白介素-18及NOD样受体蛋白3炎性小体在痤疮发病中的作用研究
|
摘要
目的探讨白介素-18(IL-18)及NOD样受体蛋白3(NLRP3)炎性小体在痤疮炎性发病机制中的作用。方法以痤疮丙酸杆菌悬液[感染复数(MOI)=0、10、20、30)]刺激人角质形成细胞(NHEK)12h、24h、36h,ELISA法检测NHEK中IL-18蛋白的表达量,real-time PCR检测NHEK中IL-18mRNA的表达。将NHEK细胞分为3组:特异性NLRP3小干扰RNA(siRNA)转染NHEK 36h后,以MOI=30的痤疮丙酸杆菌悬液刺激NHEK 36h(siRNA组);不转染、不以菌液刺激的NHEK为空白对照组;不转染、只以MOI=30的痤疮丙酸杆菌悬液刺激36h的NHEK为阳性对照组。ELISA法和real time-PCR法检测3组NHEK中IL-18蛋白和基因的表达量,Western blot法检测NLRP3炎性小体的表达情况。结果 NHEK经痤疮丙酸杆菌悬液刺激后IL-18蛋白和基因的表达量增加,具有时间相关性和剂量相关性(r均>0.75,P<0.05),以菌悬液MOI=30、刺激36h最为明显。siRNA组IL-18蛋白和基因表达量、NLRP3炎性小体的表达量较阳性对照组降低,但高于空白对照组,差异有统计学意义(P<0.05)。结论痤疮丙酸杆菌刺激NHEK分泌IL-18可能需要NLRP3炎性小体的参与。
Objective To determine the role of inlerleukin-18(IL-18)and nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3(NLRP3)in acne vulgaris.Methods We used propionibacterium acnes(P.acnes)suspensions[multiplicity of infection(MOI)=0,10,20,30]to stimulate normal human epidermal kerationocytes(NHEKs)for 12,24,and 36 h,respectively.Enzyme-linked immunosorbent assay(ELISA)was performed to detect the protein level of IL-18.Real-time quantitative PCR(real-time PCR)was adopted to detect the mRNA of IL-18.The NHEKs were divided into three groups:(1) siRNA group:NHEKs were pretreated with siRNA for 36 h,followed by 36 hexposure to MOI=30of P.ances suspensions;(2) blank control group:NHEKs free from siRNA transfection and P.ances suspensions;(3) positive control group:NHEKs free from siRNA transfection were exposed to P.ances suspensions(MOI=30).The expression of NLRP3 was detected by Western blot.Results The expressions of protein and mRNA of IL-18 increased with exposure to P.ances suspensions in a dose responsive way(r>0.75,P<0.05),with the peak effects showing for MOI=30at 36 h.The expression of IL-18 decreased in the siRNA group compared with the positive control,but was still higher than that of the blank group(P<0.05).Conclusion P.ances stimulates NHEK cells to secrete IL-18.The process possibly requires the involvement of NLRP3.
Objective To determine the role of inlerleukin-18(IL-18)and nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3(NLRP3)in acne vulgaris.Methods We used propionibacterium acnes(P.acnes)suspensions[multiplicity of infection(MOI)=0,10,20,30]to stimulate normal human epidermal kerationocytes(NHEKs)for 12,24,and 36 h,respectively.Enzyme-linked immunosorbent assay(ELISA)was performed to detect the protein level of IL-18.Real-time quantitative PCR(real-time PCR)was adopted to detect the mRNA of IL-18.The NHEKs were divided into three groups:(1) siRNA group:NHEKs were pretreated with siRNA for 36 h,followed by 36 hexposure to MOI=30of P.ances suspensions;(2) blank control group:NHEKs free from siRNA transfection and P.ances suspensions;(3) positive control group:NHEKs free from siRNA transfection were exposed to P.ances suspensions(MOI=30).The expression of NLRP3 was detected by Western blot.Results The expressions of protein and mRNA of IL-18 increased with exposure to P.ances suspensions in a dose responsive way(r>0.75,P<0.05),with the peak effects showing for MOI=30at 36 h.The expression of IL-18 decreased in the siRNA group compared with the positive control,but was still higher than that of the blank group(P<0.05).Conclusion P.ances stimulates NHEK cells to secrete IL-18.The process possibly requires the involvement of NLRP3.
引文
[1]LEEMING JP,HOLLAND KT,CUNCLIFFE WJ.The microbial colonization of inflamed acne vulgaris lesions.Br JDermatol,1988,118(2):203-208.
[2]VOWELS BR,YANG S,LEYDEN JJ.Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes:implications for chronic inflammatory acn.Infect Immun,1995,63(8):3158-3165.
[3]MIN Q,ASLAN P,MYUNGHWA K,et al.Propionibacterium acnes induces IL-1βsecretion via the NLRP3 inflammasome in human monocytes.J Invest Dermatol,2014,134(2):381-388.
[4]ASKARI N,GHAZANFARI T,YARAEE R,et al.Association between acne and serum pro-inflammatory cytokines(IL-1α,IL-1β,IL-1Ra,IL-6,IL-8,IL-12)in mustard gas-exposed patients:sardasht-iran cohort study.Arch Iran Med,2017,20(2):86-94.
[5]SALAMON M,SYSAJEDRZEJOWSKA A,LUKAMOWICE J,et al.Concentration of selected cytokines in serum of patients with acne rosacea.Przegl Lek,2008,65(9):371-374.
[6]林新瑜,罗旭松,董巍,等.痤疮患者血清白介素-1α、白介素-6、白介素-8和α-肿瘤坏死因子水平的检测分析.临床皮肤科杂志,2003,32(6):335-336.
[7]BEROLIA S,EVASA R,FREDRIK E,et al.Propionibacterium acnes activates caspase-1 in human neutrophils.APMIS,2013,121(7):652-663.
[8]AN HJ,LEE WR,KIM KH,et al.Inhibitory effects of bee venom on Propionibacterium acnes-induced inflammatory skin disease in an animal model.Int J Mol Med,2014,34(5):1341-1348.
[9]WATSON MK,DAPUL GL.Modulation of cytokine and nitric oxide production by keratinocytes,epithelial cells,and mononuclear phagocytes in a co-culture model of inflammatory acne.J Drugs Dermatol,2012,11(7):834-836.
[10]LIU PT,PHAN J,TANG D,et al.CD209(+)macrophages mediate host defense against Propionibacterium acnes.J Immunol,2008,180(7):4919-4923.
[11]KIM S,BAUERNFEIND F,ABLASSER A,et al.Listeria monocytogenes is sensed by the NLRP3 and AIM2inflammasome.Eur J Immunol,2010,40(6):1545-1551.
[12]LIU PF,HSIEH YD,LIN YC,et al.Propionibacterium acnes in the pathogenesis and immunotherapy of acne vulgaris.Current Drug Metabolism,2015,16(4):245-254.
[2]VOWELS BR,YANG S,LEYDEN JJ.Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes:implications for chronic inflammatory acn.Infect Immun,1995,63(8):3158-3165.
[3]MIN Q,ASLAN P,MYUNGHWA K,et al.Propionibacterium acnes induces IL-1βsecretion via the NLRP3 inflammasome in human monocytes.J Invest Dermatol,2014,134(2):381-388.
[4]ASKARI N,GHAZANFARI T,YARAEE R,et al.Association between acne and serum pro-inflammatory cytokines(IL-1α,IL-1β,IL-1Ra,IL-6,IL-8,IL-12)in mustard gas-exposed patients:sardasht-iran cohort study.Arch Iran Med,2017,20(2):86-94.
[5]SALAMON M,SYSAJEDRZEJOWSKA A,LUKAMOWICE J,et al.Concentration of selected cytokines in serum of patients with acne rosacea.Przegl Lek,2008,65(9):371-374.
[6]林新瑜,罗旭松,董巍,等.痤疮患者血清白介素-1α、白介素-6、白介素-8和α-肿瘤坏死因子水平的检测分析.临床皮肤科杂志,2003,32(6):335-336.
[7]BEROLIA S,EVASA R,FREDRIK E,et al.Propionibacterium acnes activates caspase-1 in human neutrophils.APMIS,2013,121(7):652-663.
[8]AN HJ,LEE WR,KIM KH,et al.Inhibitory effects of bee venom on Propionibacterium acnes-induced inflammatory skin disease in an animal model.Int J Mol Med,2014,34(5):1341-1348.
[9]WATSON MK,DAPUL GL.Modulation of cytokine and nitric oxide production by keratinocytes,epithelial cells,and mononuclear phagocytes in a co-culture model of inflammatory acne.J Drugs Dermatol,2012,11(7):834-836.
[10]LIU PT,PHAN J,TANG D,et al.CD209(+)macrophages mediate host defense against Propionibacterium acnes.J Immunol,2008,180(7):4919-4923.
[11]KIM S,BAUERNFEIND F,ABLASSER A,et al.Listeria monocytogenes is sensed by the NLRP3 and AIM2inflammasome.Eur J Immunol,2010,40(6):1545-1551.
[12]LIU PF,HSIEH YD,LIN YC,et al.Propionibacterium acnes in the pathogenesis and immunotherapy of acne vulgaris.Current Drug Metabolism,2015,16(4):245-254.