小檗碱对脑缺氧-复氧大鼠内质网应激通路的作用机制研究

Study on the mechanism of berberine on endoplasmic reticulum stress pathway in brain hypoxia reoxygenation rats

目的探讨小檗碱对脑缺氧-复氧大鼠内质网应激通路的作用机制。方法选择30只成年Sprague Dawley大鼠(200~250 g)按照随机数字表法平均分为对照组、缺氧-复氧组和小檗碱处理组,每组10只。缺氧-复氧组及小檗碱处理组于含有5%氧气的缺氧箱中缺氧处理30 min后立即转入含有100%氧气的复氧箱中复氧处理6 h后取出,建造脑缺氧-复氧模型。对照组在同等大小箱子内同等时间通入空气处理。小檗碱处理组大鼠取出2 h后,灌胃给予浓度为200 mg/(kg·day)小檗碱,对照组和缺氧-复氧组大鼠灌胃给予同等剂量生理氯化钠溶液,连续灌胃7 d,麻醉后断头取全脑进行实验。采用氯化三苯四氮唑(TTC)染色法对各组大鼠的脑梗死体积进行测算,脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)染色检测细胞凋亡。通过ELISA检测白细胞介素(IL-6)和IL-10水平检测脑组织炎症反应。蛋白印迹检测细胞介素受体拮抗剂(IL-1Ra)和IL-1β的蛋白水平。结果缺氧-复氧组脑梗面积大于对照组(P<0.01)。小檗碱处理组脑梗面积小于缺氧-复氧组(P<0.01)。小檗碱处理组脑组织细胞凋亡低于缺氧-复氧组(P<0.01)。与对照组比较,缺氧-复氧组脑组织IL-6水平上升,IL-10水平降低(P<0.01)。与对照组比较,缺氧-复氧组脑组织IL-6水平和IL-1Ra/IL-1β比值上升,IL-10水平降低(P<0.01)。与缺氧-复氧组比较,小檗碱处理组脑组织IL-6水平和IL-1Ra/IL-1β比值下降,IL-10水平升高(P<0.01)。结论小檗碱可缓解脑缺氧-复氧大脑脑梗面积,神经元凋亡及免疫炎症反应,其可以抑制脑缺氧-复氧引起的内质网应激通路。

Objective To investigate the mechanism of berberine on endoplasmic reticulum stress pathway in rats with cerebral hypoxia reoxygenation. Methods Thirty adult Sprague Dawley rats( 200-250 g) were randomly divided into three groups: control group,hypoxia reoxygenation group and berberine treatment group,with 10 rats in each group. Hypoxia reoxygenation group and berberine treatment group were treated in an hypoxia box containing 5% oxygen for 30 minutes,immediately transferred to a reoxygenation box containing 100% oxygen for 6 hours,and then taken out to build a brain hypoxia reoxygenation model. The control group was treated with air in the same size box at the same time. Two hours after removal,the rats in Berberine treatment group were given berberine at the concentration of 200 mg/( kg · day) by gavage. The rats in control group and hypoxia reoxygenation group were given the same dose of normal saline by gavage for 7 days. After anesthesia,the whole brain was cut off and taken for experiment. The cerebral infarction volume of rats in each group was measured by triphenyltetrazolium chloride( TTC) staining. Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick labeling( TUNEL). The levels of interleukin( IL-6 and IL-10) were detected by ELISA. Interleukin-1 receptor antagonist( IL-1 ra) and IL-1 were detected by Western blot βProtein levels. Results The cerebral infarction area of hypoxia reoxygenation group was larger than that of control group( P < 0. 01). The cerebral infarction area of berberine treatment group was smaller than that of hypoxia reoxygenation group( P < 0. 01). The apoptosis of brain cells in Berberine treatment group was lower than that in hypoxia reoxygenation group( P < 0. 01). Compared with the control group,the level of IL-6 in brain tissue of hypoxia reoxygenation group increased and the level of IL-10 decreased( P < 0. 01). Compared with the control group,the levels of IL-6 and IL-1 ra/IL-1 in brain tissue of hypoxia reoxygenation group were significantly higher,and the level of IL-10 decreased( P < 0. 01).Compared with hypoxia reoxygenation group,the levels of IL-6 and IL-1 ra/IL-1 in brain tissue of berberine treatment group were lower, and the level of IL-10 increased( P < 0. 01).Conclusions Berberine can alleviate cerebral infarction area, neuronal apoptosis and immune inflammatory response caused by cerebral hypoxia reoxygenation,and can inhibit endoplasmic reticulum stress pathway caused by cerebral hypoxia reoxygenation.