[1]周婷婷,李华燕,赵云超,等.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(09):36-43.
　ZHOU Tingting,LI Huayan,ZHAO Yunchao,et al.Therapeutic effects and mechanisms of cervi cornus colla on model mice with steroid-induced osteonecrosis of the femoral head:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(09):36-43.
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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()

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年09期
页码:
36-43
栏目:
膝骨关节炎
出版日期:
2025-09-20

文章信息/Info

Title:
Therapeutic effects and mechanisms of cervi cornus colla on model mice with steroid-induced osteonecrosis of the femoral head:an experimental study
作者:
周婷婷1; 李华燕2; 赵云超1; 孟郑鑫1; 董军1; 冯娜娜1; 王铁强1; 石秋玲1; 李晓明1
(1.河北省沧州中西医结合医院,河北 沧州 061001; 2.河北中医药大学研究生院,河北 石家庄 050091)
Author(s):
ZHOU Tingting1; LI Huayan2; ZHAO Yunchao1; MENG Zhengxin1; DONG Jun1; FENG Nana1; WANG Tieqiang1; SHI Qiuling1; LI Xiaoming1
1.Cangzhou Hospital of Integrated TCM-WM·Hebei,Cangzhou 061001,Hebei,China 2.Graduate School,Hebei University of Chinese Medicine,Shijiazhuang 050091,Hebei,China
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
Keywords:
femur head necrosis;  hormones;  cervi cornus colla;  bone metabolism;  OPG-RANKL-RANK signaling pathway;  oxidative stress;  mice;  animal experimentation
摘要:
目的:探讨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。方法:将40只8周龄SPF级雄性C57BL/6小鼠随机分为4组,每组10只。模型组、鹿角胶低剂量组及鹿角胶高剂量组采用臀肌注射甲泼尼龙琥珀酸钠的方式建立激素性股骨头坏死模型,空白组注射等量生理盐水。造模结束后,鹿角胶低、高剂量组以鹿角胶药液灌胃,用量分别为2 g·kg-1·d-1(生药量)和4 g·kg-1·d-1(生药量),空白组和模型组用等量生理盐水灌胃,连续给药8周。药物干预结束后,采用Micro-CT检测股骨头骨微结构,采用HE染色和Masson染色观察股骨头组织病理学变化,采用ELISA技术检测血清骨碱性磷酸酶(bone alkaline phosphatase,BALP)、骨保护素(osteoprotegerin,OPG)、丙二醛(malondialdehyde,MDA)、Ⅰ型胶原交联羧基末端肽(C-terminal telopeptide of typeⅠcollagen,CTX)、谷胱甘肽(glutathione,GSH)水平,采用抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色检测股骨头组织中破骨细胞数量,采用免疫组织化学染色和Western Blot技术检测股骨头组织中Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、OPG、核因子κB受体激活蛋白配体(receptor activator of nuclear factor κB ligand,RANKL)蛋白表达水平。结果:①股骨头骨微结构检测结果。模型组骨体积分数、骨小梁厚度、骨密度均低于空白组和鹿角胶高剂量组(骨体积分数:P=0.000,P=0.001; 骨小梁厚度:P=0.001,P=0.006; 骨密度:P=0.002,P=0.004); 模型组的骨体积分数低于鹿角胶低剂量组(P=0.010),2组骨小梁厚度、骨密度的组间差异均无统计学意义; 鹿角胶低、高剂量组骨体积分数、骨小梁厚度、骨密度的组间差异均无统计学意义。②股骨头组织病理学观察结果。HE染色结果显示,空白组股骨头组织结构完整,骨小梁完整性、连续性良好,偶可见空骨陷窝; 与空白组相比,模型组可以观察到明显骨坏死,骨小梁结构紊乱、断裂,间隙变大,骨小梁结构被纤维结缔组织代替,出现大量空骨陷窝,骨髓腔内有大量脂肪细胞堆积; 与模型组相比,鹿角胶低、高剂量组骨小梁结构相对完整,排列较规则,骨髓腔脂肪细胞堆积减少,可观察到少量空骨陷窝。模型组空骨陷窝率高于空白组、鹿角胶低剂量组、鹿角胶高剂量组(P=0.000,P=0.018,P=0.002),鹿角胶低、高剂量组空骨陷窝率的差异无统计学意义。Masson染色结果显示,空白组可见极少量的蓝色胶原纤维; 与空白组相比,模型组和鹿角胶低剂量组可见大量蓝色胶原纤维包裹在坏死骨小梁周围,鹿角胶高剂量组可观察到少量蓝色胶原染色,红色成熟骨染色增多。③血清BALP、OPG、MDA、CTX、GSH水平检测结果。模型组的血清BALP、OPG、GSH水平均低于空白组和鹿角胶高剂量组(BALP:P=0.040,P=0.015; OPG:P=0.012,P=0.004; GSH:P=0.000,P=0.001); 模型组的血清OPG、GSH水平均低于鹿角胶低剂量组(P=0.005,P=0.000),2组血清BALP水平的差异无统计学意义; 模型组的血清MDA、CTX水平均高于空白组、鹿角胶低剂量组、鹿角胶高剂量组(MDA:P=0.003,P=0.003,P=0.003; CTX:P=0.013,P=0.007,P=0.004); 鹿角胶高剂量组的血清BALP水平高于鹿角胶低剂量组(P=0.031),2组血清OPG、MDA、CTX、GSH水平的组间差异均无统计学意义。④股骨头组织中破骨细胞数量检测结果。模型组TRAP阳性染色面积大于空白组、鹿角胶低剂量组、鹿角胶高剂量组(P=0.000,P=0.000,P=0.000),鹿角胶高剂量组TRAP阳性染色面积小于鹿角胶低剂量组(P=0.001)。⑤股骨头组织中RUNX2、OPG、RANKL蛋白表达水平检测结果。模型组的RUNX2、OPG蛋白表达水平及OPG/RANKL比值均低于空白组、鹿角胶低剂量组、鹿角胶高剂量组(RUNX2:P=0.000,P=0.000,P=0.000; OPG:P=0.000,P=0.000,P=0.000; OPG/RANKL比值:P=0.000,P=0.000,P=0.000),RANKL蛋白表达水平高于空白组和鹿角胶高剂量组(P=0.002,P=0.020),模型组与鹿角胶低剂量组RANKL蛋白表达水平的差异无统计学意义; 鹿角胶高剂量组的RUNX2、OPG蛋白表达水平及OPG/RANKL比值均高于鹿角胶低剂量组(P=0.012,P=0.002,P=0.002),2组RANKL蛋白表达水平的差异无统计学意义。结论:鹿角胶可改善激素性股骨头坏死模型小鼠的骨微结构,且高剂量鹿角胶的效果较低剂量鹿角胶存在一定优势,其机制可能与调控OPG-RANKL-核因子κB受体激活蛋白信号通路和氧化应激,改善骨代谢有关。
Abstract:
Objective:To investigate the therapeutic effects of cervi cornus colla(CCC)on the model mice with steroid-induced osteonecrosis 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 succinate into the gluteal region for inducing SONFH,while the ones in blank group by injection of an equal volume of normal saline at the corresponding 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 phosphatase(BALP),osteoprotegerin(OPG),malondialdehyde(MDA),C-terminal telopeptide of typeⅠcollagen(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 osteonecrosis,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 Masson 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 expression 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.Furthermore,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.

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备注/Memo

备注/Memo:
基金项目:河北省自然科学基金项目(H2024110024); 河北省中医药管理局项目(2021319)
通讯作者:李晓明 E-mail:lixiaomingcz@126.com
更新日期/Last Update: 1900-01-01