The Mechanism Of Coxsackievirus B3 On Fractalkine Expression In Myocardial Microvascular Endothelial Cells

Background:Through studying the pathogenesis of viral myocarditis(VMC),it is known that virus infection can induce the host to produce immune response.Autoimmune therapy has become a hot topic in in current.Coxsackie virus B3(CVB3)is a common cause of viral myocarditis.First,the CVB3 must cross the barrier of myocardial microvascular endothelial cells before infecting cardiomyocytes.As the last line of defense against the external infection of cardiac myocytes,myocardial microvascular endothelial cells participate in the immunomodulatory,inflammatory reactions,and releasing inflammatory factors to resist the invasion of external pathogens.At the same time,myocardial microvascular endothelial cells can alter the functions of various endothelial cells.Fractalkine(FKN)is one of them.FKN plays a role in peripheral leukocyte migration and adhesion.FKN plays a role of adhesion without the participation of other molecules.FKN can be expressed in many kinds of cells,including endothelial cells,smooth muscle cells,macrophages and so on.When inflammatory signals are stimulated,the expression of FKN in these cells will increase greatly,especially in the early stages of a variety of diseases.And the expression of FKN is great significance in alleviating the inflammatory response and delaying the process of disease.In order to study the immunomodulatory mechanism of viral myocarditis and find an ideal autoimmunity therapy,FKN has become a hot spot as a chemokine with adhesion.However,the expression of FKN is regulated by a variety of signaling pathways.The MAPK pathway is one of them.However,the expression of FKN in the myocardial microvascular endothelial cells infected by coxsackievirus is specifically regulated by which subpathway in the MAPK.At present,the research on this aspect is less and is also the focus of this study.Objective:The MAPK regulation mechanism of FKN expression in myocardial microvascular endothelial cells after coxsackievirus infection in vitro was explored.In order to investigate the pathogenesis and autoimmune regulation of viral myocarditis.Methods:Section 1:The culture and generation of myocardial microvascular endothelial cells were carried out.The CD31 specific antigen on the surface of myocardial microvascular endothelial cells was identified by immunofluorescence.CVB3 intervention was performed on cultured myocardial microvascular endothelial cells.And morphological changes were observed under microscope.The virus was diluted with 10 times gradient of virus culture.The cultured cells were cultured from 10-1-10-8.Pathological changes were observed under the microscope.The Reed-Muench method was used to calculate the median lethal dose(TCID50)of the virus.Section 2:The CVB3 cells obtained from the first part were used to interfere with the cultivation of TCID50 cells.Then the cells were collected at 0,20,40,60,80min,respectively.The total protein was extracted.The expression of FKN and protein phosphorylation of ERK1/2,JNK and P38 were detected by Western blot.Section 3:U0126(inhibitors of protein ERK1/2),were selected to inhibit ERK1/2 pathway.First,the best concentration of U0126 was selected by CCK8.U0126 was added to the cells with CVB3.The total cell protein was extracted respectively in 0,20,40,60,and 80min.The phosphorylation of ERK1/2 and RSK90 were detected.The expression of FKN was detected by Wester blot.The virus culture medium was diluted with 10 times gradient.The cells with the best concentration of U0126 and CVB3 were added from 10-1-10-8,The Reed-Muench method was used to calculate the median lethal dose(TCID50)at the time of the virus.Section 4:Liposome plasmid transfection was used to inhibit the expression of FKN.Plasmid transfection efficiency was detected.The expression of FKN at mRNA level was detected by PCR.The expression of FKN at the protein level was detected by Western blot.The transfection results were detected.The cells of the screened positive FKN-shRNA were treated with TCID50 concentration of CVB3.The total cell protein was extracted at 0,20,40,60,and 80min.The western blot method was used to detect the protein phosphorylation of ERK1/2,JNK,P38.The virus was diluted with 10 times gradient of virus culture.The positive FKN-shRNA cells were intervened from 10-1-10-8.The Reed-Muench method was used to calculate the median lethal dose(TCID50)at the time of the virus.Results:Section 1:Myocardial microvascular endothelial cells were treated with CD31 antibody.The green fluorescence was showed on fluorescence microscope.The purity rate was very high.Under the intervention of CVB3,the cells were infected by 4-6 days.Under the microscope,the cells began to turn round and crinkle and finally float on the surface of the liquid.The median infection rate of TCID50 was 6.825Section 2:CVB3,with the final concentration of TCID50,acted on the myocardial microvascular endothelial cells.The cells were collected at 0,20,40,60 and 80min.The results showed that the phosphorylation of ERK1/2 gradually increased with the time increasing.The highest band of phosphorylation of ERK1/2 was at 40min and 60min.There was no difference between the JNK and P38 phosphorylated bands at every time.At the same time,the expression band of FKN increased gradually with time increasing.At 40min the band was deepest.There was no significant difference between 60min and 80min compared with 40min.Section 3:The best concentration of U0126 was 40ng/ml.After 40ng/ml final concentration of U0126 and TCID50 final concentration CVB3 were added to the myocardial microvascular endothelial cells.The cell protein was extracted at 0,20,40,60,80min.The phosphorylation of ERK1/2 and RSK90 were detected.The results showed that ERK1/2 pathway was effectively suppressed.After 0,20,40,60,80min,the total protein of cells with U0126 and CVB was added simultaneously was extracted.The results showed that the color of FKN bands was basically same at different times.At this time,the TCID50 of CVB3 to the cell is 7.48.Section 4:The green fluorescence was observed in the positive plasmid group under microscope.The transfection rate of 24h was reached 50%.PCR detected the low expression of FKN at mRNA level.Western blot detected FKN expression at low protein level.Therefore,FKN-shRNA was transfected into myocardial microvascular endothelial cells.Positive FKN-shRNA cells showed no change in protein phosphorylation bands of ERK1/2,JNK and P38 at 0,20,40,60,80min.At this time,the TCID50 of CVB3 to the cell is 7.99.Conclusions:When the cells are infected by Coxsackie virus,FKN can reduce the infection of CVB3 to the myocardial microvascular endothelial cells.FKN is regulated by the ERK1/2 pathway.This discovery provides a theoretical basis for further study of the immunomodulatory treatment of viral myocarditis.