Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China.
Chin J Integr Med. 2012 Oct;18(10):761-8. doi: 10.1007/s11655-011-0940-7. Epub 2012 Jan 12.
To investigate the effect of Huogu II Formula (II) with medicinal guide Radix Achyranthis Bidentatae (Ach) on bone marrow stem cells (BMSCs) homing to necrosis area after osteonecrosis of the femoral head (ONFH) frozen by liquid nitrogen in rabbit as well as to explore the mechanism of prevention and treatment for ONFH.
The animal model of ONFH was established by liquid nitrogen frozen on the rabbit left hind leg. Forty-eight Japanese White rabbits were randomly assigned to sham-operated group, model group, Huogu II group, and Huogu II plus Ach group, with 12 rabbits in each. During the course of ONFH animal model establishment, all rabbits were subcutaneously injected with recombinant human granulocyte colony-stimulating factor [rhG-CSF, 30 μg/(kg·day) for continuous 7 days]. Meanwhile, normal saline and decoction of the two formulae were administrated by gavage, respectively. White blood cells (WBC) were counted in peripheral blood before and after injection of rhG-CSF. Materials were drawn on the 2nd and 4th weeks after model built; bone glutamine protein (BGP) and bone morphogenetic protein 2 (BMP2) levels in serum were tested. Histopathologic changes were observed by hematoxylin and eosin (HE) staining. BMP2 mRNA levels were detected with in situ hybridization (ISH) staining. 5-Bromo-2'-deoxyuridine (BrdU) and stromal cell derived factor 1 (SDF-1) were measured by immunohistochemical assay in femoral head of the left hind leg.
Compared with the shamoperated group, the ratio of empty lacuna, serum BGP, and SDF-1 level in the model group increased significantly, and BMP2 in both serum and femoral head decreased significantly. However, in comparison with the model group, the empty lacuna ratio of Huogu II group and Huogu II plus Ach group decreased obviously in addition to the levels of serum BGP and BMP2, and the expressions of BMP2 mRNA, BrdU, and SDF-1 increased significantly. Above changes were particularly obvious in Huogu II plus Ach group. BGP and SDF-1 on the 2nd week and empty lacuna rate and serum BMP2 level on the 4th week in Huogu II group significantly exceeded their counterparts. On the 2nd week, only in Huogu II plus Ach group that the BrdU counting rose significantly. On the 4th week, empty lacuna rate and serum BMP2 level in Huogu II plus Ach group exceeded those in Huogu II group distinctively.
To a certain extent, the medicinal guide Ach improves the preventive and therapeutic effects of Huogu II Formula on experimental ONFH model. The possible mechanism of this is related to its promoting effect on directional homing of BMSCs to the necrosis area.
观察活骨 II 方加药用引牛膝对液氮冷冻兔股骨头坏死（ONFH）模型骨髓干细胞（BMSCs）归巢坏死区的影响，探讨其防治股骨头坏死的作用机制。
液氮冷冻法建立兔左侧后腿 ONFH 模型，48 只日本大耳白兔随机分为假手术组、模型组、活骨 II 方组、活骨 II 方加药用引牛膝组，每组 12 只。在建立 ONFH 动物模型的过程中，所有兔子均连续 7 天每天皮下注射重组人粒细胞集落刺激因子[rhG-CSF，30μg/（kg·天）]。同时，分别灌胃给予生理盐水和两种方剂的煎剂。rhG-CSF 注射前后检测外周血白细胞（WBC）计数。造模后第 2 周和第 4 周取材，检测血清骨谷氨酸蛋白（BGP）和骨形态发生蛋白 2（BMP2）水平。苏木精-伊红（HE）染色观察组织病理学变化。原位杂交（ISH）染色检测 BMP2mRNA 水平。免疫组化法检测左侧后腿股骨头内 5-溴-2'-脱氧尿苷（BrdU）和基质细胞衍生因子 1（SDF-1）。
与假手术组相比，模型组的空骨陷窝比、血清 BGP 和 SDF-1 水平显著增加，血清和股骨头内的 BMP2 水平显著降低。然而，与模型组相比，活骨 II 方组和活骨 II 方加药用引牛膝组的空骨陷窝比明显降低，血清 BGP 和 BMP2 水平以及 BMP2mRNA、BrdU 和 SDF-1 的表达明显升高。活骨 II 方加药用引牛膝组变化尤为明显。活骨 II 方组第 2 周 BGP 和 SDF-1，第 4 周空骨陷窝比和血清 BMP2 水平明显高于对照组。第 2 周，只有活骨 II 方加药用引牛膝组的 BrdU 计数明显升高。第 4 周，活骨 II 方加药用引牛膝组的空骨陷窝比和血清 BMP2 水平明显高于活骨 II 方组。
药用引牛膝在一定程度上提高了活骨 II 方对实验性 ONFH 模型的防治作用。其可能的作用机制与促进 BMSCs 向坏死区定向归巢有关。
