六味地黄丸预防激素性股骨头缺血性坏死的分子机制研究

PREVENTING STEROID-INDUCED OSTEONECROSIS OF THE FEMORAL HEAD WITH LIUWEI DIHUANG PILLS AND MOLECULAR MECHANISM

目的激素性股骨头缺血性坏死(osteonecrosis of the femoral head,ONFH)防治效果不理想,探讨六味地黄丸预防ONFH的可行性及分子机制,为预防激素性ONFH提供实验依据。方法取成年健康昆明小鼠36只,体重40～50g,平均46g,随机分为3组(n=12):空白对照组(A组)、马血清/激素组(B组)、马血清/激素/六味地黄丸组(C组)。B、C组均2次腹腔注射马血清,2次剂量分别为10mL/kg及5mL/kg,间隔2周;第2次注射马血清同时肌肉注射醋酸泼尼松龙45mL/(kg·d),共5d,制备激素性ONFH小鼠模型。于应用激素同时B组灌服生理盐水10mL/(kg·d),C组灌服六味地黄丸2g/(kg·d);A组同时间点肌肉注射及灌服等量生理盐水。观察小鼠一般情况,于第1次激素注射后2、4、8周处死小鼠,大体观察股骨头情况,取双侧股骨头及肝脏行组织学及免疫组织化学染色观察,TUNEL法检测细胞凋亡情况。结果B、C组分别于第2次腹腔注射马血清后死亡2只及1只小鼠,余小鼠均存活至实验完成。3组各时间点股骨头外观、形态及关节软骨面均正常。组织学观察示A组各时间点肝脏及股骨头均正常。B组第1次激素注射后2周肝细胞肿胀,细胞内可见小脂肪空泡,肝索结构不清,肝窦变窄;股骨头区骨小梁纤细、稀疏,部分可见断裂;且随时间推移,变化愈显著。C组各时间点肝脏及股骨头与A组相似。B组股骨头空骨陷窝率高于A、C组(P<0.01),A、C组间比较差异无统计学意义(P>0.05)。免疫组织化学染色观察,各时间点B组与A、C组比较,股骨头骨保护素局部表达阳性率下降,骨保护素配体局部表达阳性率上升,差异均有统计学意义(P<0.01)。细胞凋亡检测示B组较A、C组骨细胞凋亡指数显著升高,差异有统计学意义(P<0.01),A、C组间2、4周比较差异有统计学意义(P<0.05),8周差异无统计学意义(P>0.05)。结论六味地黄丸具有改善骨代谢、保护骨细胞、降脂等作用,可通过抑制小鼠股骨头局部骨细胞凋亡、拮抗激素影响股骨头内骨保护素/骨保护素配体表达,起到预防早期激素性ONFH的作用。

Objective To evaluate the effects and the molecular mechanism of Liuwei dihuang pills in preventing steroid-induced osteonecrosis of the femoral head(ONFH)so as to provide an expremental basis for preventing ONFH clinically.Methods Thirty-six adult Kunming mice(weighing 40-50 g,46 g on average)were randomly divided into three groups(n=12):group A(control group),group B(model group)and group C(prevention group).In groups B and C,ONFH mice models were produced by intraperitoneal injection of horse serum at first(10 mL/kg)and a second injection of horse serum intraperitoneally(5 mL/kg)and prednisolone intramuscularly45 mL/(kg?day),for 5 days2 weeks later.At the same time,the mice in group C were given Liuwei dihuang pills intragastrically2 g/(kg?day)and were given normal saline10 mL/(kg?day)in group B.In group A,mice were given normal saline intramuscularly and intragastrically as controls.The animals were sacrificed 2,4,and 8 weeks after first treatment with prednisone,and femoral heads and livers were harvested to do histopathology analysis and apoptosis assay.Results Other mice survived throughout the experiment period except two death at 7 and 11 days after second injection of horse serum intraperitoneally in group B and one death at 24 hours after second injection of horse serum intraperitoneally in group C.The appearance and shape of the femoral head and the surface of cartilages were all normal.The histological observation showed:normal structures of liver and femoral head were seen in group A at each time point;swelling liver cells with small fat vacuole,unclear structure of hepatic cords and narrower sinus hepaticus were seen,the bone trabeculae of femoral head was thin,sparse and collapsed in some regions and the changes became more obvious with time in group B;group C had similar results to group A.The percentage of empty osteocyte lacunae was significantly higher in group B than in groups A and C(P<0.01).The osteoprotegrin expression significantly decreased and the osteoprotegrin ligand expression significantly increased in group B when compared with groups A and C(P<0.01).Apoptosis analysis showed that the apoptosis index in group B was significantly higher than that in groups A and C(P<0.01).Conclusion Liuwei dihuang pills can prevent steroid-induced ONFH by improving lipid metabolism,releiving bone lesion,and protecting against cell death.