银屑病中NLRP3炎症小体通过调控Treg/Th17失衡对角质形成细胞增殖及趋化能力的影响

Effects of NLRP3 Inflammatory Bodies in Psoriasis on the Proliferation of Keratinocytes and Chemotaxis by Regulating the Treg/Th17 Imbalance

目的探讨银屑病中NLRP3炎症小体通过调控Treg/Th17失衡对角质形成细胞增殖及趋化能力的影响。方法通过qRT-PCR、Western Blot、免疫组织化学和免疫荧光化学检测NLRP3在银屑病患者受损皮肤中的表达。体外以10 ng/mL的KGF刺激HaCaT细胞建立银屑病的细胞模型,通过慢病毒介导的sh-NLRP3转染细胞,qRT-PCR和Western blot分析NLRP3表达,CCK-8和Transwell小室分别检测细胞增殖和趋化能力,ELISA检测炎性因子IL-17A、IL-23、IL-1β和趋化因子CXCL1、CXCL2、CXCL8的分泌水平。体内将慢病毒介导的sh-NLRP3注射入小鼠背部局部皮肤,再通过5%咪喹莫特(IMQ)诱导小鼠银屑病样皮损模型,qRT-PCR检测NLRP3和Cytokeratin 14在皮肤组织的表达,PASI评分评价小鼠银屑病皮损面积和疾病严重程度,HE染色观察皮肤组织病理改变,测量皮肤厚度,qRT-PCR检测炎性因子IL-17A、IL-23、IL-1β和趋化因子CXCL1、CXCL2、CXCL8的表达,流式细胞术检测Th17、Treg细胞比例。结果NLRP3在银屑病患者受损皮肤的角质形成细胞中高表达(P=0.0005<0.01),并且在银屑病细胞模型和小鼠模型中也高表达(P=0.003<0.01)。在细胞水平,敲减NLRP3降低银屑病模型中HaCaT细胞的增殖和趋化能力,下调炎性因子IL-17A(P=0.006<0.01)、IL-23(P=0.003<0.01)、IL-1β(P=0.009<0.01)和趋化因子CXCL1(P=0.003<0.01)的表达。在动物水平,敲减NLRP3改善银屑病小鼠的皮损程度,降低PASI评分和表皮厚度,下调炎性因子IL-17A(P=0.009<0.01)、IL-23(P=0.002<0.01)、IL-1β(P=0.004<0.01)和趋化因子CXCL1(P=0.005<0.01)的表达,并增加皮肤组织中Treg/Th17细胞的比例。结论炎症小体NLRP3在银屑病皮损组织中高表达,通过抑制NLRP3可以下调炎性因子IL-17A、IL-23、IL-1β和趋化因子CXCL1的表达,诱导Treg/Th17失衡,进而抑制角质形成细胞增殖及趋化能力,改善银屑病的皮损程度和表皮厚度。

Objective To explore the effect of NLRP3 inflammasome in psoriasis on the proliferation of keratinocytes and chemotaxis by regulating Treg/Th17 imbalance.Methods The expression of NLRP3 in damaged skin of patients with psoriasis was detected by qRT-PCR,Western blot,immunohistochemistry and immunofluorescence chemistry.In vitro,10 ng/mL KGF was used to stimulate HaCaT to establish a cell model of psoriasis.Cells were transfected with lentivirus-mediated sh-NLRP3,and NLRP3 expression was analyzed by qRT-PCR and Western blot.Cells were detected in CCK-8 and Transwell chambers.Proliferation and chemotactic ability,ELISA to detect cell inflammatory factors IL-17A,IL-23,IL-1βand chemokines CXCL1,CXCL2,CXCL8 secretion levels.In vivo,lentivirus-mediated sh-NLRP3 was injected into the local skin of the back of mice,and then 5%imiquimod(IMQ)was used to induce psoriasis-like skin lesions in mice.qRT-PCR detected NLRP3 and cytokeratin 14 in the skin tissue expression,PASI score to evaluate mouse psoriasis skin lesion area and disease severity,hematoxylin-eosin(HE)staining to observe skin tissue pathological changes,skin thickness measurement,qRT-PCR to detect inflammatory factors in mouse skin tissue expression of IL-17A,IL-23,IL-1βand chemokines CXCL1,CXCL2,CXCL8,flow cytometry to detect the proportion of Th17 and Treg cells in mouse skin tissue.Results NLRP3 was highly expressed in keratinocytes of damaged skin of patients with psoriasis(P=0.0005<0.01),and was also highly expressed in psoriasis cell models and mouse models(P=0.003<0.01).At the cellular level,knocking down NLRP3 reduces the proliferation of HaCaT cells and chemotaxis in psoriasis models,and down-regulates the expression of inflammatory factors IL-17A(P=0.006<0.01),IL-23(P=0.003<0.01),IL-1β(P=0.009<0.01)and chemokine CXCL1(P=0.003<0.01).At the animal level,knocking down NLRP3 improves the degree of skin lesions in psoriasis mice,reduces PASI score and epidermal thickness,down-regulates the expression of inflammatory factors IL-17A(P=0.009<0.01),IL-23(P=0.002<0.01),IL-1β(P=0.004<0.01)and chemokine CXCL1(P=0.005<0.01),and increases the ratio of Treg/Th17 cells in skin tissue.Conclusion The inflammatory body NLRP3 is highly expressed in psoriatic lesions.By inhibiting NLRP3,it can down-regulate the expression of inflammatory factors IL-17A,IL-23,IL-1βand chemokine CXCL1,and induce Treg/Th17 imbalance,inhibit the proliferation of keratinocytes and chemotactic ability and improves the degree of psoriatic lesions and epidermal thickness.