RESUMEN
Mutations or loss of function of DJ-1 and Toxoplasma gondii (T. gondii) infection has been linked to neurodegenerative diseases, which are often caused by oxidative stress. However, the relationship between DJ-1 and T. gondii infection is not yet fully understood. Therefore, this study aimed to investigate the expression of DJ-1 in the hippocampus tissue of mice or in HT22 infected with T. gondii Chinese 1 genotype Wh3 strain (TgCtwh3) and the effect of DJ-1 knockdown on neuronal apoptosis induced by TgCtwh3 tachyzoite, as well as the underlying mechanism at the cellular and molecular level. Firstly, we detected DJ-1 protein expression and cell apoptosis in the hippocampal tissue of mice infected by TgCtwh3. Then, we examined DJ-1 expression and apoptosis in HT22 challenged with TgCtwh3. Finally, we evaluated the apoptosis in HT22 with DJ-1 knockdown which was infected with TgCtwh3 and assayed the expression of NF-κBp65 and p-NF-κBp65. Our results showed that DJ-1 expression was reduced and neurons underwent apoptosis in the hippocampus of mice infected with TgCtwh3 tachyzoites. Additionally, the knockdown of DJ-1 followed by infection with TgCtwh3 tachyzoites led to increased apoptosis in HT22 cells through the NF-κB signaling pathway. Therefore, this study suggests that DJ-1 is an important target for preventing apoptosis caused by T. gondii TgCtwh3.
RESUMEN
Vertical transmission of the intracellular parasite, Toxoplasma gondii can lead to adverse pregnancy outcomes especially when infection occurs in early pregnancy. Decidual natural killer (dNK) cells accumulate at the maternal-fetal interface in large numbers during early pregnancy. Their nutritional roles during infection with T. gondii remain poorly defined. In the present study, we demonstrated that a functional deficiency of the uterine tissue-resident NK (trNK) cells, a subset of dNK cells, contributes to the adverse pregnancy outcomes induced by T. gondii in early pregnancy. Adverse pregnancy outcomes could be ameliorated by adoptive transfer of trNK cells. Moreover, fetal growth restriction could be improved after supplementation of growth-promoting factors. In addition to the widely recognized disturbance of the immune balance at the interface between the mother and the fetus, our study reveals a novel mechanism in T. gondii that contributes to the adverse pregnancy outcomes.
Asunto(s)
Toxoplasma , Toxoplasmosis , Embarazo , Femenino , Humanos , Resultado del Embarazo , Toxoplasmosis/parasitología , Decidua/parasitología , Células Asesinas Naturales , Péptidos y Proteínas de Señalización IntercelularRESUMEN
Persistent chronic inflammation of the lungs and airway remodeling are important pathological features that cannot be ignored in patients with chronic asthma. Apigenin (API) is a natural small molecule compound with good anti-inflammatory and antioxidant activity that has been widely reported in recent years, but its role in chronic asthma is not well defined. Our study began with oral gavage intervention using API (10, 20 mg/kg) or dexamethasone (DEX, 2 mg/kg) in a BALB/c mouse model of ovalbumin (OVA) sensitization. Different doses of API intervention effectively reduced airway resistance in the administered group. Additionally, inflammation was downregulated, mucus secretion was reduced, and airway remodeling was inhibited in the API intervention group compared with the model group. Asthma-related inflammatory cytokines, such as IgE, IL-4, IL-5, IL-13, and IL-17, were downregulated in alveolar lavage fluid. Moreover, the apoptosis level of the administered group was found to be lower than that of the model group in the Tunel staining experiment. By analyzing transcriptome sequencing results, we found that API may exert anti-inflammatory and anti-apoptotic effects by inhibiting the MAPK pathway. Our subsequent results supported this conclusion, showing that the phosphorylation levels of ERKs, JNKs, and p38 MAPKs were inhibited in the administered group relative to the model group. Downstream expression of the apoptosis-related protein B-cell lymphoma-2 (Bcl-2) was upregulated, and the expression of Bcl-2-associated × protein (Bax) and cleaved caspase-3 was downregulated. To further investigate the specific mechanism by which API acted, we established an in vitro model with house dust mite (HDM) stimulation, using API (10, 20 µM) for administration intervention. The results showed that API was able to improve cell viability, inhibit ROS production, and reverse HDM-induced decreases in mitochondrial membrane potential (MMP) and apoptosis in airway epithelial cells via the MAPK pathway.
Asunto(s)
Apigenina , Asma , Animales , Ratones , Apigenina/farmacología , Remodelación de las Vías Aéreas (Respiratorias) , Transcriptoma , Asma/tratamiento farmacológico , Inflamación/metabolismo , Antiinflamatorios/farmacología , Apoptosis , Células Epiteliales/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Cellular senescence, a characteristic sign of aging, classically refers to permanent cell proliferation arrest and is a vital contributor to the pathogenesis of cancer and age-related illnesses. A lot of imperative scientific research has shown that senescent cell aggregation and the release of senescence-associated secretory phenotype (SASP) components can cause lung inflammatory diseases as well. In this study, the most recent scientific progress on cellular senescence and phenotypes was reviewed, including their impact on lung inflammation and the contributions of these findings to understanding the underlying mechanisms and clinical relevance of cell and developmental biology. Within a dozen pro-senescent stimuli, the irreparable DNA damage, oxidative stress, and telomere erosion are all crucial in the long-term accumulation of senescent cells, resulting in sustained inflammatory stress activation in the respiratory system. An emerging role for cellular senescence in inflammatory lung diseases was proposed in this review, followed by the identification of the main ambiguities, thus further understanding this event and the potential to control cellular senescence and pro-inflammatory response activation. In addition, novel therapeutic strategies for the modulation of cellular senescence that might help to attenuate inflammatory lung conditions and improve disease outcomes were also presented in this research.
Asunto(s)
Enfermedades Pulmonares , Neumonía , Humanos , Senescencia Celular , Enfermedades Pulmonares/patología , Pulmón/patologíaRESUMEN
BACKGROUND: Obese asthma is one of the important asthma phenotypes that have received wide attention in recent years. Excessive oxidative stress and different inflammatory endotypes may be important reasons for the complex symptoms, frequent aggravation, and resistance to traditional treatments of obese asthma. Apigenin (API), is a flavonoid natural small molecule compound with good anti-inflammatory and antioxidant activity in various diseases and proved to have the potential efficacy to combat obese asthma. METHODS: In vivo, this study fed C57BL/6 J mice with high-fat diets(HFD)for 12 weeks and then stimulated them with OVA for 6 weeks to establish a model of chronic obese asthma, while different doses of oral API or dexamethasone were used for therapeutic interventions. In vitro, this study used HDM to stimulate human bronchial cells (HBEs) to establish the model and intervened with API or Selonsertib (SEL). RESULTS: This study clarified that OVAinduced a type of mixed granulocytic asthma with elevated neutrophils and eosinophils in obese male mice fed with long-term HFD, which also exhibited mixed TH17/TH1/TH2 inflammation. Apigenin effectively suppressed this complex inflammation and acted as a regulator of immune homeostasis. Meanwhile, apigenin reduced AHR, inflammatory cell infiltration, airway epithelial cell apoptosis, airway collagen deposition, and lung oxidative stress via the ROS-ASK1-MAPK pathway in an obese asthma mouse model. In vitro, this study found that apigenin altered the binding status of TRAF6 to ASK1, inhibited ASK1 phosphorylation, and protected against ubiquitin-dependent degradation of ASK1, suggesting that ROS-activated ASK1 may be an important target for apigenin to exert anti-inflammatory and anti-apoptotic effects. To further verify the intervention mechanism, this study clarified that apigenin improved cell viability and mitochondrial function and inhibited apoptosis by interfering with the ROS-ASK1-MAPK pathway. CONCLUSIONS: This study demonstrates for the first time the therapeutic effect of apigenin in chronic obese asthma and further clarifies its potential therapeutic targets. In addition, this study clarifies the specificity of chronic obese asthma and provides new options for its treatment.
Asunto(s)
Apigenina , Asma , Animales , Humanos , Masculino , Ratones , Apigenina/farmacología , Apoptosis , Asma/metabolismo , Células Epiteliales/metabolismo , Homeostasis , Inflamación/metabolismo , Pulmón , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Especies Reactivas de Oxígeno/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Alzheimer's disease presents an abnormal cognitive behavior. TgCtwh6 is one of the predominant T. gondii strains prevalent in China. Although T. gondii type II strain infection can cause host cognitive behavioral abnormalities, we do not know whether TgCtwh6 could also cause host cognitive behavioral changes. So, in this study, we will focus on the effect of TgCtwh6 on mouse cognitive behavior and try in vivo and in vitro to explore the underlying mechanism by which TgCtwh6 give rise to mice cognitive behavior changes at the cellular and molecular level. METHODS: C57BL/6 mice were infected orally with TgCtwh6 cysts. From day 90 post-infection on, all mice were conducted through the open field test and then Morris water maze test to evaluate cognitive behavior. The morphology and number of cells in hippocampus were examined with hematoxylin-eosin (H&E) and Nissl staining; moreover, Aß protein in hippocampus was determined with immunohistochemistry and thioflavin S plaque staining. Synaptotagmin 1, apoptosis-related proteins, BACE1 and APP proteins and genes from hippocampus were assessed by western blotting or qRT-PCR. Hippocampal neuronal cell line or mouse microglial cell line was challenged with TgCtwh6 tachyzoites and then separately cultured in a well or co-cultured in a transwell device. The target proteins and genes were analyzed by immunofluorescence staining, western blotting and qRT-PCR. In addition, mouse microglial cell line polarization state and hippocampal neuronal cell line apoptosis were estimated using flow cytometry assay. RESULTS: The OFT and MWMT indicated that infected mice had cognitive behavioral impairments. The hippocampal tissue assay showed abnormal neuron morphology and a decreased number in infected mice. Moreover, pro-apoptotic proteins, as well as BACE1, APP and Aß proteins, increased in the infected mouse hippocampus. The experiments in vitro showed that pro-apoptotic proteins and p-NF-κBp65, NF-κBp65, BACE1, APP and Aß proteins or genes were significantly increased in the infected HT22. In addition, CD80, pro-inflammatory factors, notch, hes1 proteins and genes were enhanced in the infected BV2. Interestingly, not only the APP and pro-apoptotic proteins in HT22, but also the apoptosis rate of HT22 increased after the infected BV2 were co-cultured with the HT22 in a transwell device. CONCLUSIONS: Neuron apoptosis, Aß deposition and neuroinflammatory response involved with microglia polarization are the molecular and cellular mechanisms by which TgCtwh6 causes mouse cognitive behavioral abnormalities.
Asunto(s)
Cognición , Toxoplasma , Animales , Ratones , Secretasas de la Proteína Precursora del Amiloide/genética , Proteínas Reguladoras de la Apoptosis/genética , Ácido Aspártico Endopeptidasas/genética , Modelos Animales de Enfermedad , Genotipo , Ratones Endogámicos C57BL , Toxoplasma/genéticaRESUMEN
CONTEXT: Asthma is a common respiratory system disease. Louki Zupa decoction (LKZP), a traditional Chinese medicine, presents a promising efficacy against lung diseases. OBJECTIVE: To investigate the pathogenic mechanism of asthma and reveal the intervention mechanism of LKZP. MATERIALS AND METHODS: Forty-eight female Balb/c mice were randomly divided into 6 groups: normal control group (NC), ovalbumin (OVA)/saline asthma model group, OVA/LL group, OVA/LM group, OVA/LH group and OVA/DEX group (n = 8 per group). The asthmatic mice were modelled through intraperitoneal injecting and neutralizing OVA. LKZP decoction was administrated by gavage at the challenge stage for seven consecutive days (2.1, 4.2 and 8.4 g/kg/day). We investigated the change in lung function, airway inflammation, mucus secretion and TH-1/TH-2-related cytokines. We further verify the activated status of the IL-33/ST2/NF-κB/GSK3ß/mTOR signalling pathway. RESULTS: LKZP was proved to improve asthmatic symptoms, as evidenced by the down-regulated airway resistance by 36%, 58% and 53% (p < 0.01, p < 0.001 vs. OVA/saline group), up-regulated lung compliance by 102%, 114% and 111%, decreased airway inflammation and mucus secretion by 33%, 40% and 33% (p < 0.001 vs. OVA/saline group). Moreover, the content of cytokines in BALF related to airway allergy (such as IgE) and T helper 1/T helper 2 cells (like IL-2, IL-4, IL-5, IL-13, TNF-α and IFN-γ), were also markedly reduced by 13-65% on LKZP intervention groups compared with model group. Mechanistic research revealed that the IL-33/ST2-NF-κB/GSK3ß/mTOR signalling pathway was activated in the OVA/saline group and LKZP significantly down-regulated this pathway. DISCUSSION AND CONCLUSION: LKZP improves lung function, airway inflammation, mucus secretion and correct immune imbalance by intervening with the IL-33/ST2-NF-κB/GSK3ß/mTOR signalling pathway, presenting a promising therapeutic choice for asthma.
Asunto(s)
Asma , FN-kappa B , Animales , Líquido del Lavado Bronquioalveolar , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Inflamación/patología , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Ovalbúmina , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Toxoplasma gondii is a neurotropic single-celled parasite that can infect mammals, including humans. Central nervous system infection with T. gondii infection can lead to Toxoplasma encephalitis. Toxoplasma infection can cause endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, which ultimately can lead to apoptosis of host cells. The dense granule protein GRA3 has been identified as one of the secretory proteins that contribute to the virulence of T. gondii; however, the mechanism remains enigmatic. METHODS: The expression of the GRA3 gene in RH, ME49, Wh3, and Wh6 strains was determined using quantitative real-time polymerase chain reaction (qRT-PCR). pEGFP-GRA3Wh6 was constructed by inserting Chinese 1 Wh6 GRA3 (GRA3Wh6) cDNA into a plasmid encoding the enhanced GFP. Mouse neuro2a (N2a) cells were transfected with either pEGFP or pEGFP-GRA3Wh6 (GRA3Wh6) and incubated for 24-36 h. N2a cell apoptosis and ER stress-associated proteins were determined using flow cytometry and immunoblotting. Furthermore, N2a cells were pretreated with GSK2656157 (a PERK inhibitor) and Z-ATAD-FMK (a caspase-12 inhibitor) before GRA3Wh6 transfection, and the effect of the inhibitors on GRA3Wh6-induced ER stress and apoptosis were investigated. RESULTS: GRA3 gene expression was higher in the less virulent strains of type II ME49 and type Chinese 1 Wh6 strains compared with the virulent strains of type I RH strain and type Chinese 1 Wh3 strain. Transfection with GRA3Wh6 plasmid induced neuronal apoptosis and increased the expression of GRP78, p-PERK, cleaved caspase-12, cleaved caspase-3, and CHOP compared with the control vector. Pretreatment with GSK2656157 and Z-ATAD-FMK decreased apoptosis in N2a cells, and similarly, ER stress- and apoptosis-associated protein levels were significantly decreased. CONCLUSION: GRA3 induces neural cell apoptosis via the ER stress signaling pathway, which could play a role in toxoplasmic encephalitis.
Asunto(s)
Encefalitis , Toxoplasma , Animales , Apoptosis , Caspasa 12/genética , Estrés del Retículo Endoplásmico , Humanos , Mamíferos , RatonesRESUMEN
BACKGROUND: Despite the substantial amount of efforts made to reduce morbidity and improve respiratory management, asthma control remained a major challenge for severe patients. Plant isoflavones, one of the most estrogenic compounds, are considered a potential alternative therapy for asthma. Iristectorigenin A, a naturally occurring isoflavone, is extracted from a variety of medical plants and its biological activity has not been reported previously. PURPOSE: In present study, we aim to reveal the potential therapeutic role of Iristectorigenin A against acute asthmatic mice. STUDY DESIGN: We established ovalbumin (OVA) induced asthmatic murine model and orally administrated Iristectorigenin A at concentration of 5 and 10 mg/kg and dexamethasone as a positive control substance. METHODS: Asthmatic murine model was established with OVA sensitization and challenge. Lung function was assessed with FinePoint Ventilation system recording lung resistance (RI) and lung compliance (Cydn). White cells were sorted and counted in BALF. Histopathological assessment was conducted by H&E, PAS, and Masson's trichrome staining on paraffin embedded lung tissues. BALF content of IL-4, IL-5, IL-33, IL-13, INF-γ, IL-9 and serum IgE, IgG1 were measured using ELISA kit. Expression levels of mRNAs associated with inflammatory cytokines and goblet cell metaplasia were evaluated via quantitative RT-PCR. Protein expression levels of FOXA3, MUC5AC, SPDEF were estimated by immunohistochemistry on lung tissue, while NOTCH1 and NOTCH2 expressions were evaluated by western blotting analysis. RESULTS: Iristectorigenin A resulted in improved airway hyperresponsiveness (AHR) mirrored by decreased RI and increased Cydn. With Iristectorigenin A, we also observed reduced number of BALF leukocytes, improved inflammatory cell infiltration in lung tissue, decreased content of BALF IL-4, IL-5, IL-33, but not IL-13, INF-γ, IL-9, and their mRNA levels, along with decreased levels of OVA-specific IgE, IgG1 in asthmatic mice. Additionally, Iristectorigenin A exhibited significant therapeutic potential on attenuating mucus production reflected by mitigated FOXA3 and MUC5AC immunostaining on the airway epithelium, as well as decreased mRNAs associated with goblet cell metaplasia. At last, a decrease in elevated expression level of NOTCH2, but not NOTCH1, in asthmatic mice lung tissue was observed by western blotting analysis. CONCLUSION: Our study provides strong evidence that Iristectorigenin A can be potential therapeutic agent ameliorating airway inflammation and mucus hypersecretion in allergic asthma. This is a first research reported the potential of Iristectorigenin A as an alternative therapeutic agent.
Asunto(s)
Asma , Interleucina-33 , Animales , Asma/tratamiento farmacológico , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Inmunoglobulina E , Inmunoglobulina G , Inflamación/tratamiento farmacológico , Interleucina-33/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Interleucina-9/metabolismo , Interleucina-9/uso terapéutico , Pulmón/patología , Metaplasia/metabolismo , Metaplasia/patología , Ratones , Ratones Endogámicos BALB C , Moco , Ovalbúmina , FenotipoRESUMEN
Interferon-γ (IFN-γ)-activated macrophages restrain the replication of intracellular parasites and disrupt the integrity of vacuolar pathogens. The growth of the less virulent type II strain of Toxoplasma gondii (such as ME49) was strongly inhibited by IFN-γ-activated murine macrophages. However, the mechanism of resistance is poorly understood. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Previous studies showed the cassette of autophagy-related proteins including Atg7, Atg3, and Atg12-Atg5-Atg16L1 complex, plays crucial roles in the proper targeting of IFN-γ effectors onto the parasitophorous vacuole (PV) membrane of Toxoplasma gondii and subsequent control of parasites. TgCDPK3 is a calcium dependent protein kinase, located on the parasite periphery, plays a crucial role in parasite egress. Herein, we show that the less virulent strain CDPK3 (ME49, type II) can enhance autophagy activation and interacts with host autophagy proteins Atg3 and Atg5. Infection with CDPK3-deficient ME49 strain resulted in decreased localization of IRGs and GBPs around PV membrane. In vitro proliferation and plaque assays showed that CDPK3-deficient ME49 strain replicated significantly more quickly than wild-type parasites. These data suggested that TgCDPK3 interacts with the host Atg3 and Atg5 to promote the localization of IRGs and GBPs around PV membrane and inhibits the intracellular proliferation of parasites, which is beneficial to the less virulent strain of Toxoplasma gondii long-term latency in host cells.
Asunto(s)
Parásitos , Toxoplasma , Animales , Proteínas Portadoras , Proliferación Celular , GTP Fosfohidrolasas/metabolismo , Interferón gamma/metabolismo , Macrófagos/metabolismo , Ratones , Parásitos/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Loki zupa (LKZP) decoction, a traditional Uyghur medicine prescription, has been commonly used to treat numerous respiratory ailments in the Xinjiang region of western China, especially chronic airway inflammatory diseases such as allergic asthma. Due to its complex chemical composition, however, the mechanism of action of LKZP has yet to be fully elucidated. AIM OF THE STUDY: Based on the balanced regulation theory of pro-inflammation and anti-inflammation, we tried to investigate the effectiveness of LKZP on asthma and its related protective mechanisms. MATERIALS AND METHODS: In this study, an experimental model of asthma was established using ovalbumin (OVA) in BALB/c mice to assess the effects of LKZP. The potential mechanism of LKZP anti allergic asthma were researched by the combination of in silico systems pharmacology and in vivo transcriptomics. RESULTS: Our data revealed that LKZP exerted a therapeutic effect against OVA-induced asthma by reducing airway hyperresponsiveness (AHR), peribronchial inflammation, and mucus hypersecretion. Meanwhile, LKZP downregulated the expression of OVA-induced IgE, interleukin (IL)-4, IL-5, IL-13, and tumor necrosis factor (TNF)-α and concurrently promoted the expression of interferon (IFN)-γ in serum and bronchoalveolar lavage fluid (BALF). Systems pharmacology analysis identified 10 core bioactive ingredients and 26 hub targets of LKZP against asthma. Transcriptomic analysis confirmed 246 differentially expressed genes (DEGs) after LKZP treatment. These were mainly expressed in cytokine-cytokine receptor interactions and immune and inflammatory response-related signaling pathways. Additionally, the real-time quantitative PCR (qPCR) results for the nine selected DEGs matched those of the RNA-seq analysis. Nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)-1 signaling pathways were identified as candidate targets involved in the action of LKZP on allergic asthma, which was highly consistent with the findings in silico. By qPCR, Western blot, and immunohistochemical analysis, it was verified that LKZP treatment dramatically inhibited the activation of NF-κB p65 and HIF-1α stimulated by OVA in asthmatic mice. CONCLUSIONS: Taken together, our experimental data revealed that LKZP could be a candidate for the treatment of allergic asthma via NF-κB and HIF-1 signaling pathways.
Asunto(s)
Asma , Transcriptoma , Animales , Asma/inducido químicamente , Asma/tratamiento farmacológico , Asma/genética , Líquido del Lavado Bronquioalveolar , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Pulmón , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Farmacología en Red , Ovalbúmina , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: The predominant genotype of Toxoplasma in China is the Chinese 1 (ToxoDB#9) lineage. TgCtwh3 and TgCtwh6 are two representative strains of Chinese 1, exhibiting high and low virulence to mice, respectively. Little is known regarding the virulence mechanism of this non-classical genotype. Our previous RNA sequencing data revealed differential mRNA levels of TgMIC1 in TgCtwh3 and TgCtwh6. We aim to further confirm the differential expression of TgMIC1 and its significance in this atypical genotype. METHODS: Quantitative real-time PCR was used to verify the RNA sequencing data; then, polyclonal antibodies against TgMIC1 were prepared and identified. Moreover, the invasion and proliferation of the parasite in HFF cells were observed after treatment with TgMIC1 polyclonal antibody or not. RESULTS: The data showed that the protein level of TgMIC1 was significantly higher in high-virulence strain TgCtwh3 than in low-virulence strain TgCtwh6 and that the invasion and proliferation of TgCtwh3 were inhibited by TgMIC1 polyclonal antibody. CONCLUSION: Differential expression of TgMIC1 in TgCtwh3 and TgCtwh6 may explain, at least partly, the virulence mechanism of this atypical genotype.
Asunto(s)
Moléculas de Adhesión Celular/genética , Genotipo , Proteínas Protozoarias/genética , Toxoplasma/genética , Toxoplasma/patogenicidad , Animales , China , Fibroblastos/parasitología , Humanos , Ratones , Toxoplasma/clasificación , Toxoplasma/aislamiento & purificación , VirulenciaRESUMEN
Toxoplasma gondii (T. gondii) is a neurophilic and intracellular parasite that can affect plenty of vertebrate animals, including humans. Recent researches indicate that T. gondii infection is associated with neurodegenerative diseases such as Alzheimer's disease(AD). In addition, tau hyper-phosphorylation is a crucial event leading to the formation of nerve fiber tangles in AD. Despite the efforts to understand the interactions between T. gondii and AD, there are no clear results available so far. Here, we infected mice with the T. gondii of the Chinese 1 genotype Wh6 strain (TgCtwh6) for 60 days. Then we observed the formation of tissue cysts in the brain, the damage of neuron and the increased expression of phosphorylated tau (p-tau) in the hippocampal tissue of the mice. Similarly, we also found that p-tau, glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated GSK3ß (p-GSK3ß) were upregulated in vitro in TgCtwh6 challenged hippocampal neuron cell strain, HT22 cells. We noted a down-regulated expression of GSK3ß,p-GSK3ß, and p-tau in HT22 cells, which were pretreated with LiCl, an inhibitor of GSK3ß. These data suggested that p-GSK3ß may mediate tau phosphorylation after TgCtwh6 infection. Furthermore, TgCtwh6 infection also caused the increased expression of Bax and Caspase3, the decreased expression of Bcl-XL in HT22 cells, which had both early and late apoptosis. In all, our results indicated that TgCtwh6 infection not only led to phosphorylation of tau via activating GSK3ß but also promoted hippocampal neuron apoptosis. Our research may partially reveal the mechanism with which TgCtwh6 induce neurofibrillary pathology.
Asunto(s)
Apoptosis , Glucógeno Sintasa Quinasa 3 beta/fisiología , Hipocampo/patología , Toxoplasma/clasificación , Toxoplasmosis Animal/metabolismo , Proteínas tau/metabolismo , Animales , Células Cultivadas , Genotipo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/patología , Fosforilación , Toxoplasma/genética , Toxoplasmosis Animal/patologíaAsunto(s)
Reprogramación Celular , Hepatopatías/patología , Hepatopatías/parasitología , Macrófagos/patología , Péptidos/farmacología , Toxoplasma/metabolismo , Animales , Reprogramación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/metabolismo , Toxoplasma/efectos de los fármacosRESUMEN
The rhoptry kinase 18 of Toxoplasma gondii (TgROP18) has been identified as a key virulence factor that allows the parasite to escape from host immune defences and promotes its proliferation in host cells. Although much research is focused on the interaction between host cells and TgROP18, the development of monoclonal antibodies (mAbs) against TgROP18 has not been reported till date. To produce mAbs targeting TgROP18, two hybridomas secreting mAbs against TgROP18, designated as A1 and T2, were generated using cell fusion technology. The subtypes of the A1 and T2 mAbs were identified as IgG3 λ and IgM κ, and peptide scanning revealed that the core sequences of the antigenic epitopes were 180LRAQRRRSELVFE192 and 351NYFLLMMRAEADM363, respectively. The T2 mAb specifically reacted with both T. gondii type I and Chinese I, but not with T. gondii type II, Plasmodium falciparum or Schistosoma japonicum. Finally, the sequences of heavy chain and light chain complementarity-determining regions of T2 were amplified, cloned and characterized, making the modification of the mAb feasible in the future. The development of mAbs against TgROP18 would aid the investigation of the molecular mechanisms underlying the modulation of host cellular functions by TgROP18, and in the development of strategies to diagnose and treat Toxoplasmosis.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Especificidad de la EspecieRESUMEN
Infection with helminth parasites or the administration of their antigens can prevent or attenuate autoimmune diseases. To date, the specific molecules that prime the amelioration are only limited. In this study, recombinant Schistosoma japonicum cystatin (rSjcystatin) and fructose-1,6-bisphosphate aldolase (rSjFBPA) were administered to female NOD mice via intraperitoneal (i.p.) injection to characterize the immunological response by the recombinant proteins. We have shown that the administration of rSjcystatin or rSjFBPA significantly reduced the diabetes incidence and ameliorated the severity of type 1 diabetes mellitus (T1DM). Disease attenuation was associated with suppressed interferon-gamma (IFN-γ) production in autoreactive T cells and with a switch to the production of Th2 cytokines. Following rSjcystatin or rSjFBPA injection, regulatory T cells (Tregs) were remarkably increased, which was accompanied by increased expression of interleukin-10 (IL-10) and transforming growth factor beta (TGF-ß). Our study suggests that helminth-derived proteins may be useful in strategies to limit pathology by promoting the Th2 response and upregulating Tregs during the inflammatory tissue-damage process in T1DM.
Asunto(s)
Cistatinas/administración & dosificación , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Fructosa-Bifosfato Aldolasa/administración & dosificación , Proteínas del Helminto/administración & dosificación , Factores Inmunológicos/administración & dosificación , Schistosoma japonicum/enzimología , Animales , Cistatinas/genética , Cistatinas/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Femenino , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Factores Inmunológicos/genética , Factores Inmunológicos/metabolismo , Ratones , Ratones Endogámicos NOD , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfocitos T Reguladores/inmunologíaRESUMEN
BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn's disease. AIM: To explore the beneficial effect of ToxoROP16I/III-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells. METHODS: RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) (M1 cells) were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro. The expression of ToxoROP16I/III was observed in RAW264.7 macrophages that were transfected with pEGFP-rop16 I/III. The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, transforming growth factor (TGF)-ß1, IL-10, inducible nitric oxide synthase (iNOS), and arginase-1 (Arg-1) was detected. The expression of iNOS, Arg-1, signal transducer and activator of transcription 3 (Stat3), p-Stat3, Stat6, p-Stat6, programmed death ligand-2 (PD-L2), caspase-3, -8, and -9 was analyzed by Western blotting, and Griess assays were performed to detect nitric oxide (NO). TNF-α, IL-1ß, IL-6, TGF-ß1, and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay, and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system. RESULTS: M1 cells exhibited significantly increased production of iNOS, NO, TNF-α, IL-1ß, and IL-6, while ToxoROP16I/III induced macrophage bias to M2 cells in vitro, showing increased expression of Arg-1, IL-10 and TGF-ß1 and elevated production of p-Stat3 and p-Stat6. The mixed M1 and M2 cell culture induced by ToxoROP16I/III exhibited decreased production of NO and iNOS and upregulated expression of Arg-1 and PD-L2. Accordingly, Caco-2 cells became apoptotic, and apoptosis-associated proteins such as caspase-3, -8 and -9 were dampened during co-culture of M1 and M2 cells. Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells, but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis. CONCLUSION: ToxoROP16I/III-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages. This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.
Asunto(s)
Enfermedades Inflamatorias del Intestino/fisiopatología , Enfermedades Inflamatorias del Intestino/terapia , Mucosa Intestinal/fisiopatología , Macrófagos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Animales , Apoptosis , Células CACO-2 , Técnicas de Cocultivo , Citocinas/metabolismo , Regulación hacia Abajo , Homeostasis , Humanos , Inmunoterapia , Inflamación , Lentivirus , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Ratones , Fenotipo , Células RAW 264.7RESUMEN
Bu-Shen-Yi-Qi formula (BSYQF) could suppress chronic airway inflammation according to previous studies. However, there is relatively little direct experimental evidence to evaluate the effects of BSYQF treatment on airway remodeling in chronic asthma. Recent evidence suggests that oxidative stress is involved in airway inflammation and airway remodeling in chronic asthma. BSYQF which includes various of chemical components having antioxidant effects, could be beneficial in attenuating airway remodeling in chronic asthma. The purpose of this study was to elucidate the effect of BSYQF treatment on airway remodeling and investigate its potential mechanisms in chronic asthma. To develop the murine models of chronic asthma, BALB/c mice were sensitized and challenged to ovalbumin for 8 weeks. BSYQF (5, 10, 20 g raw herbs/kg body weight) or tiotropium bromide (0.1 mM) were administered orally and intranasal instillation, respectively. The effect of BSYQF on pulmonary inflammation and remodeling was evaluated. The parameters of oxidative stress in the lung were analyzed. BSYQF treatment reduced airway hyperresponsiveness (AHR), Th2 response including IL-4, IL-13, and OVA-specific IgE and IgG1, transforming growth factor-ß (TGF-ß), vascular endothelium growth factor (VEGF), airway inflammation and airway remodeling including smooth muscle thickening and peribronchial collagen deposition. As for oxidative stress, BSYQF treatment reduced reactive oxygen species (ROS), Malondialdehyde (MDA), NO, and the expression of inducible nitric oxide synthase (iNOS), but increased significantly glutathione (GSH) /Oxidized glutathione(GSSH) ratios in the lung, restored mitochondrial ultrastructural changes of bronchial epithelia and ATP levels in the lung. In summary, this study suggested that BSYQF treatment ameliorated airway remodeling and alleviated asthmatic features in chronic asthma models. Anti-inflammatory and antioxidant effect of BSYQF may explain why BSYQF has effects on preventing airway remodeling.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Antiasmáticos/uso terapéutico , Asma/metabolismo , Medicamentos Herbarios Chinos/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Neumonía/metabolismo , Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Animales , Antiasmáticos/química , Antiasmáticos/farmacología , Asma/inducido químicamente , Asma/tratamiento farmacológico , Composición de Medicamentos , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Femenino , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo/fisiología , Neumonía/inducido químicamente , Neumonía/tratamiento farmacológico , Distribución AleatoriaRESUMEN
Icariin is a main component of the Chinese medicinal plant Epimedium brevicornu Maxim, exhibits potent activity against inflammatory diseases. Our previous data demonstrated the valid bioactivity of icariin on mitigating rodent asthma. Endoplasmic reticulum (ER) stress and nuclear factor-κB (NF-κB) pathway were involved in the pathogenesis of asthma. However, it remains poorly defined that whether icariin could inhibit ER stress and NF-κB mediated apoptosis in asthma and further influence the central neural system. Herein, we investigated the effects of icariin on primary cultured fetal rat hippocampal neurons and OVALPS-OVA induced asthma rat model. Asthma rat models were established by ovalbumin (OVA) and lipopolysaccharide (LPS) intraperitoneal injection and OVA inhalational challenge. Airway resistance was analyzed to evaluate lung function after last challenge and pathological changes were detected on lung tissues. Assessment of inflammatory cells counts in bronchoalveolar lavage fluids (BALF) were performed and ELISA was used to determine levels of interleukin (IL)-1ß, tumor necrosis factor-α, IL-6, and interferon-γ in serum. Protein expression of BiP and IRE-1α, XBP-1s and phosphorylation-IκBα (p-IκBα), IκBα, and p65 as well as cytochrome c, caspase-3 (cleaved caspase-3), and caspase-9 (cleaved caspase-9) were tested by Western blot. We found that icariin could remarkably improve pulmonary function and reduce inflammatory cells in the lung, levels of inflammatory cytokines, and ER stress related proteins as well as NF-κB were prominently suppressed by icariin. Our results suggested that icariin had an inhibitory effect on airway inflammation and neuroprotective effect on ER stress and NF-κB mediated apoptosis in asthma rats and cultured fetal rat hippocampal neurons, which may provide new mechanistic insights into the asthma prevention and treatment of icariin.