The Protective Function And Molecular Mechanism Of Dimer Thymosin β4 In Myocardial Infarction

Myocardial infarction(MI)refers to the sudden reduction or interruption of blood flow in coronary arteries due to thrombosis,which results in cardiac disease with ischemic necrosis in the corresponding myocardium.MI can easily cause heart failure and sudden cardiac death,severely affecting the patient’s quality of life.Due to the current lack of effective therapeutic drugs,MI is a clinically difficult problem.Thymosin Beta 4(Tβ4)is a 43 amino acid polypeptide in the thymosin family that is importance in embryonic development.Because of its characteristics of inducing angiogenesis and inhibiting myocardial fibrosis,it is considered to be an effective drug that can fundamentally treat MI.However,Tβ4 products entering clinical research at the present stage are all chemical synthetic products.The chemical synthesis efficiency is low,the industrial production cost is high,and the environmental pollution is serious.On the other hand,genetic engineering methods for the production of Tβ4,because of their small molecular size,are difficult to purify after prokaryotic expression and cannot achieve large-scale production.In the earlier period,our research group designed and produced recombinant thymosin beta 4(DTβ4)based on literature reports and past experience.The dimer contains two intact Tβ4monomers with a molecular weight of 9500 Da,a simple genetic engineering process,and a one-step purification rate of 98%.The product and production process obtained twonational invention patents.The research on promoting wound healing was awarded with a National Special Fund for Science and Technology(2011ZXJ091104-01B).Preclinical studies have been completed and clinical approvals will soon be applied.In view of the important role of wild-type Tβ4 in the treatment of MI,this research intends to develop the application of DTβ4 to protect the heart from MI on the basis of previous studies,and explore the molecular mechanisms of cardiac protection.To accomplish the above research objectives,we conducted the following scientific work:1.Cell scratch test,tubule formation experiment,Transwell assay,3D culture sprouting experiment,flow cytometry,Western Blot,immunofluorescence assay,etc.Detection and comparison of DTβ4 and wild-type Tβ4 in human umbilical vein endothelial cells(HUVECs),biological activity in SD rat cardiomyocytes and cardiac fibroblasts;2.An animal model of MI was established by ligation of the left anterior descending coronary artery in mice.After treatment with DTβ4 or Tβ4,the protective function of DTβ4 and wild-type Tβ4 on the heart of mice was detected by animal cardiac ultrasound,masons staining and immunofluorescence.3.RNA-seq was used to analyze the molecular mechanism of angiogenic activity increase of DTβ4 relative to wild-type Tβ4,and the sequencing results were verified by real-time quantitative PCR,RNA interference,BrdU proliferation assay,flow cytometry,and immunofluorescence.4.Transcription factor chip,Western Blot,RNA interference assay,dual luciferase reporter gene,co-immunoprecipitation(Co-IP),chromatin immunoprecipitation(ChIP),and exploration the promotes angiogenesis molecular mechanism of DTβ4 through lung adenocarcinoma metastasis-associated transcript 1(MALAT1).Through the above research work,we have reached the following conclusions:Compared with wild-type Tβ4,DTβ4 has a stronger pro-angiogenic effect and exhibits stronger cardioprotective function in MI mice;DTβ4 promotes the proliferation of vascular endothelial cells and promotes angiogenesis by up-regulating MALAT-1,while Tβ4 does not up-regulate MALAT-1,only has the effect of promoting vascular endothelial cell migration and does not have the function of promoting the proliferation of vascularendothelial cells.After MALAT-1 expression is interfered,DTβ4 has an angiogenic function that is equality with Tβ4 and promotes the disappearance of proliferative effects.DTβ4 upregulates the transcription of MALAT1 by inhibiting the ubiquitination of the transcription factor PROX1 and exerts an angiogenic effect.This research systematically studied the protective effect of DTβ4 in MI.It lays a foundation for the further development of the application of DTβ4 to protect the heart,and also provides a basis for the wide application of DTβ4.In addition,the results of this study show that the method of constructing dimer using genetic engineering is an effective means to produce biologically active small molecule polypeptides,and constructing dimers may bring new biological functions to biologically active small molecules.