复方七芍降压片对TNF-α诱导的与平滑肌细胞共培养的血管内皮细胞中炎症因子与AP-1、MCP-1蛋白表达的影响

Effects of compound Qishao Jiangya tablet on inflammatory factors,AP-1 and MCP-1 in vascular endothelial cells stimulated with TNF-αand co-cultured with smooth muscle cells

目的观察复方七芍降压片对TNF-α诱导的与平滑肌细胞共培养的血管内皮细胞中炎症因子与AP-1、MCP-1蛋白表达情况,探讨复方七芍降压片抑制血管重塑的作用机制。方法利用炎症因子TNF-α联合血管内皮细胞和血管平滑肌细胞共培养体系构建高血压炎症环境。将共培养好的细胞随机分为7组,即正常对照组(A)、模型组(B)、空白血清对照组(C)、MRS2578组(D)、厄贝沙坦片含药血清组(E)、复方七芍降压片含药血清组(F)、MRS2578+复方七芍降压片含药血清组(G)。除正常对照组外,其余各组加入炎症因子TNF-α,浓度10 ng/mL;厄贝沙坦片含药血清组、复方七芍降压片含药血清组和MRS2578+复方七芍降压片含药血清组加入10%的含药血清;MRS2578组与MRS2578+复方七芍降压片含药血清组加入10μm的P2Y6受体阻断剂MRS2578;造模与给药干预24 h后,采用CCK8法检测内皮细胞及平滑肌细胞的存活率;采用ELISA法检测培养基上清液中炎症因子IL-8的水平;采用免疫荧光、Western Blot、实时荧光定量法(qPCR)技术检测内皮细胞中AP-1、MCP-1的表达。结果与正常对照组比较,模型组和空白血清对照组内皮细胞及平滑肌细胞存活率明显降低(P<0.01),IL-8水平明显升高(P<0.01),内皮细胞中AP-1、MCP-1的荧光强度值、灰度值比值、mRNA水平均明显升高(P<0.01);与模型组比较,复方七芍降压片含药血清组内皮细胞及平滑肌细胞存活率明显升高(P<0.01),IL-8水平明显降低(P<0.05),内皮细胞中AP-1、MCP-1荧光强度值、灰度值比值、mRNA水平均明显降低(P<0.01)。结论复方七芍降压片能够改善因炎症因子TNF-α诱导的内皮细胞及平滑肌细胞的存活率,降低AP-1和MCP-1的活性,抑制内皮细胞炎症反应,改善内皮损伤,保护平滑肌细胞正常功能。

Objective To observe the expression levels of inflammatory factors,AP-1,and MCP-1 in TNF-α-stimulated vascular endothelial cells and co-cultured with smooth muscle cells following treatment with the compound Qishao Jiangya tablet,and explore the mechanism by which the compound Qishao Jiangya tablet inhibits vascular remodeling.Methods A co-culture system was established using vascular endothelial cells and smooth muscle cells.Stimulation with TNF-αwas used to mimic the hypertensive inflammatory environment.The cultured cells were randomly divided into seven groups:normal control,model,blank serum control,MRS2578,irbesartan tablet drug-containing serum,compound Qishao Jiangya tablet-containing serum,and MRS2578+compound Qishao Jiangya tablet-containing serum.Except the normal control group,all groups were stimulated with 10 ng/mL TNF-α;the irbesartan tablet drug-containing serum,compound Qishao Jiangya tablet-containing serum,and MRS2578+compound Qishao Jiangya tablet-containing serum were treated with 10%drug-containing serum;the MRS2578 and MRS2578+compound Qishao Jiangya tablet drug-containing serum groups were treated with 10μm P2Y6 receptor blocker MRS2578.After 24 hours of modeling and drug intervention,CCK8 assays were used to detect the survival rates of endothelial cells and smooth muscle cells.ELISA was used to detect the level of IL-8 in the culture supernatant.Immunofluorescence,Western Blot,and quantitative PCR were used to detect endothelial expression levels of AP-1 and MCP-1.Results Compared with the normal control group,the survival rates of smooth muscle and endothelial cells decreased significantly in the model and blank serum control groups,while the levels of IL-8 were significantly higher(P<0.01).Compared with the normal control group,AP-1 and MCP-1 in endothelial cells in the model and blank serum control groups were detected by fluorescence intensity value of immunofluorescence,gray value ratio by Western Blot,and mRNA levels by quantitative PCR were significantly increased(P<0.01).Compared with the model group,the survival rates of smooth muscle and endothelial cells were significantly increased in the compound Qishao Jiangya tablet-containing serum groups,while the levels of IL-8 were significantly lower(P<0.05).AP-1 and MCP-1 in endothelial cells were detected by fluorescence intensity value of immunofluorescence,gray value ratio by Western blotting,and mRNA levels by quantitative PCR in the compound Qishao Jiangya tablet-containing serum groups were significantly reduced compared with the model group(P<0.01).Conclusions Following stimulation with TNF-α,the compound Qishao Jiangya tablet can improve the survival rates of endothelial cells and smooth muscle cells,reduce the levels of AP-1 and MCP-1,prevent endothelial cell inflammation,improve endothelial damage,and protect normal function of smooth muscle cells.