RESUMO
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.
Assuntos
Coccidiose , Eimeria tenella , Doenças das Aves Domésticas , Animais , Apoptose , Calpaína/genética , Embrião de Galinha , Galinhas , Coccidiose/metabolismo , Coccidiose/veterinária , Eimeria tenella/genética , Doenças das Aves Domésticas/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Eimeria tenella is the most pathogenic and common coccidia that causes chicken coccidiosis. The intracellular free Ca2+ of the host cell is closely related to the invasion, development, and proliferation of intracellular parasites. To determine the dynamic changes of intracellular free Ca2+ and its function in the process of E. tenella invading host cells, we established a chick embryo cecal epithelial cells model of E. tenella infection. Chick embryo cecal epithelial cells were treated with different Ca2+ signal inhibitor, respectively, and then infected with E. tenella. The results showed that extracellular Ca2+, Ca2+ channels on the cell membrane, IP3R ion channels on the endoplasmic reticulum membrane, and RyR ion channels regulated the free Ca2+ in cecal epithelial cells. Through fluorescence labeling and invasion rate detection, we found that the intracellular Ca2+ did not change significantly during the invasion of E. tenella, but its stability was critical to the invasion of parasites.
Assuntos
Coccidiose , Eimeria tenella , Doenças das Aves Domésticas , Animais , Ceco/parasitologia , Embrião de Galinha , Galinhas , Coccidiose/veterinária , Eimeria tenella/metabolismo , Doenças das Aves Domésticas/parasitologiaRESUMO
This study investigated the role of PI3K/Akt signaling pathway on host cell apoptosis in the early infection of Eimeria tenella. Chicken cecal epithelial cells were treated with apoptosis-inducer Actinomycin D (Act D) or PI3K/Akt signaling pathway inhibitor LY294002 and then infected with E. tenella. Results demonstrated that the E. tenella-infected group had less apoptosis 4-8 h after the infection and more apoptosis 12-20 h after the infection than the control group. At 4-20 h after the infection, the apoptotic/necrotic rate and the Caspase-3 activity in the Act D + E. tenella group were lower (P < 0.01) than those in the Act D-treated group. The p-Akt and NF-κB contents in the E. tenella-infected group were higher (P < 0.01) than those in the control group 4-12 h after the infection. However, the bad content and the Caspase-9/3 activity were lower (P < 0.05) in the E. tenella-infected group than in the control group. Compared with the E. tenella-infected group, the LY294002 + E. tenella group showed decreased p-Akt content and increased apoptotic/necrotic rate, bad content, NF-κB expression, membrane permeability transition pore (MPTP) openness, and Caspase-9/3 activity. Thus, the early development of E. tenella could inhibit host cell apoptosis by downregulating the Caspase-3 activity. Upregulating this activity promoted apoptosis. In addition, activating the PI3K/Akt signaling pathway inhibited the apoptosis of E. tenella host cells in the early infection by reducing the expression of the bad content, limiting the MPTP opening, and decreasing the Caspase-9 and Caspase-3 activities.
Assuntos
Apoptose , Coccidiose/veterinária , Eimeria tenella/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Doenças das Aves Domésticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Ceco/metabolismo , Ceco/parasitologia , Galinhas , Coccidiose/metabolismo , Coccidiose/parasitologia , Coccidiose/fisiopatologia , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/genética , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/parasitologia , Doenças das Aves Domésticas/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de SinaisRESUMO
BACKGROUND: Eimeria tenella (E. tenella) is a species of Eimeria that causes haemorrhagic caecal coccidiosis, resulting in major economic losses in the global poultry industry. After E. tenella infection, the amount of ATP and Bax in host cells showed highly significant changes. Therefore, it is necessary to investigate the effects of ATP and Bax on the apoptosis of E. tenella host cells. RESULTS: The ATP-treated group and the V5-treated group had higher E. tenella infection rates than the untreated group at 24, 48, 72, 96, and 120 h after infection with E. tenella. The results of flow cytometry showed that compared with the control group, the mitochondrial permeability transition pore (MPTP) opening in the untreated group was highly significantly increased (P < 0.01) at 4, 24, 48, 72, 96, and 120 h. Moreover, results from Hoechst-Annexin V-PI staining and flow cytometry showed that the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were significantly lower (P < 0.05) or highly significantly lower (P < 0.01) than those of the control group at 4 h, while the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were higher at varying degrees than those in the control group at 24-120 h (P < 0.05 or P < 0.01). After treatment with ATP and Bax inhibitors, the rates of early apoptosis, late apoptosis, and necrosis, in addition to the MPTP opening in both the ATP-treated and V5-treated groups, were significantly lower (P < 0.05) or highly significantly lower (P < 0.01) than those in the untreated group. CONCLUSIONS: ATP and Bax play important roles in regulating the apoptosis of E. tenella host cells.
Assuntos
Trifosfato de Adenosina/farmacologia , Apoptose/efeitos dos fármacos , Eimeria tenella/efeitos dos fármacos , Proteína X Associada a bcl-2/farmacologia , Animais , Galinhas/parasitologia , Coccidiose/tratamento farmacológico , Coccidiose/microbiologia , Citometria de Fluxo/veterinária , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/parasitologiaRESUMO
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.
Assuntos
Apoptose , Ceco/citologia , Coccidiose/veterinária , Eimeria tenella/fisiologia , Mitocôndrias/metabolismo , Doenças das Aves Domésticas/fisiopatologia , Animais , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Ceco/metabolismo , Ceco/parasitologia , Embrião de Galinha , Galinhas , Coccidiose/metabolismo , Coccidiose/parasitologia , Coccidiose/fisiopatologia , Eimeria tenella/genética , Eimeria tenella/isolamento & purificação , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Marcação In Situ das Extremidades Cortadas , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/parasitologiaRESUMO
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.
Assuntos
Eimeria tenella/genética , Genes de Protozoários/fisiologia , Genes Reguladores/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Protozoários/genética , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Embrião de Galinha , Galinhas , Eimeria tenella/crescimento & desenvolvimento , Eimeria tenella/fisiologia , Hexoquinase/genética , Hexoquinase/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Distribuição Aleatória , Organismos Livres de Patógenos Específicos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
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.
Assuntos
Apoptose , Galinhas , Coccidiose/metabolismo , Coccidiose/veterinária , Eimeria tenella/fisiologia , Doenças das Aves Domésticas/metabolismo , Animais , Inibidores de Caspase/química , Ceco/metabolismo , Ceco/parasitologia , Embrião de Galinha , Coccidiose/parasitologia , Dano ao DNA , Citometria de Fluxo/veterinária , Marcação In Situ das Extremidades Cortadas/veterinária , Oligopeptídeos/química , Doenças das Aves Domésticas/patologia , Distribuição AleatóriaRESUMO
The recombinant plasmid pCI-IL-4-IL-2-EGFP containing fusion genes of chicken IL-4 and IL-2 can be used as an adjuvant to enhance the anticoccidiosis effect of the chicken coccidia live vaccine. The chickens were divided into 3 groups: blank control group, vaccine + pCI-IL-4-IL-2-EGFP adjuvant coimmunization group, and vaccine-only group to investigate the immune synergy mechanism of recombinant plasmid adjuvant pCI-IL-4-IL-2-EGFP. The expressions of IL-2, IL-4, TNF-α, and IFN-γ in chicken sera and tissues were detected by ELISA and RT-qPCR, and the proliferation of T and B lymphocytes and antigen presenting cells (APC) in chicken immune organs and intestines were detected by acid alpha-naphthalase (ANAE) staining, methyl green pyronine (MGP) staining, and immunofluorescence (IF) staining, respectively. Results showed that the mRNA expression of IL-2, IL-4, IFN-γ and the number of activated T and B lymphocytes were significantly upregulated in the spleen and cecum tonsils of chickens in vaccine + pCI-IL-4-IL-2-EGFP group compared with the vaccine-only group on 7 d after vaccination (P < 0.05). Protein contents of IL-2, IL-4 and TNF-α in vaccine + pCI-IL-4-IL-2-EGFP group were significantly increased compared to vaccine-only group on 28 d of inoculation (P < 0.05). The number of T and B lymphocytes and APC in chickens of the vaccine+ pCI-IL-4-IL-2-EGFP group was significantly higher than that of the vaccine-only group in cecum tonsils, thymus and spleen after 14 and 28 d of inoculation (P < 0.05). All results revealed that pCI-IL-4-IL-2-EGFP adjuvant enhanced the immune response of chicken coccidia live vaccine by upregulating the expression of IL-2, IL-4, TNF-α, and IFN-γ and promoting the proliferation of T, B lymphocytes and APCs in chicken intestines and immune organ sites. Moreover, our study provides a theoretical basis for the clinical application of cytogenic plasmids as adjuvants.
Assuntos
Galinhas , Coccídios , Animais , Galinhas/genética , Interleucina-2/genética , Interleucina-4/genética , Fator de Necrose Tumoral alfa/genética , Coccídios/genética , Coccídios/metabolismo , Adjuvantes Imunológicos , Plasmídeos/genéticaRESUMO
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.
RESUMO
Eimeria tenella rhoptry protein has the properties of a protective antigen. EtROP27 is a pathogenic gene that is detected via a transcriptome, but its expression pattern, immunogenicity, and potency are unknown. Therefore, a gene segment of EtROP27 was amplified and transplanted into the pET28a prokaryotic vector for the expression of the recombinant protein, and it subsequently purified for the generation of a polyclonal antibody. Then, RT-PCR and Western blotting were performed to understand the expression pattern of EtROP27. Subsequently, animal experiments were conducted to evaluate the immunoprotective effect of the recombinant protein with different immunizing doses (50, 100, and 150 µg). The results showed that the expression of EtROP27 gradually increased with the prolongation of infection time, reaching the highest level at 96 h and then decreasing. Additionally, EtROP27 is a natural antigen of coccidia that can stimulate the body to produce high levels of IgY. As with recombinant protein vaccines, the results of immune protection evaluation tests showed that the average weight gain rates of the immune challenge groups were significantly higher than that of the challenged control group, and their average lesion scores were significantly lower than that of the challenged control group. Furthermore, the oocyst excretion decreased by 81.25%, 86.21%, and 80.01%, and the anticoccidial index was 159.45, 171.47, and 166.75, respectively, for these groups. EtROP27 is a promising antigen gene candidate for the development of a coccidiosis vaccine.
RESUMO
Hexavalent chromium (Cr(â ¥)) is considered to be a common environmental pollutant, which widely exists in industrial effluents and wastes and then potentially noxious effects to the health of the poultry. Studies have reported that selenium (Se), which is one of the essential trace elements of the poultry and participates in the oxidative metabolism, can alleviate Cr(â ¥)-induced organ damage by inhibiting oxidative stress, but its specific molecular mechanism remains unclear. Herein, animal models of Cr(â ¥)- and Se-exposure were constructed using broilers to investigate the antagonistic mechanism of Se to Cr(â ¥)-induced hepatotoxicity. In this experiment, the four groups of broiler models were used as the research objects: control, Se, Se plus Cr, and Cr groups. Histopathology and ultrastructure liver changes were observed. Liver-somatic index, serum biochemistry, oxidative stress, Nrf2 pathway related factors, and autophagy-related genes were also determined. Overall, Se was found to ameliorate the disorganized structure, hepatic insufficiency, and oxidative damage caused by Cr(â ¥) exposure. Electron microscopy analysis further showed that the number of autophagosomes was obviously decreased after Se treatment compared to Cr group. Furthermore, gene and protein expression analyses illustrated that the levels of Nrf2, glutathione peroxidase 1 (GPx-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), and mechanistic target of rapamycin (mTOR) in the Se&Cr group was upregulated, along with decreased expression of Beclin 1, ATG5 and LC3 compared to the Cr group. These suggest that Se can repair the oxidative lesion and autophagy induced by Cr(â ¥) exposure in broiler livers by upregulating the Nrf2 signaling pathway.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Selênio , Animais , Selênio/farmacologia , Selênio/metabolismo , Galinhas/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Cromo/toxicidade , Cromo/metabolismo , Estresse Oxidativo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas/veterinária , Transdução de SinaisRESUMO
Intestinal mucosa injury and loss of weight gain are unavoidable while using live vaccine strain to prevent chicken coccidiosis. In this study, recombinant Lactococcus lactis NZ3900/pNZ8149-IL-4-IL-2, expressing the fusion protein of chicken IL-4 and IL-2, was constructed using food-grade NICE expression system, trying to develop a possible oral immune adjuvant to enhance the immune effect of the live vaccine against chicken coccidiosis and minimize its adverse effects. Chickens were given different doses of recombinant L. lactis together with the live vaccine, then experimently attacked with coccidia virulent strains. Results showed that weight gains of co-immunization groups, given both 1 × 109 or 1 × 1010 CFU recombinant L. lactis and the live vaccine, were significantly higher than the vaccine-only group (P<0.05), while intestinal lesion scores of duodenum, jejunum, and cecum were significantly lower than the vaccine-only group (P<0.05), so was the oocyst shedding. The anticoccidial indexes (ACI) of the co-immunized groups given 1 × 109 and 1 × 1010 CFU recombinant L. lactis were 187.85 and 193.33, respectively, higher than 174.61 of the vaccine-only group. In addition, chickens in co-immunization groups gained more body weight than the vaccine-only group before being challenged with the virulent strains (P<0.05). All the results indicated that the constructed recombinant L. lactis NZ3900/ pNZ8149-IL-4-IL-2 exhibited an immune synergistic function to coccidiosis live vaccine, and could alleviate its adverse effect affecting weight gain. The application of the recombinant L. lactis showed the potency to lift the anticoccidial efficiency of the live vaccine from a medium level to a high level.
Assuntos
Coccídios , Coccidiose , Lactococcus lactis , Doenças das Aves Domésticas , Animais , Galinhas , Interleucina-4/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Interleucina-2 , Coccidiose/prevenção & controle , Coccidiose/veterinária , Aumento de Peso , Doenças das Aves Domésticas/prevenção & controleRESUMO
This study collected 324 chicken cloacal swabs from 6 broiler farms in 4 different areas in Shanxi Province, China (i.e., Lvliang, Taiyuan, Jinzhong, and Yangquan), and analyzed the antimicrobial resistance and virulence-associated genes of the isolates to investigate the prevalence, drug resistance, and virulence gene data of Campylobacter jejuni in broilers. The population structure of C. jejuni and genetic evolutionary relationships among isolates from broiler farms in different regions were studied by using multilocus sequence typing. A total of 35 C. jejuni isolates with an infection rate of 10.8% (35/324) were obtained. The isolates were most resistant to ampicillin (85.7%) and were most sensitive to erythromycin (14.3%). Isolates with multidrug resistance accounted for 88.6% of the total isolates. In this experiment, 15 distinct sequence types were identified and included 9 new unique sequence types. cadF was present in all isolates, and ciaB had the lowest prevalence (51.4%). C. jejuni collected from broiler farms in central Shanxi had varied infection rates, and their overall positive rate was lower than of C. jejuni collected from other regions of the country. The isolates had high resistance to quinolones and ß-lactams, and multidrug resistance was prevalent. The isolates were genotypically diverse and carried 5 virulence-associated genes at high rates. Therefore, the importance of source contamination control in broiler farms is emphasized and may have considerable effects on human and animal health.
Assuntos
Infecções por Campylobacter , Campylobacter coli , Campylobacter jejuni , Animais , Humanos , Galinhas/genética , Infecções por Campylobacter/epidemiologia , Infecções por Campylobacter/veterinária , Prevalência , Virulência/genética , Fazendas , Resistência a Medicamentos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Campylobacter coli/genética , Tipagem de Sequências Multilocus/veterináriaRESUMO
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.
Assuntos
Coccidiose , Eimeria tenella , Doenças das Aves Domésticas , Animais , Embrião de Galinha , Autofagia/fisiologia , Galinhas , Coccidiose/patologia , Coccidiose/veterinária , Eimeria tenella/patogenicidade , Células Epiteliais/metabolismo , Doenças das Aves Domésticas/patologiaRESUMO
Background: Sonodynamic therapy (SDT) and its synergistic cancer therapy derivatives, such as combined chemotherapy-SDT (chemo-SDT), are promising approaches for tumor treatment. However, the main drawbacks restricting their applications are hypoxia in tumors and the reducing microenvironment or high glutathione (GSH) levels. Methods: In this study, a hybrid metal MnO2 was deposited onto nanoparticles fabricated using poly(lactic-co-glycolic acid) (PLGA), carrying docetaxel (DTX) and the sonosensitizer hematoporphyrin monomethyl ether (HMME) (PHD@MnO2) via a one-step flash nanoprecipitation (FNP) method. Characterization and in vitro and in vivo experiments were conducted to explore the chemo-SDT effect of PHD@MnO2 and evaluate the synergetic antitumor treatment of this nanosystem. Results: When low-power ultrasound is applied, the acquired PHD@MnO2, whether in solution or in MCF-7 cells, generated ROS more efficiently than other groups without MnO2 or those treated via monotherapy. Specifically, GSH-depletion was observed when MnO2 was introduced into the system. PHD@MnO2 presented good biocompatibility and biosafety in vitro and in vivo. These results indicated that the PHD@MnO2 nanoparticles overcame hypoxia in tumor tissue and suppressed the expression of hypoxia-inducible factor 1 alpha (HIF-1α), achieving enhanced chemo-SDT. Conclusion: This study provides a paradigm that rationally engineered multifunctional metal-hybrid nanoparticles can serve as an effective platform for augmenting the antitumor therapeutic efficiency of chemo-SDT.
Assuntos
Compostos de Manganês , Nanopartículas , Linhagem Celular Tumoral , Docetaxel , Glutationa , Hematoporfirinas , Humanos , Hipóxia , Nanopartículas/uso terapêutico , ÓxidosRESUMO
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.
Assuntos
Eimeria tenella , Animais , Apoptose , Galinhas/genética , Eimeria tenella/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Coccidiose , Eimeria tenella , Doenças das Aves Domésticas , Animais , Ceco/fisiologia , Embrião de Galinha , Galinhas , Coccidiose/veterinária , Eimeria tenella/fisiologia , Doenças das Aves Domésticas/parasitologiaRESUMO
The development of novel sonosensitizers with safety and efficiency is a key problem in anti-tumor sonodynamic therapy. Phycocyanin (PC) has been proved to have the singlet oxygen radicals (ROS) generation ability, and the potential of PC as a novel sonosensitizer has been investigated. To overcome the disadvantages of PC in vivo, such as poor stability and low half-life, PC nanoparticles (PCNP) were prepared by the cross-linking method. According to the results, PCNP has been found with good morphology, good particle size distribution and good stability. Human breast cancer cell line MCF-7 was used to investigate PCNP cell uptake ability. ROS generation and cytotoxicity under ultrasonic irradiation (sonotoxicity) were also studied on this cell. Under the condition of 0.75 w/cm² ultrasound, PCNP has a good ROS productivity and is equivalent to the sonotoxicity of the known sonosensitizer hematoporphyrin monomethyl Ether (HMME). In conclusion, PCNP is expected to be developed as an effective sonosensitizer for the sonodynamic therapy of tumors.
Assuntos
Nanopartículas , Neoplasias , Terapia por Ultrassom , Linhagem Celular Tumoral , Humanos , Células MCF-7 , Michigan , FicocianinaRESUMO
Combined chemotherapy and sonodynamic therapy (chemo-SDT) based on the nanoplatform/nanocarrier is a potential antitumor strategy that has shown higher therapeutic efficacy than any monotherapy. Therefore, a safe and effective multifunctional system with a concise design and simple preparation process is urgently needed. In this work, by using a one-step cross-linking method, a multifunctional nanosystem, which employs phycocyanin nanoparticles (PCNPs) as a nanocarrier to deliver the chemotherapy drug docetaxel (DTX) and a nanosonosensitizer to generate reactive oxygen species (ROS), was prepared and evaluated (PCNP-DTX). Under low-intensity ultrasound irradiation, PCNP-DTX retained the ROS generation ability of phycocyanin and caused the destruction of mitochondrial potential. PCNP was also revealed to be an acidic and ultrasound-sensitive carrier with good biocompatibility. In addition to its cumulation behavior in tumors, PCNP can achieve tumor-targeted delivery and release of DTX. PCNP-DTX has also been proven to have a significant chemo-SDT synergy effect when low-intensity ultrasound was applied, showing enhanced antitumor activity both in vitro and in vivo. This study provides a concise yet promising nanoplatform based on the natural protein phycocyanin for achieving an effective, targeted, and synergetic chemo-SDT for antitumor therapy.