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1.
Cell Physiol Biochem ; 57(1): 1-14, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36695077

RESUMO

BACKGROUND/AIMS: The ribosome-inactivating proteins include the biothreat agent, ricin toxin (RT). When inhaled, RT causes near complete destruction of the lung epithelium coincident with a proinflammatory response that includes TNF family cytokines, which are death-inducing ligands. We previously demonstrated that the combination of RT and TNF-related apoptosis inducing ligand (TRAIL) induces caspase-dependent apoptosis, while RT and TNF-α or RT and Fas ligand (FasL) induces cathepsin-dependent cell death in lung epithelial cells. We hypothesize that airway macrophages constitute a major source of cytokines that drive lung epithelial cell death. METHODS: Here, we show that RT-induced apoptosis of the monocytic cell line, U937, leads to the bystander killing of the lung epithelial cell line, A549. U937 cells were treated with ricin. Following this, A549 cells were treated with supernatants from U937 cells and death was measured by WST-1 viability assay. RESULTS: Upon RT-induced U937 cell death, released RT and FasL contributed to A549 cell death. U937 cells also released nuclear protein HMGB1. The release of RT, FasL, and HMGB1 triggered A549 cell necroptosis, rather than cathepsin-dependent killing observed previously with RT and FasL. Reactive oxygen species (ROS) were produced in A549 cells due to HMGB1 ligation of the receptor for advanced glycation end products (RAGE). CONCLUSION: These findings demonstrate the potential for bystander necroptosis of lung epithelial cells during RT toxicosis which may perpetuate or increase the proinflammatory response.


Assuntos
Proteína HMGB1 , Ricina , Humanos , Ricina/toxicidade , Células U937 , Necroptose , Apoptose , Pulmão/metabolismo , Células Epiteliais/metabolismo , Proteína Ligante Fas , Citocinas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Catepsinas , Inflamação , Receptor fas
2.
Allergol Immunopathol (Madr) ; 51(1): 54-62, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36617822

RESUMO

Acute lung injury causes severe inflammation and oxidative stress in lung tissues. In this study, we analyzed the potential regulatory role of nuclear factor erythroid-2-related factor 2 (Nrf2) on NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced inflammation and oxidative stress in human type II alveolar epithelial cells. In this study, A549 cells were transfected with Nrf2 siRNA and overexpression vectors for 6 h before being induced by TNF-α for 24 h. TNF-α upregulated the expression of NOX1 and Nrf2 in A549 cells. Furthermore, overexpression of Nrf2 could reduce TNF-α-induced NF-κB mRNA and protein expression after transfection with the Nrf2 siRNA vector, and the levels of IL-6, IL-8, ROS, and malondialdehyde (MDA) in TNF-α-induced A549 cells increased, while the level of total antioxidation capability (T-AOC) decreased. On the other hand, the overexpression of Nrf2 decreased the levels of IL-6, IL-8, ROS, and MDA, while increasing T-AOC. The mRNA and protein levels of NOX1 were dramatically increased by TNF-α, while those changes were notably suppressed by Nrf2 overexpression. Further studies demonstrated that Nrf2 suppressed NOX1 transcription by binding to the -1199 to -1189 bp (ATTACACAGCA) region of the NOX1 promoter in TNF-α-stimulated A549 cells. Our study suggests that Nrf2 may bind to and regulate NOX1 expression to antagonize TNF-α-induced inflammatory reaction and oxidative stress in A549 cells.


Assuntos
NADPH Oxidase 1 , Fator 2 Relacionado a NF-E2 , Fator de Necrose Tumoral alfa , Humanos , Células A549 , Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , NADPH Oxidase 1/genética , NADPH Oxidase 1/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , RNA Mensageiro , RNA Interferente Pequeno/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
3.
Int J Mol Sci ; 24(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36675019

RESUMO

Erinacine A (EA), a natural neuroprotectant, is isolated from a Chinese herbal medicine, Hericium erinaceus. The aim of this study was to investigate the neuroprotective effects of EA in a rat model of traumatic optic neuropathy. The optic nerves (ONs) of adult male Wistar rats were crushed using a standardized method and divided into three experimental groups: phosphate-buffered saline (PBS control)-treated group, standard EA dose-treated group (2.64 mg/kg in 0.5 mL of PBS), and double EA dose-treated group (5.28 mg/kg in 0.5 mL of PBS). After ON crush, each group was fed orally every day for 14 days before being euthanized. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined using flash visual-evoked potentials (fVEP) analysis, retrograde Fluoro-Gold labelling, and TdT-dUTP nick end-labelling (TUNEL) assay, respectively. Macrophage infiltration of ON was detected by immunostaining (immunohistochemistry) for ED1. The protein levels of phosphor-receptor-interacting serine/threonine-protein kinase1 (pRIP1), caspase 8 (Cas8), cleaved caspase 3 (cCas3), tumour necrosis factor (TNF)-α, tumour necrosis factor receptor1 (TNFR1), interleukin (IL)-1ß, inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1), and superoxide dismutase 1 (SOD1) were evaluated by Western blotting. When comparing the standard EA dose-treated group and the double EA dose-treated group with the PBS-treated group, fVEP analysis showed that the amplitudes of P1-N2 in the standard EA dose group and the double EA dose-treated group were 1.8 and 2.4-fold, respectively, higher than that in the PBS-treated group (p < 0.05). The density of RGC in the standard EA dose-treated group and the double EA dose-treated group were 2.3 and 3.7-fold, respectively, higher than that in the PBS-treated group (p < 0.05). The TUNEL assay showed that the standard EA dose-treated group and the double EA dose-treated group had significantly reduced numbers of apoptotic RGC by 10.0 and 15.6-fold, respectively, compared with the PBS-treated group (p < 0.05). The numbers of macrophages on ON were reduced by 1.8 and 2.2-fold in the standard EA dose-treated group and the double EA dose-treated group, respectively (p < 0.01). On the retinal samples, the levels of pRIP, Cas8, cCas3, TNF-α, TNFR1, IL-1ß, and iNOS were decreased, whereas those of Nrf2, HO-1, and SOD1 were increased in both EA-treated groups compared to those in the PBS-treated group (p < 0.05). EA treatment has neuroprotective effects on an experimental model of traumatic optic neuropathy by suppressing apoptosis, neuroinflammation, and oxidative stress to protect the RGCs from death as well as preserving the visual function.


Assuntos
Fármacos Neuroprotetores , Traumatismos do Nervo Óptico , Ratos , Masculino , Animais , Traumatismos do Nervo Óptico/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Ratos Wistar , Fator 2 Relacionado a NF-E2 , Receptores Tipo I de Fatores de Necrose Tumoral , Superóxido Dismutase-1 , Apoptose , Fator de Necrose Tumoral alfa/farmacologia , Modelos Teóricos , Modelos Animais de Doenças
4.
Phytomedicine ; 110: 154640, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36608498

RESUMO

BACKGROUND: Osthole (OST), a characteristic coumarin compound in Angelicae pubescentis radix (APR), has shown potent efficacy in the treatment of rheumatoid arthritis (RA), but its specific targets and potential mechanism are limited. PURPOSE: This study aimed to explore the potential targets and molecular mechanisms of OST against RA using computer-assisted techniques in combination with RA fibroblast-like synoviocytes (FLS) inflammation model and CIA rat model. METHODS: Network pharmacology and molecular docking were applied to initially predict the potential targets of OST for the treatment of RA. Thereafter, TNFα was used to stimulate FLS to build an in vitro model of inflammation, combined with RNA-seq technology and molecular biology such as qPCR to investigate the anti-inflammatory effects and related mechanisms of OST. Finally, the anti-RA effect of OST was demonstrated by establishing a CIA rat model. RESULTS: The network model results showed that the anti-RA effect of OST was mainly related to its anti-inflammatory effect, and AMPK was identified as a potential target for the potency of OST. In the TNFα-induced FLS cells, OST inhibited the secretion of FLS inflammatory factors, which was attributed to the ability of OST to activate AMPK to inhibit the activation of the NLRP3 inflammasome. Further, it was observed that the activation of AMPK by OST facilitated mitochondrial biogenesis, and corrected abnormal mitochondrial dynamics in FLS, which was favoured to the restoration of mitochondrial homeostasis, and further promoted the occurrence of apoptosis and the decrease of ROS in FLS. Consistent with in vivo studies, administration of OST significantly improved joint deformity and toe erythema, reduced arthritis index scores and inhibited synovial inflammation in CIA rats. CONCLUSION: Our study proposed for the first time that AMPK, served as a potential target of OST, positively participated in the anti-RA therapeutic effect of OST. By regulating mitochondrial homeostasis and function, OST can effectively inhibit the activation of inflammasome and the secretion of inflammatory factors in vitro and in vivo, and finally achieve beneficial effects in the treatment of RA, which provides support and greater possibility to make further efforts on pharmacological research and clinical application of OST.


Assuntos
Artrite Reumatoide , Fator de Necrose Tumoral alfa , Ratos , Animais , Fator de Necrose Tumoral alfa/farmacologia , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Quinases Ativadas por AMP , Simulação de Acoplamento Molecular , Artrite Reumatoide/tratamento farmacológico , Cumarínicos/farmacologia , Inflamação/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Fibroblastos , Células Cultivadas , Membrana Sinovial
5.
Phytomedicine ; 110: 154623, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36608504

RESUMO

BACKGROUND: Neohesperidin dihydrochalbazone (NHDC) shows a range of pharmacological actions, however, in septic acute kidney injury (AKI), the effect of NHDC is little known. PURPOSE: To assess the role of NHDC against AKI and the possible mechanisms. METHODS: In vivo, we used different concentration of NHDC (50, 100, and 200 mg/kg) treated septic AKI model of mice. Moreover, in vitro, in HK-2 cells, a lipopolysaccharide (LPS) induced cell model was treated with 10, 20, and 30 µM NHDC. Next, kidney tissue pathologic change, marker of renal injury, apoptosis, and inflammatory factors were assessed using hematoxylin and eosin staining, enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase dUTP nick end labeling, and western blot. HK-2 cell apoptosis and viability were assessed via flow cytometry and cell counting kit-8. In HK-2 cells and tissues, NLRP3, caspase 1, ASC, and P38/ERK 1/2/JNK pathway related protein levels were tested using western blot. RESULTS: NHDC (100 and 200 mg/kg) significantly attenuated kidney injury in caecal ligation and puncture (CLP)-treated mice. In CLP-treated mice, the level of BUN, Scr, KIM-1, and NAGL was reduced by 100 and 200 mg/kg NHDC. Furthermore, 100 and 200 mg/kg NHDC inhibited inflammation by reducing the production of IL-6, TNF-α, and IL-1ß, and inhibited oxidative stress by regulating the change of MDA, SOD, GSH, and CAT. NHDC (100 and 200 mg/kg) inhibited renal cell apoptosis by increasing Bcl2 protein expression and inhibiting Bax and cleaved caspase-3 protein expression. Additionally, NHDC (100 and 200 mg/kg) inhibited the protein levels of phosphorylated (p)-P38, p-JNK, p-ERK 1/2, NLRP3, caspase 1, ASC. In vitro, in LPS-stimulated HK-2 cells, NHDC (20 and 30 µM) increased cell viability, reduced cell apoptosis, restrained inflammation by reducing the content of IL-6, TNF-α, and IL-1ß, and inhibited the protein expression of caspase 1, NLRP3, ASC, p-P38, p-JNK, and p-ERK1/2. Importantly, the promotive effect of NHDC on HK-2 cell viability was reversed by DHR (an activator of P38 MAPK signaling pathway), and DHR reversed the inhibitive effects of NHDC on HK-2 cell apoptosis and inflammation. CONCLUSION: For the first time, NHDC was found to inhibit oxidative stress, inflammation, and apoptosis in AKI model, which was related to the inhibition of P38 MAPK pathways. Our findings provided the theoretical basis for NHDC on the prevention of AKI.


Assuntos
Injúria Renal Aguda , Sepse , Camundongos , Animais , Lipopolissacarídeos/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Caspase 1 , Fator de Necrose Tumoral alfa/farmacologia , Interleucina-6/farmacologia , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/metabolismo , Apoptose , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno , Sepse/metabolismo
6.
Brain Behav Immun ; 108: 162-175, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36503051

RESUMO

Exposure to inflammatory stressors during fetal development is a major risk factor for neurodevelopmental disorders (NDDs) in adult offspring. Maternal immune activation (MIA), induced by infection, causes an acute increase in pro-inflammatory cytokines which can increase the risk for NDDs directly by inducing placental and fetal brain inflammation, or indirectly through affecting maternal care behaviours thereby affecting postnatal brain development. Which of these two potential mechanisms dominates in increasing offspring risk for NDDs remains unclear. Here, we show that acute systemic maternal inflammation induced by the viral mimetic polyinosinic:polycytidylic acid (poly I:C) on gestational day 15 of rat pregnancy affects offspring and maternal behaviour, offspring cognition, and expression of NDD-relevant genes in the offspring brain. Dams exposed to poly I:C elicited an acute increase in the pro-inflammatory cytokine tumour necrosis factor (TNF; referred to here as TNFα), which predicted disruption of key maternal care behaviours. Offspring of poly I:C-treated dams showed early behavioural and adult cognitive deficits correlated to the maternal TNFα response, but, importantly, not with altered maternal care. We also found interacting effects of sex and treatment on GABAergic gene expression and DNA methylation in these offspring in a brain region-specific manner, including increased parvalbumin expression in the female adolescent frontal cortex. We conclude that the MIA-induced elevation of TNFα in the maternal compartment affects fetal neurodevelopment leading to altered offspring behaviour and cognition. Our results suggest that a focus on prenatal pathways affecting fetal neurodevelopment would provide greater insights into the mechanisms underpinning the TNFα-mediated genesis of altered offspring behaviour and cognition following maternal inflammation.


Assuntos
Transtornos do Neurodesenvolvimento , Efeitos Tardios da Exposição Pré-Natal , Ratos , Animais , Feminino , Gravidez , Humanos , Fator de Necrose Tumoral alfa/farmacologia , Comportamento Animal/fisiologia , Placenta/metabolismo , Citocinas , Poli I-C/efeitos adversos , Comportamento Materno , Inflamação/metabolismo , Modelos Animais de Doenças
7.
Mol Immunol ; 153: 160-169, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36508750

RESUMO

Cytokine release syndrome, also called cytokine storm, could cause lung tissue damage, acute respiratory distress syndrome (ARDS) and even death during SARS-CoV-2 infection. However, the underlying mechanisms of cytokine storm still remain unknown. Among these cytokines, the function of TNF-α and type I IFNs especially deserved further investigation. Here, we first found that TNF-α and IFN-ß synergistically induced human airway epithelial cells BEAS-2B death. Mechanistically, the combination of TNF-α and IFN-ß led to the activation of caspase-8 and caspase-3, which initiated BEAS-2B apoptosis. The activated caspase-8 and caspase-3 could further induce the cleavage and activation of gasdermin D (GSDMD) and gasdermin E (GSDME), which finally resulted in pro-inflammatory pyroptosis. The knock-down of caspase-8 and caspase-3 could effectively block the activation of GSDMD and GSDME, and then the death of BEAS-2B induced by TNF-α and IFN-ß. In addition, pan-caspase inhibitor Z-VAD-FMK (ZVAD) and necrosulfonamide (NSA) could inhibit BEAS-2B death induced by TNF-α and IFN-ß. Overall, our work revealed one possible mechanism that cytokine storm causes airway epithelial cells (AECs) damage and ARDS. These results indicated that blocking TNF-α and IFN-ß-mediated AECs death may be a potential target to treat related viral infectious diseases, such as COVID-19.


Assuntos
COVID-19 , Síndrome do Desconforto Respiratório , Humanos , Apoptose , Caspase 3/metabolismo , Caspase 8/metabolismo , Síndrome da Liberação de Citocina , Células Epiteliais/metabolismo , Piroptose , SARS-CoV-2/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Interferon beta
8.
Exp Cell Res ; 422(2): 113441, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36481205

RESUMO

Rheumatoid arthritis (RA) is a chronic, autoimmune and systemic inflammatory disease affecting 1% of the population worldwide. Immune suppression of the activity and progress of RA is vital to reduce the disability and mortality rate as well as improve the quality of life of RA patients. However, the immune molecular mechanism of RA has not been clarified yet. Our results indicated that exosomes derived from TNFα-stimulated RA fibroblast-like synoviocytes (RA-FLSs) suppressed chondrocyte proliferation and migration through modulating cartilage extracellular matrix (CECM) determining by MTS assay, cell cycle analysis, Transwell assay and Western blot (WB). Besides, RNA sequencing and verification by qRT-PCR revealed that exosomal long non-coding RNA (lncRNA) tumor necrosis factor-associated factor 1 (TRAF1)-4:1 derived from RA-FLSs treated with TNFα was a candidate lncRNA, which also inhibited chondrocyte proliferation and migration through degrading CECM. Moreover, RNA sequencing and bioinformatics analysis identified that C-X-C motif chemokine ligand 1 (CXCL1) was a target mRNA of miR-27a-3p while miR-27a-3p was a target miRNA of lnc-TRAF1-4:1 in chondrocytes. Mechanistically, lnc-TRAF1-4:1 upregulated CXCL1 expression through sponging miR-27a-3p as a competing endogenous RNA (ceRNA) in chondrocytes identifying by Dual-luciferase reporter gene assay. Summarily, exosomal lncRNA TRAFD1-4:1 derived from RA-FLSs suppressed chondrocyte proliferation and migration through degrading CECM by upregulating CXCL1 as a sponge of miR-27a-3p. This study uncovered a novel RA-related lncRNA and investigated the roles of RA-FLS-derived exosomes and exosomal lnc-TRAF1-4:1 in articular cartilage impairment, which might provide novel therapeutic targets for RA.


Assuntos
Artrite Reumatoide , MicroRNAs , RNA Longo não Codificante , Sinoviócitos , Humanos , Sinoviócitos/metabolismo , RNA Longo não Codificante/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Condrócitos/metabolismo , Qualidade de Vida , Fator 1 Associado a Receptor de TNF/metabolismo , Artrite Reumatoide/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Cartilagem/patologia , Fibroblastos/metabolismo , Proliferação de Células/genética , Células Cultivadas
9.
Zhongguo Zhong Yao Za Zhi ; 47(22): 6164-6174, 2022 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-36471941

RESUMO

This paper aims to explore the activity of Codonopsis canescens extract against rheumatoid arthritis(RA) based on the Toll-like receptors(TLRs)/mitogen-activated protein kinases(MAPKs)/nuclear factor kappa B(NF-κB) signaling pathways and its mechanism. The ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) was used to identify the components of C. canescens extract. Forty-eight male SD rats were randomly divided into six groups, namely the normal group, the model group, the methotrexate(MTX) tablet group, and the low, medium, and high-dose C. canescens extract(ZDS-L, ZDS-M, and ZDS-H) groups, with 8 rats in each group. The model of collagen-induced arthritis in rats was induced by injection of bovine type Ⅱ collagen emulsion. MTX(2.5 mg·kg~(-1)), ZDS-L, ZDS-M, and ZDS-H(0.3 g·kg~(-1), 0.6 g·kg~(-1), and 1.2 g·kg~(-1)) were administrated by gavage. Rats in the normal group and the model group received distilled water. MTX was given once every three days for 28 days, and the rest medicines were given once daily for 28 days. Body weight, degree of foot swelling, arthritis index, immune organ index, synovial histopathological changes, and serum levels of tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß), and interleukin-6(IL-6) were observed. Protein expressions of TLR2, TLR4, NF-κB p65, p38 MAPK, and p-p38 MAPK in rats were determined by Western blot. Thirty-four main components were identified by UPLC-Q-TOF-MS, including 15 flavonoids, 7 phenylpropanoids, 4 terpenoids, 4 organic acids, 2 esters, and 2 polyalkynes. As compared with the normal group, the body weight of the model group was significantly decreased(P<0.01), and foot swelling(P<0.05, P<0.01), arthritis index(P<0.01), and the immune organ index(P<0.01) were significantly increased. The synovial histopathological injury was obviously observed in the model group. The serum levels of inflammatory factors TNF-α, IL-1ß, and IL-6 were significantly increased(P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK in the synovial tissue were significantly increased(P<0.01) in the model group. As compared with the model group, the body weights of the ZDS dose groups were increased(P<0.01), and the degree of foot swelling(P<0.01) and the arthritis index were decreased(P<0.05, P<0.01). The immune organ index was decreased(P<0.01) in the ZDS dose groups, and the synovial tissue hyperplasia and inflammatory cell infiltration were alleviated. The serum levels of TNF-α, IL-1ß, and IL-6 were significantly decreased(P<0.05, P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK were decreased(P<0.05, P<0.01) in the ZDS dose groups. C. canescens extract containing apigenin, tricin, chlorogenic acid, aesculin, ferulic acid, caffeic acid, and oleanolic acid has a good anti-RA effect, and the mechanism may be related to the inhibition of TLRs/MAPKs/NF-κB signaling pathways.


Assuntos
Artrite Experimental , Artrite Reumatoide , Codonopsis , Masculino , Animais , Bovinos , Ratos , NF-kappa B/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Interleucina-6 , Receptor 2 Toll-Like , Ratos Sprague-Dawley , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Artrite Reumatoide/tratamento farmacológico , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Edema , Extratos Vegetais/efeitos adversos , Peso Corporal
10.
Zhonghua Nei Ke Za Zhi ; 61(12): 1318-1323, 2022 Dec 01.
Artigo em Chinês | MEDLINE | ID: mdl-36456511

RESUMO

Objective: To establish a method for detecting pancreatic ß-cell dedifferentiation using flow cytometry. Methods: Experimental study. Min6 (mouse ß cell line), αTC1-6 (mouse α cell line), HepG2 (human hepatocellular carcinoma cells) and mouse F9 cells (mouse teratocarcinoma cell) were cultured with conventional medium. Min6 cells were treated with interleukin-1ß (IL-1ß) in combined with tumor necrosis factor α (TNFα), or palmitic acid (PA) overnight and stained with anti-chromogranin A (ChgA), anti-insulin (Ins), anti-glucagon (Gcg), anti-SRY-box transcription factor 9 (Sox9) and anti-octamer binding transcription factor 4 (Oct4) antibodies, respectively. Flow cytometry was applied to detect the pression of ChgA, Ins, Gcg, Sox9, and Oct4 in the cells, respectively. Unpaired Student t test was used for statistical analysis. Results: Flow cytometry analyses showed that Ins and ChgA were highly expressed in Min6 cells, Gcg was highly expressed in αTC1-6, Sox9 was highly expressed in HepG2, and Oct4 was highly expressed in F9 cells, respectively (around 90%). Treatment of Min6 cells with IL-1ß+TNFα significantly decreased Ins positive staining cells (92.775%±1.702% vs. 97.125%±0.246%, P=0.045), while increased Sox9 positive staining cells (41.675%±0.390% vs. 25.875%±3.348%, P=0.003). No significant changes in ChgA and Oct4 expression could be viewed (both P>0.05). PA treatment elevated the number of Gcg positive staining cells (54.500%±3.597% vs. 41.160%±3.007%, P=0.022). The levels of mRNA expression by qPCR of the above proteins were in consistent with the levels of protein expression by flow cytometry in Min6 cells. Conclusion: Flow cytometry can be used to detect proteins expressed in dedifferentiated models of ß cells, which provides a new method for identify dedifferentiation of pancreatic ß cells.


Assuntos
Células Secretoras de Insulina , Humanos , Animais , Camundongos , Citometria de Fluxo , Fator de Necrose Tumoral alfa/farmacologia , Desdiferenciação Celular , Glucagon , Insulina , Anticorpos
11.
Int J Mol Sci ; 23(24)2022 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-36555705

RESUMO

Cell migration is an essential part of the complex and multistep process that is the development of cancer, a disease that is the second most common cause of death in humans. An important factor promoting the migration of cancer cells is TNF-α, a pro-inflammatory cytokine that, among its many biological functions, also plays a major role in mediating the expression of MMP9, one of the key regulators of cancer cell migration. It is also known that TNF-α is able to induce the Warburg effect in some cells by increasing glucose uptake and enhancing the expression and activity of lactate dehydrogenase subunit A (LDHA). Therefore, the aim of the present study was to investigate the interrelationship between the TNF-α-induced promigratory activity of cancer cells and their glucose metabolism status, using esophageal cancer cells as an example. By inhibiting LDHA activity with sodium oxamate (SO, also known as aminooxoacetic acid sodium salt or oxamic acid sodium salt) or siRNA-mediated gene silencing, we found using wound healing assay and gelatin zymography that LDHA downregulation impairs TNF-α-dependent tumor cell migration and significantly reduces TNF-α-induced MMP9 expression. These effects were associated with disturbances in the activation of the ERK1/2 signaling pathway, as we observed by Western blotting. We also reveal that in esophageal cancer cells, SO effectively reduces the production of lactic acid, which, as we have shown, synergizes the stimulating effect of TNF-α on MMP9 expression. In conclusion, our findings identified LDHA as a regulator of TNF-α-induced cell migration in esophageal cancer cells by the ERK1/2 signaling pathway, suggesting that LDHA inhibitors that limit the migration of cancer cells caused by the inflammatory process may be considered as an adjunct to standard therapy in esophageal cancer patients.


Assuntos
Neoplasias Esofágicas , Fator de Necrose Tumoral alfa , Humanos , Lactato Desidrogenase 5 , Fator de Necrose Tumoral alfa/farmacologia , L-Lactato Desidrogenase/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células
12.
J Mol Neurosci ; 72(12): 2497-2506, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36527597

RESUMO

It is known that neurotrophic factors are a major source of the neuroprotective effects of olfactory ensheathing cells (OECs). However, the form of neurotrophic factors that originate from OECs is not fully understood. Our previous study demonstrated that OECs could secrete exosome (OECs-Exo), which provided neuroprotection by switching the phenotype of macrophages/microglia. Considering that exosomes could also be taken up by neurons, we explored the direct effect of OECs-Exo on neuronal survival and the underlying mechanism. Electron microscopy, nano-traffic analysis, and Western blotting were applied to identify the OECs-Exo. The effect of OECs-Exo on neuronal survival was tested by flow cytometry and TUNEL staining. Western blotting and ELISA were used to detect neurotrophic factors in purified OECs-Exo. We first isolated OECs-Exo and found that OECs-Exo exerted protective effects on neuronal survival in response to TNF-α challenge. Brain-derived neurotrophic factor (BDNF) was then identified in OECs-Exo, and its receptor TrkB in neurons was activated by OECs-Exo treatment. Furthermore, we demonstrated that OECs prevented TNF-α-induced apoptosis in neurons partially through exosome-derived BDNF. Our data showed that OECs attenuated TNF-α-induced apoptosis in neurons partially through OEC-Exo-derived BDNF, which might provide a novel strategy for the neuroprotective effect of OEC-Exo-based treatment.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Exossomos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Bulbo Olfatório , Neurônios , Apoptose
13.
Endocrinology ; 164(2)2022 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-36477465

RESUMO

The inflammatory eye disease Graves' orbitopathy (GO) is the main complication of autoimmune Graves' disease. In previous studies we have shown that hypoxia plays an important role for progression of GO. Hypoxia can maintain inflammation by attracting inflammatory cells such as macrophages (MQ). Herein, we investigated the interaction of MQ and orbital fibroblasts (OF) in context of inflammation and hypoxia. We detected elevated levels of the hypoxia marker HIF-1α, the MQ marker CD68, and inflammatory cytokines TNFα, CCL2, CCL5, and CCL20 in GO biopsies. Hypoxia stimulated GO tissues to release TNFα, CCL2, and CCL20 as measured by multiplex enzyme-linked immunosorbent assay (ELISA). Further, TNFα and hypoxia stimulated the expression of HIF-1α, CCL2, CCL5, and CCL20 in OF derived from GO tissues. Immunofluorescence confirmed that TNFα-positive MQ were present in the GO tissues. Thus, interaction of M1-MQ with OF under hypoxia also induced HIF-1α, CCL2, and CCL20 in OF. Inflammatory inhibitors etanercept or dexamethasone prevented the induction of HIF-1α and release of CCL2 and CCL20. Moreover, co-culture of M1-MQ/OF under hypoxia enhanced adipogenic differentiation and adiponectin secretion. Dexamethasone and HIF-1α inhibitor PX-478 reduced this effect. Our findings indicate that GO fat tissues are characterized by an inflammatory and hypoxic milieu where TNFα-positive MQ are present. Hypoxia and interaction of M1-MQ with OF led to enhanced secretion of chemokines, elevated hypoxic signaling, and adipogenesis. In consequence, M1-MQ/OF interaction results in constant inflammation and tissue remodeling. A combination of anti-inflammatory treatment and HIF-1α reduction could be an effective treatment option.


Assuntos
Oftalmopatia de Graves , Humanos , Oftalmopatia de Graves/metabolismo , Adipogenia , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Órbita/metabolismo , Órbita/patologia , Inflamação/metabolismo , Fibroblastos/metabolismo , Hipóxia/metabolismo , Dexametasona/farmacologia , Células Cultivadas
14.
Front Immunol ; 13: 1036196, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36531989

RESUMO

Introduction: Bacteroides vulgatus is one of the predominant Bacteroides species in the human gut and exerts a series of beneficial effects. The aim of this study was to investigate the protective role of B. vulgatus Bv46 in a dextran sodium sulfate (DSS) induced colitis mouse model. Methods: Female C57BL/6J mice were given 3% DSS in drinking water to induce colitis and simultaneously treated with B. vulgatus Bv46 by gavage for 7 days. Daily weight and disease activity index (DAI) of mice were recorded, and the colon length and histological changes were evaluated. The effects of B. vulgatus Bv46 on gut microbiota composition, fecal short chain fatty acids (SCFAs) concentration, transcriptome of colon, colonic cytokine level and cytokine secretion of RAW 264·7 macrophage cell line activated by the lipopolysaccharide (LPS) were assessed. Results and Discussion: B. vulgatus Bv46 significantly attenuated symptoms of DSS-induced colitis in mice, including reduced DAI, prevented colon shortening, and alleviated colon histopathological damage. B. vulgatus Bv46 modified the gut microbiota community of colitis mice and observably increased the abundance of Parabacteroides, Bacteroides, Anaerotignum and Alistipes at the genus level. In addition, B. vulgatus Bv46 treatment decreased the expression of colonic TNF-α, IL-1ß and IL-6 in DSS-induced mouse colitis in vivo, reduced the secretion of TNF-α, IL-1ß and IL-6 in macrophages stimulated by LPS in vitro, and downregulated the expression of Ccl19, Cd19, Cd22, Cd40 and Cxcr5 genes in mice colon, which mainly participate in the regulation of B cell responses. Furthermore, oral administration of B. vulgatus Bv46 notably increased the contents of fecal SCFAs, especially butyric acid and propionic acid, which may contribute to the anti-inflammatory effect of B. vulgatus Bv46. Supplementation with B. vulgatus Bv46 serves as a promising strategy for the prevention of colitis.


Assuntos
Colite , Microbioma Gastrointestinal , Animais , Feminino , Humanos , Camundongos , Bacteroides , Colite/induzido quimicamente , Colite/microbiologia , Colite/terapia , Citocinas/farmacologia , Sulfato de Dextrana , Ácidos Graxos Voláteis/farmacologia , Imunidade , Interleucina-6/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/farmacologia
15.
BMC Cardiovasc Disord ; 22(1): 539, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36494772

RESUMO

BACKGROUND: Titin phosphorylation contributes to left ventricular (LV) diastolic dysfunction. The independent effects of inflammation on the molecular pathways that regulate titin phosphorylation are unclear. METHODS: We investigated the effects of collagen-induced inflammation and subsequent tumor necrosis factor-α (TNF-α) inhibition on mRNA expression of genes involved in regulating titin phosphorylation in 70 Sprague-Dawley rats. LV diastolic function was assessed with echocardiography. Circulating inflammatory markers were quantified by enzyme-linked immunosorbent assay and relative LV gene expression was assessed by Taqman® polymerase chain reaction. Differences in normally distributed variables between the groups were determined by two-way analysis of variance (ANOVA), followed by Tukey post-hoc tests. For non-normally distributed variables, group differences were determined by Kruskal-Wallis tests. RESULTS: Collagen inoculation increased LV relative mRNA expression of vascular cell adhesion molecule 1 (VCAM1), pentraxin 3 (PTX3), and inducible nitric oxide synthase (iNOS) compared to controls, indicating local microvascular inflammation. Collagen inoculation decreased soluble guanylate cyclase alpha-2 (sGCα2) and soluble guanylate cyclase beta-2 (sGCß2) expression, suggesting downregulation of nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling. Inhibiting TNF-α prevented collagen-induced changes in VCAM1, iNOS, sGCα2 and sGCß2 expression. Collagen inoculation increased protein phosphatase 5 (PP5) expression. Like LV diastolic dysfunction, increased PP5 expression was not prevented by TNF-α inhibition. CONCLUSION: Inflammation-induced LV diastolic dysfunction may be mediated by a TNF-α-independent increase in PP5 expression and dephosphorylation of the N2-Bus stretch element of titin, rather than by TNF-α-induced downregulation of NO-sGC-cGMP pathway-dependent titin phosphorylation. The steady rise in number of patients with inflammation-induced diastolic dysfunction, coupled with low success rates of current therapies warrants a better understanding of the systemic signals and molecular pathways responsible for decreased titin phosphorylation in development of LV diastolic dysfunction. The therapeutic potential of inhibiting PP5 upregulation in LV diastolic dysfunction requires investigation.


Assuntos
Fator de Necrose Tumoral alfa , Disfunção Ventricular Esquerda , Ratos , Animais , Guanilil Ciclase Solúvel , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Ratos Sprague-Dawley , GMP Cíclico/metabolismo , Inflamação , Disfunção Ventricular Esquerda/genética , Colágeno , RNA Mensageiro/metabolismo
16.
Molecules ; 27(21)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36364190

RESUMO

Cancer chemotherapy-induced cognitive impairment (chemobrain) is a major complication that affects the prognosis of therapy. Our study evaluates the nootropic-like activity of levetiracetam (LEVE) against doxorubicin (DOX)-induced memory defects using in vivo and molecular modelling. Rats were treated with LEVE (100 and 200 mg/kg, 30 days) and chemobrain was induced by four doses of DOX (2 mg/kg, i.p.). Spatial memory parameters were evaluated using an elevated plus maze (EPM) and Y-maze. Additionally, acetylcholinesterase (AChE) and the neuroinflammatory biomarkers cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), nuclear factor-κB (NF-κB), and tumor necrosis factor-alpha (TNF-α) were analyzed using brain homogenate. PharmMapper was used for inverse docking and AutoDock Vina was used for molecular docking. LEVE treatment significantly diminished the DOX-induced memory impairment parameters in both the EPM and Y-maze. In addition, the drug treatment significantly reduced AChE, COX-2, PGE2, NF-κB, and TNF-α levels compared to DOX-treated animals. The inverse docking procedures resulted in the identification of AChE as the potential target. Further molecular modelling studies displayed interactions with residues Gly118, Gly119, and Ser200, critical for the hydrolysis of ACh. Analysis of the results suggested that administration of LEVE improved memory-related parameters in DOX-induced animals. The 'nootropic-like' activity could be related to diminished AChE and neuroinflammatory mediator levels.


Assuntos
Comprometimento Cognitivo Relacionado à Quimioterapia , Nootrópicos , Animais , Ratos , Simulação de Acoplamento Molecular , Nootrópicos/farmacologia , Levetiracetam/farmacologia , Acetilcolinesterase/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , NF-kappa B/farmacologia , Ciclo-Oxigenase 2 , Doenças Neuroinflamatórias , Dinoprostona , Doxorrubicina/efeitos adversos , Colinérgicos/farmacologia , Estresse Oxidativo
17.
Biomed Pharmacother ; 156: 113922, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36411615

RESUMO

BACKGROUND: Although Shenhuang plaster (SHP) from traditional Chinese medicine prescriptions, has the potential to promote the recovery progression of postoperative ileus (POI), the underlying mechanism remains elusive. Along these lines, in this work, both in vivo and in vitro studies were conducted to systematically explore the regulatory effect and mechanism of SHP on the inflammatory response of the intestinal basal layer in the POI model mice. METHODS: Intestinal manipulation in mice was utilized for the POI model. The impact of SHP in response to POI was evaluated by carrying fluorescein-labeled dextran, histomorphology, immunohistochemistry, in combination with flow cytometry analysis and transcriptome RNA sequencing in vivo. Besides, the cytotoxicity of the SHP treatment on RAW264.7 cells was detected by cell counting kit-8 (CCK-8), the biological effects were assessed by polymerase chain reaction (PCR) and the potential influences on the PI3K/Akt/NF-κB pathway were identified through detecting the expression levels of P85, AKT, IKK and P65 by western blot in vitro. RESULTS: The implementation of the SHP treatment could significantly reduce the expressions of interleukin (IL)- 1ß and tumor necrosis factor (TNF)-α in the intestine, whereas the recovery of gastrointestinal motility is promoted. In addition, SHP can regulate the polarization of macrophages, indicating that the proportion of the M2 type is increased after the application of the SHP treatment. In addition, SHP inhibited the activity of PI3K/AKT/NF-κB signaling pathway-related proteins. CONCLUSION: SHP can significantly ameliorate the inflammatory response of POI and at the same time promote the recovery of gastrointestinal motility. Its mechanism may be mediated by the polarization of macrophages through the PI3K/AKT/NF-κB signaling pathway.


Assuntos
Íleus , NF-kappa B , Camundongos , Animais , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inflamação/tratamento farmacológico , Íleus/tratamento farmacológico , Fator de Necrose Tumoral alfa/farmacologia
18.
Arch Oral Biol ; 144: 105573, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36341994

RESUMO

OBJECTIVE: This study aimed to investigate the effect of infliximab on orthodontic tooth movement in rats. MATERIALS AND METHODS: Sixty male Sprague-Dawley rats were randomly divided into two groups: saline group and infliximab group. The two groups of rats received weekly intraperitoneally injection of saline or infliximab (5 mg/kg), respectively. After four weeks of injection, five rats in each group were euthanized and orthodontic appliances were placed in the other twenty-five rats each. On days 1, 3, 7, 14 and 21, five rats from each group were euthanised. Maxillae of all the rats were collected and examined by micro-computed tomography, haematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, and immunohistochemical staining of tumour necrosis factor (TNF)-α, receptor activator of nuclear factor κB ligand (RANKL), receptor activator of nuclear factor κB (RANK), and osteoprotegerin (OPG). All data were analysed with Mann-Whitney test. RESULTS: Infliximab inhibited orthodontic tooth movement and decreased osteoclastogenesis on the compression side during orthodontic tooth movement. The elevated TNF-α level, induced by orthodontic force, was decreased by infliximab. Furthermore, infliximab reduced the expression of RANKL and RANK, while increased the expression of OPG on the compression side. CONCLUSION: Infliximab inhibits orthodontic tooth movement by reducing levels of TNF-α, RANKL, and RANK, while increasing level of OPG, and decreasing osteoclastogenesis on the compression side of periodontium.


Assuntos
Técnicas de Movimentação Dentária , Fator de Necrose Tumoral alfa , Ratos , Masculino , Animais , Técnicas de Movimentação Dentária/métodos , Infliximab/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Microtomografia por Raio-X , Osteoclastos , Ratos Sprague-Dawley , Ligante RANK/metabolismo , Osteoprotegerina/metabolismo , Receptor Ativador de Fator Nuclear kappa-B
19.
Molecules ; 27(21)2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36364277

RESUMO

NF-κB signaling is a key regulator of inflammation and atherosclerosis. NF-κB cooperates with bromodomain-containing protein 4 (BRD4), a transcriptional and epigenetic regulator, in endothelial inflammation. This study aimed to investigate whether BRD4 inhibition would prevent the proinflammatory response towards TNF-α in endothelial cells. We used TNF-α treatment of human umbilical cord-derived vascular endothelial cells to create an in vitro inflammatory model system. Two small molecule inhibitors of BRD4-namely, RVX208 (Apabetalone), which is in clinical trials for the treatment of atherosclerosis, and JQ1-were used to analyze the effect of BRD4 inhibition on endothelial inflammation and barrier integrity. BRD4 inhibition reduced the expression of proinflammatory markers such as SELE, VCAM-I, and IL6 in endothelial cells and prevented TNF-α-induced endothelial tight junction hyperpermeability. Endothelial inflammation was associated with increased expression of the heparin-binding growth factor midkine. BRD4 inhibition reduced midkine expression and normalized endothelial permeability upon TNF-α treatment. In conclusion, we identified that TNF-α increased midkine expression and compromised tight junction integrity in endothelial cells, which was preventable by pharmacological BRD4 inhibition.


Assuntos
Aterosclerose , Proteínas Nucleares , Humanos , Proteínas Nucleares/metabolismo , NF-kappa B/metabolismo , Células Endoteliais , Midkina , Fator de Necrose Tumoral alfa/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Fatores de Transcrição , Proteínas de Ciclo Celular
20.
Viruses ; 14(11)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36366477

RESUMO

Yellow fever (YF) may cause lesions in different organs. There are no studies regarding the in situ immune response in the human lung and investigating immunopathological aspects in fatal cases can help to better understand the evolution of the infection. Lung tissue samples were collected from 10 fatal cases of human yellow fever and three flavivirus-negative controls who died of other causes and whose lung parenchymal architecture was preserved. In YFV-positive fatal cases, the main histopathological changes included the massive presence of diffuse alveolar inflammatory infiltrate, in addition to congestion and severe hemorrhage. The immunohistochemical analysis of tissues in the lung parenchyma showed significantly higher expression of E-selectin, P-selectin, ICAM-1, VCAM-1 in addition to cytokines such as IL-4, IL-10, IL-13, TNF- α, IFN-γ and TGF-ß compared to the negative control. The increase in immunoglobulins ICAM-1 and VCAM-1 results in strengthening of tissue transmigration signaling. E-selectin and P-selectin actively participate in this process of cell migration and formation of the inflammatory infiltrate. IFN-γ and TNF-α participate in the process of cell injury and viral clearance. The cytokines IL-4 and TGF-ß, acting in synergism, participate in the process of tissue regeneration and breakdown. The anti-inflammatory cytokines IL-4, IL-10 and IL-13 also act in the reduction of inflammation and tissue repair. Our study indicates that the activation of the endothelium aggravates the inflammatory response by inducing the expression of adhesion molecules and cytokines that contribute to the rolling, recruitment, migration and eliciting of the inflammatory process in the lung parenchyma, contributing to the fatal outcome of the disease.


Assuntos
Molécula 1 de Adesão Intercelular , Febre Amarela , Humanos , Molécula 1 de Adesão de Célula Vascular/análise , Molécula 1 de Adesão de Célula Vascular/metabolismo , Interleucina-13 , Interleucina-10 , Interleucina-4 , Citocinas/farmacologia , Endotélio/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Pulmão/metabolismo , Fator de Crescimento Transformador beta
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