目的 探讨复方泽漆冲剂含药血清对银屑病细胞模型PI3K/Akt/NF-κB通路及NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体的调控作用以阐明该药对银屑病的干预机制。方法 制备复方泽漆冲剂大鼠含药血清,体外以10 ng·mL^-1的角质形成细胞生长因子(KGF)刺激人永生化角质形成细胞系HaCaT建立银屑病细胞模型,将细胞分为对照组、模型组及复方泽漆冲剂5 %、10 %、20 %含药血清组,CCK-8法检测不同体积分数含药血清作用于HaCaT细胞0、24、48、72 h时的细胞活力,确定复方泽漆冲剂含药血清最佳体积分数及作用时间。体外以10 ng·mL^-1的KGF刺激HaCaT细胞建立银屑病细胞模型,以siRNA基因沉默HaCaT细胞NLRP3,将细胞分为对照组、模型组、10 %含药血清组、si-NLRP3-NC组(质粒阴性对照)、si-NLRP3组,CCK-8法检测复方泽漆冲剂含药血清对银屑病模型细胞相对活力的影响;ELISA检测含药血清干预后白细胞介素-1β(IL-1β)、IL-17α、IL-23、IL-8和趋化因子配体(CXCL)1、CXCL2的分泌水平及乳酸脱氢酶(LDH)释放量;实时荧光定量PCR (qRT-PCR)分析NLRP3、半胱氨酸蛋白酶(Caspase-1)、凋亡相关斑点样蛋白(ASC)、磷脂酰激醇激酶(PI3K)、丝氨酸/苏氨酸激酶(Akt)、核因子-κB (NF-κB)的mRNA表达水平;Western blotting分析NLRP3、Caspase-1、ASC、p-PI3K、p-Akt、p-NF-κB、cleaved-Caspase-1、Gasdermin D (GSDMD)的蛋白表达水平;免疫荧光检测p-PI3K、p-Akt、p-NF-κB、ASC、Caspase-1的表达与分布。结果 10 %复方泽漆冲剂含药血清及作用48 h为含药血清最佳作用体积分数和时间;在KGF刺激HaCaT细胞增殖的银屑病细胞模型中,与对照组比较,IL-17α、IL-23、IL-1β、IL-8和CXCL1、CXCL2的分泌水平升高,LDH释放量显著升高;与模型组相比较,10 %复方泽漆冲剂含药血清干预48 h后,银屑病细胞模型中IL-17α、IL-23、IL-1β、IL-8和CXCL1、CXCL2表达显著降低,LDH释放量显著下降;NLRP3、ASC、Caspase-1、PI3K、Akt、NF-κB的mRNA表达水平也有所降低;含药血清处理后,NLRP3、Caspase-1、ASC、p-PI3K、p-Akt、p-NF-κB、cleaved-Caspase-1、GSDMD的蛋白表达水平下降明显,p-PI3K、p-Akt、p-NF-κB、ASC、Caspase-1的表达与分布也有所减少。结论 复方泽漆冲剂含药血清通过下调PI3K/Akt/NF-κB信号通路及NLRP3炎症小体发挥对银屑病细胞模型的干预作用。

Objective To investigate the regulatory effect of Compound Zeqi Punch-containing serum on PI3K/Akt/NF-κB pathway and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome in psoriasis cell model, so as to elucidate the intervention mechanism of Compound Zeqi Punch on psoriasis. Methods Preparation of Compound Zeqi Punch rat serum, NLRP3 gene silencing was performed on human immortalized keratinocyte cell line HaCaT cells, HaCaT cells were stimulated with 10 ng·mL^-1 keratinocyte growth factor (KGF) in vitro to establish a psoriasis cell model. The cells were divided into control group, model group, Compound Zeqi Punch-containing serum at 5 %, 10 % and 20 % dose groups. CCK-8 method was used to detect the cell viability of HaCaT cells treated with different concentrations of Compound Zeqi Punch-containing serum at 0 h, 24 h, 48 h and 72 h, and the optimal concentration and action time of Compound Zeqi Punch-containing serum were determined. ELISA was used to detect the secretion levels of inflammatory factors IL-1β, IL-17α, IL-23, IL-8, chemokine CXCL1 and CXCL2 and the release of LDH before and after the intervention of Compound Zeqi Punch-containing serum. The mRNA expression levels of NLRP3, cysteinyl aspartate specific proteinase (Caspase-1), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), PI3K, Akt and NF- κ B were analyzed by qRT-PCR. The protein expression levels of NLRP3, Caspase-1, ASC, p-PI3K, p-Akt, p-NF- κB, cleaved-Caspase-1 and gasdermin D (GSDMD were analyzed by Western blotting. The expression and distribution of p-PI3K, p-Akt, p-NF-κB, ASC and Caspase-1 were detected by immunofluorescence. Results The serum containing 10 % Compound Zeqi Punch and 48 h were selected as the optimal concentration and time of drug-containing serum. In the psoriasis cell model of KGF-stimulated HaCaT cell, compared with the control group, the secretion levels of inflammatory factors IL-17α, IL-23, IL-1β, IL-8 and chemokines CXCL1 and CXCL2 were increased, and the release of lactate dehydrogenase (LDH) was significantly increased. Compared with the model group, the expression of inflammatory factors IL-17α, IL-23, IL-1β, IL-8 and CXCL1 and CXCL2 in the psoriasis cell model was significantly decreased after 48 h of intervention with 10 % Compound Zeqi Punchcontaining serum, and the release of LDH was significantly decreased. The mRNA expression levels of NLRP3, ASC, Caspase-1, PI3K, Akt and NF- κB were also decreased. After treatment with the optimal concentration of Compound Zeqi Punch-containing serum, the protein expression levels of NLRP3, Caspase-1, ASC, p-PI3K, p-Akt, p-NF- κB, cleaved-Caspase-1 and GSDMD decreased significantly, and the expression and distribution of p-PI3K, p-Akt, p-NF-κB,ASC and Caspase-1 also decreased. Conclusion Compound Zeqi Punch-containing serum exerts an intervention effect on the psoriasis cell model by affecting the PI3K/Akt/NF-κB signaling pathway and NLRP3 inflammasome.

R285.5

安徽中医药大学第一附属医院临床科学研究项目(2020yfyzc18)