刺猬信号通路在慢性氟中毒大鼠关节软骨损害中作用

Role of hedgehog signaling pathway on cartilage tissue damage in chronic fluorosis rats

目的探讨刺猬(hedgehog,Hh)信号通路在慢性氟中毒大鼠关节软骨损害中作用。方法 36只健康SD大鼠随机分为对照组(自来水含氟量<1 mg/L),低、高氟组(饮水含氟量分别为5、50 mg/L),每组12只。饲养6个月后,应用氟离子电极法检测各组大鼠尿氟、骨氟含量;苏木素–伊红(HE)染色观察关节软骨组织病理形态学改变;免疫组织化学(IHC)、蛋白印迹法(Wb)检测大鼠关节软骨组织中音速刺猬蛋白(Shh)、跨膜蛋白(Smo)、骨形态发生蛋白–2(BMP-2)及B细胞淋巴瘤基因/白血病基因–2(Bcl-2)、B细胞淋巴瘤/白血病基因伴随蛋白x(Bax)蛋白表达情况。结果与对照组比较,低、高氟组大鼠尿氟含量[分别为(3.66±0.43)、(8.05±0.60)mg/L]与骨氟含量[分别为(402.38±33.77)、(935.12±49.60)mg/kg]明显升高,差异均有统计学意义(P<0.05);染氟组大鼠后肢膝关节干骺端软骨厚度变薄,软骨细胞数减少,具有硬化性氟骨症改变;与对照组比较,低、高氟组大鼠关节软骨组织中Shh、Smo、BMP-2及Bax蛋白表达[分别为(0.86±0.09)、(0.92±0.11)、(1.02±0.14)、(0.91±0.14);(1.11±0.15)、(1.17±0.14)、(1.13±0.12)、(0.92±0.11)]均明显升高,Bcl-2蛋白表达[分别为(0.78±0.03)、(0.57±0.09)]明显降低,差异均有统计学意义(P<0.05)。结论 Hh信号通路的激活及其下游靶基因过度表达,参与慢性氟中毒关节软骨损伤的发病过程。

Objective To investigate the role of hedgehog （Hh） signaling pathway on cartilage tissue damage in rats with chronic fluorosis.Methods Thirty-six healthy Sprague-Dawley （SD） rats were randomly divided into three groups （6 males and 6 females in each group）：a control group （supplied with drinking water containing sodium fluoride[NaF] of 〈 1 mg/L） and two fluorosis groups （supplied with drinking water containing NaF of 5 and 50 mg/L）. By the end of 6 months＇ treatment, fluorine content in urine and bone of the rats were detected with fluorin-ion electrode method. Histological changes in cartilage tissues were observed with light microscope using hematoxylin and eosin （HE） staining. Protein components of the Hh signaling pathway, including sonic hedgehog （Shh）, smoothened （Smo） and bone morphogenetic protein-2 （BMP-2）, as well as apoptosis-regulating proteins B cell lymphoma/leukemia-2 （Bcl-2） and B cell lymphoma/leukemia gene associated x protein （Bax）, were detected using immunohistochemistry staining and Western blot.Results The fluorine contents in the urine （3.66 ±0.43 and 8.05 ±0.60 mg/L） and bone （402.38 ±33.77 and 935.12 ±49.60 mg/kg） of the rats of low and high-dose fluoride groups were higher than those of the rats of control group （P 〈 0.05 for all）. Thinned metaphysic cartilage of stifles, decreased count of chondrocytes, and signs of sclerotic skeletal fluorosis were observed in the rats with fluorine treatment. Compared with those of the control rats, significantly increased protein expression of Shh （0.86 ±0.09 and 1.11 ±0.15）, Smo （0.92 ±0.11 and 1.17 ±0.14）, BMP-2 （1.02 ±0.14 and 1.13 ±0.12）, Bax （0.91 ±0.14 and 0.92 ±0.11） and decreased protein expression of Bcl-2 （0.78 ±0.03 and 0.57 ±0.09） were detected in the rats treated with low and high dose fluorine （P 〈 0.05 for all）.Conclusion The activation of Hh signaling pathway and excessive expression of the downstream target genes may become pathogenesis of chondrocyte damage in chronic fluorosis in rats.