The Effect And Mechanism Of Death-receptor Adapter Protein In Eimeria Tenella-induced Chick Embryo Cecal Epithelial Cells Apoptosis

In order to explore the effect and mechanism of death-receptor adapter protein in E.tenella-induced chick embryo cecal epithelial cells apoptosis,the primary chick embryo cecum epithelial cell culture technique,gene silencing technology,ELISA activity assay,spectrophotometer technology,Q-PCR and Hoechst-Annexin V-PI fluorescence labeled technique were used to detect the apoptosis rate,the development of the parasite,the mRNA and protein expressions of Fas,TNFR1,TRAIL,TRADD,FADD,Daxx,RIP1,ASK1 and TRAF-2 in host cells that were treated or untreated with Fas,TNFR1,TRAIL,TRADD,FADD,Daxx,and RIP1 SiRNA.The results showed that:(1)Compared with the control group(group C),at 4h after infection with E.tenella,the rates of early apoptosis,late apoptosis,and necrosis in the infection group(group TO)decreased significantly(P<0.05),the mRNA and protein expressions of TRADD,RIP-1,and TRAF-2 increased highly significantly(P<0.01);at 24,72,and 120h,both the rates of early apoptosis,late apoptosis,and necrosis and the mRNA and protein expressions of FADD,TRADD,and RIP-1 in group TO increased significantly or highly significantly(P<0.05 or P<0.01);at 72 and 120h,the mRNA and protein expressions of Daxx and ASK1 increased significantly or highly significantly(P<0.05 or P<0.01);the mRNA and protein expression of TRAF-2 increased significantly(P<0.05)at 24h.The results indicated that E.tenella could lead to the significant expressions of death-receptor adapter protein FADD,TRADD,Daxx,ASK1,RIP-1,and TRAF-2 of host cells.(2)Compared with the group C,at 4-120h after infection,the mRNA and protein expression of Fas in group TO increased significantly or highly significantly(P<0.05 or P<0.01).Compared to these of the NC group(group T8),at 24,72,and 120h after infection,both the protein expressions of FADD,and Daxx and the apoptosis rate of E.tenella host cells in Fas SiRNA-treated group decreased significantly(P<0.05).The res?lts showed that in the middle and later developmental stages of E.tenella,the high expression of Fas co?ld increase the E.tenella host cell apoptosis rate and promote the protein expressions of FADD and Daxx.(3)Compared with the group C,at 72,120h after infection,the mRNA and protein expression of TRAIL in group TO increased significantly or highly significantly(P<0.05 or P<0.01).Compared to these of the group T8,at 72,120h after infection,the FADD protein expression and the apoptosis rate of E.tenella host cells in TRAIL SiRNA-treated group decreased significantly(P<0.05).The results showed that in the middle and later developmental stages of E.tenella,the high expression of TRAIL could increase the E.tenella host cell apoptosis rate and promote FADD protein expression.(4)Compared with the group C,at 4-120h after infection,the mRNA and protein expression of TNFR1 in group TO increased highly significantly(P<0.01).Compared to these of the group T8,at 4h after infection,the protein expression of TRADD in TNFR1 SiRNA-treated group decreased significantly(P<0.05)and the apoptosis rate of E.tenella host cells increased significantly(P<0.05);at 24,72,and 120h after infection,both the TRADD protein expression and the apoptosis rate in TNFR1 SiRNA-treated group decreased significantly(P<0.05).The results showed that in the early developmental stages of E.tenella,the TNFR1 high expression could inhibit the E.tenella host cell apoptosis by promoting TRADD protein expression;in the middle and later developmental stages of E.tenella,the TNFR1 high expression could promote the E.tenella host cell apoptosis and increase TRADD protein expression.(5)Compared to these of the group T8,at 4h after infection,the protein expression of RIP 1 in TRADD SiRNA-treated group decreased significantly(P<0.05)and the apoptosis rate of E.tenella host cells increased significantly(P<0.05);at 24,72,and 120h after infection,the protein expression of FADD and the apoptosis rate in TRADD SiRNA-treated group decreased significantly(P<0.05).The results showed that in the early developmental stages of E.tenella,the TRADD high expression could inhibit the E.tenella host cell apoptosis by promoting RIP1 protein expression;in the middle and later developmental stages of E.tenella,the TRADD high expression could promote the E.tenella host cell apoptosis by increasing FADD protein expression.(6)Compared to these of the group T8,at 24,72,and 120h after infection,the caspase-8 activity of E.tenella host cells in FADD SiRNA-treated group had been inhibited significantly(P<0.05)and the apoptosis rate decreased significantly(P<0.05).The results showed that in the middle and later developmental stages of E.tenella,E.tenella could promote the host cell apoptosis by FADD-caspase-8 pathway.(7)Compared to these of the group T8,at 72,120 h after infection,both the protein expressions of ASK1,and RIP1 and the apoptosis rate of E.tenella host cells in Daxx SiRNA-treated group decreased significantly(P<0.05).The results showed that in the middle and later developmental stages of E.tenella,E.tenella promoted the E.tenella host cells apoptosis by Daxx-ASK1 apoptotic pathway.(8)Compared to these of the group T8,at 4 h after infection,the protein expressions of TRAF-2 and p-NIK of E.tenella host cells in RIP1 SiRNA-treated group decreased significantly or highly significantly(P<0.05 or P<0.01),and the apoptosis rate increased significantly(P<0.05);at 24 h after infection,the protein expressions of TRAF-2 and p-MEKK1 of E.tenella host cells in RIP1 SiRNA-treated group decreased significantly(P<0.05),and the apoptosis rate decreased significantly(P<0.05).The results showed that in the early developmental stages of E.tenella,the RIP1 high expression could inhibit the E.tenella host cell apoptosis by promoting the high protein expressions of TRAF-2 and p-NIK;in the middle developmental stages of E.tenella,the RIP1 high expression could promote the E.tenella host cell apoptosis by increasing the high protein expressions of TRAF-2 and p-MEKK1.