A Novel Anti-thrombosis And Anti-fibrosis Mechanism Of Chinese Herbs Resina Draconis And Toddalia Asiatica

Plasminogen activator inhibitor-1(PAI-1)is a member of the serine protease inhibitor gene family and is the major inhibitor of both tissue-type plasminogen activator(tPA)and urokinase-type plasmanogen activator(uPA)invivo.Previous studies indicated that PAI-1over-expressed could induce spontaneous thrombosis in transgenic mice,while PAI-1 gene knockout mice manifested resistance to thrombosis induced by endotoxin or chemical damages.PAI-1 inhibitor RG1 could prevent and therapy of thrombosis in animal models.In2012,Xiao and colleagues isolated two PAI-1 inhibitors STC and CTC from the traditional Chinese anticoagulant herb salvia miltiorrhiza,they proposed that PAI-1 inhibition was a possible mechanism for the anticoagulant activity of the herb.Motivated by these considerations,we hypothesize that PAI-1 inhibitors may ubiquitously exist in anticoagulant herbs and PAI-1 activity inhibition may be a common mechanism for these herbs.Resina Draconis and Zanthoxylum nitidum have long been used for blood circulation promotion in the traditional Chinese medicine.Although efficiency of Resina Draconis and Zanthoxylum nitidum in blood promotion have been fully confirmed,molecular mechanisms of the herbs remains elusive.We are curiously to know whether there are PAI-1 inhibitors exist in the herbs.In the present study,Two active compounds Loureirin B(LB)and Toddalolactone(TOD)with PAI-1 inhibition activities were isolated from Resina Draconis and Zanthoxylum nitidum under activity-directed purification strategy.Molecular pharmacological activities and in vivo effects of the compounds were investigated.The results showed that:(1)Both LB and TOD dose-dependently inhibited PAI-1 activities with IC50 values of26.10±2.13 ?M(LB)and 37.31±3.23 ?M(TOD),respectively,as shown in the chromogenic method.(2)SDS-PAGE analysis indicated that LB and TOD could prevent the formation of PAI-1/uPA complex.(3)In vitro study showed that LB and TOD accelerated the process of fibrin clots lysis.(4)In the CCl4-induced mouse liver fibrosis model,intraperitoneally injection(ip)of LB and TOD(1mg/kg)per day for 2 weeks could significantly decrease plasma hydroxyproline levels to Loureirin B(LB)and Toddalolactone(TOD)compared to PBS in the control group(P<0.05),and the degrees of liver fibrosis were also significantly decreased in the tests groups.(5)In the FeCl3-induced mouse model of arterial thrombosis,LB and TOD(1 mg kg-1 ip)could significantly prolonged tail bleeding time from 11±3.3 min(control group)to 15±6.5 min(LB)and 17±5.3 min(TOD)min,which accompanied reduced arterial thrombus weight to 7.4±3 mg(LB)and 7.2±5 mg(TOD)from 8.8±3 mg(control)(P<0.05).In conclusion,the present study identified two PAI-1 inhibitors(LB and TOD)from the traditional anticoagulant herbs Resina Draconis and Zanthoxylum nitidum under activity-directed purification method.In vitro and in vivo studies indicated that both LB and TOD could significantly inhibited the activity of PAI-1,prolonged tail bleeding time,reduced arterial thrombus and decreased liver fibrosis in mouse model.The results showed a novel anti-thrombosis mechanism of Chinese herbs Resina Draconis and Zanthoxylum nitidum.