[关键词]
[摘要]
目的 探讨柚皮苷通过P38 MAPK/NF-κB通路对脂多糖(LPS)致HaCaT细胞炎症损伤的抑制作用。方法 LPS(0、0.1、1.0、10.0、20.0 μg/mL)孵育24 h刺激人永生化角质细胞HaCaT,模拟银屑病模型,MTT法观察细胞存活率变化,Western bloting法检测白介素(IL)-6和NF-κB蛋白表达;MTT法观察柚皮苷(0、5、10、20、40、80、160、320μmol/L)作用24 h对HaCaT存活率的影响;选择LPS、柚皮苷最佳作用浓度。柚皮苷(20和40 μmol/L)作用银屑病模型HaCaT细胞24 h,实时荧光定量PCR(qRT-PCR)法检测IL-6、IL-1β、IL-17和肿瘤坏死因子-α(TNF-α)的mRNA表达水平变化;Western blotting法检测细胞核内转录因子NF-κB p65、P38 MAPK磷酸化蛋白水平以及炎症因子IL-6蛋白水平。结果 LPS增加HaCaT细胞存活率,并上调NF-κB p65和IL-6蛋白表达,呈剂量相关性,炎症反应在20 μg/mL达到高峰;5~160 μmol/L柚皮苷对HaCaT细胞无明显毒性作用。与模型组比较,柚皮苷能够显著抑制炎症因子IL-6、IL-1β、IL-17和TNF-α的转录水平(P<0.05);同时能够显著抑制LPS诱导的NF-κB p65和P38 MAPK磷酸化蛋白水平、抑制IL-6蛋白的表达(P<0.05)。结论 柚皮苷抑制脂多糖致HaCaT细胞炎症反应,发挥改善银屑病的作用,机制可能与抑制P38 MAPK/NF-κB信号通路有关。
[Key word]
[Abstract]
Objective To investigate the inhibitory effects of naringin on HaCaT cells inflammation induced by lipopolysaccharide (LPS) through NF-κB/P38 pathway. Methods LPS (0, 0.1, 1.0, 10.0, 20.0 μg/mL) incubated for 24 hours stimulated human immortalized keratinocytes HaCaT to simulate psoriasis model. MTT method was used to observe the changes of cell survival rate, and Western blotting method was used to detect the expression of IL-6 and NF-κB protein. MTT method was used to observe the effect of naringin (0, 5, 10, 20, 40, 80, 160, 320 μmol/L) on the survival rate of HaCaT cells. The optimal concentration of LPS and naringin was selected. Naringin (20 and 40 μmol/L) acted on HaCaT cells of psoriasis model for 24 hours. Reverse transcription polymerase chain reaction (qRT-PCR) was performed to determine the mRNA expression levels of IL-6, IL-1β, IL-17 and TNF-α. Western blotting were used to detect the phosphorylation levels of P38 MAPK and NF-κB p65 and the inflammatory factor IL-6. Results LPS increased the survival rate of HaCaT cells, and up-regulated the protein expression of NF-κB p65 and IL-6, which was dose-related, and the inflammatory response peaked at 20 μg/mL. 5~160 μmol/L naringin had no obvious toxic effect on HaCaT cells. Compared with model group, naringin can significantly inhibit the transcription of inflammatory cytokines IL-6, IL-1β, IL-17 and TNF-α (P<0.05). Meanwhile, naringin can significantly inhibit LPS-induced phosphorylation of P38 MAPK and NF-κB p65, inhibiting the expression of IL-6 protein (P<0.05). Conclusion Naringin inhibits inflammation of HaCaT cells induced by lipopolysaccharide and plays a role in improving psoriasis. The mechanism may be related to the inhibition of P38 MAPK/NF-κB signaling pathway.
[中图分类号]
R962.2
[基金项目]
黑龙江省属高等学校基本科研业务费科研项目(2015-KYYWF-002);牡丹江医学院研究生创新科研项目(2017YJSCX-20MY)