基于网络药理学及实验验证探讨淫羊藿苷干预强直性脊柱炎的分子机制

Exploration of the Molecular Mechanism of Icariin Intervention in Ankylosing Spondylitis Based on Network Pharmacology and Experimental Verification

目的基于网络药理学及实验验证确定淫羊藿苷(icariin,ICA)干预强直性脊柱炎(ankylosing spondylitis,AS)的潜在作用靶点。方法应用网络药理学方法和分子对接预测ICA治疗AS的可能靶点和途径。招募15名活动期AS患者和15名性别、年龄匹配的健康志愿者,提取外周血单个核细胞(peripheral blood mononuclear cell,PBMC)进行体外实验,检测经不同浓度ICA干预后蛋白酪氨酸激酶2/信号转导与转录激活子3(janus kinase 2/signal transducer and activator of transcription 3,JAK2/STAT3)信号通路相关分子的表达。结果转录因子p65(transcription factor p65,RELA)、一氧化氮合成酶(nitric oxide synthase,NOS)2、丝裂原活化蛋白激酶(mitogen-activated Protein Kinase,MAPK)14、MAPK1、胰岛素样生长因子(insulin-like growth factor,IGF)1、热休克70 kDa蛋白(heat shock 70 kDa protein,HSPA)1A、热休克90 kDa蛋白α(HSP90A)A1、JAK2和蛋白激酶Cβ(protein kinase C beta,PRKCB)可能是ICA调控AS的潜在作用靶点。体外实验结果表明,活动期AS患者PBMC中JAK2和STAT3的磷酸化水平明显高于对照组(P<0.05),经ICA体外干预后AS患者PBMC中JAK2和STAT3磷酸化水平显著降低(P<0.05)。特异性转录因子维甲酸相关孤儿核受体(retinoic acid related orphan receptor,ROR)γt蛋白和其编码基因RORc在活动期AS患者中的表达显著高于对照组(P<0.05),ICA可下调RORγt蛋白和RORc mRNA的表达(P<0.05)。结论ICA可能通过RELA、NOS2、MAPK14、MAPK1、IGF1、HSPA1A、HSP90AA1、JAK2等关键靶点和JAK2/STAT3信号通路发挥抗AS的作用。

Objective To explore the potential target of icariin(ICA)in the intervention of ankylosing spondylitis(AS)by using network pharmacology and experimental verification.Methods Network pharmacology and molecular docking were used to predict possible targets and pathways for ICA treatment of AS.A total of 15 active AS patients and 15 gender-and age-matched healthy volunteers were recruited to extract peripheral blood mononuclear cells(PBMCs)for in vitro experiments to detect the expression of janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway related molecules after different concentrations of ICA intervention.Results Transcription factor p65(RELA),nitric oxide synthase(NOS)2,mitogen-activated Protein Kinase(MAPK)14,MAPK 1,insulin-like growth factor(IGF)1,heat shock 70 kDa protein(HSPA)1A,heat shock 90 kDa protein alpha(HSP90A)A1,JAK2 and protein kinase C beta(PRKCB)may be potential targets for ICA regulation of AS.The results showed that the phosphorylation levels of JAK2 and STAT3 in PBMCs of active AS patients were significantly higher than those of control group(P<0.05),the phosphorylation levels of JAK2 and STAT3 in PBMCs of AS patients were significantly decreased after ICA in vitro intervention(P<0.05).The expression of specific transcription factors retinoic acidrelated orphan receptor(ROR)γt and its coding gene RORc in active AS patients was significantly higher than that in the control group(P<0.05),and ICA could down-regulate the expression of RORc protein and RORc mRNA(P<0.05).Conclusion ICA may play a role in the treatment of AS through key targets such as RELA,NOS2,MAPK14,MAPK1,IGF1,HSPA1A,HSP90AA1,JAK2 and JAK2/STAT3 signaling pathway.