中医正骨2025，Vol.37Issue(9)：26-35,10.
鹿角胶干预激素性股骨头坏死模型小鼠的效果及作用机制研究
Therapeutic effects and mechanisms of cervi cornus colla on model mice with steroid-induced osteonecrosis of the femoral head:an experimental study
摘要
Abstract
Objective:To investigate the therapeutic effects of cervi cornus colla(CCC)on the model mice with steroid-induced osteo-necrosis of the femoral head(SONFH),and to explore its underlying mechanisms.Methods:Forty 8-week-old SPF-grade male C57BL/6 mice were selected and randomized into model group,low-dose CCC(L-CCC)group,high-dose CCC(H-CCC)group,and blank group,with 10 ones in each group.All mice but the ones in blank group were modeled by intramuscular injection of methylprednisolone sodium succi-nate into the gluteal region for inducing SONFH,while the ones in blank group by injection of an equal volume of normal saline at the corre-sponding sites.After successful modeling,the mice in L-CCC group and H-CCC group were intervened by intragastric administration with CCC solution at a crude dose of 2 and 4 g/kg/day,respectively,while the ones in blank group and model group with the equivalent volume of normal saline for consecutive 8 weeks.Following the drug intervention,the bone microstructure of the femoral head was examined using Micro-CT,and the histopathological changes were observed via HE and Masson staining.In addition,the serum levels of bone alkaline phos-phatase(BALP),osteoprotegerin(OPG),malondialdehyde(MDA),C-terminal telopeptide of type Ⅰ collagen(CTX),and glutathione(GSH)were detected by ELISA,and the number of osteoclasts in the femoral head tissues was quantified by tartrate resistant acid phosphatase(TRAP)staining.Furthermore,the protein expression levels of Runt-related transcription factor 2(RUNX2),OPG,and receptor activator of nuclear factor κB ligand(RANKL)in the femoral head tissues were determined by using immunohistochemical staining and Western Blot.Results:①Findings from the Micro-CT analysis of femoral head bone microstructure.The bone volume/tissue volume(BV/TV),trabecular thickness(Tb.Th),and bone mineral density(BMD)were all lower in model group compared to blank group and H-CCC group(BV/TV:P=0.000,P=0.001;Tb.Th:P=0.001,P=0.006;BMD:P=0.002,P=0.004).The BV/TV was also lower in model group compared to L-CCC group(P=0.010),while,the Tb.Th and BMD showed no significant difference between the 2 groups.Furthermore,no significant differences in BV/TV,Tb.Th,or BMD were observed between L-CCC group and H-CCC group.②Histopathological observations of femoral head tissues.The HE staining revealed that,in blank group,the femoral head tissues exhibited an intact architecture with well-preserved and continuous trabeculae,with occasional empty lacunae observed.Compared to the blank group,the model group exhibited significant osteone-crosis,which mainly manifesting as disorganized and fractured trabeculae with enlarged intertrabecular spaces,whose structure was replaced by fibrous connective tissues,meanwhile,numerous empty lacunae and substantial adipocyte accumulation within the bone marrow cavities were observed in the model group.Compared to the model group,both L-CCC group and H-CCC group showed relatively intact and more regularly arranged trabeculae,and these two groups showed a reduction in adipocyte accumulation within the bone marrow cavity,with only occasional empty lacunae observed.The empty lacunae rate was significantly higher in model group compared to blank group,L-CCC group and H-CCC group(P=0.000,P=0.018,P=0.002),with no significant difference between the 2 treatment groups.In addition,the Mas-son staining showed minimal blue-stained collagen fibers in the blank group.While,compared to the blank group,both model group and L-CCC group exhibited extensive blue-stained collagen fibers enveloping the necrotic trabeculae,in contrast,only a small amount of blue-stained collagen,accompanied by increased red-stained mature bone,were observed in the H-CCC group.③Serum levels of BALP,OPG,MDA,CTX and GSH.The serum levels of BALP,OPG,and GSH were lower in model group compared to blank group and H-CCC group(BALP:P=0.040,P=0.015;OPG:P=0.012,P=0.004;GSH:P=0.000,P=0.001).The serum levels of OPG and GSH were also lower in model group compared to L-CCC group(P=0.005,P=0.000),while that of BALP showed no significant difference between the 2 groups.In contrast,the serum levels of MDA and CTX were higher in model group compared to blank group,L-CCC group and H-CCC group(MDA:P=0.003,P=0.003,P=0.003;CTX:P=0.013,P=0.007,P=0.004).Furthermore,the serum level of BALP was higher in H-CCC group compared to L-CCC group(P=0.031),while those of OPG,MDA,CTX and GSH showed no significant difference between the 2 groups.④Quantification of osteoclasts in femoral head tissues.The TRAP-positive stained area was significantly larger in model group compared to blank group,L-CCC group and H-CCC group(P=0.000,P=0.000,P=0.000),in contrast,it was smaller in H-CCC group compared to L-CCC group(P=0.001).⑤Protein expression levels of RUNX2,OPG,and RANKL in femoral head tissues.The protein ex-pression levels of RUNX2 and OPG,as well as the OPG/RANKL ratio,were all lower in model group compared to blank group,L-CCC group and H-CCC group(RUNX2:P=0.000,P=0.000,P=0.000;OPG:P=0.000,P=0.000,P=0.000:OPG/RANKL ratio:P=0.000,P=0.000,P=0.000),conversely,the protein expression level of RANKL was significantly higher in model group compared to blank group and H-CCC group(P=0.002,P=0.020),but it showed no significant difference between model group and L-CCC group.Fur-thermore,the protein expression levels of RUNX2 and OPG,as well as the OPG/RANKL ratio,were all higher in H-CCC group compared to L-CCC group(P=0.012,P=0.002,P=0.002),while that of RANKL showed no significant difference between the 2 groups.Conclusion:CCC can ameliorate the bone microarchitecture in SONFH model mice,with H-CCC demonstrating superior efficacy to L-CCC.It may work by modulating OPG-RANKL-receptor activator of nuclear factor κB signaling pathway and mitigating oxidative stress,thereby enhancing bone metabolism.关键词
股骨头坏死/激素类/鹿角胶/骨代谢/OPG-RANKL-RANK信号通路/氧化性应激/小鼠/动物实验Key words
femur head necrosis/hormones/cervi cornus colla/bone metabolism/OPG-RANKL-RANK signaling pathway/oxidative stress/mice/animal experimentation引用本文复制引用
周婷婷,李华燕,赵云超,孟郑鑫,董军,冯娜娜,王铁强,石秋玲,李晓明..鹿角胶干预激素性股骨头坏死模型小鼠的效果及作用机制研究[J].中医正骨,2025,37(9):26-35,10.基金项目
河北省自然科学基金项目(H2024110024) （H2024110024）
河北省中医药管理局项目(2021319) （2021319）
