目的 探討復(fù)方澤漆沖劑含藥血清對銀屑病細(xì)胞模型PI3K/Akt/NF-κB通路及NOD樣受體熱蛋白結(jié)構(gòu)域相關(guān)蛋白3(NLRP3)炎癥小體的調(diào)控作用以闡明該藥對銀屑病的干預(yù)機(jī)制。方法 制備復(fù)方澤漆沖劑大鼠含藥血清,體外以10 ng·mL^-1的角質(zhì)形成細(xì)胞生長因子(KGF)刺激人永生化角質(zhì)形成細(xì)胞系HaCaT建立銀屑病細(xì)胞模型,將細(xì)胞分為對照組、模型組及復(fù)方澤漆沖劑5 %、10 %、20 %含藥血清組,CCK-8法檢測不同體積分?jǐn)?shù)含藥血清作用于HaCaT細(xì)胞0、24、48、72 h時(shí)的細(xì)胞活力,確定復(fù)方澤漆沖劑含藥血清最佳體積分?jǐn)?shù)及作用時(shí)間。體外以10 ng·mL^-1的KGF刺激HaCaT細(xì)胞建立銀屑病細(xì)胞模型,以siRNA基因沉默HaCaT細(xì)胞NLRP3,將細(xì)胞分為對照組、模型組、10 %含藥血清組、si-NLRP3-NC組(質(zhì)粒陰性對照)、si-NLRP3組,CCK-8法檢測復(fù)方澤漆沖劑含藥血清對銀屑病模型細(xì)胞相對活力的影響;ELISA檢測含藥血清干預(yù)后白細(xì)胞介素-1β(IL-1β)、IL-17α、IL-23、IL-8和趨化因子配體(CXCL)1、CXCL2的分泌水平及乳酸脫氫酶(LDH)釋放量;實(shí)時(shí)熒光定量PCR (qRT-PCR)分析NLRP3、半胱氨酸蛋白酶(Caspase-1)、凋亡相關(guān)斑點(diǎn)樣蛋白(ASC)、磷脂酰激醇激酶(PI3K)、絲氨酸/蘇氨酸激酶(Akt)、核因子-κB (NF-κB)的mRNA表達(dá)水平;Western blotting分析NLRP3、Caspase-1、ASC、p-PI3K、p-Akt、p-NF-κB、cleaved-Caspase-1、Gasdermin D (GSDMD)的蛋白表達(dá)水平;免疫熒光檢測p-PI3K、p-Akt、p-NF-κB、ASC、Caspase-1的表達(dá)與分布。結(jié)果 10 %復(fù)方澤漆沖劑含藥血清及作用48 h為含藥血清最佳作用體積分?jǐn)?shù)和時(shí)間;在KGF刺激HaCaT細(xì)胞增殖的銀屑病細(xì)胞模型中,與對照組比較,IL-17α、IL-23、IL-1β、IL-8和CXCL1、CXCL2的分泌水平升高,LDH釋放量顯著升高;與模型組相比較,10 %復(fù)方澤漆沖劑含藥血清干預(yù)48 h后,銀屑病細(xì)胞模型中IL-17α、IL-23、IL-1β、IL-8和CXCL1、CXCL2表達(dá)顯著降低,LDH釋放量顯著下降;NLRP3、ASC、Caspase-1、PI3K、Akt、NF-κB的mRNA表達(dá)水平也有所降低;含藥血清處理后,NLRP3、Caspase-1、ASC、p-PI3K、p-Akt、p-NF-κB、cleaved-Caspase-1、GSDMD的蛋白表達(dá)水平下降明顯,p-PI3K、p-Akt、p-NF-κB、ASC、Caspase-1的表達(dá)與分布也有所減少。結(jié)論 復(fù)方澤漆沖劑含藥血清通過下調(diào)PI3K/Akt/NF-κB信號通路及NLRP3炎癥小體發(fā)揮對銀屑病細(xì)胞模型的干預(yù)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

安徽中醫(yī)藥大學(xué)第一附屬醫(yī)院臨床科學(xué)研究項(xiàng)目(2020yfyzc18)