| Chicken coccidiosis is a globally prevalent parasitic disease that seriously endangers the healthy development of poultry farming,and its infection is very common.To improve the damage mechanism of coccidia on the body,could provide new ideas and theoretical support for the research of drugs and vaccines.The aim of this study was explored the effects of Eimeria tenella(E.tenella)infection and E.tenella microsome-like 4 EGF-like(EtMIC4 EGF-like)on epidermal growth factor receptor(EGFR)signaling pathway and pathway-related molecules in host cells.And the role of EtMIC4 EGF-like in regulating host cell apoptosis through EGFR signaling pathway and the relationship between host cell EGFR signaling pathway and E.tenella infection was investigated.The E.tenella cecal epithelial cell culture model in vitro was used.The cell infection and apoptosis rate,expression and activity of related factors of the group treated with E.tenella-sporophyte,EtMIC4 EGF-like protein,si RNA EGFR or untreated were tested with techniques of recombinant protein expressing in Pichia pastoris,Annexin V-FITC/PI/Hoechst fluorescent labeling,gene interferenced(si RNA),Q-PCR,Western blot and H&E staining.The results showed that:(1)After inoculation with E.tenella for 4 h,the m RNA and protein phosphorylation of EGFR,PI3 K,AKT,PKC and ERK genes,the m RNA expression of Ras and Raf genes,and the total protein of PKC,Ras and Raf in E.tenella group were significantly(P < 0.01)higher than those in si RNA group EGFR+E.tenella group and blank control group.The m RNA expressions of p38 and JNK genes and the phosphorylation of Raf,p38 and JNK proteins in si RNA EGFR+E.tenella group were significantly(P <0.01)lower than those in si RNA EGFR+E.tenella group and the blank control group.The m RNA and protein phosphorylation of EGFR,PI3 K,AKT,PKC and ERK genes in E.tenella group were significantly(P < 0.01)higher than those in si RNA EGFR+E.tenella group at 24 h after E.tenella infection.The phosphorylation of EGFR,AKT and PKC proteins,the m RNA expression of Ras,Raf,PI3 K,PKC,ERK,p38 and JNK genes,and the total protein of Ras and Raf were significantly(P < 0.01)lower than those in the blank control group.The phosphorylation of Raf protein was significantly(P < 0.01)higher than that in si RNA EGFR+E.tenella group and blank control group.The phosphorylation of p38 and JNK proteins were significantly(P < 0.01)lower than those in si RNA EGFR+E.tenella group,and significantly(P < 0.01)higher than those in blank control group.At 96 h after E.tenella inoculation,the m RNA expression and protein phosphorylation of EGFR,PI3 K,AKT,PKC and ERK genes in E.tenella group were significantly(P < 0.01)higher than those in si RNA EGFR+E.tenella group.The m RNA and protein phosphorylation of p38 and JNK in si RNA EGFR+E.tenella group were significantly(P < 0.01)lower than those in the blank control group,and significantly(P < 0.01)higher than those in si RNA EGFR+E.tenella group.The expression of Ras and Raf genes were consistent with the results at 24 h after inoculation.(2)EtMIC4 EGF-Like was added for 4-24 h,the m RNA and protein phosphorylation of EGFR,PI3 K,AKT,PKC and ERK genes in EtMIC4 EGF-Like group were higher(P < 0.05)or significantly(P < 0.01)higher than those in si RNA EGFR+ EtMIC4 EGF-Like group and blank control group.The m RNA expressions of p38 and JNK genes and the phosphorylation of Raf,p38 and JNK proteins were significantly(P < 0.01)lower than those in si RNA EGFR+EtMIC4 EGF-Like group and blank control group.E.tenella infection and EtMIC4 EGF-Like supplementation for 4 h,in E.tenella+EtMIC4 EGF-Like group,the m RNA expression and protein phosphorylation of EGFR,PI3 K,AKT,PKC and ERK genes,the m RNA expression and total protein of Ras gene and the total protein of PKC were significantly(P < 0.01)higher than those in blank control group.The m RNA expression and total protein of Raf gene was significantly(P< 0.01)higher than that in blank control group.The m RNA and protein phosphorylation of p38 and JNK genes and the phosphorylation of Raf protein were significantly(P < 0.01)lower than those in si RNA EGFR+E.tannella+EtMIC4 EGF-Like group and blank control group.E.tenella infection and EtMIC4EGF-Like supplementation for 24 h,in E.tenella+EtMIC4 EGF-Like group,the m RNA expression and protein phosphorylation of EGFR,PI3 K,AKT and ERK genes and the total protein of PKC were significantly(P < 0.01)higher than those in si RNA EGFR+E.tannella+EtMIC4 EGF-Like group and blank control group.The m RNA expression and protein phosphorylation of PKC gene,the m RNA expression of Ras,Raf,p38 and JNK genes were significantly(P < 0.01)lower than those in blank control group.The protein phosphorylation of Raf,p38 and JNK were significantly(P < 0.01)higher than those in blank control group.E.tenella infection for 24-96 h and EtMIC4 EGF-Like supplementation for 24 h,in E.tenella+EtMIC4 EGF-Like group,the m RNA expression and protein phosphorylation of EGFR,PI3 K,AKT,PKC and ERK genes were significantly(P < 0.01)higher than those in si RNA EGFR+E.tannella+EtMIC4 EGF-Like group and significantly(P < 0.01)lower than those in blank control group.The m RNA expression and total protein of Ras and Raf genes and the total protein level of PKC were significantly(P < 0.01)lower than those in blank control group.The m RNA expression and protein phosphorylation of p38 and JNK genes and the protein phosphorylation of Raf were significantly(P < 0.01)lower than those in si RNA EGFR+E.tannella+EtMIC4 EGF-Like group,but significantly(P < 0.01)higher than those in blank control group.(3)E.tenella infection and EtMIC4 EGF-Like supplementation for 4 h,the early apoptosis rate,late apoptosis rate and necrosis rate in E.tenella group and EtMIC4 EGF-Like+E.tenella group were significantly(P < 0.01)lower than si RNA EGFR +E.tenella group,si RNA PI3K+E.tenella group,si RNA ERK+E.tenella group,and si RNA EGFR+EtMIC4 EGF-Like+E.tenella group,si RNA PI3 K +E.tenella+EtMIC4 EGF-Like group,si RNA ERK E.tenella+EtMIC4 EGF-Like group and blank control group,respectively.EtMIC4 EGF-Like supplementation for 4-24 h,the results of Et MIC EGF-Like group were consistent with the E.tenella infection and EtMIC4 EGF-Like supplementation for 4 h.After E.tenella infection for 24-96 h and EtMIC4 EGF-Like supplementation for 24 h,the early apoptosis rate,late apoptosis rate and necrosis rate in E.tenella group and EtMIC4 EGF-Like+E.tenella group were significantly(P < 0.01)lower than si RNA EGFR +E.tenella group,si RNA PI3K+E.tenella group,si RNA ERK+E.tenella group and si RNA EGFR+EtMIC4 EGF-Like+E.tenella group,si RNA PI3 K +E.tenella+EtMIC4 EGF-Like group,si RNA ERK E.tenella+EtMIC4 EGF-Like group,respectively,but significantly(P < 0.01)higher than in blank control group.(4)E.tenella infection and EtMIC4 EGF-Like supplementation for 4 h,the early apoptosis rate,late apoptosis rate and necrosis rate in E.tenella+Act D group and Et MIC EGF-Like+E.tenella+Act D group were significantly(P < 0.01)lower than si RNA EGFR +E.tenella+Act D group,si RNA EGFR+EtMIC4EGF-Like+E.tenella+Act D group and blank control group,but significantly(P < 0.01)higher than E.tenella group and EtMIC4 EGF-Like+E.tenella group.EtMIC4 EGF-Like supplementation for 4-24 h,the results of Et MIC EGF-Like+Act D group were consistent with the E.tenella infection and EtMIC4EGF-Like supplementation for 4 h.After E.tenella infection for 24-96 h and EtMIC4 EGF-Like supplementation for 24 h,the early apoptosis rate,late apoptosis rate and necrosis rate in E.tenella+Act D group and Et MIC EGF-Like+E.tenella+Act D group were significantly(P < 0.01)lower than si RNA EGFR +E.tenella+Act D group and si RNA EGFR+EtMIC4 EGF-Like+E.tenella+Act D group,but significantly(P < 0.01)higher than E.tenella group,EtMIC4 EGF-Like+E.tenella group and blank control group.(5)After E.tenella sporozoites inoculation for 4-96 h,the infection rate of EtMIC4 EGF-Like+E.tenella group was significantly(P < 0.01)higher than that of si RNA EGFR+EtMIC4 EGF-Like+E.tenella group and si RNA PI3K+EtMIC4 EGF-Like+E.tenella group,si RNA ERK+EtMIC4 EGF-Like+E.tenella group and E.tenella group.After E.tenella sporozoites inoculation for 4-96 h,the infection rate of E.tenella group was significantly(P < 0.01)lower than that of EtMIC4 EGF-Like+E.tenella group.The infection rate of E.tenella+Act D group and E.tenella+EtMIC4 EGF-Like+Act D group were significantly(P < 0.01)higher than that of si RNA EGFR+E.tenella+Act D group and si RNA EGFR+E.tenella+EtMIC4EGF-Like+Act D group,but significantly(P < 0.01)lower than E.tenella group and EtMIC4 EGF-Like+E.tenella group.The results indicated that E.tenella could activate EGFR through EtMIC4 EGF-Like in the early stage of infection.At the same time,it could up-regulate the expression of host cell EGFR gene and activate EGFR protein,thereby up-regulating the anti-apoptotic genes(PI3K,AKT,PKC)expression and activating the PI3 K pathway.It also increased the expression and activity of Ras,Raf and ERK genes,and activated ERK/MAPK signaling pathway.In addition,it can reduce the expression and activity of p38 and JNK genes in E.tenella host cells,inhibit the JNK and p38/MAPK signaling pathways,and then inhibit the apoptosis of host cells.The opposite was observed in the middle and late stages of E.tenella infection.The activation of host cell EGFR signaling pathway could promote the invasion and development of E.tenella,while host cell apoptosis could inhibit the invasion and development. |
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