Absorption Characteristics And Thrombolytic Efficacy Of Oral Recombinant Subtilisin QK-2

As a serious global medical problem,thrombotic diseases are endangering human health with an alarming incidence.The age of onset has gradually become younger in recent years,and it has also caused huge economic losses and social burdens.At present,there are various drawbacks in the clinically used thrombus treatment methods,such as limited patient type(surgical thrombectomy),short effective time,common bleeding side effects(plasminogen activator thrombolytic drugs),etc.There is an urgent need to find a new type of high quality thrombolytic drug.Subtilisin QK is a serine protease with similar function to nattokinase.It has the characteristics of targeting degradation of cross-linked fibrin,and can exert fibrinolytic effect in vitro and in vivo.But the Subtilisin QK obtained by natural fermentation of Bacillus subtilis shows low yield,purity and thrombolytic activity.Therefore,in the previous work,Wang Yefu's research team constructed a Pichia pastoris genetically engineered strain that efficiently expressed Subtilisin QK-2,and obtained recombinant Subtilisin QK-2 lyophilized powder with high-purity(98.1%),and high-activity(800,000 IU/g),which was realized by the fermentation process amplification workshop and separation/purification platform of Wuhan Zhenfu Pharmaceutical Co.,Ltd..The thrombolytic effect of Subtilisin QK-2 in vitro and in vivo and its effect on intravenous thrombosis have been observed,but its oral thrombolytic effect is still unclear.On this basis,the aim of this study is to explore the absorption characteristic of oral recombinant Subtilisin QK-2,and verify the thrombolytic effect on animal thrombus model,and finally explore its effect in human umbilical vein endothelial cells(HUVEC).The research of fibrinolytic mechanism provides a theoretical basis for the development of Subtilisin-like drugs and provides new choices for the prevention and treatment of thrombotic diseases.The specific research content can be divided into the following three aspects:(1)It was verified in vitro that recombinant Subtilisin QK-2 is capable of specifically dissolving cross-linked fibrin(an important component of thrombus)and is highly resistant to artificial intestinal digestive juice(mainly trypsin);In vitro everted sacs model showed that recombinant Subtilisin QK-2 can be absorbed through different parts of the small intestine(duodenum,jejunum,ileum),and still has obvious thrombolytic activity after absorption.In addition,the in vivo imaging results of oral administration of recombinant Subtilisin QK-2 in animals also showed that recombinant Subtilisin QK-2 has a good absorption effect in vivo.(2)It was proved that recombinant Subtilisin QK-2 has an activation effect on fibrinolytic system by evaluating the changes of plasma fibrinogen/fibrin degradation products(FDP)and D-dimer levels in healthy rats after oral administration of recombinant Subtilisin QK-2 for one week.In addition,the rat thrombus model was successfully established by carrageenan-induced inflammation.Based on this model,the morphological thrombus clearance of rat blood vessels was evaluated and significant thrombus improvement was detected at the histological level.(3)To explore the fibrinolytic mechanism of recombinant Subtilisin QK-2 in human umbilical vein endothelial cells(HUVEC),related experiments were carried out at the cellular level.In a certain dose range of recombinant Subtilisin QK-2,the expression of tissue plasminogen activator(t-PA)in HUVEC cells was significantly up-regulated at mRNA and protein levels,while type 1 plasminogen activator inhibitor(PAI-1)showed a completely opposite trend.Recombinant Subtilisin QK-2 can activate the fibrinolytic system by up-regulating plasminogen activator,and can also attenuate the inhibitory effect of plasminogen activator inhibitor and achieve high-efficiency thrombolysis.This study found that the recombinant Subtilisin QK-2 has the potential for oral absorption as a protein thrombolytic drug,which was confirmed in vitro and in vivo.Recombinant Subtilisin QK-2 has a good absorption effect and can maintain obvious thrombolytic activity after being absorbed.Related animal experiments have demonstrated that oral recombinant Subtilisin QK-2 can significantly reduce the risk of thrombosis in vivo,and has a good improvement effect on the thrombus already produced in the body.Finally,it was verified at the cellular level that recombinant Subtilisin QK-2 has a regulatory effect on the components of fibrinolytic system and can effectively activate the thrombolytic activity in vivo.The recombinant Subtilisin QK-2 is a new drug-using idea for the treatment of thrombotic diseases,and provides a theoretical basis for the fibrinolytic mechanism and efficacy of protein-based oral thrombolytic drugs.