铅对大鼠铜稳态失衡的影响及槲皮素的干预作用

Disorder of copper homeostasis induced by lead exposure among mice and intervention effect of quercetin

目的 探讨铅暴露对大鼠体内铜及铜金属酶的影响和槲皮素的干预作用.方法 选用SPF级健康雄性SD大鼠24只,随机分为3组：对照组、醋酸铅组和醋酸铅＋槲皮素组,每组8只.醋酸铅组大鼠采用1g/L醋酸铅每日饮水染毒,对照组采用等量的醋酸钠饮水,共8周；醋酸铅＋槲皮素组大鼠在醋酸铅饮水染毒同时腹腔注射槲皮素30 mg/kg·d,共8周.应用Morris水迷宫实验测试大鼠学习记忆功能；应用石墨炉原子吸收法测定大鼠血清、海马、皮质和骨骼中铅、铜水平；应用试剂盒检测血清、海马中晚期糖基化终末产物（AGEs）的含量、超氧化物歧化酶（Cu/ZnSOD）活力以及铜蓝蛋白（CP）的含量和活性的变化.用HE染色法观察大鼠海马组织病理变化.结果 Morris水迷宫测试结果表明醋酸铅组大鼠的潜伏期为（52.50±12.04）s,高于对照组（28.08±7.31）s,差异有统计学意义（P＜0.05）,同时穿越平台的次数低于对照组；铅在皮质、海马中的蓄积量是对照组的2.72和3.79倍,铜在皮质、海马和血清自由铜中的蓄积量分别是对照组的1.15、1.48、6.44倍；海马中CP活性为（0.78±0.08） U/mg pro,低于对照组的（1.23±0.40） U/mg pro；此外CP的含量、Cu/ZnSOD的活力比对照组下降31.81％、19.49％；血清中自由铜含量与大鼠潜伏期、血铅、皮质铅和海马铅含量呈正相关（r=0.895,P=0.048;r=0.936,P=0.044;r＝0.960,P=0.040;r=0.988,P=0.012）；应用槲皮素干预后,大鼠寻找平台的潜伏期缩短了42.15％,差异有统计学意义（P＜0.05）.槲皮素组大鼠皮质、海马中的铅水平（0.246±0.58,0.202±0.049） μg/g,明显低于醋酸铅组[（0.391±0.49,0.546±0.120）μg/g],但未见海马、皮质铜水平及自由铜明显降低；槲皮素组大鼠海马中Cu/Zn SOD活力和CP的含量高于醋酸铅组.光学显微镜下可见,醋酸铅组海马组织细胞数量减少,排列紊乱；槲皮素干预后,海马组织受损情况好转.结论 铅暴露能引起机体铜稳态失调,槲皮素对铅引起的损伤有一定缓解作用.

Objective To investigate the effect of lead exposure on copper and copper metalloenzyme and the intervention effect of quercetin.Methods Twenty-four specific pathogen-free male Sprague-Dawley rats of good health were randomly divided into control group （n=8）,lead acetate group （n=8）,and lead acetate＋ quercetin group （n=8）.The rats in lead acetate group were poisoned by drinking water with 1 g/L lead acetate for 8 weeks,while the rats in control group were fed by drinking water with sodium acetate of the same volume for 8 weeks; the rats in lead acetate＋quercetin group were intraperitoneally injected with quercetin （30 mg· kg1 ·d-1） for 8 weeks while drinking water with lead acetate.The Morris water maze was used to test the learning and memory abilities of rats.The lead and copper levels in the serum,hippocampus,cortex,and bone were measured by graphite furnace atomic absorption spectrometry.The level of advanced glycation end products,activity of Cu/Zn superoxide dismutase （SOD）,and content and activity of ceruloplasmin （CP） in the hippocampus and serum were measured using a test kit.HE staining was performed to observe the pathological changes in the hippocampus.Results The Morris water maze test showed that the latency in lead acetate group （52.50± 12.04 s） was significantly longer than that in control group （28.08±7.31 s） （P＜0.05）,and the number of platform crossings was significantly lower in the lead acetate group than in the control group.Compared with those in the control group,the lead levels in the cortex and hippocampus in lead acetate group increased 2.72-fold and 3.79-fold,and the copper in the cortex and hippocampus,and serum free copper levels in lead acetate group increased 1.15-fold,1.48-fold,and 6.44-fold.Compared with the control group,the lead acetate group had a lower content of CP in the hippocampus （1.23±0.40 U/mg pro vs 0.78±0.08 U/mg pro） and 31.81％ and 19.49％ decreases in CP content and Cu/Zn SOD activity.Free copper level in serum was positively correlated with the latency and lead levels in the serum,cortex,and hippocampus.The escape latency of rats in lead acetate＋quercetin group was decreased by 42.15％ （P＜0.05）.The lead levels in the cortex and hippocampus in lead acetate＋quercetin group （0.246±0.58 μg/g and 0.202±0.049 μg/g） were significantly lower than those in lead acetate group （0.391±0.49 μg/g and 0.546±0.120 μg/g）,but the free copper and copper levels in the hippocampus and cortex were not significantly reduced.The lead acetate ＋quercetin group had higher Cu/Zn SOD activity and CP content in the hippocampus than the lead acetate group （P＜0.05）.The light microscope observation showed that the number of cells in the hippocampus was reduced with disordered arrangement in the lead acetate group;with quercetin intervention,the hippocampus damage was reduced.Conclusion Lead exposure results in disorder of copper homeostasis,while quercetin may alleviate the damage induced by lead to some extent.