长期应用糖皮质激素对大鼠骨组织中HMGB1、RAGE、OPG和RANKL表达的影响

Influence of long term application glucocorticoid on expressions of HMGB1,RAGE,OPG and RANKL in bone tissue of rats

目的:观察长期应用糖皮质激素(GC)对大鼠骨组织中高迁移率族蛋白B1(HMGB1)、晚期糖化终产物受体(RAGE)、骨保护素(OPG)和核因子κB受体活化因子配体(RANKL)表达的影响,探讨其导致激素性骨质疏松症的相关机制。方法:将48只健康成年SD大鼠随机分为低、中、高剂量GC组和对照组,每组12只。低、中、高剂量GC组大鼠分别给予肌肉注射醋酸泼尼松龙7.0、12.5、25.0mg·kg-1,对照组大鼠同时给予相同部位注射等量生理盐水。所有大鼠每周注射2次,干预4周后取材。ELISA法检测大鼠血清中HMGB1、OPG和RANKL水平,HE染色检测大鼠骨组织病理形态学,免疫组织化学和Western blotting法检测大鼠骨组织中HMGB1、RAGE、OPG和RANKL蛋白的表达水平。结果 :ELISA检测,各剂量GC组大鼠血清中HMGB1水平与对照组比较差异无统计学意义(P>0.05);OPG水平均较对照组降低,且随GC剂量增加OPG水平呈下降趋势,其中高剂量GC组与对照组和低、中剂量GC组OPG水平比较差异有统计学意义(P<0.05);各剂量GC组大鼠血清中RANKL水平均较对照组升高,且随GC剂量增加呈上升趋势,其中高剂量GC组与对照组和低、中剂量GC组RANKL水平比较差异有统计学意义(P<0.05)。HE染色,低剂量GC组大鼠股骨组织接近正常骨组织,中剂量GC组大鼠股骨组织镜下见软骨下区出现骨小梁变细,骨小梁间隙内出现肉芽纤维组织,可见坏死骨细胞及空骨陷窝;高剂量GC组大鼠股骨组织骨小梁紊乱并存在骨折,脂肪细胞空泡变性,骨髓腔水肿,造血细胞稀少,骨小梁周边成骨细胞数量减少,多核破骨细胞增多。免疫组织化学和Western blotting法检测,各剂量GC组大鼠骨组织中HMGB1蛋白表达水平均较对照组明显升高(P<0.05),各剂量GC组大鼠HMGB1蛋白表达水平比较差异有统计学意义(P<0.05);各组大鼠骨组织中RAGE蛋白表达水平比较差异无统计学意义(P>0.05);各剂量GC组大鼠骨组织中OPG蛋白表达水平均较对照组明显降低(P<0.05),各剂量GC组大鼠OPG蛋白表达水平比较差异有统计学意义(P<0.05);中和高剂量GC组大鼠骨组织中RANKL蛋白表达水平均较对照组明显升高(P<0.05),中剂量GC组大鼠骨组织中RANKL表达低于高剂量组(P<0.05)。结论:大鼠使用过量GC后,骨组织中HMGB1水平升高,OPG/RANKL比例降低,这可能是激素性骨质疏松症的发病机制之一。

Objective To observe the influence of glucocorticoid（GC）on the expressions of high mobility group protein B1（HMGB1）,receptor of advanced glycation endproducts（RAGE）,osteoprotegerin（OPG）and receptor activator of nuclear factor-κB ligand（RANKL）in bone tissue of the rats,and to discuss the related mechanism in glucocorticoid-induced osteoporosis.Methods 48 healthy adult male SD rats were randomly divided into low dose of GC（LGC）group（n=12）,medium dose of GC（MGC）group（n=12）,high dose of GC（HGC）group（n=12）,and control group（n=12）.The rats in LGC,MGC and HGC groups were treated by intramuscular injection of prednisolone 7.0,12.5and 25.0mg·kg^-1,respectively,and the rats in control group were only treated by the same volume of normal saline.All the rats receieved the injection twice a week.After 4weeks of intervention,all the rats were sacrificed.ELISA method was used to detect the serum HMGB1,OPG,and RANKL levels of the rats; HE staining method was used to detect the pathomorphology of bone tissue of the rats;Immunohistochemistry and Western blotting method were used to detect the expression levels of HMGB1,RAGE,OPG and RANKL protein in the bone tissue of rats.Results The ELISA results showed that the serum HMGB1 levels of the rats in GC groups had no significant differences compared with control group;the OPG levels were lower than that in control group,and they were decreased with the increase of the GC dose,and there were significant differences between HGC group and control,LGC,MGC groups（P〈0.05）;the serum RANKL levels of the rats in GC groups were higher thanthat in control group and they were increased in a dose-dependent manner;there were significant differences between HGC group and control,LGC, MGC groups（P〈0.05）.The HE staning results showed that the femur tissue of the rats in LGC group was close to normal bone tissue;a lot of bone trabecular which already had a reduced thickness in MGC group were found,and replaced by granulation tissue,the dead osteocytes and empty osteocyte lacunae were easy to encounter;the bone trabecula of the rats in HGC group were seriously compromised,and the adipocytes almost replacing the hematopoietic cells in the marrow cavity were hyperplasia and hypertrophy which render them abnormal appearance,and the necrotic bone marrow and discontinuous bone chips were also found.The immunohistochemistry and Western blotting results showed that the HMGB1 protein levels in bone tissue of the rats in GC groups were increased compared with control group（P〈0.05）,and there were significant differences between different doses of GC groups（P〈0.05）;the RAGE protein levels in bone tissue of the rats had no significant differences between various groups;the OPG protein levels in bone tissue of the rats in GC groups were decreased compared with control group（P〈0.05）,and there were significant differences between different doses of GC groups（P〈0.05）;the RANKL protein levels in bone tissue of the rats in MGC and HGC groups were increased compared with control group（P〈0.05）,and the RANKL protein level in MD group was lower than that in HGC group（P〈0.05）.Conclusion After the rats are treated with excess GC,the levels of HMGB1 in bone tissue is increased and the proportion of OPG/ RANKL is decreased which may be one of the pathogenesis of osteoporosis.