Wnt信号通路在氟中毒肾脏中表达及其意义

Expressions of protein and mRNA relevant to Wnt / β-catenin signaling pathway in rats with experimental fluorosis

目的观察Wnt/β-catenin信号通路及其相关信号分子蛋白及mRNA表达水平在实验性氟中毒大鼠肾组织中的变化,探讨氟中毒发病机制。方法选择健康成年SD大鼠36只,雌雄各半,随机分为:对照组(饮水含氟量<1 mg/L),低、高氟组(饮水加氟量5、50 mg/L),每组12只,观察大鼠氟斑牙发生情况,测定尿氟、骨氟含量及肾功能变化;光镜观察肾组织病理形态学变化;采用实时荧光定量PCR和免疫组化方法检测肾组织中Wnt4、β-连环蛋白(β-catenin)、E-粘钙蛋白(E-cadherin)蛋白及mRNA表达。结果低氟组大鼠氟斑牙检出率为75%(9/12),高氟组为91.7%(11/12),与对照组比较,染氟组大鼠尿氟、骨氟含量随染氟浓度增加而逐渐升高,差异有统计学意义(P<0.05);染氟组大鼠血清肌酐及尿素氮明显高于对照组(均P<0.05);光镜观察染氟组大鼠肾小管上皮细胞明显水肿、渗出、坏死,细胞间界限不清,且随着染氟浓度增高,损伤加重;低、高氟组大鼠肾组织中Wnt4、β-catenin、E-cadherin蛋白表达分别为[(7.78±1.17)、(6.66±0.59)、(3.23±0.82)]和[(17.46±3.18)、(12.46±0.89)、(1.23±0.35)],mRNA表达分别为[(3.01±0.81)、(1.86±0.43)、(0.276±0.12)]和[(5.79±0.86)、(5.84±1.19)、(0.12±0.46)],与对照组比较,氟中毒组大鼠肾组织中Wnt4、β-catenin蛋白和mRNA表达升高,而E-cadherin蛋白及mRNA表达降低。结论慢性氟中毒时,氟通过刺激Wnt信号通路中Wnt4、β-catenin过表达、E-cadherin低表达,导致肾小管上皮细胞间充质转分化(EMT)。

Objective To examine the expressions of protein and mRNA related to Wnt / β-catenin signaling pathw ay in rats with experimental fluorosis. Methods Thirty-six healthy adult Sprague-Daw ley（ SD） rats were randomly divided into a control group（ drinking w ater fluoride 1 mg / L）,a low fluoride group（ drinking w ater fluoride = 5 mg / L）,and a high fluoride group（ drinking w ater fluoride = 50 mg / L）（ 6 male and 6 female rats in each group）. The occurrence of dental fluorosis in the rats was observed. The fluoride content in urine and bone was determined using fluoride ion selective electrode method. Renal function was tested using automatic biochemical analyzer. Pathological changes in renal tissues were observed with optical microscopy. The mRNA and protein expressions of Wnt4,b-catenin,E-cadherin in kidney tissues were detected with real-time fluorescent quantitative PCR（ real-time PCR） and immunohistochemistry（ IHC）.Results The overall detection rate of dental fluorosis in the fluoride-exposed rats was 83. 3%（ 20 /24）（ 75%[9 /12]for the low fluoride group and 91. 7%[11 /12]for the high fluoride group）. The urinary and bone fluoride concentrations increased with the increase of fluoride exposure（ P〈0. 05） and the differences in the concentrations between the fluoride exposure groups and control group were significant（ P〈0. 05）. Creatinine and urea were significantly increased in the fluoride-exposed groups. Light microscopic observation found edema in tubular epithelial cells; luminal stenosis and obvious interstitial congestion of renal tubules were observed in the rats of fluoride-exposed groups and the damages in tubular epithelial cells aggravated with the increase of fluoride concentration. Protein expressions of Wnt4,β-catenin,and E-cadherin in the low fluoride group and high fluoride group were 7. 78 ± 1. 17,6. 66 ± 0. 59,and 3. 23 ± 0. 82 and17. 46 ± 3. 18,12. 46 ± 0. 89,and 1. 23 ± 0. 35,respectively; and the mRNA expressions of the three proteins were 3. 01 ±0. 81,1. 86 ± 0. 43,and 0. 276 ± 0. 12 and 5. 79 ± 0. 86,5. 84 ± 1. 19,and 0. 12 ± 0. 46,respectively. The immunohistochemistry and real-time fluorescent quantitive PCR show ed that the protein and mRNA expressions of Wnt4 and b-catenin increased but those of E-cadherin decreased in kidney tissue of the rats of fluorosis group compared with those of the control group. Conclusion Excessive fluoride may activate Wnt signaling pathw ay and then induce epithelial-mesenchymal transdifferentiation（ EM T） of renal tubules in rat.