The Research On The Relationship Between Host Prdx6 And Brucella Intracellular Parasitism In Reproductive System Related Cells

Brucella can be parasitic in phagocytic cells and non-phagocytic cells,causing a widespread epidemic of infectious Brucellosis in China.In recent years,with the rapid development of animal husbandry,the population and stocking density have increased year by year,resulting in a sudden increase in the infection rate and incidence of brucellosis among animals.The disease is not easy to be detected in the early stage of infection,and once it enters the chronic phase,it can cause damage to various organs and systems.Brucellosis in animals mainly causes reproductive system diseases such as abortion and stillbirth in male orchitis;the main manifestations of brucellosis patients are male orchitis,prostatitis,and female oophoritis.The prevalence of brucellosis seriously threatens the development of the aquaculture industry and human health,while causing huge economic losses.Peroxiredoxin 6,a member of the cysteine-dependent peroxidase family,has three enzyme activities,including Peroxidase activity,Phospholipase A2 activity,lyso-Phosphatidylcholine acyl transferase activity.In our previous study,we found that after the infection of sheep by Brucella,the expression level of Prdx6 in sheep changed significantly.In order to further explore the relationship between Prdx6 and intracellular parasitics of Brucella,our research group successfully expressed Prdx6 recombinant protein in sheep and mice,and prepared anti-prdx6 rabbit polyclonal antibody and mouse monoclonal antibody.It was found that the host Prdx6 plays a promoting role in the parasitism of Brucella in macrophages,and the change of its expression was significantly correlated with inflammatory reaction and leukocyte migration.There are no reports on the study of the relationship between Brucella infection and host Prdx6 in other cells.Based on the important of Prdx6 and Brucella parasites in macrophages,combined with Brucella infection mainly invading the reproductive system,this study used HPT-8,TM4 and CHO reproductive system related cells as research objects,using over-expression,silencing and gene knockout techniques to further explored the relationship between host Prdx6 and intracellular parasitic ability of Brucella in reproductive system related cells,and to analyze the difference in the expression of Prdx6,which caused the differential expression of cytokines in reproductive system related cells of Brucella infection.To provide a scientific basis for us to study the molecular mechanism of Brucella causing host reproductive system diseases.In this study,Brucella suis S2 was used to infect reproductive system related cells,and the expression of intracellular Prdx6 protein was analyzed by Western Blot.The results showed that the expression of Prdx6 was irregular at 0 h-12 h,and the expression of Prdx6 was up-regulated at 12-48 h in HPT-8 TM4 and CHO cells.It is indicated that Brucella infection can cause differential expression of reproductive system related cells Prdx6.The expression level of Prdx6 in HPT-8,TM4 and CHO cells was regulated by RNA interference vector and overexpression vector,and the effect of differential expression of Prdx6 on the survival of Brucella in the reproductive system related cells was analyzed.At the same time,the Prdx6 gene of HPT-8,TM4 and CHO cell lines was knocked out by CRISPR/Cas9 technology,and the constructed Prdx6-pSpCas9(BB)-2A-Puro-sgRNA vector was transferred into cells by non-liposome transfection.A series of screening tests confirmed that the HPT-8 cell line with the Prdx6 gene deletion was successfully established,and the HPT-8 cells lacking the Prdx6 gene were infected with Brucella suis S2.The results showed that overexpression of Prdx6 increased the parasitic ability of Brucella in the reproductive system related cells.The knockdown and deletion of Prdx6 can attenuate the viability of Brucella in the cell.After overexpressing and interfering with Prdx6 in HPT-8,TM4 and CHO,the bacterial infection experiment was carried out,and the relevant cytokines DLL4,ICAM-1,JAM-A,MCSF,MIP-1b and Progranulin were detected by real-time PCR.The results showed that when Prdx6 was up-regulated,Progranulin showed down-regulated expression,MCSF showed up-regulated expression,and the opposite was observed in the Prdx6 knockdown group.There was no significant difference between DLL4,ICAM-1,JAM-A and MIP-1B.Progranulin and MCSF may be related to host Prdx6 in response to Brucella infection,but further studies are needed.This study found that the expression of host Prdx6 is conducive to the parasitization of Brucella in the reproductive system related cells.Under Brucella infection,the differential expression of Prdx6 causes changes in the expression of Progranulin and MCSF cytokines.To provide a new entry point for in-depth exploration of the infection and parasitic mechanism of Brucella in the reproductive system related cells,it provides a basis for further research on prevention and treatment of Brucellosis.