山奈酚调控细胞衰老治疗类风湿性关节炎的体外研究

Kaempferol exerts therapeutic effect on rheumatoid arthritis by regulating cell senescence:a study based on network pharmacology and in vitro experiments

目的 基于网络药理学以及体外实验探讨山奈酚治疗类风湿性关节炎(rheumatoid arthritis, RA)的机制。方法 通过TCMSP及SwissTarget数据库查询山奈酚靶点,检索GeneCards和DisGeNET数据库,获得RA相关功能靶点。山奈酚与RA靶点通过Venny2.1.0交叉后,利用STRING数据库进行蛋白互作(protein-protein interaction, PPI)分析,并对山奈酚和靶点进行分子对接。随后用脂多糖(lipopolysaccharide, LPS)与山奈酚处理小鼠单核细胞白血病细胞RAW264.7进行验证,分组为:Control组、LPS组、LPS+Kaempferol组。MTT法检测山奈酚对RAW264.7细胞毒性的影响。使用LPS诱导RAW264.7细胞构建炎症细胞模型,检测各个处理组衰老和衰老相关炎症因子表达的影响。通过SA-β-Gal检测各组RAW264.7细胞β-半乳糖苷酶水平的变化。细胞免疫荧光检测各组IL-6的表达。结果 共得到138个山奈酚的作用靶点,山奈酚抗RA的潜在作用靶点76个。PPI分析结果显示TNF、EGFR、PTGS2、AKT1可能是山奈酚抗RA的核心靶点。分子对接结果表明,山奈酚与核心靶点TNF、EGFR、PTGS2、AKT1能形成较稳定的复合物。MTT结果证明,在24 h时,0.125~16μmol/L山奈酚对RAW264.7细胞增殖均具有促进作用(P<0.05)。在48 h时,0.125~8μmol/L山奈酚能够促进RAW264.7细胞增殖(P<0.05)。而72 h时,0.125~16μmol/L山奈酚对RAW264.7细胞增殖没有显著影响。qPCR和Westerm blot结果表明山奈酚能够降低LPS诱导的IL-1β、IL-6以及TNF-α的表达(P<0.05)。山奈酚也降低了细胞衰老标志基因p16和p21的表达,同时促进细胞周期标志物CCND1和CCNE1的表达(P<0.05)。免疫荧光结果表明山奈酚能够降低IL-6表达水平(P<0.05)。SA-β-Gal染色结果显示,LPS处理可以促进RAW264.7细胞β-半乳糖苷酶的水平(P<0.05),而山奈酚可以缓解LPS诱导的β-半乳糖苷酶水平增加(P<0.05)。结论 山奈酚可能通过调控炎症因子IL-1β、IL-6和TNF-α与p16/p21衰老相关基因,防止免疫细胞过早衰老,发挥治疗RA的作用。

Objective To investigate the mechanism of kaempferol in the treatment of rheumatoid arthritis(RA)based on network pharmacology and in vitro experiments.MethodsKaempferol targets were queried by TCMSP and SwissTarget databases,and RA related functional targets were searched in the GeneCards and DisGeNET databases.After the cross between kaempferol and RA targets through Venny2.1.0,STRING database was used for proteinprotein interaction(PPI)analysis,and molecular docking between kaempferol and the targets was conducted.Mouse mononuclear leukemia RAW264.7 cells were treated by lipopolysaccharide(LPS)and kaempferol,and divided into control group,LPS group,LPS+Kaempferol group.MTT assay was used to determine the cytotoxicity of RAW264.7 cells after kaempferol treatment.LPSinduced RAW264.7 cells were used to construct a cellular inflammatory model.The change in aging and agingrelated inflammatory factors were detected in each treatment group.The content of β-galactosidase was detected by SA-β-Gal,and the expression of IL-6 was detected by cellular immunofluorescence assay.ResultsA total of 138 targets of kaempferol and 76 potential targets of kaempferol against RA were obtained.PPI analysis showed that TNF,EGFR,PTGS2 and AKT1 may be the core targets of kaempferol against RA.The results of molecular docking indicated that kaempferol could form stable complexes with the core targets of TNF,EGFR,PTGS2 and AKT1.The results of MTT assay displayed that the treatment of 0.125 to 16μmol/L kaempferol for 24 h promoted the proliferation of RAW264.7 cells(P<0.05),the doses of 0.125 to 8μmol/L for 48 h exerted similar effect on the proliferation(P<0.05),but the dose of 0.125 to 16μmol/L for 72 h had no such significant effect.The results of qPCR and Western blotting showed that kaempferol decreased the expression of cell senescence markers p16 and p21 and promoted the expression of cell cycle markers CCND1 and CCNE1(P<0.05).Cellular immunofluorescence assay indicated that kaempferol reduced the expression of IL-6(P<0.05).SA-β-Gal staining displayed that LPS treatment promoted β-galactosidase level in RAW264.7 cells(P<0.05),and this effect was reversed by kaempferol treatment(P<0.05).ConclusionKaempferol may regulate the expression of IL-1β,IL-6 and TNFαand p16/p21 senescence related genes,prevent premature aging of immune cells,and thus play a therapeutic role for RA.