氟对小鼠骨组织白细胞介素-6／糖蛋白130信号通路表达的影响

Effect of fluoride on the protein expression of interleukin-6/glycoprotein 130 signaling pathway in bone tissue of mice

目的观察氟对小鼠骨组织白细胞介素-6（IL-6）、糖蛋白130（gp130）、磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）表达的影响，探讨IL-6／gpl30信号通路在氟致骨骼损伤中的作用机制。方法64只雄性Balb／c小鼠，按体质量采用随机数字表法分成4组，每组16只。对照组饮用蒸馏水，低、中、高氟组分别饮用含氟量为25、50、100mg／L的蒸馏水。饲养3个月，建立饮水型氟中毒模型，观察小鼠氟斑牙发生情况，镜下观察氟中毒小鼠骨组织形态学改变及细胞超微结构变化。采用氟离子选择电极法检测脊柱骨氟含量；微量酶标法检测血清碱性磷酸酶（ALP）和酸性磷酸酶（AcP）活性；酶联免疫吸附法检测血清IL-6含量；实时荧光定量PCR（real-time PCR）检测长骨组织IL-6、gp130、PI3K、Akt的mRNA表达水平；蛋白免疫印迹法（Western blot）检测长骨组织gp130、PI3K、Akt的蛋白表达水平。结果低、中、高氟组小鼠氟斑牙检出率分别为62．5％（10／16）、100．0％（15／15）、100．0％（16／16），均明显高于对照组（0，0／15，P均〈0．05）。骨氟含量随着染氟剂量增加而增加（F=309．716，P〈0．05）。光镜下，染氟组骨小梁明显增粗、紊乱，密度增加，呈骨质硬化；骨小梁面积百分比随着染氟剂量增加而增加（F=25．161。P〈0．05）。电镜下，高氟组成骨细胞、破骨细胞明显肿胀，内质网扩张，染色质边集，细胞超微结构明显受损。血清ALP、ACP活性随染氟剂量增加而增加（F=19．586、13．329，P均〈0．05），其中高氟组[（8．05±1．10）、（10．95±1．85）金氏单位]、中氟组[（6．48±0．92）、（9．18±0．76）金氏单位]明显高于对照组[（5．15±0．73）、（7．49±0．66）金氏单位，P均〈0．05]。对照组和低、中、高氟组血清IL-6水平[（5．98±1．43）、（7．54±2．16）、（5．25±1．97）、（6．31±1．36）ng／L]组间比较差异有统计学意义（F=3．840，P〈0．05），其中低氟组明显高于对照组（P〈0．05），中氟组明显低于低氟组（P〈0．05）。长骨组织IL-6、gp130、PI3K、Akt mRNA表达组间比较差异有统计学意义（F=9．952、8．954、6．997、21．504，P均〈0．05），其中高氟组（0．59±0．42、0．46±0．22、0．55±0．30、0．61±0．10）均明显低于对照组（1．10±0．54、1．16±0．67、1．20±0．84、1．06±0.37）和低氟组（1．86±0．63、1．61±0．64、1．74±0．65、1．70±0．58，P均〈0．05），除gp130外，各指标低氟组均明显高于对照组（P均〈0．05）。长骨组织gp130、PI3K、Akt蛋白表达水平组间比较差异有统计学意义（F=4．777、7．479、3．535，P均〈0．05），其中低氟组（0．66±0．10、0．69±0．12、0．92±0．09）均明显高于对照组（0．51±0．11、0．47±0．06、0．66±0．14，P均〈0．05），高氟组（0．42±0．11、0．38±0．06、0．67±0．14）均明显低于低氟组（P均〈0．05）。结论氟中毒小鼠骨组织IL-6、gp130、PI3K、Akt的mRNA和蛋白表达均有变化，提示IL-6／gp130信号通路可能参与了氟致骨骼损伤机制。

Objective To observe the expression of interleukin-6 （IL-6）, glycoprotein 130 （gp130）, phosphatidylinositol 3- kinase （PI3K） and protein kinase B （Akt） in bone tissue of fluorosis mice and explore the mechanisms of IL-6/gp130 signaling pathway in the pathogenesis of bone injury caused by fluoride. Methods Sixty-four male Balb/c mice were divided into 4 groups based on body weight by random number table method and 16 mice were in each group. The mice in control group were fed with distilled water, experimental animals in low, middle and high dose groups were fed with distilled water containing NaF 25, 50 and 100 mg/L, respectively. The mice were fed for three months to establish a drinking water fluorosis model. Mice dental fluorosis was observed and the fluoride contents in spine were detected by fluorine-ion selective electrode method. The changes of morphology and cell ultra structure in fluorosis bone tissue were observed by optical and electronic microscope. The activities of serum alkaline phosphatase （ALP） and acid phosphatase （ACP） were detected by micro enzyme labeled method. The levels of serum IL-6 were detected by enzyme-linked immunosorbent assay. The mRNA expression levels of IL-6, gp130, PI3K and Akt in long bone tissue were detected by real-time fluorescence quantitative PCR （real-time PCR）. The protein expression levels and activity of gp130, PI3K and Akt were detected by Western blotting. Results The rates of dental fluorosis in low fluoride group, middle fluoride group and high fluoride group were 62.5% （10/16）, 100.0% （15/15） and 100.0% （16/16）, respectively, and all the fluoride groups were higher than control group （0, 0/15, all P 〈 0.05）. Fluoride contents of bone were significantly increased with increase of fluoride doses （F = 309.716, P 〈 0.05）. The bone trabecular thickness and density in fluorosis mice were remarkably increased and disorder, and bone sclerosis was observed under optical microscope. The percentage of trabecular bone area was significantly increased with increase of fluoride doses （F = 25.161, P 〈 0.05）. Ultra structurally, swelling of cells, dilation of endoplasmic reticulum, chromatin margination in osteoblasts and osteoclasts of high fluoride group were observed which indicated that cells ultra structure was apparently damaged. The activities of serum ALP and ACP were significantly increased with increase of fluoride doses （F = 19.586, 13.329, all P 〈 0.05）, and both high fluoride group [（8.05 ± 1.10）, （10.95 ± 1.85） King unit] and middle fluoride group [（6.48 ± 0.92）, （9.18 ± 0.76） King unit] were significantly higher than control group [（5.15 ± 0.73）, （7.49 ± 0.66） King unit, all P 〈 0.05]. The levels of serum IL-6 in control group, low fluoride group, middle fluoride group and high fluoride group were （5.98 ± 1.43）, （7.54 ± 2.16）, （5.25 ± 1.97）, （6.31 ± 1.36） ng/L, respectively, the differences were statistically significant （F = 3.840, P 〈 0.05）, and low fluoride group was significantly higher than control group （P 〈 0.05）, and middle fluoride group was significantly lower than low fluoride group （P 〈 0.05）. The differences of mRNA expression levels of IL-6, gp130, PI3K and Akt in long bone tissue between groups were statistically significant （F = 9.952, 8.954, 6.997, 211504, all P 〈 0.05）, and high fluoride group （0.59 ± 0.42, 0.46 ± 0.22, 0.55 ± 0.30, 0.61 ± 0.10） was lower than control group （1.10 ± 0.54, 1.16 ± 0.67, 1.20 ± 0.84, 1.06 ± 0.37） and low fluoride group （1.86 ± 0.63, 1.61 ± 0.64, 1.74 ± 0.65, 1.70 ± 0.58, all P 〈 0.05）, and all mRNA expression except gp130 of low fluoride group was higher than that of control group （all P 〈 0.05）. The differences of protein expression levels and activities of gp130, PI3K and Akt in long bone tissue between groups were statistically significant （F = 4.777, 7.479, 3.535, all P 〈 0.05）, and low fluoride group （0.66 ± 0.10, 0.69 ± 0.12, 0.92 ± 0.09） was significantly higher than control group （0.51 ± 0.11, 0.47 ± 0.06, 0.66 ± 0.14, all P 〈 0.05）, and high fluoride group （0.42 ± 0,11, 0.38 ± 0.06, 0.67 ± 0.14） was lower than low fluoride group （all P 〈 0.05）. Conclusion The mRNA and protein expression levels of IL-6, gp130, PI3K and Akt in bone tissue of fluorosis mice have changed, which indicates that IL-6/gp130 signaling pathway may be involved in the pathogenesis of bone injury caused by fluoride.