铝对大鼠海马细胞内钙水平和钙通道蛋白基因表达的影响

Effects of aluminum on the intracellular calcium concentration and the expression of calcium channels in the hippocampus of rats

目的观察铝对大鼠海马细胞内游离钙（[Ca^2＋]i）水平和钙通道蛋白表达的影响。方法以64只健康Wistar大鼠为研究对象，按体质量将大鼠分为16组，经口灌胃给予氯化铝（AlCl3），按A1C]，剂量（mg／kg）分为0（对照）、37．3、74．7、248．7组；染铝时间为45、75、120d，其中120d的大鼠再正常饲养30d；分别在实验45、75、120、150d处死大鼠，取脑分离海马。应用荧光分光光度计测定海马[Ca^2＋]i，应用RT-PCR方法检测海马中Ryanodine受体2（RyR2）和L-型钙通道α1C亚基（L—Ca^2＋α1C）mRNA表达情况。结果AlCl3剂量和实验时间均可增加大鼠海马[Ca^2＋]i（F值分别为23．136、19．089，P〈0．01），二者具有交互作用（F=2．270，P〈0．05）；实验120、150d时，大鼠海马[Ca^2＋]i，37.3、74．7、248．7mg／kg组[（299．3±48．7）、（342．7±35．3），（391．2±47．9）、（408．1±42．8），（397．9±55．8）、（405．2±22．7）nmol／L]均较对照组[（195．1±29．9）、（209．1±30．6）nmol／L]明显升高（P〈0．01）。AlCl3可使大鼠海马RyR2、L—Ca^2＋α1C mRNA表达增加（F值分别为23．301、60．812，P〈0．01）。实验时间对大鼠海马中RyR2 mRNA表达无影响（F=1．361，P〉0．05），但可下调L—Ca^2＋α1C mRNA的表达（F=6．088，P〈0．01）；AlCl3剂量和实验时间对L-Ca^2＋α1C mRNA表达有交互作用（F=5．876，P〈0．01）。实验75、120、150d时，大鼠海马L—Ca^2＋α1C mRNA表达水平74．7、248．7mg／kg组（1．03±0．16、1．18±0．18、0．92±0．11，1．89±0．26、1．25±0．10、1．07±0．14）均较同时间对照组（0．63±0．09、0．78±0．16、0．69±0．11）明显增加（P〈0．05或〈0．01）。实验45、75、120、150d时，大鼠海马RyR2 mRNA表达水平，74．7、248．7mg／kg组（0．49±0．06、0．51±0．07、0．57±0．11、0．47±0．11，0．47±0．03、0．52±0．09、0．70±0．10、0．78±0．09）均较同时间对照组组（0．24±0．07、0．32±0．04、0．30±0．06、0．27±0．06）明显升高（P〈0．05或〈0．01）。结论铝可以通过上调RyR2 mRNA和L—Ca^2＋α1C mRNA的表达而增加海马[Ca^2＋]i，发挥不可恢复性的神经毒性作用。

Objective To dynamically investigate the effects of aluminum on the concentration of free intracellular Ca^2＋（[Ca^2＋]i） and the expression of calcium channels in the hippocampus of rats. Methods Healthy 64 Wistar rats were taken as the experimental objects. And these rats were randomly divided into 16 groups according to their weights, and were instilled with AlCl3 at 0（control）, 37.3,74.7 and 248.7 mg/kg respectively. The experimental time exposed to AlCl3 was 45,75,120 d, among which the rats were given AlCl3 for 120 d fed normally for 30 d. The hippocampus were segregated on day 45,75,120 and 150 d and the [ Ca^2＋]i of hippocampus of rats were detected by fluorospectrophotometer. The expression of Ryanodine receptor 2 （RyR2） mRNA and α 1C ubunit of L-type calcium channels（L-Ca^2＋ α1C） mRNA were detected by RT-PCR analysis. Results [Ca^2＋]i was increased by AlCl3 in a dose- and time-dependant manner（F = 23.136 and 19.089, P 〈 0.01）. There was a synergistic effect between the dose and time in [Ca^2＋]i （F = 2.270, P 〈 0.05）. In time of 120,150 days, the [Ca^2＋]i of rats hippocampus in 37.3 [ （299.3 ± 48.7）, （342.7 ± 35.3）nmol/L], 74.7[ （391.2 ± 47.9）, （408.1± 42.8）nmol/L] and 248.7 mg/kg group[ （397.9 ± 55.8）, （405.2 ±22.7）nmol/L] significantly increased compared with control group [（195.1 ± 29.9）, （209.1 ±30.6）nmol/L; P 〈 0.01]. Tile expression of RyR2 mRNA and L-Ca^2＋α1C mRNA were increased by AlCl3（F = 23.301 and 60.812, P 〈 0.01 ）. The experimental time could lower the expression of L-Ca^2＋ α1C mRNA（F = 6.088, P 〈 0.01 ）, but had no influences on the expression of RyR2 mRNA（F = 1.361, P 〉 0.05）. There was interaction between the dose of AlCl3 and the time in the expression of L-Ca^2＋ α1C mRNA （F = 5.876,P 〈 0.01）. On day 75,120 and 150 of the experiment, the expression of L-Ca^2＋ α1C mRNA in rat hippoeampus of 74.7 （1.03 ±0.16,1.18 ±0.18,0.92±0.11） and 248.7mg/kggroup（1.89 ±0.26,1.25 ± 0.10,1.07± 0.14） also increased compared with control group（0.63± 0.09,0.78 ± 0.16,0.69 ± 0.11; P 〈 0.05 or 〈 0.01 ）. On day 45,75, 120 and 150 of the experiment, the expression of RyR2 mRNA in 74.7（0.49 ± 0.06,0.51 ±0.07,0.57 ± 0.11, 0.47 ± 0.11 ）, 248.7（0.47 ± 0.03,0.52 ± 0.09, 0.70 ± 0.10, 0.78 ± 0.09）mg/kg AlCl3 groups was highly increased compared with control group （0.24 ± 0.07, 0.32 ±0.04, 0.30 ± 0.06, 0.27 ± 0.06; P 〈 0.05 or 〈 0.01）. Conclusion Al inereases [Ca^2＋]i by increasing the expression of the RyR2 mRNA and L-Ca^2＋α1C mRNA, thus exerts an irreversible neuronal toxicity.