燃煤污染型氟中毒大鼠Mn-超氧化物歧化酶mRNA和蛋白水平的改变

Effect of fluoride on oxidative stress and Mn-SOD expression in rats with endemic fluorosis of coal burning

目的研究燃煤污染型氟中毒对大鼠氧化应激及Mn-超氧化物歧化酶（SOD）mRNA和蛋白水平的影响。方法SD大鼠22只分成对照组和染氟组（以病区煤烘玉米为主要饲料，饲料含氟量为17mg／kg），每组11只，雌雄各半。观察氟斑牙发生情况，用氟离子选择电极法检测尿氟含量，并检测血清和肝组织中丙二醛（MDA）含量、SOD和谷胱甘肽还原酶（GR）活性，以及血清中反映肝功能的指标。应用逆转录聚合酶链反应和Western blot检测肝组织Mn-SOD基因及蛋白表达水平。结果染氟大鼠出现氟斑牙，检H{比例为11／11。尿氟含量（3．50±2．58）mg／L明显高于对照组的（1．42±0．38）mg／L（P〈0．05）。染氟动物肝功能异常，天冬氨酸转氨酶（223．74±71．51）U／L高于对照组的（169．28±53．74）U／L（P〈0．05）；总蛋白（72．43±5．59）g／L低于对照组的（82．36±7．31）g／L（P〈0．05）。染氟肝组织中MDA（10．41±0．59）μmol／gprot比对照组（5．80±1．31）μmol／gprot升高（P〈0．01）；SOD和GR[分别为（62．60±8．65）U／mgprot和（1．17±0．66）U／gprot]活性均比对照组[（117．28±8．64）U／mgprot和（8．80±1．59）U／gprot]明显降低（P〈0．05，P〈0．01）。染氟肝组织中Mn-SOD[（14．83±2．50）U／mgprot]活性比对照组[（34．05±5．22）U／mgprot]下降（P〈0．01），Mn—SOD mRNA及其蛋白表达[（0．64±0．15）和（0．84±0．13）]均明显低于对照组[（0．86±0．21）和（1．04±0．14）；P〈0．05，P〈0．01]。结论氟中毒时Mn—SOD活性降低是由于Mn—SOD基因转录活性的降低导致其蛋白表达减少的分子机制之一。氟中毒时Mn—SOD的下调导致的氧化应激加剧在氟中毒中起着重要作用。

Objective To study the effect of fluoride on the oxidative stress of the rats in endemic fluorosis of coal burning and Mn-SOD expression at mRNA and protein levels. Methods SD rats were divided into 2 groups （ the number of female and male in each group was the same ） ： control group and fluorosis group. All rats of the fluorosis group were fed corn dried by burning coal from endemic fluorosis areas with high fluoride content （fluoride 17 mg/kg in feed） to establish an animal model of fluorosis. In these rats, dental fluorosis was evaluated. The fluoride content in the urine was measured by fluorine ion-elective electrode method. The hepatic tissue and serum level of malonaldehyde （MDA） and the activities of superoxide dismutase （SOD） and glutathion reductase （GR） were measured by biochemical methods. The index signs of liver function were also measured from the serum. Reverse transcription- polymerase chain reaction （RT-PCR） and Western blot were performed to detect the alterations of Mn-SOD expression in the liver at mRNA and protein levels. Results The dental fluorosis was observed in the fluorosis group, and the incidence was 11/11. The fluoride contents [ （3.50 ±2. 58） mg/L] in the urine of fluorosis rats were increased as compared with the control [ （ 1.42 ± 0. 38 ） mg/L ] （ P 〈 0. 05 ） . AST [ （223.74 ± 71.51 ） U/L ] and total protein [ （ 72.43 ± 5.59 ） g/L ] of the hepatic function index in fluorosis rats showed obviously abnormal as compared with the control [ （ 169. 28 ± 53.74 ） U/L and （82. 36 ±7.31） g/L], respectively （P 〈0.05）. In the liver the content of MDA [（10.41 ±0.59） μmol/g protein] increased as eompared to the control [ （5.80 ± 1.31 ） μmol/g protein,P 〈0. 01 ], and the aetivities of SOD [ （ 62. 60 ± 8.65 ） U/nag protein ] and GR [ （ 1.17 ± 0. 66 ） U/g protein ] markedly decreased in the fluorosis group compared to the control [ SOD （117.28 ± 8.64） U/mg protein and GR [ （8. 80 ±1. 59） U/g protein; P 〈0.05, P〈0.01]. The level of Mn-SOD in the liver was markedly decreased in the fluorosis group [ （14. 83 ± 2. 50） U/mg protein ] as compared with the control [（34.05±5.22） U/rag protein, P 〈 0.01 ] . The levels of mRNA （0.64 ± 0.15） and protein （0. 84 ±0. 13） of Mn-SOD were markedly deereased in the fluorosis group as compared with the control [（0.86±0.21） and （1.04±0.14）], respeetively （P〈0.05,P〈0.01）. Conclusions Fluorosis ean decrease the activities of Mn-SOD, which is associated with decreased levels of mRNA and protein of Mn-SOD. Down-regulation of Mn-SOD expression may play an important role in the aggravation of oxidative stress in endemic fluorosis.