摘要
目的 观察细胞外调节蛋白激酶（Erk）1／2、磷酸化（phospho）-Erk1／2、基质金属蛋白酶（MMP）-2和MMP-9在实验性慢性氟中毒大鼠脑组织中的改变及硫酸软骨素的治疗作用与机制。方法 采用成组设计及细胞培养方法，将人神经母细胞瘤SH-SY5Y细胞分为对照组（正常培养液）、染氟组（氟离子：4.0 mmol／L）和硫酸软骨素处理组（氟离子：4.0 mmol／L、硫酸软骨素：0.4 g／L）。染氟24 h，电子显微镜观察SH-SY5Y细胞超微结构变化；染氟72 h，锥虫蓝染色观察细胞存活数量并计算细胞存活率。15只清洁级SD大鼠，按体质量（100 - 120 g）采用随机数字表法分为对照组（饮用自来水，含氟量 〈 0.5 mg／L）、染氟组（氟离子：10.0 mg／L）和硫酸软骨素处理组（氟离子：10.0 mg／L，染氟90 d后0.66 mg／kg硫酸软骨素连续腹腔注射5 d），每组5只，实验周期为95 d。采用Morris水迷宫方法检测大鼠行为学变化，蛋白质免疫印迹法检测脑组织中Erk1／2、phospho-Erk1／2、MMP-2和MMP-9的蛋白表达水平，免疫组织化学法检测大鼠脑组织海马CA2区phospho-Erk1／2、MMP-2、MMP-9的表达及分布。结果 细胞体外培养结果显示，电镜下硫酸软骨素处理组细胞内吞饮小泡较多，有线粒体及内质网肿胀但相对染氟组较少。硫酸软骨素处理组SH-SY5Y细胞存活率［（92 ± 23）%］和细胞数［（7.83 ± 1.38） × 10^6个／ml］明显高于染氟组［（55 ± 2）%、（2.19 ± 1.26） × 10^6个／ml，P均 〈 0.05］。动物实验结果显示，对照组与硫酸软骨素处理组大鼠趋向式搜索次数（2.20 ± 1.09、3.40 ± 1.34）高于染氟组（0.40 ± 0.54，P均 〈 0.05）。染氟组phospho-Erk1／2的蛋白表达（3.26 ± 0.88）较对照组和硫酸软骨素处理组升高（1.53 ± 0.28、2.36 ± 0.87，P均 〈 0.05）。免疫组织化学结果显示，硫酸软骨素处理组phospho-Erk1／2平均灰度值（220.20 ± 3.09）显著高于对照组和染氟组（100.00 ± 0.00、130.98 ± 1.27，P均 〈 0.05）；染氟组MMP-2平均灰度值（294.52 ± 5.18）显著高于对照组和硫酸软骨素处理组（100.00 ± 0.00、117.95 ± 1.55，P均 〈 0.05）；染氟组和硫酸软骨素处理组MMP-9平均灰度值（993.64 ± 3.66、1 167.30 ± 2.39）均显著高于对照组（100.00 ± 0.00， P均 〈 0.05）。结论 Erk1／2通路可能通过调节MMP-2和MMP-9表达维持细胞生存内环境稳定。硫酸软骨素对慢性氟中毒所致的神经损害具有一定的拮抗作用。
Objective To investigate the changes of extracellular regulated protein kinases （Erk）1/2, phospho-Erk1/2, matrix metalloproteinases （MMP）-2 and MMP-9 in rats with experimental chronic fluorosis and the role of chondroitin sulfate in treatment of rat with experimental chronic fluorosis. Methods Using a group design and cell culture methods, the SH-SY5Y cells were divided into control group （culture medium with 0.0 mmol/L fluoride ion）, fluoride group （fluoride ion： 4.0 mmol/L） and chondroitin sulfate group （fluoride ion： 4.0 mmol/L, chondroitin sulfate： 0.4 g/L）. The ultrastructural changes of the SH-SY5Y cells were observed through electron microscope after 24 h treatment. The SH-SY5Y cells were cultured for 72 h, the number of cells survived in three groups were detected after stained by trypan blue. Fifteen clean grade SD rats with body weight of 100 - 120 g were divided into control group （tap water： fluorine content less than 0.5 mg/L）, fluoride group （fluoride ion： 10.0 mg/L） and chondroitin sulfate group （fluoride ion： 10.0 mg/L,the rats were performed intraperitoneal injection with 0.66 mg/kg chondroitin sulfate for 5 days after intaking fluoride for 90 days） on the basis of random number table. Five rats were in each group, and the experiment was carried out for 95 days. The capability of learning and memory of rats were tested by Morris water maze test; the expression of phospho-Erk1/2, Erk1/2, MMP-2 and MMP-9 protein in brain tissue was detected by Western blotting; the expression of phospho-Erk1/2, MMP-2 and MMP-9 in hippocampus CA2 area of brain was detected by immunohistochemistry. Results More vesicles and swelling of mitochondria or endoplasmic reticulum were observed in SH-SY5Y cell treated with fluoride through electron microscope, but relatively less in chondroitin sulfate group. Survival rate and amount of SH-SY5Y treated with chondroitin sulfate [（92 ± 23）% and （7.83 ± 1.38） × 10^6/ml] were significantly higher than that of fluoride group [（55 ± 2）%, （2.19 ± 1.26） × 10^6/ml, P 〈 0.05]. Animal experiment results showed that most rats in control group and chondroitin sulfate group used spatial direct search strategy, and the amount of this search strategy （2.20 ± 1.09, 3.40 ± 1.34） was more than that in fluoride group （0.40 ± 0.54, P 〈 0.05）. The expression of phospho-Erk1/2 in brain tissue of rats in fluoride group （3.26 ± 0.88） was significantly higher than that in control group （1.53 ± 0.28） and chondroitin sulfate group （2.36 ± 0.87, P 〈 0.05）. Immunohistochemistry results showed that average gray value of phospho-Erk1/2 in chondroitin sulfate group （220.20 ± 3.09） was significantly higher than that of the control group and the fluoride group （100.00 ± 0.00, 130.98 ± 1.27, P 〈 0.05）. The average gray value of MMP-2 in the fluoride group （294.52 ± 5.18） was significantly higher than that in control group and chondroitin sulfate group （100.00 ± 0.00, 117.95 ± 1.55, P 〈 0.05）. The average gray value of MMP-9 protein of the fluoride group （993.64 ± 3.66） and the chondroitin sulfate group （1 167.30 ± 2.39） was significantly higher than that of control group （100.00 ± 0.00, P 〈 0.05）. Conclusions Erk1/2 pathway possibly maintains the stability of cell survival by regulating the expression of MMP-2 and MMP-9. Chondroitin sulfate can protective nerve cells and reduce the nervous damage caused by fluorosis to some certain extent.
作者
王胜远
邱志伟
官志忠
刘艳洁
Wang Shengyuan;Qiu Zhiwei;Guan Zhizhong;Liu Yanjie(Department of Pathology, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China （Wang SY, Qiu ZW, Guan ZZ, Liu YJ;Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education, Key Laboratory of Molecular Biology, Guizhou Medical University, Guiyang 550004, China （Guan ZZ)
出处
《中华地方病学杂志》
CAS
CSCD
北大核心
2018年第4期271-277,共7页
Chinese Journal of Endemiology
基金
国家自然科学基金（81260417）
贵州省科技厅贵阳医学院联合基金（黔科合LG[2012]006号）
