Study On The Effect And Mechanism Of MAPK Signaling Pathway In The Infection Of Sarcocystis Cruzi In Yak

Sarcocystis is an obligate intracellular parasite of the apicomplexan protozoa.It is a common zoonotic parasite that parasitizes humans and animals.Among them,Sarcocystis cruzi(S.cruzi)is the most serious infection of cattle.Mitogen-activated protein kinase(MAPK)is a kind of serine/threonine protein kinase,which is an important molecule in the process of apicomplexan infection.After apicomplex an parasite infects the host,it can cause the phosphorylation of MAP kinase in host cells,thereby regulating cell proliferation and differentiation.However,the interaction between Sarcocystis spp and the MAPK signaling pathway is still unclear.This study took S.cruzi isolated and identified from yak myocardium in plateau areas as the research object,and used four different culture cell lines Vero cells,PAM cells,Hela cells and PK15 cells to cultivate S.cruzi in vitro,for screening out the most suitable cell line for culturing S.cruzi,and establishing an in vitro culture method for S.cruzi.Real-time fluorescent quantitative PCR(RT-PCR)and fully automated protein a nalysis system(Wes)method were used to determine the m RNA expression l evels of ERK1,ERK2,GRB2,TAK1 and MKP1 genes and changes in the expression of ERK,p38,JNK,GRB2 and TAK1 proteins in the MAPK signaling pathway.The inhibitors PD98059 and U0126 were used to evaluate the changes in the host MAPK signaling pathway in Vero cells infected with S.cruzi tachyzoites,respevtively.The results showed that S.cruzi could grow in four different cell lines,and it grew faster in Vero cells.The worms began to grow to a peak on the 5th to 6th day of culture,and the peak period lasted for 2 days.It shows that Vero cells are suitable for the in vitro culture of S.cruzi.The changes of MAPK signaling pathway after parasite infection were measured by RT-PCR and Wes test.The results showed that within 1hour after S.cruzi invaded the host,the relative expression level of related genes in the cellular MAPK signaling pathway could be up-regulated(P <0.01),which can trigger the initiation of the MAPK signaling pathway,in which ERK,JNK and p38 are phosphorylated between 10 min and 60 min after the worm invasion(P<0.01).ERK phosphorylation was significantly inhibited(P<0.0001)after blocking with specific inhibitors of ERK1/2 phosphorylation PD98059 or U0126,respectively.The effects of the two inhibitors on S.cruzi infection of Vero cells were detected by Giemsa staining.It was found that the amount of tachyzoites in Vero cells treated with the inhibitor PD95059 at 10 μM and 20 μM for 24 h decreased by 19.34% and31.24%(P<0.0001),respectively.When adding the inhibitor U0126 10μM and 20μM to co-culture the cells for 24 h,the amount of infected tachyzoites in the cells decreased by 28.19% and 44.17% on average compared with the control group(P<0.0001).In conclusion,the invasion of S.cruzi can activate the phosphorylation of ERK,JNK and p38 proteins in the MAPK signaling pathway of Vero cells,thereby promoting cell growth and facilitating the infection and growth of tachyzoites.The use of MAPK inhibitor not only effectively prevented the phosphorylation of ERK caused by S.cruzi infection,but also had a certain inhibitory effect on S.cruzi infected cells and intracellular proliferation.