Roles of calpain in the apoptosis of Eimeria tenella host cells at the middle and late developmental stages.

This study investigated the role of calpain in Eimeria tenella-induced host cell apoptosis. Chick embryo cecal epithelial cell culture technology, flow cytometry, enzyme-linked immunosorbent assays, and fluorescence quantitative PCR were used to detect the E. tenella host cell apoptotic rate, Bax and Bid expression levels, and calpain activity. The results demonstrated that Bax, Bid, and calpain levels were upregulated and apoptosis was increased following E. tenella infection at 24-120 h. Calpain levels were reduced by pharmacological inhibition of calpain using SJA6017 or by blocking Ca2+ entry into the cell using BAPTA/AM at 24-120 h. The mRNA and protein levels of Bax and Bid, the E. tenella infection rate, and the early apoptotic and late apoptotic (necrosis) rates were decreased by using SJA6017 at 24-120 h. These results indicated that E. tenella-promoted host cell apoptosis is regulated by calpain via Bid and Bax at 24-120 h. Thus, manipulation of calpain levels could be used to manage E. tenella infection in chickens in the middle and late developmental stages.

Mitochondrial pathways are involved in Eimeria tenella-induced apoptosis of chick embryo cecal epithelial cells.

Accumulating evidence suggests that Eimeria tenella severely damages the intestinal mucosa in infected poultry, resulting in deadly haemorrhagic typhlocolitis and major economic losses. Damage to host tissue is believed to arise mainly from apoptosis, which is, in general, intimately related to mitochondrial function. However, it is unclear whether mitochondria-dependent apoptotic pathways are specifically involved in parasite-induced apoptosis of chick embryo cecal epithelial cells. Because the mitochondrial permeability transition pore (MPTP) and caspase-9 are important elements in these pathways, we studied the effects of their respective inhibitors (i.e., cyclosporine A [CsA] and Z-LEHD-FMK, respectively) in primary cultures of chicken embryonic cecum epithelial cells using histopathological techniques, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays, flow cytometry (FCM) and ELISA. Results indicated that the inhibitors significantly decreased (p < 0.01) DNA injury, apoptosis and caspase-9 and caspase-3 activity of chick embryo cecal epithelial cells at 24, 48, 72, 96 and 120 h after E. tenella infection. Thus, our data supported that mitochondria-dependent apoptotic pathways were involved in apoptosis of parasitised chick embryo cecal epithelial cells.

Dynamic changes in the main regulatory genes of mitochondrial permeability transition pore in Eimeria tenella host cells.

The purpose of the present study was to investigate the dynamic changes in the main regulatory genes of the mitochondrial permeability transition pore in E. tenella host cells. Primary chick embryo cecum epithelial cell culture techniques, spectrophotometer technology, Hoechst-Annexin V-PI apoptosis staining and ELISA were used to detect the apoptosis rate and dynamic changes of Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, HK-II, and ATP content in E. tenella host cells at 4, 24, 48, 72, 96, and 120 h. The rates of early apoptosis, late apoptosis, and necrosis of group T0 were significantly lower (P < 0.05) or highly significantly lower (P < 0.01) than those of group C at 4 h, but higher (P < 0.05 or P < 0.01) at varying degrees than those of the same group at 24-120 h. Compared to group C, the amount of Bcl-2, ATP, Bax and Bad in group T0 were visibly lower (P < 0.05 or P < 0.01) at 4 h, whereas Bcl-xl/Bax was highly significantly higher (P < 0.01) at 4 h. In addition, group T0 had less ATP at 24-120 h than group C, whereas the amount of Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad and HK-II in group T0 inversely increased in varying degrees at 24-120 h compared with group C. Moreover, Bcl-2/Bax was lower (P < 0.01) at 24, 48, and 96 h, and Bcl-xl/Bax was lower (P < 0.05) at 48 h in group T0 than in group C, respectively. Taken together, these observations indicate that in the early developmental stages of E. tenella, the host-cell apoptosis rate decreased; although the amount of anti- and pro-apoptotic genes in host cells decreased, the ratios of anti-apoptotic to pro-apoptotic bcl-2 gene-family members increased. In the middle and later developmental stages of E. tenella, the host-cell apoptosis rate increased; the amount of anti- and pro-apoptotic genes increased, while the ratios of anti-apoptotic to pro-apoptotic bcl-2 gene-family members decreased. In addition, ATP decreased at all developmental stages of E. tenella.

Relationship between Eimeria tenella development and host cell apoptosis in chickens.

Coccidiosis causes considerable economic losses in the poultry industry. At present, the pathology of coccidiosis is preventable with anticoccidials and vaccination, although at considerable cost to the international poultry industry. The purpose of the present study was to elucidate the relationship between Eimeria tenella development and host cell apoptosis in chickens, which provides a theoretical basis for further study of the injury mechanism of E. tenella and the prevention and treatment of coccidiosis. Cecal epithelial cells from chick embryo were used as host cells in vitro. In addition, flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling, and histopathological assays were used to detect the dynamic changes in E. tenella infection rates, DNA injury rates, and apoptosis rates in groups treated with and without the caspase-9 inhibitor Z-LEHD-FMK. Following E. tenella infection, we demonstrated that untreated cells had less apoptosis at 4 h and, inversely, more apoptosis at 24 to 120 h compared with control cells. Furthermore, after the application of Z-LEHD-FMK, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays, and translation of phosphatidyl serines to the host cell plasma membrane surface, the treated group chick embryo cecal epithelial cells exhibited decreased apoptosis and DNA injuries (P<0.01) at 24 to 120 h. However, light microscopy showed that E. tenella infection rates of treated cells were higher (P<0.01) than untreated cells during the whole experimental period. Together, these observations suggest that E. tenella can protect host cells from apoptosis at early stages of development but can promote apoptosis during the middle to late stages. In addition, the inhibition of host cell apoptosis can be beneficial to the intracellular growth and development of E. tenella.

Dynamic changes in key factors of the blood-brain barrier in early diabetic mice.

Chronic hyperglycemia can result in damage to the hippocampus and dysfunction of the blood-brain barrier (BBB), potentially leading to neurological disorders. This study examined the histological structure of the hippocampus and the expression of critical genes associated with the BBB at 2 early stage time points in a streptozotocin-induced diabetes mellitus (DM) mouse model. Routine histology revealed vascular congestion and dilation of Virchow-Robin spaces in the hippocampal CA1 region of the DM group. Neuronal alterations included rounding and swelling and reduction in Nissl bodies and increased apoptosis. Compared to the control group, TJP1 mRNA expression in the DM group was significantly lower (P < .05 or P < .01), while mRNA levels of JAM3, TJP3, CLDN5, CLDN3, and OCLN initially increased and then decreased. At 7, 14, and 21 days, mRNA levels of the receptor for advanced glycation end products (AGER) were greater in the DM group than in the control group (P < .05 or P < .01). These findings indicate that early-stage diabetes may cause structural and functional impairments in hippocampal CA1 in mice. These abnormalities may parallel alterations in the expression of key BBB tight junction molecules and elevated AGER expression in early DM patients.

Pathogenic mechanism of Eimeria tenella autophagy activation of chicken embryo cecal epithelial cells induced by Eimeria tenella.

Eimeria tenella mainly invades and develops into cecal epithelial cells of chickens, resulting in cecal epithelial cell damage. Infectious intracellular pathogens possibly act by influencing the autophagy process after invading cells. The interaction between E. tenella and the autophagy of host cells was explored by infecting E. tenella with chick embryo cecal epithelial cells. Transmission electron microscopy, laser confocal microscopy, and Western blot analysis were used to demonstrate that E. tenella infection could induce autophagy in host cells. Results showed that infection with E. tenella induced the formation of autophagosomes in cells. The expression of ATG 5, Beclin-1, and LC3B-II proteins were significantly (P < 0.01) increased after E. tenella infected host cells. Expression of p62 protein levels were significantly (P < 0.01) decreased in host cells infected with E. tenella. Chloroquine (CQ) significantly (P < 0.01) increased the expression levels of LC3B-II and P62 in E. tenella-infected host cells. Rapamycin (RAPA) induced autophagy in host cells, thus reducing the intracellular infection of E. tenella. By contrast, the infection rate of E. tenella increased in cells treated with 3-Methyladenine (3-MA). Hence, E. tenella sporozoite infection could induce autophagy activation in chick embryo cecal epithelial cells, and enhanced autophagy could reduce the infection rate of E. tenella.

The role of Ca2+ in the injury of host cells during the schizogenic stage of E. tenella.

Cecal epithelial cell damage is a key factor in host injure during the development of E. tenella. The intracellular free Ca2+ of the host cell is closely related to the invasion, development and proliferation of intracellular parasites, and cell damage. To determine the relationship between Ca2+ and host cell damage in the schizogenic stage of E. tenella, we established a chick embryo cecal epithelial cells model of E. tenella infection. Fluorescence staining, flow cytometry, transmission electron microscopy, inhibition and blocking experiments were used to detect the damage effect and mechanism of host cells during the schizogenic stage of E. tenella. The results showed that the host cells cytoskeletal remodeling, cell and organelle structure was destroyed, and apoptosis and necrosis were increased during the schizont stage of E. tenella. Furthermore, the above-mentioned effects of the schizogenic stage of E. tenella on cells can be alleviated by reducing the intracellular Ca2+ concentration in the host cells. These observations indicate that the effect of host cell injury was closely related to Ca2+ during schizont stage of E. tenella.

Role of EtMIC4 EGF-like in regulating the apoptosis of Eimeria tenella host cells via the EGFR pathway.

This study aimed to explore the role and key point of EtMIC4 EGF-like recombinant protein in regulating the apoptosis of Eimeria tenella host cells via the epidermal growth factor receptor (EGFR) pathway. The cells were treated with EtMIC4 EGF-like protein, EGFR-specific siRNA, or both. Infection and apoptosis rates as well as dynamic changes in the key genes and proteins of the EGFR signaling pathway in the host cells were determined. Results showed that the E. tenella and EtMIC4 EGF-like group had the highest infection rate (P < 0.01). In cells treated with EtMIC4 EGF-like for 4 to 24 h, the apoptosis rate was significantly decreased (P < 0.01) and the relative mRNA expression and protein phosphorylation levels of EGFR, protein kinase B (AKT), and extracellular regulated protein kinases (ERK) were significantly increased (P < 0.01). In E. tenella sporozoites infected for 4 to 96 h, the rate of host cell apoptosis induced by E. tenella infection was significantly (P < 0.01) reduced by EtMIC4 EGF-like. The relative mRNA expression and protein phosphorylation levels of EGFR, AKT, and ERK in the host cells of E. tenella + EtMIC4 EGF-like group were significantly increased (P < 0.01). These results indicated that E. tenella could activate the EGFR pathway through EtMIC4 EGF-like and regulate the expression of key genes in the AKT and ERK signaling pathways, thereby inhibiting cell apoptosis.

Comparison of the host cells apoptosis induced by precocious strains and virulent strains of Eimeria tenella.

The present study aims to investigate the similarities and differences between the host cells apoptosis induced by virulent line of Eimeria tenella (Tsx) and precocious line (PTsx), which can provide a theoretical basis for the study of drugs and vaccines against coccidiosis. HE staining, Hoechst 33342/AnnexinV-FITC/PI composite staining, and ELISA were used to detect the infection rate, apoptosis rate, and Caspase-3 enzyme activity of host cells infected by PTsx or Tsx, respectively. The apoptotic rates and Caspase-3 absorbance of the inoculation groups were lower (P < 0.05 or P < 0.01) than those of the control group at 4 h, whereas the apoptotic rates and Caspase-3 absorbance of the inoculation groups were higher (P < 0.05 or P < 0.01) than those of the control groups at 24 to 120 h. At the same inoculation dose, there was no significant difference in the infection rate, apoptosis rate or Caspase-3 absorbance between Tsx groups and PTsx groups after E. tenella inoculation for 4 to 72 h (P > 0.05). However, these indicators of PTsx groups were lower (P < 0.01) than those of the same dose inoculated Tsx groups at 120 h. The apoptosis rates of cecal and glandular epithelial cells in the inoculated groups were higher (P < 0.01) than those in the control group after inoculated E. tenella 5 D in vivo, and the apoptosis rates of cecal and glandular epithelial cells in PTsx group was lower (P < 0.01) than that in the same dose inoculated Tsx group. These observations indicate that both Tsx and PTsx inhibit host cell apoptosis in the early development of E. tenella, induce host cell apoptosis in the middle and late stages, and the apoptosis-inducing effect on host cells increases with increasing dose. However, when the same dose of oocysts was inoculated, the amount of apoptosis induced by PTsx in late development was less than Tsx.

Dynamic expression of death receptor adapter proteins tradd and fadd in Eimeria tenella-induced host cell apoptosis.

The present study aimed to investigate the dynamic expression patterns of death-receptor adapter proteins TNF-receptor-associated death-domain protein (TRADD) and Fas-associated death-domain protein (FADD) in E. tenella-induced host-cell apoptosis. Culture techniques for primary chick embryo cecum epithelial cells, ELISA, hematoxylin-eosin staining, fluorescence quantitative PCR techniques, and Hoechst-Annexin V-PI apoptosis staining were used to detect the apoptosis rates and dynamic expression patterns of TRADD and FADD in E. tenella host cells at 4, 24, 48, 72, 96, and 120 h. The rates of early apoptosis, late apoptosis, and necrosis of E. tenella-infected group (group T0) were significantly lower (P < 0.05) or highly significantly lower (P < 0.01) than those of control group (group C) at 4 h, but higher (P < 0.05 or P < 0.01) at varying degrees than those of the same group at 24 to 120 h. Compared with group C, both the mRNA and protein expression levels of TRADD in the group T0 cells increased highly significantly (P < 0.01) at 4 and 24 h, and significantly (P < 0.05) at 48, 72, and 120 h. Compared with the mRNA expression in the group C cells, that of TRADD in the group T0 cells increased significantly (P < 0.05) at 96 h. Both the mRNA and protein expression of FADD in the group T0 cells displayed no significant difference (P > 0.05) from those in the group C cells at 4 h. However, FADD expression in the group T0 cells were significantly (P < 0.05) or highly significantly higher (P < 0.01) than those in the group C cells at 24 to 120 h. These observations indicate that in the early developmental stages of E. tenella, the host-cell apoptosis rate decreased, and TRADD expression increased. In the middle and later developmental stages of E. tenella, the host-cell apoptosis rate increased and expression of TRADD and FADD increased. The variation trends of TRADD and FADD expression were significantly positively correlated with the change rule of the host-cell apoptosis rate, respectively. These results indicate that TRADD and FADD may play an important role in E. tenella-induced host-cell apoptosis.

The effect of the mitochondrial permeability transition pore on apoptosis in Eimeria tenella host cells.

Although the mitochondrial permeability transition pore (MPTP) is associated with cellular apoptosis and necrosis, its effect in host response to Eimeria infections is not well understood. In an effort to better understand the effect of MPTP on apoptosis in Eimeria tenella host cells, an MPTP inhibitor (cyclosporin A) was used to inhibit MPTP opening in vitro. Cecal epithelial cells from chick embryos, which were either treated or non-treated with cyclosporin A, were used as Eimeria tenella host cells. In addition, primary chick embryo cecum epithelial cell culture techniques and flow cytometry were used to detect the dynamic changes in MPTP opening, mitochondrial transmembrane potential, and cell apoptosis rate of Eimeria tenella host cells. Compared with the control group, cytometric techniques showed that untreated host cells exhibited a significantly higher (P < 0.01) degree of MPTP opening but lower (P < 0.01 or P < 0.05) mitochondrial transmembrane potential. Moreover, untreated group cells had less apoptosis (P < 0.01) at 4 h and more apoptosis (P < 0.05 or P < 0.01) at 24 to 120 h as compared with control group cells. After the application of cyclosporin A, the degree of MPTP opening in the treated group was significantly lower (P < 0.01) at 4 to 120 h compared to the untreated group, whereas the treated group had higher (P < 0.05 or P < 0.01) mitochondrial transmembrane potentials at 24 to 120 h. Flow cytometry assays also showed that there was less (P < 0.05 or P < 0.01) apoptosis after 24 h in the treated group than in the untreated group. Taken together, these observations indicate that MPTP is a key node that plays a predominant role in the mitochondrial apoptosis pathway in the host cell induced by Eimeria tenella.

Calcium homeostasis in mitochondrion-mediated apoptosis of chick embryo cecal epithelial cells induced by Eimeria tenella infection.

In this study, the process of Eimeria tenella-induced apoptosis and the effect of calcium homeostasis were investigated in chick embryo cecal epithelial cells. In particular, we examined cytochrome c release into the cytoplasm, mitochondrial permeability transition pore (MPTP) opening, and changes in [Ca(2+)]c and apoptosis in host cells. Apoptosis, MPTP opening, cytochrome c release, and [Ca(2+)]c in host cells increased following infection. This trend was reversed by blocking the increase in [Ca(2+)]c using BAPTA/AM and EGTA (intra- and extracellular chelators of Ca(2+), respectively) and by applying heparin sodium and ryanodine (blockers of the inositol triphosphate and ryanodine receptors of the endoplasmic reticulum, respectively). These results indicate that [Ca(2+)]c plays a significant role in host cell mitochondrial apoptosis, which is induced via modulation of extracellular Ca(2+) levels and endoplasmic reticulum Ca(2+) channels. Thus, agents that restore Ca(2+) homeostasis may be useful for managing E. tenella infection in chickens.

The effect of mitochondrial ATP-sensitive potassium channels on apoptosis of chick embryo cecal cells by Eimeria tenella.

The objective of this study was to investigate the effect of mitochondrial ATP-sensitive potassium (mitoKATP) channels on apoptosis induced by Eimeria tenella. At 24, 48, 72, 96 and 120 h after Eimeria tenella infection, TUNEL assays and translation of phosphatidyl serines to the host cell plasma membrane surface showed that diazoxide-treated chick embryo cecal cells underwent less apoptosis (P <0.05), while light microscopy showed that infection rates of treated cells were higher (P <0.01) than untreated cells. Caspase 9 and caspase 3 of infected cells were activated less (P <0.01) in diazoxide-treated cells than untreated cells. These results indicate that opening mitoKATP channels can protect chick embryo cecal cells from mitochondria-dependent apoptosis induced by Eimeria tenella by inhibiting activations of caspase 9 and caspase 3.