Kv1.5蛋白基于PI3K/Akt信号通路影响内毒素致大鼠血管内皮细胞损伤

Mechanism of Kv1.5 Protein Affecting Endotoxin-induced Injury of Rat Vascular Endothelial Cells Based on PI3K/Akt Signaling Pathway

目的研究Kv1.5蛋白对内毒素致大鼠血管内皮细胞损伤的机制。方法雄性SD大鼠60只,随机分为Control组、LPS组、DPO-1(Kv1.5特异性通道阻滞剂)低、中、高剂量组,每组12只。Control组尾静脉注射生理盐水,LPS组尾静脉注射LPS 5 mg/kg,DPO-1低、中、高剂量组静脉注射LPS 5 mg/kg前30 min腹腔分别注射不同浓度DPO-1(1、3、5 mg/kg)。于LPS注射后6 h,采集股静脉血样,测定血清硫酸肝素(heparin sulfate,HS)、多配体蛋白聚糖1抗体(syndecan-1,SDC-1)、肿瘤坏死因子-α(TNF-α)及白细胞介素-6(IL-6)水平。取血样后处死大鼠取胸主动脉内膜组织,实时荧光定量PCR(qPCR)及Western blot检测主动脉内膜组织PI3K、p-Akt mRNA及蛋白表达情况。结果LPS组较Control组SDC-1、HS、TNF-α、IL-6含量明显升高(均P<0.01),DPO-1干预后,SDC-1、HS、TNF-α、IL-6含量降低(均P<0.01);Western blot检测结果显示,与Control组比较,LPS组PI3K、p-Akt蛋白表达水平明显下降(均P<0.01),而DPO-1高、中、低剂量组较LPS组PI3K、p-Akt蛋白表达水平上升(均P<0.01);qPCR检测结果显示,LPS组较Control组PI3K、p-Akt的mRNA表达下调,而DPO-1可抑制这种效应,上调PI3K、p-Akt的mRNA表达。结论Kv1.5特异性通道阻滞剂可能通过激活PI3K/Akt信号通路参与内毒素致大鼠血管内皮细胞损伤。

Objective To study the mechanism underlying the influence of Kv1.5 protein on endotoxin-induced injury of rat vascular endothelial cells.Methods Totally,60 male SD rats were randomly divided into Control group,LPS group,DPO-1(specific channel blocker of Kv1.5)low,medium,and high dose groups,12 rats in each group,and the control group was injected with normal saline through the tail vein;LPS(5 mg/kg)was injected into the tail vein in the LPS group;DPO-1 low,medium,and high-dose groups were injected intraperitonelly with different concentrations of DPO-1(1,3,5 mg/kg)30 min before intravenous injection of LPS;6 h after LPS injection,blood samples from femoral vein were collected,and serum heparin sulfate and syndecan-1 antibodies(syndecan-1,SDC-1),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)levels were detected;after blood samples were harvested,the rats were sacrificed and the thoracic aorta intima tissues were obtained for detecting PI3 K and p-Akt mRNA and protein expression in the aortic intima tissue by real-time fluorescent quantitative PCR(qPCR)and Western blotting.Results The levels of SDC-1,HS,TNF-αand IL-6 in the LPS group were significantly higher than those in the Control group(all P<0.01),after the intervention of DPO-1,the contents of SDC-1,HS,TNF-α,and IL-6 decreased(all P<0.01).Western blotting results showed that compared with the Control group,the expression levels of PI3 K and p-Akt protein in the LPS group were significantly decreased(all P<0.01),while the expression levels of PI3 K and p-Akt protein in the DPO-1 high,medium,and low dose groups were higher than those in the LPS group(all P<0.01).The qPCR results showed that the mRNA expression of PI3 K and p-Akt in the LPS group was down-regulated compared with the Control group,while DPO-1 could inhibit this effect and up-regulate the mRNA expression of PI3 K and p-Akt.Conclusion Kv1.5 specific channel blocker may participate in endotoxin-induced injury of rat vascular endothelial cells by activating PI3 K/Akt signaling pathway.