基于Notch信号通路探讨银屑平丸含药血清对TNF-α诱导的人表皮角质形成细胞相关蛋白及炎症因子的影响

Exploring the Effect of Yinxieping Pills-Containing Serum on TNF-α-Induced Human Epidermal Keratinocyte-Related Protein and Inflammatory Factors Based on Notch Signaling Pathway

目的基于调节Notch信号通路探讨银屑平丸含药血清对肿瘤坏死因子α(TNF-α)诱导的人表皮角质形成细胞相关蛋白及炎症因子的影响。方法根据中药血清药理学方法制备银屑平丸含药血清、阿维A含药血清,将血清分为5%、10%、20%3个浓度对人表皮角质形成细胞进行干预,采用CCK-8试剂筛选含药血清最佳作用浓度;建立TNF-α诱导的银屑病细胞损伤模型,设置空白组、模型组、银屑平丸含药血清组、阿维A组、模型+γ-分泌酶抑制剂(DAPT)组、银屑平丸含药血清+DAPT组。采用ELISA法检测各组细胞上清液中TNF-α、白细胞介素6(IL-6)、白细胞介素8(IL-8)水平,采用Real-timePCR法检测Notch信号通路Notch1,Jagged-1表达,免疫荧光法检测各组细胞内皮蛋白(Involucrin)表达水平。结果TNF-α最佳作用浓度为10 ng·mL^(-1),银屑平丸含药血清最佳作用浓度为20%。与空白组比较,模型组可使各组细胞上清TNF-α、IL-6、IL-8水平升高(均P<0.0001),Notch1、Jagged-1表达上调(均P<0.0001),并使细胞Involucrin荧光表达增强(P<0.0001);与模型组比较,银屑平丸含药血清组可使细胞上清TNF-α、IL-6、IL-8水平降低(P<0.05,P<0.01),使Notch信号通路Notch1、Jagged-1表达下调(均P<0.0001),并使细胞Involucrin荧光表达减弱(P<0.05);与银屑平丸含药血清组比较,银屑平丸含药血清+DAPT组细胞上清TNF-α、IL-6、IL-8水平降低(P<0.0001,P<0.05,P<0.01),Notch1、Jagged-1表达下调(均P<0.0001),细胞Involucrin荧光表达减弱(P<0.05)。结论银屑平丸含药血清可抑制TNF-α诱导的人表皮角质形成细胞增殖,降低炎症因子表达水平及人表皮角质形成细胞相关蛋白表达,其机制可能与调节Notch信号通路有关。

Objective To explore the effect of Yinxieping Pills-containing serum on tumor necrosis factor-alpha(TNF-α)-induced human epidermal keratinocyte-related protein and inflammatory factors by regulating Notch signaling pathway.Methods YinxiepingPills-containing serum and acitretin-containing serum were prepared according to the serum pharmacology method of the traditional Chinese medicine.The serum was divided into three concentrations of 5%,10%and 20%to intervene on human epidermal keratinocytes,and CCK-8 reagent was used to screen the optimal concentration of drug-containing serum.After the establishment of a TNF-α-induced psoriasis cell injury model,cells were divided into blank group,model group,Yinxieping Pills-containing serum group,acitretin-containing serum group,model+γ-secretase inhibitor(DAPT)group,Yinxieping Pills-containing serum+DAPT group.ELISA was used to detect TNF-α,interleukin-6(IL-6)and interleukin-8(IL-8)levels in the supernatant of each group.Real-time PCR was used to detect the expressions of Notch1 and Jagged-1 of Notch signaling pathway,and immunofluorescence was used to detect the expression level of involucrin in each group of cells.ResultsThe optimal action concentration of TNF-αwas 10 ng·mL^(-1),and the optimal action concentration of Yinxieping Pills-containing serum was 20%.Compared with the blank group,the model group could increase TNF-α,IL-6,IL-8 levels(P<0.0001),up-regulate the expression of Notch1,Jagged-1(P<0.0001)and enhance the fluorescent expression of cellular involucrin in the supernatant of each group(P<0.0001).Compared with the model group,Yinxieping Pills-containing serum group could decrease TNF-α,IL-6,IL-8 levels(P<0.05,P<0.01),down-regulate the expression-of Notch1,Jagged-1 of Notch signaling pathway(P<0.0001)and weaken the fluorescent expression of involucrin(P<0.05)in the cell supernatant.Compared with the Yinxieping Pillscontaining serum group,the Yinxieping Pills-containing serum+DAPT group decreased the levels of TNF-α,IL-6,IL-8(P<0.0001,P<0.05,P<0.01),down-regulated Notch1 and Jagged-1 expressions(P<0.0001),and weakened the fluorescent expression of involucrin(P 0.05).Conclusion YinxiepingPills-containing serum inhibited TNF-α-induced proliferation of human epidermal keratinocytes as well as reduced the expression levels of inflammatory factors and human epidermal keratinocyte-related protein.Its mechanism may be associated with the regulation of Notch signaling pathway.