Objective: To investigate the effects of rapamycin target protein (mTOR) pathway and autophagy on bone formation and bone resorption in fluorosis osteoporosis in rats. Methods: In September 2018, a rat model of skeletal fluorosis was established by intragastric administration of fluorine. The experimental animals were divided into control group, 10 mgF(-)/kg group, 20 mgF(-)/kg group, 2 mg/kg rapamycin (RAPA) +10 mgF(-)/kg group and 2 mg/kg RAPA+20 mgF(-)/kg group, 20 per group. The experiment lasted for 3 months. The changes of bone tissue in rats were observed by hematoxylin-eosin (HE) staining. Bone mineral density (BMD) and biomechanical indexes, such as Modulus of elasticity, Stiffness, Maximum stress and Maximum load, were measured by BMD and biomechanical biometer. Serum levels of alkaline phosphatase (ALP) , osteocalcin (BGP) , osteoprotectin (OPG) , type I procollagen amino-terminal peptide (PINP) , tartrate-resistant acid phosphatase (TRACP) and nuclear factor kappa B receptor activator ligand (RANKL) were determined by enzymatic linked immunosorbent assay (ELISA) . Bone tissue phosphorylated mTOR (p-mTOR) , autophagy-related index selective autophagy adaptor protein p62, microtubule associated protein II (LC3-II) , ALP, osteoblastic transcription factor (Osterix) , and RNT Expression of related transcription factor 2 (Runx2) and bone resorption indicator RANKL were detected by Western blotting. Results: Compared with the control group, dental fluorosis in the 10 mgF(-)/kg and 20 mgF(-)/kg groups was significantly increased, periosteum thickness and absorption lacunae appeared, and BGP, OPG, PINP, TRACP and RANKL in serum contents were increased (P<0.05) , BMD, Modulus of elasticity, Stiffness, Maximum stress and Maximum load of bone tissue decreased significantly (P<0.05) , and the expressions of p-mTOR and p62 were decreased (P<0.05) , also the expressions of ALP, Osterix, Runx2 and RANKL were increased (P<0.05) . Compared with 10 mgF(-)/kg and 20 mgF(-)/kg groups, there were no obvious dental fluorosis symptoms in 2 mg/kg RAPA+10 mgF(-)/kg group and 2 mg/kg RAPA+20 mgF(-)/kg group, and serum ALP, BGP and OPG levels were significantly increased (P<0.05) . TRACP and RANKL contents were significantly decreased (P<0.05) . BMD, Modulus of elasticity, Stiffness, Maximum stress and Maximum load were significantly increased (P<0.05) . The levels of p-mTOR, p62 and RANKL in bone tissues were decreased (P<0.05) , and the expressions of LC3-II, LC3-II/LC3-I, ALP, Osterix and Runx2 were increased (P<0.05) . Conclusion: RAPA may activate autophagy by inhibiting mTOR phosphorylation, and inhibit bone resorption while promoting bone formation, thus alleviating early osteoporosis in skeletal fluorosis rats.
目的： 探讨哺乳动物雷帕霉素靶蛋白（mTOR）通路与自噬对大鼠氟骨症骨质疏松中骨形成和骨吸收的影响。 方法： 于2018年9月，采用灌胃给氟建立大鼠氟骨症模型，设立对照组、10 mgF(-)/kg组、20 mgF(-)/kg组、2 mg/kg雷帕霉素（RAPA）+10 mgF(-)/kg组和2 mg/kg RAPA+20 mgF(-)/kg组，每组20只，实验周期3个月。苏木素-伊红（HE）染色观察大鼠骨组织的变化，骨密度仪和力学生物仪检测骨组织骨密度和生物力学指标弹性模量、刚度、最大应力及最大承受力，酶联免疫吸附测定（ELISA）法检测血清内碱性磷酸酶（ALP）、骨钙素（BGP）、骨保护素（OPG）、I型前胶原氨基端原肽（PINP）、抗酒石酸酸性磷酸酶（TRACP）和核因子κB受体活化因子配体（RANKL）含量，蛋白免疫印迹（Western blotting）法检测骨组织磷酸化mTOR（p-mTOR）、自噬相关指标选择性自噬接头蛋白p62和微管相关蛋白II（LC3-II）、ALP、成骨细胞转录因子（Osterix）、Runt相关转录因子2（Runx2）和骨吸收指标RANKL的蛋白表达。 结果： 与对照组比较，10 mgF(-)/kg和20 mgF(-)/kg组大鼠氟斑牙表现明显，骨膜厚度增加且出现吸收陷窝，血清中BGP、OPG、PINP、TRACP和RANKL含量上升（P<0.05），骨组织骨密度、弹性模量、刚度、最大应力和最大承受力明显降低（P<0.05），骨组织p-mTOR和p62表达减弱（P<0.05），ALP、Osterix、Runx2和RANKL表达增加（P<0.05）。与10 mgF(-)/kg和20 mgF(-)/kg组比较，2 mg/kg RAPA+10 mgF(-)/kg和2 mg/kg RAPA+20 mgF(-)/kg组无明显氟斑牙症状出现，血清中ALP、BGP和OPG水平明显升高（P<0.05），TRACP和RANKL含量降低（P<0.05），骨组织骨密度、弹性模量、刚度、最大应力和最大承受力明显上升（P<0.05），骨组织p-mTOR、p62和RANKL表达减弱（P<0.05），LC3-II、LC3-II/LC3-I、ALP、Osterix和Runx2表达增加（P<0.05）。 结论： RAPA可能通过抑制mTOR磷酸化激活自噬，促进骨形成的同时抑制骨吸收，从而缓解氟骨症大鼠早期骨质疏松。.
Keywords: Autophagy; Bone formation; Fluorine; Fluorosis; Mammalian target of rapamycin (mTOR); Osteoporosis; Rats; Skeletal fluorosis.