摘要
目的:探讨甜菜碱在大鼠脑微血管内皮细胞(BMECs)氧糖剥夺损伤中的作用,阐明甜菜碱对磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)通路的调节机制。方法:选取5只7日龄SD大鼠,获取大鼠BMECs,在低氧低糖条件下制备BMECs氧糖剥夺模型,分为模型组,低、中和高剂量甜菜碱组及阳性对照组,另设空白对照组(不进行造模),空白对照组和模型组大鼠BMECs给予新鲜培养基,阳性对照组大鼠BMECs给予终浓度为10μmol·L^(-1)的尼莫地平,低、中和高剂量甜菜碱组大鼠BMECs分别给予终浓度为0.5、1.0及2.0 mmol·L^(-1)的甜菜碱。采用CCK-8法检测培养12、24和48 h时各组大鼠BMECs存活率,试剂盒检测各组大鼠BMECs中乳酸脱氢酶(LDH)活性和三磷酸腺苷(ATP)水平,酶联免疫吸附试验(ELISA)法检测各组大鼠BMECs上清液中肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6、IL^(-1)β和IL^(-1)8水平,试剂盒检测各组大鼠BMECs中超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平,使用跨内皮电阻(TEER)分析仪检测各组大鼠BMECs的TEER值,使用插入式细胞培养器检测各组大鼠BMECs的辣根过氧化物酶(HRP)通透率,缺口末端标记(TUNEL)染色法检测各组大鼠BMECs凋亡率,Western blotting法检测各组大鼠BMECs中磷酸化PI3K(p-PI3K)/PI3K和磷酸化AKT(p-AKT)/AKT比值。结果:与空白对照组比较,模型组大鼠BMECs存活率,SOD活性,ATP水平和TEER值及大鼠BMECs中p-PI3K/PI3K和p-AKT/AKT比值均明显降低(P<0.05);LDH活性,TNF-α、IL-6、IL^(-1)β、IL^(-1)8和MDA水平,BMECs凋亡率和HRP通透率均明显升高(P<0.05)。与模型组比较,低、中和高剂量甜菜碱组及阳性对照组大鼠BMECs存活率,SOD活性,ATP水平和TEER值及大鼠BMECs中p-PI3K/PI3K和p-AKT/AKT比值均明显升高(P<0.05);LDH活性、TNF-α、IL-6、IL^(-1)β、IL^(-1)8和MDA水平,BMECs凋亡率和HRP通透率均明显降低(P<0.05)。结论:甜菜碱能够修复大鼠BMECs氧糖剥夺损伤,抑制BMECs氧化损伤及凋亡,改善大鼠BMECs通透性,其机制可能与调节PI3K/AKT通路有关。
Objective:To investigate the effect of betaine in oxygen-glucose deprivation injury of rat brain microvascular endothelial cells(BMECs),and to clarify the regulatory effect of betaine on phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)pathway.Methods:Five SD rats aged 7 d were selected and the rat BMSEs were obtained.The oxygen-glucose deprivation model of rat BMECs was prepared under hypoxic and hypoglycemic conditions;the experiment was divdided into model group,and low dose,medium dose,and high dose of betaine groups and positive control group,at the same time,blank control group(without modeling)was set up.The BMECs in blank control group and model group were treated with fresh medium,the BMECs in positive control group were given a final concentration of 10μmol·L^(-1)nimodipine,and the BMECs in low,medium and high doses of betaine groups were treated with betaine at final concentration of 0.5,1.0 and 2.0 mmol·L^(-1),respectively.The survival rates of BMECs in various groups were determined by CCK-8 method at 12,24 and 48 h after culture;the activities of lactate dehydrogenase(LDH)and the levels of adenine ribonucleoside triphosphate(ATP)in the rat BMECs in various groups were determined using kits,and the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL^(-1)β,and IL^(-1)8 in supernatants of the BMECs in various groups were determined by enzyme-linked immunosorbent assay(ELISA);the activities of superoxide dismutase(SOD)and levels of malondialdehyde(MDA)in the BMECs in various groups were determined by kits;the transendothelial resistance(TEER)values of rat BMSCs in various groups were determined by TEER analyzer,and the horseradish peroxidase(HRP)permeabilities of BMECs in various groups were determined by an insertion cell culture apparatus.TUNEL staining was used to determine the apoptotic rates of rat BMECs in vaisous groups,and Western blotting method was used to determine the ratios of phosphory lated PI3K(p-PI3K)/PI3K and phosphorylated AKT(p-AKT)/AKT in the rat BMECs in various groups.Results:Compared with blank control group,the survival rate of BMECs,activity of SOD,and level of ATP,value of TEER,and ratios of p-PI3K/PI3K and p-AKT/AKT of the rat BMECs in model group were significantly decreased(P<0.05),while the activity of LDH,the levels of TNF-α,IL-6,IL^(-1)β,IL^(-1)8,and MDA,the apoptotic rate of the BMECs,and HRP permeability were significantly increased(P<0.05).Compared with model group,the survival rates of the BMECs,activities of SOD,and levels of ATP,values of TEER,and ratios of p-PI3K/PI3K and p-AKT/AKT of the BMECs in low,medium,and high doses of betaine groups and positive control group were significantly increased(P<0.05),while the activities of LDH,the levels of TNF-α,IL-6,IL^(-1)β,IL^(-1)8,and MDA,the apoptotic rates of BMECs and HRP permeabilities were significantly decreased(P<0.05).Conclusion:Betaine can significantly repair the oxygen-glucose deprivation/reperfusion injury in the rat BMECs,inhibit the oxidative damage and apoptosis of BMECs,and improve the permeability of the cells;its mechanism may be related to the regulation of the PI3K/AKT pathway.
作者
陈敏
朱慧艳
陶静
徐奕鹏
CHEN Min;ZHU Huiyan;TAO Jing;XU Yipeng(Department of Rehabilitation Medicine,People’s Hospital,Xinjiang Uygur Autonomous Region,Urumqi 830001,China)
出处
《吉林大学学报(医学版)》
北大核心
2025年第1期96-104,共9页
Journal of Jilin University:Medicine Edition
基金
新疆维吾尔自治区科技厅自然科学基金项目(2022D01C113,2022D01C125)。
关键词
甜菜碱
氧糖剥夺
再灌注损伤
磷脂酰肌醇3激酶
蛋白激酶B
Betaine
Oxygen and sugar deprivation
Reperfusion injury
Phosphoinositide 3-kinase
Protein kinase B
