燃煤型氟中毒大鼠脑组织神经型尼古丁受体mRNA及蛋白表达水平改变

Expression levels of brain nicotinic acetylcholine receptor mRNA and protein in coal-burning type of fluorosis rats

目的 观察燃煤型氟中毒大鼠学习记忆能力变化,测定大鼠脑组织神经型尼古丁受体（nAChR）mRNA和蛋白表达水平,探讨大鼠学习记忆能力改变的发生机制.方法 健康SD大鼠24只,体质量100～120 g,按体质量随机分为3组,每组8只.对照组饲以常规饲料,低氟组和高氟组以燃煤型氟中毒重病区燃煤烘烤的当地玉米为主要饲料（含氟量分别为11.30、104.20 mg/kg）来复制慢性氟中毒大鼠模型,染氟时间为6个月.染氟结束后,用Morris水迷宫方法检测大鼠行为学变化,处死动物取脑,用匀浆-氟离子选择电极法测定脑组织含氟量,实时荧光定量PCR法检测nAChR mRNA水平,蛋白印迹法测定nAChR蛋白表达水平.结果 低氟组和高氟组大鼠逃避潜伏期时间[（12.42±8.03）、（17.48±8.05）s]较对照组[（7.04±3.29）s]显著延长（P均〈0.05）,高氟组第7天穿过平台次数[（1.62±0.87）次]和逗留平台象限时间[（16.70±5.02）s]较对照组[（3.53±1.67）次、（23.33±5.35）s]降低（P均〈0.05）.低氟组和高氟组大鼠脑组织含氟量[（1.14±0.04）、（1.79±0.04）mg/kg]显著高于对照组[（0.52±0.05）mg/kg,P均〈0.05],且高氟组大鼠脑组织含氟量高于低氟组（P〈0.05）.高氟组大鼠脑组织nAChR α3、α4、α7亚单位mRNA水平（1.51±0.20、1.45±0.06、1.63±0.08）较对照组（1.79±0.11、1.66±0.14、1.83±0.06）显著降低（P均〈0.05）,而低氟组（1.65±0.17、1.59±0.09、1.71±0.03）与对照组比较无明显改变（P均〉0.05）.低氟组和高氟组大鼠脑组织nAChR α3、α4、α7亚单位蛋白表达水平（0.58±0.13、0.16±0.03、1.41±0.38和0.56±0.23、0.08±0.02、0.51±0.16）较对照组（1.48±0.42、0.57±0.21、2.56±0.26）显著降低（P〈0.05或〈0.01）.结论 燃煤型氟中毒大鼠学习记忆能力降低可能与脑组织nAChR蛋白表达及mRNA水平降低有关,nAChR表达改变可能是引起动物学习记忆能力降低的主要机制.

Objective To observe the learning and memory changes in coal-burning type of fluorosis rats, detect the expressions of neuronal nicotinic acetylcholine receptor（nAChR） at mRNA and protein levels in rat brains and to reveal the mechanism of changed learning and memory ability. Methods Twenty-four healthy SD rats, weighting 100 - 120 g, were randomly divided into three groups（8 in each）. Control group was fed with normal diet, and low- and high-dose fluoride groups were fed with corn polluted with high fluoride （fluoride were 11.30,104.20 mg/kg, respectively） during drying processes with local burning-coal from the areas of endemic fluorosis to established rat model of chronic fluorosis. After exposed to fluoride for 6 months, behavioral changes were measured by Morris water maze. Animals were sacrificed, the brain was taken, after homogenizing the fluoride content of brain tissue was determined by fluoride ion selective electrode. The α3, α4 and α7 nAChR subunits at mRNA and protein levels were analyzed by real-time PCR and Western blotting, respectively. Results For rats in low- and high-fluoride groups, the escape latency time[（12.42 ± 8.03）,（17.48 ± 8.05）s] was significantly longer than that in the control[（7.04 ± 3.29）s, all P〈 0.05]. For rats in high-fluoride group, the numbers of crossing the platforms （1.62 ± 0.87） and the time of staying at the platforms[（16.70 ± 5.02）s] were significantly decreased as compared to that of control[3.53 ± 1.67, （23.33 ± 5.35）s, all P 〈 0.05]. The fluoride content in rat brain tissue in low- or high-fluoride groups [（1.14 ± 0.04）, （1.79 ± 0.04）mg/kg] was significantly higher than that of control [ （0.52 ± 0.05） mg/kg, all P 〈 0.05]; in addition, the amount of fluoride in brain tissue of high-fluoride group was significantly higher than that of low-fluoride group（P 〈 0.05）. In high-fluoride group, the mRNA expressions of α3, α4 and α7 nAChR subunits in rat brains（1.51 ± 0.20,1.45 ± 0.06,1.63 ± 0.08） were significantly lower as compared to controls （1.79 ± 0.11,1.66 ± 0.14,1.83 ± 0.06, all P〈 0.05）; whereas there were no significant changes in mRNA levels of these receptor subunits of the rat brains between low-fluoride group（1.65 ± 0.17,1.59 ± 0.09,1.71 ± 0.03） and controls （all P 〉 0.05）. Furthermore, the protein levels of α3, α4 and α7 nAChR subunits in rat brains of highfluoride group（0.58 ± 0.13,0.16 ± 0.03,1.41 ± 0.38） and low-fluoride group（0.56 ± 0.23,0.08 ± 0.02,0.51 ± 0.16） were significantly lower than those of controls（ 1.48 ± 0.42,0.57 ± 0.21,2.56 ± 0.26, P〈0.05 or 〈 0.01）. Conclusions Decreased ability of learning and memory in coal-burning type of fluorosis rats may be associated with declined expressions of nAChR at proteins and mRNA levels, which might be the main mechanism of the behavior change.