丹蓝仙硼疗氟胶囊对氟中毒大鼠骨保护蛋白配体和巨噬细胞集落刺激因子基因及其蛋白表达水平的影响

目的 研究丹蓝仙硼疗氟胶囊对饮水型氟中毒大鼠骨组织中骨保护蛋白配体(OPGL)和巨噬细胞集落刺激因子(M-CSF)mRNA及其蛋白质表达的影响.方法 以72只SD大鼠为研究对象,按体重均衡随机分为6组(组内雌雄各半):氟中毒组、高剂量药物干预组、中剂量药物干预组、低剂量药物干预组、对照组、硼砂治疗组.除对照组外,各实验组均饮用50 mg/L含氟水,复制氟中毒动物模型.各药物组应用中药丹蓝仙硼疗氟胶囊,高、中、低剂量药物干预组按大鼠每千克体质量每天分别给药0.8、0.4和0.2 g,阳性药物对照(硼砂)剂量为大鼠每千克体质量每天0.8 g.氟电极法检测尿氟、骨氟含量,HE切片观察骨骼病变,并测定骨小梁厚度、骨小梁密度及骨皮质厚度.应用逆转录聚合酶链式反应(RT-PCR)方法和免疫组织化学(EnVision法)染色,观察骨组织中OPGL和M-CSFmRNA及其蛋白表达的变化.结果 (1)氟中毒大鼠10/12发生氟斑牙;低剂量药物干预大鼠2/12发生氟斑牙.中、高剂量药物干预组,正常对照组及硼砂治疗组均无氟斑牙发生.氟中毒组骨氟尿氟明显高于对照组,高中剂量干预组及硼砂治疗组显著降低.(2)与对照组比较,氟中毒大鼠骨小梁厚度、骨皮质厚度及骨小梁密度均明显减少,而各药物组大鼠骨小梁厚度、骨皮质厚度及骨小梁密度均与对照组无明显差异.(3)与对照组比较,氟中毒大鼠骨组织OPGL、M-CSF mRNA表达均增高,差异有统计学意义(P＜0.05).与氟中毒组比较,高、中剂量药物干预组及硼砂治疗组OPGL mRNA表达降低,差异有统计学意义(P＜0.05);高、中、低剂量药物干预组及硼砂治疗组M-CSFmRNA表达降低,差异有统计学意义(P＜0.05).(4)与对照组比较,氟中毒大鼠OPGL蛋白表达增高,M-CSF蛋白表达降低;与氟中毒大鼠比较,高、中、低剂量药物干预组及硼砂治疗组OPGL蛋白表达降低,M-CSF蛋白表达有所升高,差异均有统计学意义(P＜0.05).结论 氟可能通过上调OPGL和M-CSFmRNA及其蛋白的共同表达而影响骨代谢;丹蓝仙硼疗氟胶囊能通过下调OPGL mRNA和M-CSF mRNA的表达影响骨重建.

The influence of fluoride on expression of OPGL and M-CSF genes and their proteins in rats with experimental fluorosis and the therapeutic effect of Danlan Xianpeng Liaofu caspule

Objective To investigate mRNA and protein expressions of OPGL and M-CSF mRNA in bones of rats with experimental fluorosis induced by intake of fluoride in the drinking water, and to study the antagonizing effects of Danlan Xianpeng Liaofu capsules on the gene expression. Methods Totally 72 SD rats were randomly assorted into 6 groups including the control group, the fluoride group, the high-dosage (0.8 g/kg·d), mid-dosage (0.4 g/kg·d) and low dosage(0.2 g/kg·d) medication groups and the borax group (borax, 0.8 g/kg·d). The distribution of female and male rats in each group was divided up on a fifty-fifty basis. Except the control group, a NaF containing water (NaF 50 mg/L in concentration) was supplied as the drinking water for all the experimental rats in order to establish experimental fluorosis. The thickness and density of trabecula and the thickness of bone cortex were measured by light microscopy. The fluoride content in urine and bone were analyzed by using fluoride ion selective electrode method. Expressions of OPGL and M-CSF mRNA and protein were studied using RT-PCR and immuno histochemistry, respectively. Results (1) 10/12 of the experimental fluorosis rats developed dental fluorosis, and 2/12 of dental fluorosis rats occurred in the low-dosage medication group. Fluoride content in urine and bone of the fluorosis rats increased (P＜0.05). (2) Compared with that of the control rats, the bone trabecular depth,cortical thickness and trabecular density in experimental fluorosis rats were remarkably reduced. (3) Compared with that of the control group, mRNA expression of both OPGL and M- CSF was increased in the fluoride group rats. The difference was statistically significant ( P＜0.05).(4) Compared with that of the fluoride group animals, the expression intensity of OPGL mRNA decreased in animals of the control group, the high, mid- and low- dosage medication groups and the borax group. Among them, except the low-dosage group, the difference between all the other groups and the fluoride group was statistically significant, respectively (P＜0.05).There was also a decrease of M-CSF mRNA in all the 3 medication groups and the borax group animals in comparing with that of the fluoride group and the difference was also statistically significant(P＜0.05 ), respectively. ( 5) Compared with that of the control group. There were an increase of OPGL and a decrease of M-CSF protein expression; and in addition, there were a decrease of OPGL and an increase of M-CSF protein expression in all 3 medication groups and the borax groupi in comparing with that of the fluoride group anima ( P ＜ 0. 05 ). Conclusions Excessive fluoride induces an accelerated bone turnover and may promote the absorption activity of osteoclasts by increasing the expression of OPGL and M-CSF.Danlan Xianpeng Liaofu capsule may be capable of regulating bone remodeling through a down-regulation on OPGL and M-CSF expression.