文献类型：期刊文献

中文题名：大蒜素对幼年铅中毒大鼠海马细胞凋亡损伤的抑制作用

英文题名：Inhibitory effects of Allicin on apoptosis injury in hippocampus of young rats with lead poisoning

作者：胡爽[1];郜振彦[2];应晓兰[2];周灿灿[2];颜崇淮[2];刘建文[1]

机构：[1]华东理工大学药学院,上海200237;[2]上海交通大学医学院附属新华医院环境与儿童健康重点实验室,200092

年份：2018

卷号：33

期号：21

起止页码：1652

中文期刊名：中华实用儿科临床杂志

外文期刊名：Chinese Journal of Applied Clinical Pediatrics

收录：CSTPCD;;北大核心:【北大核心2017】；CSCD:【CSCD_E2017_2018】；

基金：国家重点基础研究发展计划（2012CB525001）

语种：中文

中文关键词：大蒜素;铅中毒;海马细胞凋亡;线粒体凋亡通路;天冬氨酸特异性半胱氨酸蛋白酶家族

外文关键词：Allicin;Lead poisoning;Apoptosis;Mitochondrial pathway;Cysteine - containing aspartate - specific proteases family

摘要：目的探讨大蒜素对铅中毒大鼠海马细胞凋亡损伤的抑制作用。方法60只雄性3周龄SD大鼠按随机数字表法分成6组，每组10只，分别为低剂量（A－L）、中剂量（A－M）和高剂量（A－H）大蒜素组，铅暴露（Pb）组，二巯基丁二酸（DMSA）组和空白对照组。空白对照组提供超纯水，其余5组饮用1.0 g/L的醋酸铅水溶液，20 d后换为超纯水并进行灌胃给药，A－L组、A－M组和A－H组大蒜素用药剂量分别为2.7 mg/kg、5.4 mg/kg、10.8 mg/kg，DMSA剂量为10.8 mg/kg，Pb组予9 g/L盐水。模型建立后处死大鼠收集全血和海马组织。检测血铅和海马组织铅浓度，采用TUNEL染色法检测海马组织细胞凋亡水平，实时荧光定量PCR（qPCR）和Western blot法检测天冬氨酸特异性半胱氨酸蛋白酶－3（caspase－3）、天冬氨酸特异性半胱氨酸蛋白酶－9（caspase－9）和聚腺苷酸二磷酸核糖转移酶（PARP）的mRNA和激活型蛋白表达量，采用免疫荧光技术检测细胞色素C在胞质内的表达情况。结果1．A－L组、A－M组和A－H组大鼠血铅和海马组织铅水平[（190.54±11.33） μg/L，（0.28±0.03） μg/L；（159.55±16.94） μg/L，（0.22±0.06） μg/L；（116.62±8.85） μg/L，（0.19±0.01） μg/L]显著低于Pb组[（ 271.34±21.23） μg/L，（0.31±0.04） μg/L]，差异均有统计学意义（均P〈0.05）；DMSA组大鼠血铅和海马组织铅水平[（50.12±7.44） μg/L，（0.15±0.03） μg/L]低于A－L组、A－M组和A－H组。2.TUNEL染色结果显示，A－L组、A－M组和A－H组大鼠海马细胞凋亡水平显著低于Pb组[（2.81±0.17）%, （2.08±0.28）%，（1.33±0.08）%比（4.23±0.17）%]，差异均有统计学意义（均P〈0.05）；DMSA组大鼠海马细胞凋亡水平[（2.63±0.32）%]高于A－M组和A－H组，低于Pb组。3.qPCR结果表明，与Pb组比较，A－H组caspase－3、caspase－9和PARP的mRNA均显著下调（1.07±0.05，1.02±0.02，1.11±0.02比1.34±0.02，1.26±0.05，1.93±0.07），差异均有统计学意义（均P〈0.05）；A－L组、A－M组caspase－3和PARP mRNA表达量下调（1.21±0.05，1.43±0.12、1.16±0.02，1.20±0.06比1.34±0.02，1.93±0.07），差异均有统计学意义（均P〈0.05），caspase－9 mRNA无明显变化；A－H组caspase－3、caspase－9和PARP的mRNA水平（1.07±0.05，1.02±0.02，1.11±0.02）均低于DMSA组（1.14±0.02，1.15±0.08，1.32±0.05）。4.Wes－tern blot结果：与Pb组比较，A－H组激活型caspase－3、caspase－9和PARP蛋白表达量显著下调（A－H组：0.44±0.15、0.58±0.25、0.31±0.19和0.23±0.07比Pb组：0.69±0.13、0.72±0.22、0.55±0.21和0.43±0.10），A－M组激活型caspase－9蛋白表达量低于Pb组（A－M组：0.59±0.18比Pb组：0.72±0.22），差异均有统计学意义（均P〈0.05）; A－H组激活型caspase－3和RARP蛋白表达低于DMSA组。5.荧光染色结果显示A－L组、A－M组和A－H组细胞色素C在胞质中的表达量均显著低于Pb组及DMSA组。结论大蒜素在降低SD大鼠海马组织铅的同时，可通过线粒体通路抑制铅中毒大鼠海马组织细胞凋亡，大蒜素抑制凋亡损伤的作用可能优于DMSA。
ObjectiveTo investigate the inhibitory effect of Allicin on the apoptosis of hippocampal neurons induced by lead in rats.MethodsSixty male Sprague-Dawley rats aged 3 weeks were randomly divided into 6 groups, 10 rats in each group, which were low dose group（A-L）, medium-dose group（A-M） and high dose （A-H） Allicin group and lead exposure group（Pb group）, dimercaptosuccinic acid （DMSA） group and blank control group.The blank control group animals were treated with ultrapure water, and the other 5 groups received 1.0 g/L lead acetate aqueous solution instead of ultrapure water after 20 days and they were treated them with compounds by oral gavage.The doses of Allicin in group A-L, A-M group and A-H group were 2.7 mg/kg and 5.4 mg/kg, and 10.8 mg/kg, respectively.The DMSA dose was 10.8 mg/kg, and the Pb group was given 9 g/L saline.After the model was established, the rats were sacrificed to collect whole blood and hippocampus.Blood lead and tissue lead concentrations were measured, and the level of apoptosis in hippocampus was observed by TUNEL staining.The levels of cysteine-containing aspartate-specific proteases（caspase）-3, caspase-9, poly adenosine diphosphate-ribose polymerase （PARP） mRNA and caspase-3, caspase-9, PARP activated protein and cytochromes C distribution in the hippocampus cells were detected by using real-time quantitative PCR （qPCR）, Western blot, and immunofluorescence staining.Results（1）Lead levels in the blood lead and hippocampus of rats in A-L group, A-M group and A-H group [（190.54±11.33） μg/L, （0.28±0.03） μg/L; （159.55±16.94） μg/L, （0.22±0.06） μg/L; （116.62±8.85） μg/L, （0.19±0.01） μg/L] were lower than those in Pb group [（271.34±21.23） μg/L, （0.31±0.04） μg/L], and there were significant differences （all P〈0.05）. The blood lead and hippocampal lead levels in the DMSA group [（50.12±7.44） μg/L, （0.15±0.03） μg/L] were lower than those in the A-L group, A-M group and A-H group.（2） The results of TUNEL staining showed that the apoptosis levels of hippocampus in A-L group, A-M group and A-H group were lower than that in Pb group [（2.81±0.17）%, （2.08±0.28）%, （1.33±0.08）% vs. （4.23±0.17）%], and there were significant differences （all P〈0.05）; the apoptosis level of hippocampus in the DMSA group [（2.63±0.32）%] was higher than that in the A-M group and the A-H group, which was lower than that in the Pb group.（3） qPCR results showed that the levels of caspase-3, caspase-9 and PARP mRNA in A-H group were down-regulated compared with Pb group （1.07±0.05, 1.02±0.02, 1.11±0.02 vs.1.34±0.02, 1.26±0.05, 1.93±0.07）. The differences were statistically significant （P〈0.05）. The expression levels of caspase-3 and PARP mRNA in A-L group and A-M group were down-regulated （1.21±0.05, 1.43±0.12, 1.16±0.02, 1.20±0.06 vs.1.34±0.02, 1.93±0.07）, and there were significant differences （all P〈0.05）, and there was no significant change in caspase-9 mRNA; the mRNA levels of caspase-3, caspase-9 and PARP in A-H group （1.07±0.05, 1.02±0.02, 1.11±0.02） were lower than those in DMSA group （1.14±0.02, 1.15±0.08, 1.32±0.05）. （4） Western blot results： compared with Pb group, the expression levels of activated caspase-3, caspase-9 and PARP protein in A-H group were down-regulated （A-H group： 0.44±0.15, 0.58±0.25 and 0.31±0.19, 0.23±0.07 vs. Pb group： 0.69±0.13, 0.72±0.22 and 0.55±0.21, 0.43±0.10）, the expression of activated caspase-9 protein in A-M group was lower than that in Pb group （A-M group： 0.59±0.18 vs. Pb group： 0.72±0.22）, and there were significant differences （all P〈0.05）; the expression of activated caspase-3 and RARP protein in A-H group was lower than that in DMSA group.（5） Fluorescence staining showed that the expression of cytochrome C in cytoplasm of A-L group, A-M group and A-H group were significantly lower than that of Pb group and DMSA group.ConclusionAllicin can inhibit the apoptosis of hippocampus cells in rats with lead poisoning through mitochondrial pathway.The effect of Allicin on apoptosis inhibition may be better than DMSA.