantly lower in the 4th week, the third month after treatment (P<0.05, P<0.01), treatment for the third month, the 6th month of gastrointestinal adverse reaction cases and gastrointestinal adverse reaction integrals were significantly reduced (P<0.05, P<0.01).Conclusion:Tonifying Qi and activating blood circulation combined with western medicine can reduce platelet aggregation rate, improve the symptoms of angina pectoris, Qi deficiency and blood stasis syndrome and decrease gastrointestinal adverse reaction induced DAPT.Experimental study:1. Objective:To observe the anti-thrombosis effect of PQS combined with PNS and AMI in acute myocardial infarction (AMI) rats, and to study the possible mechanism based on the COX/PG pathway.Methods:AMI model on Wistar Rats were created by ligating left anterior descending coronary artery(LAD). Model animals were randomly divided into model group, DAPT PQS+DAPT), PNS+DAPT, PQS+PNS+DAPT.The sham group underwent the same procedure except for LAD ligation. Each group consists of 15 rats. Rats were intra-gastrically administered for 14 days. Myocardial infarct(MI)areas were calculated after HE, TTC stain. We measured the platelet aggregation rate by turbidimetry, rate of CD62p activation by flow cytometry(FCM)and concentration of thromboxane B2(TXB2) in plasma,6-Ketoprosta-glandin F1 alpha(6-Keto-PGF1α) in plasma, tissue plasminogen activator(t-PA)in serum, plasminogen activator inhibitor-1(PAI-1) and D-dimer in serum by radioinununo-assay and calculated ratio of TXB2/6-Keto-PGF1α.Results:(1) compared to the sham group, AMI rats markedly increased MI area, reduced platelet aggregation rate, CD62p activation rate, concentration of TXB2,6-Keto-PGF1α in plasma, ratio of TXB2/6-Keto-PGF1α, PAI-1 and D-dimer content in serum (P<0.05, P<0.01), decrease the content of plasma t-PA content (P<0.01); (2) compared to the model group, administration of DAPT to AMI rats significantly decreased MI area, platelet aggregation rate, CD62p activation rate, the concentration of TXB2 in plasma,6-Keto-PGF1α, D-dimer content(P<0.05, P<0.01); (3) compared to the DAPT, administration of PQS+DAPT to AMI rats significantly decreased MI area, the concentration of TXB2 in plasma, PAI-1 in serum, ratio of TXB2/6-Keto-PGF1α and increased remarkably the concentration of 6-Keto-PGF1α in plasma, t-PA in serum(P<0.05), administration of PNS+DAPT, PQS+PNS+DAPT to AMI rats significantly decreased MI area, platelet aggregation rate, CD62p activation rate, the concentration of TXB2 in plasma, ratio of TXB2/6-Keto-PGFla and PAI-1 content in serum (P<0.05, P<0.01), increased 6-Keto-PGFIa in plasma content of t-PA in serum (P<0.05, P<0.01); (4) compared with the PQS+DAPT, administration of PQS+PNS+DAPT reduced platelet aggregation rate, CD62p activation rate and the ratio of TXB2/6-Keto-PGF1α (P<0.05); compared with the PNS+DAPT, administration of PQS+PNS+DAPT increased the ratio of TXB2/6-Keto-PGF1α(P<0.05).Conclusion:(1) the PQS and PNS combined can enhance the inhibition of MI and anti-thrombotic effects of DAPT therapy in rats. The mechanism may be related to the role of PQS for its synergetic inhibitory effect to the anti-thrombotic effect of aspirin on the COX-1/TXA2 pathway of platelet, and up-regulating COX/PGI2 pathways and activating fibrinolytic system; (2) the combination of PQS and PNS is superior to PQS or PNS alone with DAPT in the area of MI, anti-platelet effect superior to PQS in combination with DAPT therapy.2.Objective:Through COX/PG pathways, we further explore how PQS combined with PNS improve the gastric mucosal injury caused by DAPT drugs in AMI rat model.Methods:AMI model on Wistar Rats were created by ligating left anterior descending coronary artery(LAD), and were randomly divided into the model group, DAPT, PQS+DAPT. Sham group underwent the same procedure except for LAD ligation. Each group including 15 rats. Rats were intra-gastrically administered for 14 days. Gastric mucosal ultrastructure was observed by scanning electron microscopy. Expression of Prostaglandin E2(PGE2),6-Keto-PGF1α in gastric tissue were detected by RIA, and Cyclooxygenase-1(COX-1), Cyclooxyge nase-2 (COX-2), Vascular endothelial growth factor(VEGF)in gastric tissue were detected by Immunohistochemistry (ICH). COX-1, COX-2 in gastric tissue also were detected by western-blot.Result:(1) Compared to the sham group, GAS and MTL in serum were decreased, the level of the COX-2 and TNF-a raised significantly in the model group(P<0.05, P<0.01); (2) compared to the sham group, we observed gastric damage on epidermis and substance layer in DAPT group, the level of GAS, MTL in serum, the expression of PGE2, COX-1/-2, and VEGF in gastric tissue was deceased(P<0.05, P<0.01), the expression of TNF-a was increased significantly (P<0.01); (3) compared to DAPT group, gastric epidermis and substance layer damage and TNF-a expression was lowed, GAS, MTL content, the COX-1/-2, PGE2, VEGF expression was increased(P<0.05, P<0.01) in the PQS+DAPT, PNS+DAPT and PQS+PNS+DAPT, except for the expression of COX-1 in gastric tissue in the PQS+DAPT was not significant(P>0.05); (4) compared to the PQS+PNS+DAPT group, we observed in PQS+DPAT group with suppressed COX-1 expression, non-significantly different gastric tissue COX-2 expression (P>0.05) and elevated PNS+DAPT gastric tissue TNF-a expression (P<0.05).Conclusion:(1) gastric mucosal injury associated with DAPT in AMI rat model may be associated with decreased serum GAS and MTL level and gastric tissue of COX-1, COX-2, PGE2 and VEGF expression and elevated gastric tissue TNF-a expression; (2) PQS and PNS could alleviate gastric mucosa lesions caused by DAPT drug in AMI rats, which may be related to up-regulating the expressions of COX-2/PGI2-PGE2 and VEGF in gastric tissue.(3) the PQS, PNS combined with DAPT in gastric tissue was increased COX-1, COX-2, PGE2 and VEGF expression and inhibition of TNF-a expression than the PQS or PNS alone combined with DAPT.3.Objective:To study the relationship between ROCK/RhoA and the PI3K/Akt/Racl pathway, based on the effects of and PQS、PNS on the cerebral microcirculation in rats with cerebral ischemia reperfusion (I/R) model.Methods:Suture-occluded method was used to create MCA I/R in Wistar rats, and the suture was removed 2h later to preprae I/R model. Rats with mNSS scores of 7-12 points on postoperative 1day were included in the experiment. Grouping was same as experiment one. Cranial window method was used to observe the diameters and velocities of rat pia mater arteriole and venule, and red blood cells and white blood cells in vessels, so as to evalute the effects of drugs on cerebral microcirculation of I/R rats. Cerebral infarction area was taken, the expression was measured p-Akt/GAPDH, GTP-Racl/Total-Racl, GTP-RhoA/Total-RhoA, ROCK/GAPDH and p120-Catenin/VE-cadherin from rat brain tissue by Western blot. The cerebral infarct areas were calculated after TTC stain.Results:(1) compared with the sham group, pial arterioles and venules significant contracted (P<0.01), meningeal microcirculation, cerebral infarction area were increased (P<0.01), brain tissue p120-Catenin/VE-cadherin, the expression of GTP-RhoA in brain tissue were increased, the expression of ROCK and p-Akt was increased (P<0.01), the expression of GTP-Racl in brain tissue was not significantly different (P>0.05); (2) compare-d with the model group,the pial arteries and vein diameter were significantly expanded (P<0.05), the cerebral pial microcirculation was alleviated; the cerebral infarction area were decreased (P<0.05), the expression of p120-Catenin/VE-cadherin ratio in brain tissue were increased (P>0.05), the expression of GTP-RhoA, ROCK in brain tissue were decreased significantly (P<0.01), the expression of P-Akt, GTP-Rac-1 were no significant in the DAPT group (P>0.05); (3) compared with the DAPT, PQS+DAPT, PNS+DAPT.the pial arteries and vein diameter were enlarged, the area of cerebral infarction were reduced (P<0.05, P<0.01), the expression of brain tissue p120-Catenin/VE cadherin ratio, p-Akt, GTP-Racl were increased significantly (P<0.05, P<0.01), the expression of ROCK, RhoA in brain tissue were decreased in PQS+PNS+DAPT group (P<0.05,P>0.01); (4) compared with the PQS+DAPT, PQS+PNS+DAPT group, the expression of p-Akt and GTP-Rac in the brain tissue was increased (P<0.05, P<0.01), and the other indexes had no significant difference (P>0.05).Conclusion:(1) We observed cerebral microcirculation disturbance and partial brain necrosis in rats with brain I/R, where p120-Catenin/VE cadherin ratio were reduced in brain tissue and RhoA, ROCK expressions were significantly raised in RhoA/ROCK pathway;(2) DAPT can alleviate cerebral microcirculation disturbance and brain necrosis in brain I/R rats, and inhibit RhoA expressions of RhoA/ROCK pathway; but the down-regulation of RhoA expression is not associated with the activation of PI3K/Akt/Racl pathway. (3) PQS+PNS could promote DAPT to improve cerebral microcirculation disturbance and to inhibit brain necrosis in brain I/R rats. They could also promote DAPT to upregulate p120-Catenin/VE-cadherin ratio in brain tissues of brain I/R rat activation PI3K/Akt/Racl pathway protein expression, inhibition RhoA/ROCK signal pathway protein expression.