辽宁中医药大学学报

目的 探讨生骨再造丸治疗早期激素性股骨头坏死（SONFH）的可能作用靶点，验证生骨再造丸治疗早期SONFH的临床疗效。方法 实验以雄性SD大鼠为研究对象，随机抽取15只大鼠作为空白对照组（NC组），实验组75只采用脂多糖联合糖皮质激素（GC）肌肉注射建立早期SONFH大鼠模型。从中随机抽取8只大鼠采用空气栓塞法处死并取出双侧股骨头标本制作病理切片，证实早期SONFH造模成功，然后进行分组。将剩余的SONFH大鼠模型按照随机数字表法分为5组：激素模型组（Model组）11只，生骨再造丸高剂量组（SGZZ-H组）、生骨再造丸中剂量组（SGZZ-M组）、生骨再造丸低剂量组（SGZZ-L组）各10只，阳性对照组（JGSW组）11只。按照预先的实验设计进行药物灌胃治疗，灌胃期间密切观察并记录各组SD大鼠一般情况的变化，直至给药第8周末治疗结束后2 h，各组大鼠称重、取血，离心后分离血清并在-20℃保存。采用空气栓塞法处死大鼠，无菌条件下用手术剪及手术刀取出双侧的股骨头标本，剔除软骨，生理盐水漂洗，滤纸拭干，称重完成后左侧股骨头标本迅速放入液氮中速冻过夜后转移至-80℃冰箱保存，采用ELISA法检测大鼠血清相关炎性细胞因子（IL-1β、IL-6、TNF-α）、血清碱性磷酸酶（ALP）、血清钙（S-Ca）、血清磷（S-P）、骨钙素（BGP）、降钙素（CT）、抗酒石酸酸性磷酸酶（TRACP）等骨代谢相关指标的含量。结果 （1）大鼠行为学观察：经生骨再造丸灌胃治疗至第8周末，SGZZ-H组、SGZZ-M组、SGZZ-L组与JGSW组较Model组精神状况、活动量、饮食饮水、后肢肌力及跛行状况均有不同程度改善，其中以SGZZ-H组和JGSW组症状改善更加明显，但较NC组未恢复正常。（2）股骨头标本形态学观察：Model组股骨头明显出现皲裂、缺损且血运较差，有骨质疏松、坏死的表现，SGZZ-H组、SGZZ-M组、SGZZ-L组与JGSW组较Model组均有不同程度改观。（3）ELISA试验检测血清IL-1β、IL-6、TNF-α、ALP、S-Ca、S-P、BGP、CT和TRACP指标水平：Model组大鼠血清IL-1β、IL-6、TNF-α、ALP、CT和TRACP表达水平相对NC组均明显升高（P<0.001）,Model组大鼠血清S-Ca、S-P和BGP表达水平相对NC组均明显下降（P<0.001），且各治疗组大鼠血清IL-1β、IL-6、TNF-α、S-Ca、S-P、BGP、ALP、CT和TRACP表达水平相对Model组均有不同程度改善，其中以SGZZ-H组、SGZZ-M组及JGSW组大鼠血清IL-1β、IL-6、TNF-α、ALP、CT和TRACP表达水平降低最为明显（P<0.01）,SGZZ-H组及JGSW组大鼠血清S-Ca、S-P、BGP表达水平升高最为明显（P<0.001）。SGZZ-H组与JGSW组大鼠血清IL-1β、IL-6、TNF-α、S-Ca、S-P、BGP、ALP、CT和TRACP表达水平比较差异均无统计学意义（P>0.05）。结论 （1）该实验证明采用脂多糖联合GC肌肉注射可以成功诱导SD大鼠早期SONFH的发生。（2）生骨再造丸可通过抑制IL-1β、IL-6、TNF-α、ALP、CT和TRACP的表达，促进S-Ca、S-P和BGP的表达，改善骨代谢，防治早期SONFH。

Objective To explore Shenggu Zaizao Pills （生骨再造丸） in the treatment of early steroidinduced avascular necrosis of the femoral head(SONFH) possible targets,and verify Shenggu Zaizao Pills treatment clinical curative effect of early SONFH. Methods This experiment in male SD rats as the research object,randomly selected 15 rats as blank control(NC)group,the experimental group 75lipopolysaccharide combined GC muscle injection is adopted to establish the early SONFH. From a random sample were executed by air embolism method in 8 rats and remove the bilateral femoral head specimens made pathological section,confirmed the early SONFH building success,and then to group. The remaining SONFH rats model according to random number table method is divided into 5 groups:11 rats in hormone model group,Shenggu Zaizao Pills high dose(SGZZ-H)group,Shenggu Zaizao Pills middle dose(SGZZ-H)group,Shenggu Zaizao Pills low dose(SGZZ-H)group 10 rats in each,positive control(JGSW)group 11 rats. According to the advance of experimental design for drug lavage treatment,lavage closely observe and record each SD rats during the change of the general situation,until the end of therapy on the dosing 8 weekend after 2 h,groups of rats weighing,take the blood,after centrifugal separation of serum and preserved in-20 ℃. Put to death by air embolism method in rats,aseptic conditions with surgical scissors and a scalpel to take out the bilateral femoral head specimens,remove cartilage,physiological saline rinse,dry filter paper,weighing on the left side of the femoral head specimens after the completion of rapid in liquid nitrogen frozen after the night shift to-80 ℃ refrigerator,the mRNA and protein expression levels of Cbfα1 and Osterix factors were determined by PCR and Western blot,the right side of the femoral head specimens fixed in 4% paraformaldehyde solution for histopathological detection after 24 h. Using ELISA method to detect the rat serum inflammatory cytokines(IL-1β,IL-6,TNF-α),serum alkaline phosphatase(ALP),serum calcium(S-Ca),serum phosphorus(S-P),osteocalcin(BGP),calcitonin(CT),tartrate resistant acid phosphatase(TRACP)and so on the content of bone metabolism related indicators. Results （1）In the rat behavior observation:The Shenggu Zaizao Pills lavage treatment to 8th weekend,SGZZ-H group,SGZZ-M group,SGZZ-L group and JGSW group compared with model group mental condition,activity,diet drink,hind leg muscle and limp condition are improved to varying degrees.Among them,the symptoms of SGZZ-H group and JGSW group improved more obviously,but the NC group did not back to normal.(2)Specimens of femoral head morphological observation:Model group chapping,defect is apparent in the femoral head and poor blood supply,has the show of osteoporosis and necrosis,SGZZ-H group,SGZZ-M group,SGZZ-L group and JGSW group compared with model group are improved to varying degrees.(3)Serum levels of IL-1β,IL-6,TNF-α,ALP,S-Ca,S-P,BGP,CT and TRACP were detected by ELISA:The expression levels of serum IL-1β,IL-6,TNF-α,ALP,CT and TRACP in model group were significantly higher than those in NC group(P<0.001),compared with NC group,the expression levels of S-Ca,S-P and BGP in serum of model group decreased significantly(P<0.001),compared with the model group,the expression levels of serum IL-1β,IL-6,TNF-α,S-Ca,S-P,BGP,ALP,CT and TRACP in each treatment group were improved to some extent,the expression levels of serum IL-1β,IL-6,TNF-α,ALP,CT and TRACP in SGZZ-H group,SGZZ-M group and JGSW group decreased most obviously(P<0.01),the expression levels of serum S-Ca,S-P and BGP in SGZZ-H group and JGSW group increased most obviously(P<0.001). There was no significant difference in serum IL-1β,IL-6,TNF-α,S-Ca,S-P,BGP,ALP,CT and TRACP expression between SGZZ-H group and JGSW group(P>0.05). Conclusion （1）The experiment prove that the lipopoly-saccharide joint GC muscle injection can be successfully induced SD rats occurred early SONFH;(2)Shenggu Zaizao Pills can inhibit the expression of IL-1β,IL-6,TNF-α,ALP,CT and TRACP,and promote the expression of S-Ca,S-P and BGP to improve the bone metabolism,the prevention and treatment of early SONFH.