新风胶囊含药血清通过抑制VEGFA/SDF-1/CXCR4通路缓解强直性脊柱炎患者血小板活化的机制

Xinfeng capsule-containing serum relieves platelet activation by inhibiting VEGFA/SDF-1/CXCR4 pathway in patients with ankylosing spondylitis

目的通过体外实验研究新风胶囊(XFC)对VEGFA/SDF-1/CXCR4通路的影响,探讨其改善强直性脊柱炎(AS)患者血小板活化的的机制。方法分离外周血单核细胞(PBMC),分为正常组(NC)、模型组(MC)、新风胶囊组(XFC)、AMD3100组,MTT法检测XFC最佳浓度,免疫荧光法检测CD62p、CD40L、PDGFA的表达,ELISA法检测TNF-ɑ、IL-1β、IL-4、IL-10、VEGFA及VEGFR的表达,实时荧光定量PCR(RT-qPCR)法检测SDF-1、CXCR4、VEGFA mRNA的表达,Western blot法检测SDF-1、CXCR4、VEGFA、VEGFR蛋白的表达。结果中剂量XFC在24 h时对细胞抑制作用最强。与正常组相比,模型组CD62p、CD40L、PDGFA、IL-1β、TNF-α、VEGFA、VEGFR、SDF-1、CXCR4表达均升高,IL-4、IL-10降低(P<0.05,P<0.01)。与模型组相比,XFC组CD62p、CD40L、PDGFA、IL-1β、TNF-α、VEGFA、VEGFR、SDF-1、CXCR4表达均降低,IL-4、IL-10升高(P<0.05,P<0.01)。结论 XFC具有类AMD3100样作用,通过抑制VEGFA/SDF-1/CXCR4通路的表达,下调IL-1β、TNF-α,上调IL-4、IL-10,调节免疫炎症反应,从而降低AS患者血小板活化。

This study was designed to investigate the effect of Xinfeng Capsule(XFC) on VEGFA/SDF-1/CXCR4 pathway in vitro and to explore the mechanism of relieving platelet activation in patients with ankylosing spondylitis(AS). Peripheral blood mononuclear cells(PBMC) from normal subjects and AS patients were divided into normal group(NC), model group(MC), Xinfeng capsule group(XFC) and AMD3100 group. The optimal concentration of XFC was detected by MTT assay;the expression levels of CD62 p, CD40 L and PDGFA were detected by fluorescence method;the expression levels of TNF-ɑ, IL-1β, IL-4, IL-10, VEGFA and VEGFR were detected by ELISA. The mRNA and protein expression of SDF-1, CXCR4 and VEGFA were detected by real-time fluorescent quantitative PCR(RT-qPCR) and Western blotting, respectively. MTT results showed that medium dose XFC had the strongest inhibitory effect on cells at 24 h. Compared with the normal group, the expression of CD62 p,CD40 L, PDGFA, IL-1β, TNF-α, VEGFA, VEGFR, SDF-1 and CXCR4 increased, while IL-4 and IL-10 decreased in model group(P<0.05, P<0.01), and XFC could reverse all these changes(P<0.05, P<0.01). In conclusion, XFC may relieve platelet activation in AS patients by inhibiting the expression of VEGFA/SDF-1/CXCR4 pathway, downregulating IL-1β, TNF-α, up-regulating IL-4 and IL-10, and regulating immune inflammatory response.