The Dynamic Changes And Roles Of Mitochondria Released Key Factors In The Host Cell Of E.Tenella

In order to explore the role of released key protein from mitochondria in E.tenella host cell apoptosis, we utilized in vitro experiments applying chicken primary cecum epithelial cells as host cells to detect apoptosis, caspase9activity and expressions of Cyt-c、Smac、AIF、 Endo G in mitochondria and cytoplasm by ELISA and flow cytometry from4h to120h after E.tenella infection that treated or untreated with Cyt-c inhibitor the cyt-c inhibitor(TMPD、 Sodium ascorbate)、the inhibitor of PARP-1(4-AN) or thr inhibitor of MPTP(CSA),the results show that:1. At4h, in the untreated group(TO) mitochondrial cyt-c, smac and Endo G expressions were significantly (P<0.05) or extremely significantly (P<0.01) lower than that of control group(CO), whilst mitochondrial AIF expression were extremely significantly (P<0.01) higher than that of control group. The total and cytoplasmic cyt-c, smac, Endo G and AIF expressions were all significantly (P<0.05) or extremely significantly (P<0.01) lower than that of group C0. The data indicate that the expressions of cyt-c, smac, Endo G, AIF in the host cells, especially in cytoplasm were declined in the early state of E.tenella infecting host cells.2. The untreated groups at48h to120h, mitochondrial cyt-c, smac, Endo G and AIF expressions were significantly (P<0.05) or extremely significantly (P<0.01) lower than that of the control group and not increased with the insect development; At24h to120h the cytoplasmic and total cyt-c, smac, Endo G and AIF expressions were increased with the insect development, whilst the total cyt-c, Endo G and AIF expressions and cytoplasmic cyt-c, smac, Endo G and AIF expressions were significantly (P<0.05) or extremely significantly (P<0.01) lower than that of the control group. The data indicate that in the late state of E.tenella infecting host cells, the expressions of cyt-c, smac, Endo G and AIF in the mitochondria were declined, whereas the expressions were increased in host cells, especially in cytoplasm.3. At4h-120h, in the treated groups(T1), mitochondrial cyt-c, smac, Endo G and AIF expressions were scarcely higher than that of untreated groups, whilst total cyt-c, Endo G and AIF expressions and cytoplasmic cyt-c, smac, Endo G and AIF expressions were all significantly (P<0.05) or extremely significantly (P<0.01) lower than that of the uninfected groups. The data indicate that, in process of apoptosis induced by E.tenella, cyt-c, smac, Endo G and AIF is released from mitochondria to the cytoplasm with MPTP opening.4. In the treated groups(T2), the apoptosis rate and the activity of caspase-9were all lower than that of untreated groups, whilst in the treated groups(T2),the apoptosis rate and the activity of caspase-9were significantly (P<0.05) or extremely significantly (P<0.01) lower that of the untreated group and were increased with the insect development. All show that cyt-c through the caspase-dependent pathway regulates the apoptosis caused by E.tenella.5. At24h to120h, mitochondrial smac expressions of group T2were significantly (P<0.05) or extremely significantly (P<0.01) higher than that of the untreated groups; cytoplasmic smac expressions were all extremely significantly (P<0.01) than that of the untreated groups, and the total smac expressions were no significantly (P>0.05) lower than that of the treated groups, showing that the reduction of oxidation state of cytosolic cyt-c block smac expressions which were released from mitochondria to cytoplasm on the course of E.tenella inducing apoptosis6. At4h to96h, apoptosis rates of the treated group(T3) in the host cells were all significantly (P<0.05) or extremely significantly (P<0.01) lower than the untreated groups, indicating that in the process of apoptosis induced by E.tenella, AIF is released from mitochondria to the cytoplasm with MPTP opening.