| Coccidiosis caused by Eimeira tenella(E. Tenella)is a serious poultry disease responsible for enormous economic losses to the global poultry industry. E. tenella represents the most pathogenic species causing severe cecal hemorrhage and death in poultry coccidiosis. Chick cecal cells are host cells of E. tenella, so a lot of host cell death occurs in E. tenella infecting process. Apoptosis is one of the main ways upon cell death. There are three pathways in apoptosis. Mitochondrial pathway is the base and core among the apoptotic pathways. Oxidative stress is the result of E. tenella infection. What the relationship is between oxidative stress and mitochondria mediating apoptosis, and how to make effect for oxidative stress on mitochondria mediating apoptosis are the aims of the present experiments.Chick embryo primary cecal cell culture, flow cytometry, real-time quantitative PCR, ELISA and spectrophotometer were employed in the study to understand the effect and mechanism of oxidatice stress on apoptotic mitochondrial pathway. Apocynin, febuxostat, L-NMMA, NAC and diazoxide were utilized to treat the infeced chick embryo primary cecal cells for checking the levels of reactive oxygen species (ROS) and nitric oxide (NO) in cells and culturing mediator, activities of nicotinamide adenine dinucleotide phosphate oxidase, xanthine oxidase, xanthine oxidase and caspase 9, extent of opening mitoKATP channels and expression levels of B cell lymphoma 2 and BCL-2 associated X protein in mRNA and protein. The results of the study are listed as below:(1) There were no differences (P>0.05) between T1 and T groups in E. tenella infection rates at all the time points, the same as T2 and T groups, which showed there was not relationship between inhibiting the activities of ROS producing enzyme and E. tenella infection rate. At 96 h and 120 h after infection, the infection rates of T3 group were significantly (P<0.05) or more significantly (P<0.01) higher than T gruop, which showed inhibiting NO producing is good at development of E. tenella in host cells. At 24 h,96 h and 120 h after infection, the infection rates of T4 group were significantly (P<0.05) or more significantly (P<0.01) lower than T gruop, which showed cleaning ROS could reduce infection rate of E. tenella in host cell. At 96 h and 120 h after infection, the infection rates of T5 group were more significantly (P<0.01) higher than T gruop, which showed opening mitoKATP channels could inhibit host cells apoptosis, lengthen E. tenella development time and enhance E. tenella infection rate in host cells.(2) At 24 h to 120 h and 24 h to 96 h after infection respectively, the levels of ROS and NO in T group were more significantly (P<0.01) higher than C gruop, which showed E. tenella infection could induce oxidative stress in host cells.(3) At 4 h to 120 h and 24 h to 120 h after infection respectively, the levels of ROS and the activities of NOX in T1 group were more significantly (P<0.01) lower than T gruop; at 24 h to 120 h after infection, the levels of ROS and the activities of XO in T2 group were more significantly (P<0.01) lower than T gruop; at 4 h,24 h,72h,120 h and 4 h to 120 h after infection respectively, the levels of NO and the activities of NOS in T3 group were significantly (P<0.05) or more significantly (P<0.01) lower than T gruop, which showed origins of ROS and NO resulting from E. tenella infection are NOX, XO and NOS.(4) At 24 h to 120 h after infection, the levels of H2O2 and NO in T1 group were significantly (P<0.05) or more significantly (P<0.01) higher than C gruop in culturing mediator, which showed ROS and NO could be released into culturing mediator.(5) At 24 h to 120 h and 24 h,72 h,96 h,120 h after infection respectively, the early apoptosis rates in T3 and T4 groups were significantly (P<0.05) or more significantly (P<0.01) lower than T gruop, which showed inhibiting NO production and eliminating ROS could reduce early apoptosis rates in host cells.(6) At 48 h and 24 h after infection respectively, the degrees of opening MPTP in T3 and T4 groups were more significantly (P<0.01) lower than T gruop, which showed oxidative stress affected MPTP partly.(7) At 24 h to 120 h and 48 h to 120 h after infection respectively, the activities of caspase 9 in T3 and T4 groups were significantly (P<0.05) or more significantly (P<0.01) lower than T gruop; at 24 h,48 h, 96 h,120 h and 48 h,96 h,120 h after infection respectively, the levels of cyt c in mitochondria in T3 and T4 groups were significantly (P<0.05) or more significantly (P<0.01) higher than T gruop; at 24 h to 120 h after infection, the levels of cyt c in plasm both in T3 and T4 groups were significantly (P<0.05) or more significantly (P<0.01) lower than T gruop, which showed oxidative stress resulting from E.tenella infection induced apoptosis by mitochondrial pathway.(8) At 4 h to 120 h and 4 h,24 h,48 h,72 h,120 h after infection respectively, the levels of P-JNK and P-p38 both in T3 and T4 groups were significantly (P<0.05) or more significantly (P<0.01) lower than T gruop; at 4 h,48 h,72 h,120 h and 4 h to 72 h after infection respectively, the levels of BCL-2 both in T3 and T4 groups were significantly (P<0.05) or more significantly (P<0.01) higher than T gruop; at 24 h to 96 h after infection, the levels of BAX both in T3 and T4 groups were more significantly (P<0.01) lower than T gruop; at 24 h to 120 h and 24 h,72 h,96 h,120 h after infection respectively, the levels of BCL-2 mRNA both in T3 and T4 groups were more significantly (P<0.01) higher than T gruop; at 4 h,48 h,72 h, 96 h,120 h and 24 h to 120 h after infection respectively, the levels of BAX mRNA both in T3 and T4 groups were significantly (P<0.05) or more significantly (P<0.01) lower than T gruop, which showed oxidative stress affected mitochondria-mediated apoptosis by activating JNK and p38 and regulating BCL-2 and BAX.(9) At 24 h,72 h,96 h,120 h and 48 h to 120 h after infection respectively, the levels of early apoptosis, late apoptosis and necrosis in T5 group were significantly (P<0.05) or more significantly (P<0.01) lower than T gruop; at 24 h to 120 h after infection respectively, the levels of DNA injury, caspase 9 activity and caspase 3 activity in T5 group were lower significantly (P<0.01) than T gruop more, which showed opening mitoKATP channels could reduce the apoptosis induce by E.tenella through inhibiting the activities of caspase 9 and caspase 3.(10) At 24 h,48 h,72 h,120 h and 4 h,48 h,72 h after infection respectively, the levels of ROS and NO in T5 group were significantly (P<0.01) lower than T gruop more; at 4 h to 120 h and 48 h,72 h after infection respectively, the levels of H2O2 and NO in culturing mediator in T5 group were significantly (P<0.01) lower than T gruop more, which showed opening mitoKATP channels could reduce the levels of ROS and NO indicating that there were relationships between mitoKATP channels and ROS, NO respectively. |
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