紫草素通过调控PI3K/AKT通路减轻脑缺血再灌注损伤的机制研究

The underling regulation mechanisms of shikonin by regulating PI3K/AKT pathway in ischemia-reperfusion injury

目的探讨脑缺血再灌注损伤后PI3K/AKT信号通路的活性变化及紫草素发挥相应作用的调控机制。方法采用改良的Longa线栓法制作小鼠大脑中动脉缺血再灌注模型。成年雄性CD-1小鼠随机被分为5组:假手术组(Sham)、缺血再灌注组(Vehicle)、低剂量紫草素干预组(L-Shi)、高剂量紫草素干预组(H-Shi)、紫草素+LY294002组(H-Shi+LY294002)。各组小鼠取材前先进行神经功能评分、取材后进行脑梗死体积、脑组织含水量的测定,并对超氧化物歧化酶(SOD)的活性和丙二醛(MDA)含量的变化进行检测,运用免疫组化,蛋白印迹法和实时定量PCR等方法检测缺血侧脑组织中PI3K、AKT、HO-1、Nrf2蛋白和mRNA水平的变化;以及紫草素在脑缺血再灌注损伤中的神经保护作用及其对PI3K/AKT信号转导通路的作用。结果H-Shi组可明显改善神经功能缺损、降低脑含水量、减小脑梗死体积;免疫组化、Western blot结果显示H-Shi组在24 h和72 h均可明显升高p-PI3K、p-AKT、HO-1、Nrf2的表达,但这种作用同样可被LY294002阻断。H-Shi对于HO-1、Nrf2的mRNA表达与免疫组化和Western blot结果一致。结论PI3K/AKT通路参与了紫草素的脑保护作用,为其临床进一步应用提供了理论基础。

Objective This study is to evaluate PI3K/AKT pathway activity in ischemia injury and explore the underling regulation mechanisms of shikonin.Methods A modified Longa line suppository method was used to make transient focal cerebral ischemia/reperfusion model.Adult male CD-1 mice were randomly divided into five subgroups:sham operation group(Sham),Vehicle,low-dose shikonin intervention group(L-Shi)(10 mg·kg^(-1)),high-dose shikonin intervention group(H-Shi)(25mg·kg^(-1)),shikonin+LY294002 group(H-Shi+LY294002).Each group of mice was scored for nerve function before taking materials,and the volume of cerebral infarction and the water content of brain tissue were measured after taking materials.The activity of superoxide dismutase(SOD)and the change of malondialdehyde(MDA)content were detected.Immunohistochemistry,RT-qPCR and Western blot were used to analyse the expression of HO-1,p85(PI3-kinase)and p-AKT and Nrf2.The neuroprotective effect of shikonin in cerebral ischemia-reperfusion injury and its effect on PI3K/AKT pathway were studied.Results Shikonin reduced infarct volume and behavioral deficits caused by MCAO.Immunohistochemical and Western blot results showed that shikonin could significantly increase the number of positive cells of p-PI3K,p-Akt,HO-1 and Nrf2 at 24 and 72 hours,but this effect could also be blocked by LY294002.The mRNA expression of HO-1 and Nrf2 by shikonin was consistent with the results of immunohistochemistry and Western blot,but this improvement can be blocked by PI3K inhibitor LY294002.Conclusion PI3K/AKT pathway is involved in the cerebral protective effects of shikonin,providing a theoretical basis for its clinical application for cerebral ischemic therapies.