NGF介导Fabp4/LCN-2蛋白对骨质疏松大鼠骨微结构及骨强度的影响

Effect of NGF mediated FABP4/LCN-2 protein on bone microstructure and strength in osteoporosis rats

目的 探讨NGF通过介导血清脂质运载蛋白2(Fabp4/LCN-2)的表达进而改善骨质疏松(osteoporosis,OP)大鼠骨微结构,增加骨强度的作用。方法 60只大鼠随机被分为正常组、OP组、NGF组和干预对照组,每组15只,OP组、NGF组及干预对照组大鼠均采用维甲酸混悬液连续灌胃,每日一次,每次70 mg/kg,连续灌胃4周后建立骨质疏松症大鼠模型。建模成功后,对NGF组及干预对照组分别行腹腔注射NGF、MSCs,剂量为2.0μ/kg,正常组及OP组均经腹腔注射等体积的PBS生理盐水,每日一次,连续注射3 d。通过ELISA检测血清中生化指标;分离股骨组织采用HE染色检测组织病理形态;通过micro CT测量股骨骨微结构观察组织形态指标水平;通过Hologic双能X线骨密度仪测量股骨组织骨强度;运用免疫印迹法检测股骨组织Fabp4/LCN-2蛋白含量。结果 OP组大鼠血清BGP、ALP及TRAP表达水平均升高,和正常组比较差异具有统计学意义(P<0.05),NGF组及干预对照组大鼠血清上述指标水平低于OP组,组间比较差异具有统计学意义(P<0.05);正常组大鼠股骨骨小梁呈现红紫色,粗细均匀、排列紧致,OP组大鼠股骨组织骨小梁断裂及骨髓腔表变大,NGF组和干预对照组骨小梁数目增多,分布较规则,结构和正常组相似;OP组大鼠股骨组织TB·Th、TB·N、BMD及BMC含量降低,TB·Sp含量升高,与正常组比较差异具有统计学意义(P<0.05),NGF组和干预对照组上述指标水平,和OP组比较差异具有统计学意义(P<0.05),其余组别无差异(P>0.05);OP组大鼠股骨组织中Fabp4蛋白水平为1.80±0.33(升高),LCN-2蛋白水平为1.06±0.26(降低),与正常组Fabp4蛋白水平(1.39±0.15)及LCN-2蛋白水平(2.06±0.55)比较存在差异(P<0.05),NGF组Fabp4蛋白为1.52±0.20(降低),LCN-2蛋白为1.80±0.33(升高),与OP组比较存在差异(P<0.05),Fabp4抑制剂组Fabp4蛋白为1.36±0.18(降低),与NGF组比较存在差异(P<0.05)。结论 骨质疏松大鼠腹腔注射NGF能够改善大鼠股骨微结构,增加骨小梁占比而提高骨强度,研究机制可能与NGF激活LCN-2表达、抑制Fabp4进而发挥抗骨质疏松的作用有关。

Objective To investigate the effect of NGF on improving bone microstructure and increasing bone strength in osteoporosis rats by mediating the expression of serum lipid carrier protein 2(FABP4/LCN-2).Effect of NGF mediated FABP4/LCN-2 protein(serum lipid carrier protein 2)on bone microstructure and strength in osteoporosis rats.Methods Sixty rats were randomly divided into normal group,OP group,NGF group and intervention control group,with 15 rats in each group.Rats in OP group,NGF group and intervention control group were given retinoic acid suspension for continuous intragastric administration,once a day,70 mg/kg each time,for 4 weeks,osteoporosis model was established.After successful modeling,NGF group and intervention control group were intraperitoneally injected NGF and MSCs with a dose of 2.0μ/kg,respectively.Normal group and OP group were intraperitoneally injected with equal volume of PBS normal saline once a day for 3 consecutive days,biochemical indexes in serum were detected by ELISA.The femoral tissues were separated and the histopathological morphology was detected by HE staining.Micro CT was used to measure the microstructure of femur bone to observe the level of tissue morphology indexes.The bone strength of femoral tissue was measured by Hologic dual-energy X-ray absorptiometry.The protein content of FABP4/LCN-2 in femoral tissues was detected by western blot.Results The expression levels of BGP,ALP and TRAP in serum of rats in OP group were increased,and there were differences compared with normal group(P<0.05).The levels of above indexes in serum of rats in NGF group and intervention control group were lower than that in OP group,and there were differences between groups(P<0.05).In the normal group,the femoral trabecula was red and purple with uniform thickness and compact arrangement.In the OP group,the fracture of femoral trabecula and the surface of bone marrow cavity were enlarged.In the NGF group and the intervention control group,the number of bone trabecula was increased,the distribution was more regular,and the structure was similar to that of the normal group.The contents of Tb·Th,Tb·N,BMD and BMC in OP group decreased,while the contents of Tb·SP increased,which were different from those in normal group(P<0.05).The levels of the above indexes in NGF group and intervention control group were different from those in OP group(P<0.05),but no difference was found in other groups(P>0.05).The level of FABP4 protein in the femoral tissue of the OP group was increased(1.80±0.33),while the level of LCN-2 protein was decreased(1.06±0.26),which was different from that of the normal group(1.39±0.15)and LCN-2 protein was 2.06±0.55(P<0.05).FABP4 protein decreased by 1.52±0.20 and LCN-2 protein increased by 1.80±0.33 in the NGF group and the intervention control group,compared with the OP group(P<0.05).FABP4 protein decreased by 1.36±0.18 in the FABP4 inhibitor group,which was different from that in the NGF group(P<0.05).Conclusion Intraperitoneal injection of NGF in osteoporosis rats can improve the femoral microstructure,increase the proportion of bone trabecula and increase bone strength.The mechanism of this study is that NGF can activate the expression of LCN-2 and inhibit FABP4,thus playing an anti-osteoporosis role.