GCDH^-/-大鼠海马氧化应激损伤机制研究

目的探讨高赖氨酸饮食的戊二酰辅酶A脱氢酶(GCDH)基因缺陷(GCDH^-/-)大鼠氧化应激损伤机制及可能通路。方法4周龄雄性SD大鼠按照随机数字表法分为6组:野生型标准饮食(WT)组(n=6)、纯合子标准饮食(GCDH^-/-)组(n=11)、野生型高赖氨酸(WT+Lys)组(n=8)、纯合子高赖氨酸(GCDH^-/-+Lys)组(n=13)、野生型高赖氨酸加维生素E(WT+Lys+VE)组(n=7)、纯合子高赖氨酸加维生素E(GCDH^-/-+Lys+VE)组(n=12)。WT组和GCDH^-/-组给予标准饮食(常规大鼠饲料),余4组给予4.7%高赖氨酸加强饲料,自由进食水。WT+Lys+VE和GCDH^-/-+Lys+VE组于每天上午10点维生素E100 mg/(kg·d)]灌胃1次,其余各组等量生理盐水灌胃。观察各组大鼠体质量及存活情况。干预28 d后腹腔注射100 g/L水合氯醛麻醉后断头取脑获取海马组织,通过HE染色观察大鼠海马病理形态学改变;ELISA法检测海马氧化应激指标谷胱甘肽过氧化物酶(GPx)、丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、还原型谷胱甘肽(GSH)含量/活性;Western blotting实验检测海马P38、c-Jun N-氨基端激酶(JNK)、细胞外调节蛋白激酶(ERK)蛋白表达情况。结果(1)一般情况:GCDH^-/-+Lys组大鼠存活比例为9/13,GCDH^-/-+Lys+VE组大鼠为11/12。干预第7天开始,GCDH^-/-+Lys组、GCDH^-/-+Lys+VE组大鼠体质量均显著低于WT组,差异有统计学意义(P<0.05)。(2)应激指标检测结果:与WT组相比,GCDH^-/-+Lys组和GCDH^-/-+Lys+VE组大鼠海马组织MDA含量显著增加,差异有统计学意义(P<0.05)。与WT组比较,GCDH^-/-+Lys组大鼠GPx活性、CAT活性、SOD活性显著减弱,GSH含量显著减少,差异有统计学意义(P<0.05);与GCDH^-/-+Lys组比较,GCDH^-/-+Lys+VE组大鼠GPx活性、CAT活性、SOD活性显著增强,GSH含量显著增加,差异有统计学意义(P<0.05)。(3)Western blotting实验结果:与WT组比较,GCDH^-/-+Lys组和GCDH^-/-+Lys+VE组大鼠海马P38蛋白表达显著增加,差异有统计学意义(P<0.05);与GCDH^-/-+Lys组比较,GCDH^-/-+Lys+VE组P38蛋白表达减少,差异有统计学意义(P<0.05)。结论高赖氨酸饮食GCDH^-/-大鼠海马存在氧化应激损伤,其可能机制与激活P38启动丝裂原活化蛋白激酶信号通路有关;维生素E可降低P38表达,减轻氧化应激损伤。

Mechanism of oxidative stress injury in the hippocampus of glutaryl CoA dehydrogenase^-/- rats

Objective To investigate the mechanism of oxidative stress injury and possible pathways in glutaryl CoA dehydrogenase deficient(GCDH^-/-)rats with high lysine diet(Lys).Methods Four-week-old rats were randomly divided into 6 groups:wild type+standard diet group(WT,n=6),GCDH^-/-+standard diet group(GCDH^-/-,n=11),WT+Lys group(n=8),GCDH^-/-+Lys group(n=13),WT+Lys+vitamin(V)E group(n=7),and GCDH^-/-+Lys+VE group(n=12);rats in the WT group and GCDH^-/-group were given standard diet,and rats in the WT+Lys group,GCDH^-/-+Lys group,WT+Lys+VE group and GCDH^-/-+Lys+VE group were given high lysine diet(4.7%Lys);rats in the WT+Lys+VE and GCDH^-/-+Lys+VE group were given VE(100 mg/kg·d])by intragastric administration once per d,and rats in other groups were given normal saline by intragastric administration once per d.The body mass and survival of rats in each group were observed.Twenty-eight d after intervention,rats were injected intraperitoneally with 10%chloral hydrate and anesthetized;their brains were severed to obtain hippocampal tissues;and pathomorphological changes were observed by HE staining;the content/activity of glutathione peroxidase(GPx),malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and reduced glutathione(GSH)in the hippocampus were detected by ELISA;the protein expressions of P38,c-Jun N-terminal kinase(JNK)and extra-celluar regulated protein kinase(ERK)in the hippocampus were detected by Western blotting.Results(1)The survival ratio of rats in the GCDH^-/-+Lys group was 9/13,and that in the GCDH^-/-+Lys+VE group was 11/12.From the 7th d of intervention,the body mass of rats in the GCDH^-/-+Lys group and GCDH^-/-+Lys+VE group was significantly lower than that in the WT group(P<0.05).(2)As compared with that in the WT group,MDA content in hippocampal tissues of rats in the GCDH^-/-+Lys+VE group and GCDH^-/-+Lys+VE group was significantly increased(P<0.05).As compared with WT group,GCDH^-/-+Lys group had significantly decreased GPx activity,CAT activity and SOD activity,and statistically decreased GSH content(P<0.05).As compared with those in the GCDH^-/-+Lys+VE group,the GPx activity,CAT activity,SOD activity,and GSH content in the GCDH^-/-+Lys+VE group were significantly increased(P<0.05).(3)Western blotting showed that as compared with that in the WT group,the P38 protein expression in the hippocampus of rats in GCDH^-/-+Lys group and GCDH^-/-+Lys+VE group was significantly increased(P<0.05);as compared with GCDH^-/-+Lys+VE group,the P38 protein expression in the GCDH^-/-+Lys+VE group was statistically decreased(P<0.05).Conclusion There is oxidative stress injury in the hippocampus of GCDH^-/-rats with Lys,whose possible mechanism is to activate P38 and initiate MAPK signaling pathway;VE protects GCDH^-/-hippocampal cells from oxidative stress by decreasing P38 expression.