The Effect And Mechanism Of Calcium Homeostasis In Mitochondrion-mediated Apoptosis Of Chick Embryo Cecal Epithelial Cells Induced By Eimeria Tenella Infection

In order to explore the effect and mechanism of calcium homeostasis in mitochondrion-mediated apoptosis of chick embryo cecal epithelial cells induced by Eimeria tenella, the primary chick embryo cecum epithelial cell culture technique, ELISA activity assay, Spectrophotometer technology, RT-PCR and flow cytometry were used to detect the apoptosis, the development of the parasite, the opening of MPTP and the dynamic changes of Ca²⁺-ATPase activity, cytoplasm Cytc and Ca²⁺([Ca²⁺]c), calpain, the mRNA expression of Bax and Bid, CaN, Bad and 14-3-3 in E.tenella host cells that were treated or untreated with BAPTA/AM, EGTA (intra-and extracellular chelators of Ca²⁺, respectively), heparin sodium and ryanodine （blockers of the inositol triphosphate and ryanodine receptors, respectively, of the endoplasmic reticulum （ER））, SJA6017（inhibitor of Calpain） TFP （inhibitor of CaM）. These results showed that:（1） At 4h to 120h after infection, [Ca²⁺]c was increased in infected/untreated group （group TO） relative to uninfected/untreated controls （group C） （P< 0.05 or P< 0.01）. All the results indicated that infection with E.tenella resulted in an increase of [Ca²⁺]c in the host cells.（2） At each time point after infection, [Ca²⁺]c was higher in group TO than in intracellular chelators of Ca²⁺BAPTA/AM （group T3） （P< 0.05 or P< 0.01） excepting 4h and 120h. At 4h after infection, the apoptosis rate was higher in group T3 than in group TO （P< 0.05）. at 48,96, and 120 h after infection, there were more early apoptotic cells in group TO than in group T3 （P< 0.05）. Similarly, the late apoptosis rates and necrosis were higher in group TO than in group T3 （P< 0.01） at 4h to 120h after infection. The probability of MPTP opening was higher in group TO at 96 and 120h than in group T3 （P< 0.05）. At 4h after infection, cytochrome c levels were higher in groups T3 than in group TO （P< 0.05）. The cytochrome c release was lower in groups T3 （at 24,96, and 120 h） than in group TO （P< 0.05）. These results provided additional evidence that the host cell apoptosis were regulated by [Ca²⁺]c, and intracellular chelators prevented the opening of MPTP and cytochrome c release.（3） At 48,72, and 96h post-infection, [Ca²⁺]c was lower in those treated with the extracellular Ca²⁺ chelator EGTA （group T2） than in group TO （P< 0.01）; at 48,96, and 120h, [Ca²⁺]c was higher in group TO than in cells treated with RyR and IP₃R blockers （group T1） （P< 0.05）. These results suggested that inhibiting Ca²⁺influx into the cell and ER Ca²⁺release blocks the increase in [Ca²⁺]c induced by E. tenella infection.（4） At 4h after infection, a higher cell apoptosis rate and cytochrome c release from mitochondria were observed in group T1 and T2 than in group T0. At 24-120h after infection, host cell apoptosis and cytochrome c release were lower in group T1 and T2 than in group T0. At 4-120h after infection, the probability of MPTP opening was higher in group T1 and T2 than in group T0. At 4h after infection, Ca²⁺-ATPase activity were lower in groups T1 and T2 than in group T0. Howerver, at 24-120h after infection, Ca²⁺-ATPase activity were higher in groups T1 and T2 than in group T0. These results suggest that blocking extracellular Ca²⁺entring into cell and ER Ca²⁺release abrogated the opening of the MPTP, cytochrome c release and Ca+-ATPase activity caused by E. tenella infection.（5） At 4h after infection, the levels of calpains were lower in group TO than in group C （P< 0.01）. But the levels of calpains was higher in group TO than in group C at 24-120h after infection（P< 0.01）. At 4h after infection, calpains activity was higher in group T3 than in group TO （P< 0.01）. At 24,72,96 and 120h post-infection, calpains activity was higher in group TO than in group T3（P< 0.05）. It shows that the activity of calpains was decreased at early stage of E. tenella infection, and then increased later, blocking extracellular Ca²⁺ entry into the cell result in abrogated calpains activity.（6） The probability of MPTP opening was higher in group TO than in cells treated with calpains inhibitor （group T4） at 4,48,96 and 120h post-infection. At 4h after infection, the apoptosis rates were higher in group T4 than in group TO （P< 0.01）.Conversely, the rate of early apoptosis at 48,96 and 120h after infection and the rates of late apoptosis and necrosis at 24-120h were higher in group TO than in group T4 （P< 0.01）. These results suggested that apoptosis were regulated by calpains, since treatment with calpains inhibitor prevented the opening of MPTP.（7） At 4h after infection, the expression of Bax and Bid gene were lower in group TO than group C （P < 0.05）. The expression of Bax and Bid gene were higher in group T4 than group T0, and the expression of Bax mRNA was higher in group T4 than group TO （P< 0.05）. Conversely, at 24-120h post-infection, the expression of Bax and Bid gene were higher in group TO than group C （P< 0.05）. and the expression of Bax and Bid gene were lower in group T4 than group T0;the expression of Bax mRNA was lower in group T4 than group TO at 24-72h （P< 0.01）;the expression of Bid mRNA was lower in group T4 than group TO at 24-120h after infection （P< 0.05）. The protein levels of Bax （at 48h） and Bid （at 48,72 and 120h） were lower in group T4 than group TO （P< 0.05）. These results suggested that Eimeria tenella infection inducing mitochondrion-mediated apoptosis were regulated by calpains, since being treated with calpains inhibitor prevented the the expression of Bax and Bid gene.（8） At 4h post-infection, the levels of calcineurin were lower in group TO than in group C （P< 0.01）. Conversely, at 24-120h after infection, the levels of CaN were higher in group TO than in group C excepting 96h （P< 0.05）. The levels of calcineurin were higher in group T3 than in group TO at 4h. Conversely, at 24,48 and 120h, the levels of calcineurin were lower in group T3 than in group T0. These results suggested that the levels of calcineurin were decreased at early stage of Eimeria tenella infection, and increased at late stage, and the levels of calcineurin were controlled by [Ca²⁺]c.（9） At 120h after infection, the probability of MPTP opening was lower in those treated with CaM inhibitor （group T5） than in group TO （P< 0.05）. Apoptosis rate was higher in group T5 than in groupTO （P < 0.05）. Conversely, at 24-120h after infection, the rate of early apoptosis was lower in group T5 than in group TO （P< 0.05）. The rates of late apoptosis and necrosis was lower in group T5 than in group TO （P< 0.01） at 48-120h. These results suggest that apoptosis were regulated by CaM, since it may control the levels of calcineurin and the probability of MPTP opening.（10） At 4h post-infection, the levels of Bad and 14-3-3 were lower in group TO than in group C （P< 0.05）, and the levels of Bad and 14-3-3 were higher in group T5 than in group TO （P> 0.05）. Conversely, at 48-120h after infection, the levels of Bad were higher in group TO than in group C （P< 0.05）, and at 24h, 72h,96h and 120h, the levels of Bad were higher in group TO than in group C （P< 0.05）. The levels of Bad were lower in group T5 than in group TO （P< 0.05）. These results suggested the levels of Bad and 14-3-3 were increased in cells infected by E. tenella, Eimeria tenella infection induced mitochondrion-mediated apoptosis are regulated by CaM, since treatment with CaM inhibitor prevented the the protein expression of 14-3-3 and Bad.