基于PI3K/Akt/Nrf2信号通路探讨地龙蛋白对自发性高血压大鼠的血管内皮功能保护机制

Earthworm Protein Protects Vascular Endothelial Function in Spontaneously Hypertensive Rats via PI3K/Akt/Nrf2 Signaling Pathway

目的:观察地龙蛋白对自发性高血压大鼠(SHR)主动脉磷脂酰肌醇3-激酶/蛋白激酶B/核因子E2相关因子2(PI3K/Akt/Nrf2)通路相关因子表达的影响,探讨其治疗高血压血管内皮功能障碍(VED)的作用机制。方法:选取10只10周龄Wistar Kyoto(WKY)大鼠及50只SHR适应性喂养1周,WKY大鼠作为正常组,50只SHR按体质量随机分为模型组、缬沙坦组(8×10^(-3) g·kg^(-1)·d^(-1))、地龙蛋白高剂量组(0.2g·kg^(-1)·d^(-1))、地龙蛋白中剂量组(0.1g·kg^(-1)·d^(-1))、地龙蛋白低剂量组(0.05 g·kg^(-1)·d^(-1)),正常组和模型组大鼠用等体积双蒸水灌胃。药物干预期间观察各组大鼠一般情况并分别于给药前及给药后每隔1周特定时间监测大鼠血压。药物干预8周后酶联免疫吸附测定法(ELISA)检测各组大鼠血清中血管紧张素-Ⅱ(Ang-Ⅱ)、内皮素-1(ET-1)活性水平;生化试剂盒法检测一氧化氮(NO)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GPX)水平,超氧化物歧化酶(SOD)活性、亚铁离子(Fe2+)浓度;苏木素-伊红(HE)染色观察主动脉内膜变化情况;实时荧光定量聚合酶链式反应(Real-time PCR)检测主动脉组织PI3K、Akt、Nrf2、血红素加氧酶1(HO-1)、谷胱甘肽过氧化物酶4(GPX4)mRNA表达情况;蛋白免疫印迹法(Western blot)检测胸主动脉中p-PI3K(Tyr467/199)、PI3K、p-Akt(Ser473)、Akt、Nrf2、HO-1、GPX4蛋白表达水平。结果:与正常组比较,模型组大鼠体质量偏低,大鼠较烦躁易激惹,内皮损伤程度较重;模型组大鼠血压、血清Ang-Ⅱ、ET1、MDA、Fe2+均显著升高(P<0.01),NO显著下降(P<0.01),主动脉组织中p-PI3K(Tyr467/199)、PI3K、p-Akt(Ser473)、Akt、Nrf2、HO-1、GPX4蛋白及mRNA表达显著降低(P<0.01);与模型组比较,各治疗组大鼠体质量变化不明显,大鼠相对安静,内皮损伤程度改善;各治疗组大鼠血压、血清Ang-Ⅱ、ET1、MDA、Fe2+均显著下降(P<0.01),NO明显升高(P<0.05),p-PI3K(Tyr467/199)、PI3K、p-Akt(Ser473)、Akt、Nrf2、HO-1、GPX4蛋白及mRNA表达明显升高(P<0.05)。结论:地龙蛋白可通过改善SHR内皮功能障碍起到降压作用,其机制可能与调控PI3K/Akt/Nrf2相关信号通路、抑制氧化应激及铁死亡相关。

Objective:To observe the effect of earthworm protein on the expression of phosphatidylinositol 3-kinase/protein kinase B/nuclear factor E2-related factor 2(PI3K/Akt/Nrf2)pathway in the aorta of spontaneously hypertensive rats(SHR)and explore mechanism of earthworm protein in treating hypertensive vascular endothelial dysfunction(VED).Method:Ten 10-week-old Wistar Kyoto(WKY)rats and fifty SHR rats were selected for a week of adaptive feeding.WKY rats were selected as the normal group,and fifty SHR rats were randomized according to body weight into model,valsartan(8×10^(-3) g·kg^(-1)·d^(-1)),and high-,medium-,and low-dose(0.2,0.1,0.05 g·kg^(-1)·d^(-1),respectively)earthworm protein groups.The normal and model groups were administrated with equal volume of double distilled water by gavage.During the drug intervention period,the general situations of rats in each group were observed and their blood pressure was monitored at specific time points every other week before and after administration.After 8 weeks of drug intervention,enzyme-linked immunosorbent assay was employed to measure the levels of angiotensin-Ⅱ(Ang-Ⅱ)and endothelin-1(ET-1)in the serum of rats in each group.The corresponding kits were used to determine the levels of nitric oxide(NO),malondialdehyde(MDA),glutathione peroxidase(GPX),superoxide dismutase(SOD),and ferrous ion(Fe2+).Hematoxylin-eosin(HE)staining was employed to observe the changes in the intima of the aorta.Fluorescence quantitative polymerase chain reaction(Real-time PCR)was employed to measure the mRNA levels of PI3K,Akt,Nrf2,heme oxygenase-1(HO-1),and glutathione peroxidase 4(GPX4)in the aortic tissue.Western blotting was used to determine the protein levels of p-PI3K(Tyr467/199),PI3K,p-Akt(Ser473),Akt,Nrf2,HO-1,and GPX4 in the thoracic aorta.Result:Compared with the normal group,the model group had decreased body mass,increased irritability,severe endothelial damage,elevated blood pressure and serum levels of Ang-Ⅱ,ET1,MDA,and Fe2+(P<0.01),lowered NO level(P<0.01),and down-regulated mRNA and protein levels of p-PI3K(Tyr467/199),PI3K,p-Akt(Ser473),Akt,Nrf2,HO-1,and GPX4 in the aortic tissue(P<0.01).Compared with the model group,drug intervention caused no significant change in the body mass,calmed the rats,alleviated the endothelial damage,lowered blood pressure and serum levels of Ang-Ⅱ,ET1,MDA,and Fe2+(P<0.01),elevated the NO level(P<0.05),and up-regulated the mRNA and protein levels of p-PI3K(Tyr467/199),PI3K,p-Akt(Ser473),Akt,Nrf2,HO-1,and GPX4(P<0.05).Conclusion:The earthworm protein can exert antihypertensive effects by ameliorating VED in SHR.Specifically,it may regulate the PI3K/Akt/Nrf2 signaling pathway to inhibit oxidative stress and ferroptosis.