瑞香素对TNF-α刺激的HaCaT角质形成细胞炎症因子表达的影响
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摘要
目的观察瑞香素对TNF-α刺激的HaCaT角质形成细胞炎症因子表达的影响,并初步探讨其机制。方法细胞活力检测采用CCK-8检测试剂盒。实时荧光定量PCR检测炎症因子IL-1β、IL-6、TNF-α、IL-8和MCP-1 mRNA的水平。Western blotting检测p65和p-p65的蛋白水平变化。结果 20μM以下瑞香素处理HaCaT角质形成细胞24小时未表现出明显的细胞毒性。TNF-α刺激能显著促进HaCaT角质形成细胞IL-1β(P<0.05)、IL-6(P<0.001)、TNF-α(P<0.05)、IL-8(P<0.05)和MCP-1(P<0.05)的表达;而瑞香素(20μM)联合处理能减弱TNF-α刺激所引起的炎症因子表达升高。TNF-α处理能够诱导p65磷酸化,而瑞香素(20μM)联合处理能够抑制p65的磷酸化。结论瑞香素对TNF-α诱导的HaCaT角质形成细胞炎症反应具有抑制作用,而这种抑制作用与NF-κB信号通路的抑制相关。
Objective To observe the effects of daphnetin on the expression of inflammatory factors in TNF-α-stimulated HaCaT keratinocytes and explore its possible mechanism. Methods Cell viability was measured using CCK-8. qRT-PCR was performed to measure the mRNA levels of inflammatory factors IL-1β, IL-6, TNF-α, IL-8 and MCP-1. Western blotting was used to detect the protein levels of p65 and p-p65. Results No significant toxicity was observed in HaCaT keratinocytes treated with daphnetin at concentrations below 20 μM for 24 hours. TNF-α stimulation significantly induced the expression of IL-1β(P<0.05), IL-6(P<0.001), TNF-α(P<0.05),IL-8(P<0.05) and MCP-1(P<0.05). Daphnetin treatment partially decreased the inflammatory factors expression in TNF-α-stimulated HaCaT keratinocytes. TNF-α stimulation induced p65 phosphorylation; while daphnetin treatment could inhibit p65 phosphorylation.Conclusion Daphnetin can inhibit the inflammatory response in TNF-α-stimulated HaCaT keratinocytes, which possibly is associated to the inhibition of NF-κB signaling pathway.
Objective To observe the effects of daphnetin on the expression of inflammatory factors in TNF-α-stimulated HaCaT keratinocytes and explore its possible mechanism. Methods Cell viability was measured using CCK-8. qRT-PCR was performed to measure the mRNA levels of inflammatory factors IL-1β, IL-6, TNF-α, IL-8 and MCP-1. Western blotting was used to detect the protein levels of p65 and p-p65. Results No significant toxicity was observed in HaCaT keratinocytes treated with daphnetin at concentrations below 20 μM for 24 hours. TNF-α stimulation significantly induced the expression of IL-1β(P<0.05), IL-6(P<0.001), TNF-α(P<0.05),IL-8(P<0.05) and MCP-1(P<0.05). Daphnetin treatment partially decreased the inflammatory factors expression in TNF-α-stimulated HaCaT keratinocytes. TNF-α stimulation induced p65 phosphorylation; while daphnetin treatment could inhibit p65 phosphorylation.Conclusion Daphnetin can inhibit the inflammatory response in TNF-α-stimulated HaCaT keratinocytes, which possibly is associated to the inhibition of NF-κB signaling pathway.
引文
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[13]GILLITZER R,BERGER R,MIELKE V,et al.Upper keratinocytes of psoriatic skin lesions express high levels of NAP-1/IL-8 mRNA in situ[J].Journal of Investigative Dermatology,1991,97(1):73-79.
[14]QAZI B S,TANG K,QAZI A.Recent advances in underlying pathologies provide insight into interleukin-8 expression-mediated inflammation and angiogenesis[J].International journal of inflammation,2011.
[15]GILLITZER R,WOLFF K,TONG D,et al.MCP-1 mRNA expression in basal keratinocytes of psoriatic lesions[J].Journal of Investigative Dermatology,1993,101(2):127-131.
[16]LEMBO S,CAPASSO R,BALATO A,et al.MCP-1 in psoriatic patients:effect of biological therapy[J].Journal of Dermatological Treatment,2014,25(1):83-86.
[17]BELL S,DEGITZ K,QUIRLING M,et al.Involvement of NF-κBsignalling in skin physiology and disease[J].Cell Signal,2003,15(1):1-7.
[18]HAYDEN M S,GHOSH S.Shared principles in NF-κB signaling[J].Cell,2008,132(3):344-362.
[2]ALBANESI C,PASTORE S.Pathobiology of chronic inflammatory skin diseases:interplay between keratinocytes and immune cells as a target for antiinflammatory drugs[J].Curr Drug Metab,2010,11(3):210-227.
[3]YU W W,LU Z,ZHANG H,et al.Anti-inflammatory and protective properties of daphnetin in endotoxin-induced lung injury[J].J Agric Food Chem,2014,62(51):12315-12325.
[4]LV X,LI Y,XIAO Q,et al.Daphnetin activates the Nrf2-dependent antioxidant response to prevent arsenic-induced oxidative insult in human lung epithelial cells[J].Chem Biol Interact,2019,
[5]HE Z,DONG W,YAO K,et al.Daphnetin inhibits proliferation and glycolysis in colorectal cancer cells by regulating the PI3K/Akt signaling pathway[J].RSC Advances,2018,8(60):34483-34490.
[6]YANG L,YANG Q,ZHANG K,et al.Neuroprotective effects of daphnetin against NMDA receptor-mediated excitotoxicity[J].Molecules,2014,19(9):14542-14555.
[7]LI M,SHI X,CHEN F,et al.Daphnetin inhibits inflammation in the NZB/W F1 systemic lupus erythematosus murine model via inhibition of NF-κBactivity[J].Experimental and therapeutic medicine,2017,13(2):455-460.
[8]WANG D,LU Z,ZHANG H,et al.Daphnetin alleviates experimental autoimmune encephalomyelitis via regulating dendritic cell activity[J].CNSneuroscience&therapeutics,2016,22(7):558-567.
[9]MAHE E,DESCAMPS V.Anti-TNF alpha in dermatology;proceedings of the Ann Dermatol Venereol,F,2002[C].
[10]ANDREA CAVANI C A,GIROLOMONI G.Interferon-γ-stimulated human keratinocytes express the genes necessary for the production of peptide-loaded MHC class II molecules[J].Journal of Investigative Dermatology,1998,110(2):138-142.
[11]ALBANESI C,CAVANI A,GIROLOMONI G.IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes:synergistic or antagonist effects with IFN-γand TNF-α[J].The Journal of Immunology,1999,162(1):494-502.
[12]ALBANESI C,SCARPONI C,SEBASTIANI S,et al.IL-4 enhances keratinocyte expression of CXCR3 agonistic chemokines[J].The Journal of Immunology,2000,165(3):1395-1402.
[13]GILLITZER R,BERGER R,MIELKE V,et al.Upper keratinocytes of psoriatic skin lesions express high levels of NAP-1/IL-8 mRNA in situ[J].Journal of Investigative Dermatology,1991,97(1):73-79.
[14]QAZI B S,TANG K,QAZI A.Recent advances in underlying pathologies provide insight into interleukin-8 expression-mediated inflammation and angiogenesis[J].International journal of inflammation,2011.
[15]GILLITZER R,WOLFF K,TONG D,et al.MCP-1 mRNA expression in basal keratinocytes of psoriatic lesions[J].Journal of Investigative Dermatology,1993,101(2):127-131.
[16]LEMBO S,CAPASSO R,BALATO A,et al.MCP-1 in psoriatic patients:effect of biological therapy[J].Journal of Dermatological Treatment,2014,25(1):83-86.
[17]BELL S,DEGITZ K,QUIRLING M,et al.Involvement of NF-κBsignalling in skin physiology and disease[J].Cell Signal,2003,15(1):1-7.
[18]HAYDEN M S,GHOSH S.Shared principles in NF-κB signaling[J].Cell,2008,132(3):344-362.