乳铁蛋白通过调节IGF-1介导的PI3K/mTOR信号对糖皮质激素性骨质疏松大鼠的治疗作用

Therapeutic effect of lactoferrin on glucocorticoid-induced osteoporosis in rats by regulating IGF-1-mediated PI3K/mTOR signaling pathway

摘要:

目的： 观察乳铁蛋白对糖皮质激素性骨质疏松大鼠的治疗作用和机制。方法： SPF级Wistar雌性大鼠60只，随机分为对照组、模型组，乳铁蛋白低剂量组、中剂量组、高剂量组和阳性药组（阿仑膦酸钠组），肌内注射地塞米松磷酸钠注射液建立糖皮质激素性骨质疏松大鼠模型，造模成功后灌胃给予药物治疗。放射学检测骨形态和骨密度（bone mineral density，BMD）；酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）检测血清中1型前胶原氨基端延长肽（procollagen type 1 amino-terminal propeptide，P1NP）、骨碱性磷酸酶（bone alkaline phosphatase，bALP）、骨钙素（osteocalcin，OC）、β-胶原降解产物（β-crosslaps，β-CTX）、胰岛素样生长因子-1（insulin like growth factor 1，IGF-1）、胰岛素样生长因子结合蛋白（IGF binding protein，IGFBP）-3、IGFBP-4、IGFBP-5表达水平；Western blot检测股骨组织中胰岛素样生长因子-1受体（IGF-1 receptor，IGF-1R）和磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）/哺乳动物雷帕霉素复合物（mammalian target of rapamycin，mTOR）信号相关蛋白表达。结果： 与对照组比较，模型组大鼠的BMD降低（P=0.000），骨形态破坏；血清中P1NP、bALP、OC、β-CTX、IGF-1、IGFBP-3、IGFBP-4、IGFBP-5表达水平明显升高（P=0.000）；股骨组织中IGF-1R、p-PI3K/PI3K、p-Akt/Akt和p-mTOR蛋白表达无统计学差异（P>0.05）。与模型组比较，乳铁蛋白中剂量组和高剂量组大鼠的BMD增加（P=0.030，P=0.008），骨形态改善；血清中P1NP（P=0.009，P=0.000）、bALP（P=0.002，P=0.000）、OC（P=0.007，P=0.000）和β-CTX（P=0.020，P=0.009）表达水平均降低；IGF-1（P=0.007，P=0.003）、IGFBP-3（P=0.030，P=0.007）、IGFBP-4（P=0.020，P=0.009）、IGFBP-5（P=0.040，P=0.008）表达水平呈剂量依赖性增加；大鼠股骨组织中IGF-1R（P=0.020，P=0.006）、p-PI3K/PI3K（P=0.040，P=0.005）、p-Akt/Akt（P=0.003，P=0.007）和p-mTOR（P=0.010，P=0.000）蛋白表达明显上调。结论： 乳铁蛋白呈剂量依赖性改善糖皮质激素性骨质疏松大鼠的骨代谢，且与调控IGFBP及IGF-1介导的PI3K/mTOR信号激活有关，具有潜在的抗骨质疏松活性。

Abstract:

Objective: To observe the therapeutic effect and mechanism of lactoferrin on glucocorticoid-induced osteoporosis (GIOP) in rats. Methods: Sixty female SPF Wistar rats were randomly divided into control group, model group, low-dose lactoferrin group, medium-dose group, high-dose group and alendronate group. Dexamethasone sodium phosphate injection was intramuscularly injected to establish GIOP model rats. After the molding being completed, drug intervention was given to them by gavage. The bone morphology and bone mineral density (BMD) were detected by radiological examinations. The levels of procollagen type 1 amino-terminal propeptide (P1NP), bone alkaline phosphatase (bALP), osteocalcin (OC), β-crosslaps (β-CTX), insulin like growth factor-1 (IGF-1), IGF binding protein-3 (IGFBP-3), IGFBP-4, and IGFBP-5 in serum were detected by enzyme linked immunosorbent assay (ELISA). Western blot was used to detect the expression of IGF-1 receptor (IGF-1R) and phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signal-related proteins in femoral tissue. Results: Compared with control group, the BMD value of rats in the model group was decreased (P=0.000) and bone morphology was destroyed. The serum levels of P1NP, bALP, OC, β-CTX, IGF-1, IGFBP-3, IGFBP-4 and IGFBP-5 were increased significantly (P=0.000). There was no significant difference in the protein expression of IGF-1R, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR in femoral tissue (P>0.05). Compared with those of model group, the BMD (P=0.030, P=0.008) and bone morphology of rats in middle and high-dose of lactoferrin groups increased; serum levels of P1NP (P=0.009, P=0.000), bALP (P=0.002, P=0.000), OC (P=0.007, P=0.000) and β-CTX (P=0.020, P=0.009) decreased; serum levels of IGF-1 (P=0.007, P=0.003), IGFBP-3 (P=0.030, P=0.007), IGFBP-4 (P=0.020, P=0.009), and IGFBP-5 (P=0.040, P=0.008) increased dose-dependently; the protein expression of IGF-1R (P=0.020, P=0.006), p-PI3K/PI3K (P=0.040, P=0.005), p-Akt/Akt (P=0.003, P=0.007) and p-mTOR (P=0.010, P=0.000) was significantly up-regulated in middle and high-dose lactoferrin groups in femoral tissues. Conclusion: Lactoferrin can improve the bone metabolism of GIOP in rats, and it is related to the regulation of IGFBP and IGF-1-mediated PI3K/mTOR signal activation. Lactoferrin has potential anti-osteoporosis activity.