RESUMO
OBJECTIVE: To explore the molecular immune mechanism of HPV-infected HaCaT cells in vitro based on TLRs signaling pathway by analyzing the effects of interfering TLRs on inflammatory and immune factors in the signaling pathway. METHODS: FCM was used to analyze the proportion of Th1, Th2, Th17, and Treg cells in blood samples. HPV-infected HaCaT cells were divided into five groups: A, B, C, D, and E. Group A added TLR3 antagonist, group B added TLR9 antagonist, group C added equivalent saline, group D added IRF3 agonist, and group E added IRF3 inhibitor. Immunohistochemistry was used to analyze the expression of TLR3 and TLR9 in HaCaT cell model; ELISA was used to analyze the expression of inflammatory factors IL-2, TNF-a, and IFN-beta; WB was used to analyze the expression of TRAF3, IKK epsilon, and TBK1; RT-PCR was used to analyze the expression of IRF3 and IRF7 in each cell model. RESULTS: The proportion of blood immune cells in patients with HPV infection was Th1, Th17, Th2, and Treg, with statistical significance (P < .05); the expression of TLR3 and TLR9 in HPV-infected cells was higher than that in negative control group, with statistical significance (P < .05); TLR3 was higher than TLR9, with no significant difference (P > .05); the expression of IL-2, TNF-alpha, IFN-beta in each group, TLR3, and TLR9 was higher than that in negative control group (P < .05). The expression of TRAF3, IKK epsilon, and TBK1 in the control group was higher than that in the TLR3 and TLR9 inhibitor groups, and the expression of IRF3 and IRF7 in the TLR9 inhibitor group was higher than that in the TLR3 inhibitor group (P < .05); the expression of IRF3 and IRF7 in the TLR3i and TLR9i inhibitor groups was lower than that in the TLR3 inhibitor group (P < .05). Compared with the control group, IRF3a group was higher than the control group, IRF3i group was lower than the control group, the difference was statistically significant (P < .05). CONCLUSION: TLR3 and TLR9, the key factors of TLRs, are highly expressed in HaCaT cells infected with HPV. Through TLRs-IKK-e-IRFs-IFN signaling pathway, they can induce high expression of inflammatory factors, IKK-e, IRFs, and IFN, and improve immunity.
Assuntos
Células HaCaT/imunologia , Células HaCaT/virologia , Infecções por Papillomavirus/imunologia , Transdução de Sinais , Receptores Toll-Like/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Mediadores da Inflamação/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Modelos Biológicos , Infecções por Papillomavirus/sangue , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T/imunologia , Fator 3 Associado a Receptor de TNF/metabolismoRESUMO
OBJECTIVE: We investigated whether purpurin inhibits various pathways of inflammation leading to atopic dermatitis. INTRODUCTION: 1,2,4-Trihydroxyanthraquinone, commonly called purpurin, is an anthraquinone that is a naturally occurring red/yellow dye. Purpurin is a highly antioxidative anthraquinone and previous studies have reported antibacterial, anti-tumor, and anti-oxidation activities in cells and animals. However, the skin inflammatory inhibition activity mechanism study of purpurin has not been elucidated in vitro. METHODS: In this study, we investigated the anti-inflammatory activity of purpurin in HaCaT (human keratinocyte) cell lines stimulated with a mixture of tumor necrosis factor-alpha (TNF-α)/Interferon-gamma (IFN-γ). The inhibitory effect of Purpurin on cytokines (IL-6, IL-8, and IL-1ß) and chemokine (TARC, MDC, and RANTES) was confirmed by ELISA and RT-qPCR. We investigated each signaling pathway and the action of inhibitors through western blots. RESULTS: The expression levels of cytokines and chemokines were dose-dependently suppressed by purpurin treatment in TNF-α/IFN-γ-induced HaCaT cells from ELISA and real-time PCR. Purpurin also inhibited protein kinase B (AKT), mitogen-activated protein kinase (MAPKs), and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) activation in TNF-α/IFN-γ-stimulated HaCaT cells. Additionally, there was a synergistic effect when purpurin and inhibitor were applied together, and inflammation was dramatically reduced. CONCLUSION: Therefore, these results demonstrate that purpurin exhibits anti-inflammatory and anti-atopic dermatitis activity in HaCaT cells.
Assuntos
Antraquinonas , Dermatite Atópica , Células HaCaT , Interferon gama , Fator de Necrose Tumoral alfa , Animais , Antraquinonas/farmacologia , Citocinas , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/imunologia , Células HaCaT/imunologia , Humanos , Inflamação , Interferon gama/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The present study aimed to investigate the effects of Solanum nigrum Linne (SNL) in a model of 1chloro2,4dinitrobenzene (DNCB)induced atopic dermatitis (AD) and in TNFα/IFNγstimulated HaCaT cells. AD is a chronic inflammatory skin disease and is characterized by erythema, edema, increased pruritus and eczema. Steroids are most commonly used for antiinflammatory therapy; however, their longterm use is limited due to sideeffects, such as osteoporosis, brittle skin, muscle weaknesses and diabetes. Therefore, patients with AD require alternative treatment strategies. In previous studies, SNL has been reported to be effective against oxidants and cancer. However, to the best of our knowledge, the effects of SNL on AD have not yet been investigated. The present study examined the effects of SNL ethanol extract on a model of DNCB induced AD and on TNFα/IFNγstimulated HaCaT cells. The skin tissue was sectioned to measure the thicknesses of the epidermis and dermis, as well as the numbers of eosinophils, mast cells and CD8 infiltration by H&E, toluidine blue, Masson's trichrome and IHC staining. ELISA was performed using serum to measure IgE levels. The present study also examined the expression of various inflammatory cytokines, MAPK and NFκB in TNFα/IFNγstimulated HaCaT cells. SNL significantly reduced the levels of cytokines released from HaCaT cells stimulated with TNFα/IFNγ. SNL also significantly reduced the levels of pp38 at 30 min and significantly reduced the activation of NFκB in a time course experiment. In addition, SNL significantly reduced the level of serum IgE and dermal thickness and the infiltration of mast cells and CD8 in the BALB/c mouse model of DNCBinduced AD. The results of the current study suggest that SNL exerts a suppressive effect on proinflammatory cytokines in vitro and in vivo through the regulation of the immune system.