Study On The Role Of Heme Oxygenase-1 Suppressing Duck Tembusu Virus Replication In Vitro

Duck Tembusu virus disease,is a kind of important infectious disease caused by Duck Tembusu virus,which can induce bradygenesis,sudden drop in egg production and paralysis in duck.DTMUV belongs to positive single-strand RNA virus,and it is a member of the Flaviridae family,Flaviridae genus and Entaia virus group.It can infect many animals such as ducks,chickens and geese,and furthermore,it may be a health threat to human beings.Heme Oxygenase is an initiating enzyme that catalyzes the degradation of heme and a very important rate-limiting enzyme in cells.Heme Oxygenase-1 is a subtype of HO,which is an important endogenous protective protein normally present in the body.Its activity is closely related to the body's anti-inflammatory,antioxidant,anti-apoptosis and other functions,and it plays a very important regulatory role in the process of virus infection and replication.However,its regulatory role is discrepant in different viruses.Whether HO-1 interact with DTMUV's infection in cells is still unclear.Therefore,this paper aims to clarify the role and mechanism of HO-1 in the process of DTMUV infection on cells and provide theoretical basis for the prevention and treatment of the disease through regulating the expression of HO-1 and detecting the effect on DTMUV infection in host cells.Firstly,the effect of DTMUV infection on HO-1 gene expression in cells was tested.The Duck Embryo Fibroblasts cells were inoculated with DTMUV strains,and the non-infected control group was set up simultaneously.The infected cells and control group cells at different time points were collected respectively.Total RNA of each group were extracted for reverse transcription.The expression level of HO-1 was detected by q PCR.The results showed that the expression of HO-1 in cells increased briefly at first and then decreased rapidly to a lower level with the extension of infection time of DTMUV,which indicated that DTMUV infection could inhibit the expression of HO-1 gene in cells in total.Then the effect of upregulation of HO-1 expression on DTMUV infection was tested.In order to study the role of HO-1 in DTMUV infection,cobalt protoporphyrin,a specific inducer of HO-1,and a specific expression plasmid were used to treat cells to induce HO-1gene expression and detect its effect on DTMUV infection.The results showed that specific plasmids and CoPP could significantly up-regulate expression of HO-1 gene and protein expression without affecting cell activity.The gene expression of HO-1 was positivelycorrelated with the specific plasmid transfection content and CoPP concentration,while the E gene expression level of DTMUV in cells was negatively correlated with the specific plasmid transfection content and CoPP concentration.After transfected with HO-1 overexpression plasmid,the proliferation of intracellular virus was significantly suppressed.In this experiment,the optimal concentration of specific plasmid and CoPP were used to treat cells to up-regulate the expression of HO-1 in the cells significantly.With the extension of infection time,the expression of E gene of DTMUV in cells gradually decreased,which indicated that up-regulation of HO-1 expression could effectively inhibit DTMUV infection.Thirdly,the effect of downregulation of HO-1 expression on DTMUV infection was detected.To clarify the role of HO-1,we silenced the HO-1 gene specifically by si RNA to reduce the expression of HO-1,and then detected the E gene expression of DTMUV in cells.The results showed that the expression of HO-1 gene in cells transfected with si RNA decreased significantly,while the expression of E gene in DTMUV cells increased significantly.With the increase of si RNA concentration,the expression of HO-1 protein decreased gradually.The content of virus increased significantly,which indicated that the specific reduction of HO-1 expression could promote the proliferation of DTMUV in cells.Finaly,the inhibitory mechanism of HO-1 on DTMUV infection was explored.In order to clarify the mechanism of HO-1 inhibiting DTMUV infection,DEF cells were treated with CoPP before and after DTMUV infection respectively.The results showed that DTMUV infection was inhibited regardless of treating time,and the results showed there has no significant difference.The biliverdin,CORM and Fe Cl3 solutions were used to simulate the three catalytic products of HO-1,biliverdin,CO and Fe²⁺ respectively to treat cells.The results showed that only the cells treated with Fe Cl3 have a significant increase in the expression of HO-1 m RNA with a significant decrease in the expression of E gene,which indicated that the effect of CoPP on the inhibition of DTMUV infection in cells was not related to the period of viral infection,while the inhibition of virus infection in cells was mediated by catalytic product Fe²⁺ of HO-1.