| In order to investigate the regulation mechanism of death receptor pathway on apoptosis in E.tenella host cells and the effect of E.tenella-infected culture medium on apoptosis in E.tenella host cells, we used primary culture of chick embryo cecal epithelial cells, Annexin V and PI assays, ELISA activity assays, in situ terminal transferase enzyme maker technology and hematoxylin and eosin stain to detect the apoptotic cells, the activities of Caspase-8, Caspase-3, sFas, sFasL, mFas, mFasL and E.tenella infection rate, via treating or untreating with the Caspase inhibitor (Z-IETD-FMK) and different E.tenella infected or uninfected cultured medium. The results show that:At 4 h after sporozoits inoculation, the early apoptosis rate, the late apoptosis and necrosis rate in E.tenella-infected group were lower than E.tenella-uninfected group, but the differences were not significant (P > 0.05). The DNA damage rate of sporozoits-inoculated host cells was significantly (P< 0.05) lower than sporozoits-uninoculated host cells; The early apoptosis rate, the late apoptosis and necrosis rate of sporozoits-inoculated host cells were extremely significantly (P<0.01) lower than sporozoits-uninoculated host cells at 24 h to 72 h and 120 h after sporozoits inoculation. DNA damage rate of sporozoits-inoculated host cells was extremely significantly (P< 0.01) higher than sporozoits-uninoculated host cells at 24 h to 96 h after sporozoits inoculation; The apoptosis rate of host cell increased along with the length of the E.tenella-uninfected time. These results showed that the apoptosis of host cells were inhibited at early sporozoits-inoculating phase and were accelerated at late sporozoits-inoculating phase.The early apoptosis rate, the late apoptosis and necrosis rate, DNA damage rate of host cells and E.tenella infection rate had no significant differences (P> 0.05) between treated (with Z-IETD-FMK) and untreated groups at 4 h; The early apoptosis rates in the treated groups were significantly (P< 0.05) or extremely significantly (P< 0.01) lower than untreated groups at 48 h to 96 h after sporozoits inoculation; At 48 h to 96 h after sporozoits inoculation, the late apoptosis and necrosis rates in the treated groups were lower than untreated groups, and specially at 72 h,96 h the differences were extremely significant (P<0.01); DNA damage rates in the treated groups were significantly (P<0.05) or extremely significantly (P<0.01) lower than untreated groups at 24 h to 96 h after sporozoits inoculation;The E.tenella infection rates in the treated groups were extremely significantly (P<0.01) higher than the untreated groups at 24 h to 120 h after sporozoits inoculation. These results showed that death receptor pathway took part in the E.tenella-induced apoptotic pathway, and inhibiting death receptor pathway was benifical to the development of E.tenella.The activities of Caspase-3 in host cells at 4 h after sporozoits inoculation were extremely significantly (P< 0.01) lower than control group; The activities of Caspase-3 and Caspase-8 at 24 h to 120 h were extremely significantly (P<0.01) or significantly (P<0.05) higher than control groups; The activity of Caspase-8 in treated group was higher than untreated group at 4 h after sporozoits inoculation; The activities of Caspase-3, Caspase-8 in treated groups were extremely significantly (P< 0.01) lower than untreated groups at 24 h to 120 h. These results showed that E.tenella regulated the death receptor apoptotic pathway by activating Caspase-8 and Caspase-3.The activities of sFas in host cells culture supernatant were extremely significantly (P<0.01) or significantly (P<0.05) lower in sporozoits-infected groups than control groups at 4 h-120 h except 48 h; Moreover, the activities of mFas of host cells of E.tenella were extremely significantly (P< 0.01) or significantly (P< 0.05) lower than control groups; The activities of sFasL in host cell culture supernatant were extremely significantly (P<0.01) or significantly (P<0.05) lower in sporozoits-infected groups than control groups than control groups; The activities of mFasL of host cells of E.tenella were extremely significantly (P < 0.01) or significantly (P< 0.05) higher than control group at 4 h-120 h except 48 h. These data showed that the higher activity of Fas/FasL was one of merchasnism in the apoptosis of host cell after sporozoits inoculationThe early apoptosis rate of host cell in treated (with 48 h sporozoits-infected cell culture supernatant) group was significantly (P< 0.05) higher than treated (with 48 h sporozoits-uninfected cell culture supernatant) group; The late apoptosis and necrosis rate of host cell in treated group (with 12 h sporozoits-infected cell culture supernatant) was extremely significantly (P< 0.01) lower than treated group (with 12 h sporozoits-uninfected cell culture supernatant). The late apoptosis and necrosis rate of host cell in treated groups (with 4 h,24 h-120 h sporozoits-infected cell culture supernatant) were higher than treated groups (with 4 h,24 h-120 h sporozoits-uninfected cell culture supernatant), and the differences were extremely significantly (P< 0.01) or significantly (P<0.05)in groups with 24 h and 120h sporozoits-infected cell culture supematantafter. The activities of Caspase-8 of host cell between treated groups and untreated groups had no significant differences (P> 0.05). The activities of Caspase-9 of host cell in treated groups were higher than untreated groups at 4 h,24 h after sporozoits inoculation. The data showed that E.tenella secretions affected the apoptosis of uninfected host cell after sporozoits inoculation, and mitochondrial apoptotic pathway was the main way to trigger the apoptosis of E.tenella host cell. |
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