[1]李雪枫,王玉杭,邹凯奥,等.肾髓安康饮干预小鼠膝骨关节炎的效果和安全性及作用机制研究[J].中医正骨,2025,37(02):4-12.
　LI Xuefeng,WANG Yuhang,ZOU Kaiao,et al.Efficacy,safety,and mechanism of Shensui Ankang Yin(肾髓安康饮)against knee osteoarthritis in mice:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(02):4-12.
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肾髓安康饮干预小鼠膝骨关节炎的效果和安全性及作用机制研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年02期
页码:
4-12
栏目:
基础研究
出版日期:
2025-02-20

文章信息/Info

Title:
Efficacy,safety,and mechanism of Shensui Ankang Yin(肾髓安康饮)against knee osteoarthritis in mice:an experimental study
作者:
李雪枫1; 王玉杭1; 邹凯奥1; 温经渊1; 陈文哲1; 袁文华2; 肖鲁伟2; 金红婷2
(1.浙江中医药大学第一临床医学院,浙江 杭州 310053; 2.浙江省中医院,浙江 杭州 310006)
Author(s):
LI Xuefeng1; WANG Yuhang1; ZOU Kaiao1; WEN Jingyuan1; CHEN Wenzhe1; YUAN Wenhua2; XIAO Luwei2;  JIN Hongting2
1.The First Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 2.Zhejiang Provincial Hospital of Traditional Chinese Medicine,Hangzhou 310006,Zhejiang,China
关键词:
骨关节炎; 膝;  肾髓安康饮;  疼痛;  敏化;  软骨细胞;  神经生长因子;  小鼠
Keywords:
osteoarthritis; knee;  Shensui Ankang Yin;  pain;  sensitization;  chondrocytes;  nerve growth factor;  mice
摘要:
目的:探讨肾髓安康饮干预小鼠膝骨关节炎(knee osteoarthritis,KOA)的效果、安全性及作用机制。方法:将30只C57BL/6J小鼠随机分为假手术组、模型组和肾髓安康饮组。模型组和肾髓安康饮组小鼠采用内侧半月板失稳术在右后肢构建KOA模型,假手术组小鼠切开右后肢皮肤、暴露膝关节后缝合。自造模手术后第2天起,肾髓安康饮组小鼠以肾髓安康饮浓缩液(生药浓度1.6 g·mL-1)灌胃,每次0.5 mL,每天1次,连续灌胃8周; 模型组和假手术组小鼠以等量生理盐水灌胃。干预8周后,分别采用Up-down法和热板实验测定小鼠机械痛阈值和热痛阈值,采用DigiGait步态分析系统分析小鼠步态; 采用Micro-CT扫描仪扫描小鼠右膝关节,测定胫骨平台软骨下骨的骨体积分数; 制备小鼠膝关节软骨组织切片,采用阿尔辛蓝-苏木素/橙黄G染色观察膝关节软骨退变情况,采用国际骨关节炎研究学会(Osteoarthritis Research Society International,OARSI)评分标准评价膝关节软骨退变情况,采用免疫组织化学染色法检测膝关节软骨中Ⅱ型胶原、基质金属蛋白酶(matrix metalloproteinase,MMP)-13的表达情况; 制备小鼠膝关节软骨下骨和L4右侧背根神经节组织切片,采用免疫荧光染色法检测小鼠膝关节软骨下骨和L4右侧背根神经节内神经生长因子(nerve growth factor,NGF)的表达情况; 取小鼠心脏、肝脏、脾脏、肺脏、肾脏,制备组织切片,采用HE染色法观察小鼠内脏组织病变情况。结果:①疼痛阈值测定结果。模型组小鼠50%缩足阈值低于假手术组(P=0.000),右后足反应时间短于假手术组(P=0.000); 肾髓安康饮组小鼠50%缩足阈值高于模型组(P=0.000),右后足反应时间长于模型组(P=0.000)。②步态分析结果。模型组小鼠右后肢的摆动相时间长于假手术组(P=0.001),支撑相时间短于假手术组(P=0.000),步幅和足底接触面积均小于假手术组(P=0.000,P=0.000); 肾髓安康饮组小鼠右后肢的摆动相时间短于模型组(P=0.001),支撑相时间长于模型组(P=0.000),步幅和足底接触面积均大于模型组(P=0.000,P=0.000)。③膝关节Micro-CT分析结果。模型组小鼠胫骨平台软骨下骨异常骨重塑明显,肾髓安康饮组小鼠胫骨平台软骨下骨异常骨重塑较模型组明显减少。模型组小鼠胫骨平台软骨下骨骨体积分数大于假手术组(P=0.000),肾髓安康饮组小鼠胫骨平台软骨下骨骨体积分数小于模型组(P=0.000)。④膝关节软骨组织病理学观察结果。模型组小鼠膝关节软骨磨损严重,软骨层变薄、缺失,裂隙深达软骨下骨; 肾髓安康饮组小鼠膝关节软骨损伤较模型组有明显好转,软骨表面部分损伤、基质结构较完整。模型组小鼠OARSI评分高于假手术组(P=0.000),肾髓安康饮组小鼠OARSI评分低于模型组(P=0.000)。⑤膝关节软骨Ⅱ型胶原和MMP-13表达的检测结果。模型组小鼠膝关节软骨Ⅱ型胶原阳性表达面积比小于假手术组(P=0.000),MMP-13阳性表达面积比大于假手术组(P=0.000); 肾髓安康饮组小鼠膝关节软骨Ⅱ型胶原阳性表达面积比大于模型组(P=0.000),MMP-13阳性表达面积比小于模型组(P=0.000)。⑥膝关节软骨下骨和L4右侧背根神经节内NGF表达的检测结果。模型组小鼠膝关节软骨下骨和L4右侧背根神经节内NGF阳性表达面积比均大于假手术组(P=0.000,P=0.000),肾髓安康饮组小鼠膝关节软骨下骨和L4右侧背根神经节内NGF阳性表达面积比均小于模型组(P=0.000,P=0.000)。⑦小鼠内脏组织病理学观察结果。3组小鼠心脏、肝脏、脾脏、肺脏、肾脏均未见病理性改变。结论:肾髓安康饮能够有效减轻KOA小鼠疼痛敏化和软骨退化,且安全性高,其作用机制可能与其能够抑制NGF表达、调节软骨细胞外基质代谢有关。
Abstract:
Objective:To observe the outcomes and safety of Shensui Ankang Yin(肾髓安康饮,SSAKY)against knee osteoarthritis(KOA)in mice,and to explore its mechanism.Methods:Thirty C57BL/6J mice were selected and randomized into sham-operated group,model group,and SSAKY group.The mice in model group and SSAKY group were subjected to destabilization of the medial meniscus(DMM)on the right hind limbs to build KOA models,while the ones in sham-operated group were merely incised the skin at the corresponding site and then sutured after exposing the knee joints.From day 2 after the modeling surgery,the mice in SSAKY group were intervened by intragastric administration with SSAKY concentrate(the crude drug concentration was 1.6 g/mL),once a day,0.5 mL at a time for consecutive 8 weeks; while the ones in model group and sham-operated group with the same dosage of normal saline.After 8-week intervention,the mechanical and thermal pain thresholds of the mice were measured by Up-down method and hot plate test,respectively,and the gait of the mice was analyzed using the DigiGait gait analysis system.After that,the mice were executed,and the knee joints were harvested from their right hind limbs,and scanned by Micro-CT scanner to measure the bone volume fraction(BVF)of the subchondral bone in tibial plateau.Furthermore,the knee joint cartilage tissue sections were prepared and stained with alcian blue-hematoxylin/orange G(ABH/OG)to observe the degeneration of knee joint cartilage,and then evaluate by using Osteoarthritis Research Society International(OARSI)scoring criteria,apart from that,the expression of typeⅡcollagen and matrix metalloproteinase(MMP)-13 in the knee joint cartilages was detected by employing immunohistochemical staining.In addition,the tissue sections of the subchondral bone of mice knee joints and the right dorsal root ganglion(DRG)of L4 were prepared for detecting the expression of nerve growth factor(NGF)by immunofluorescence staining; besides,the heart,liver,spleen,lung and kidney of mice were harvested to make tissue sections for observing the histopathological changes via HE staining.Results:①The pain threshold.The 50% paw withdrawal threshold(PWT)was lower,and the reaction time of the right hind paw was shorter in model group compared to sham-operated group(P=0.000,P=0.000); while,the 50% PWT was higher,and the reaction time of the right hind paw was longer in SSAKY group compared to model group(P=0.000,P=0.000).②The gait.The model group exhibited longer swing phase duration,shorter stance phase duration,smaller stride length and paw contact area in the right hind limbs compared to the sham-operated group(P=0.001,P=0.000,P=0.000,P=0.000); while,the SSAKY group showed shorter swing phase duration,longer stance phase duration,greater stride length and paw contact area compared to the model group(P=0.001,P=0.000,P=0.000,P=0.000).③Micro-CT analysis on the knee joints.The obviously abnormal subchondral bone remodeling was observed in the tibial plateau in mice of model group,while that was significantly improved in SSAKY group compared to model group.The BVF of subchondral bone in tibial plateau was greater in model group compared to sham-operated group,while it was smaller in SSAKY group compared to model group(P=0.000,P=0.000).④The pathological observation of knee joint cartilage tissues.The changes,manifesting as severely worn cartilage with thinned and lost cartilage layer and fissures extending deep into the subchondral bone,were observed in the knee joints of mice in model group; while,compared with that of model group,the knee joint cartilage injury in mice of SSAKY group was significantly improved,only presented as partially damaged cartilage surface and a relatively intact matrix structure.The OARSI score was higher in model group compared to sham-operated group,while it was lower in SSAKY group compared to model group(P=0.000,P=0.000).⑤The expression of typeⅡcollagen and MMP-13 in the knee joint cartilage.The positive expression area ratio of typeⅡcollagen in knee joint cartilage was smaller,while that of MMP-13 was greater in model group compared to sham-operated group(P=0.000,P=0.000); The positive expression area ratio of typeⅡcollagen in knee joint cartilage increased,while that of MMP-13 decreased in SSAKY group compared to mo-del group(P=0.000,P=0.000).⑥The expression of NGF in the subchondral bone of knee joint and the right DRG of L4.The positive expression area ratio of NGF in the subchondral bone of knee joint and the right DRG of L4 was greater in model group compared to sham-operated group(P=0.000,P=0.000),while it was smaller in SSAKY group compared to model group(P=0.000,P=0.000).⑦The pathological observation of visceral tissues in mice.The pathological changes failed to be observed in the heart,liver,spleen,lung,or kidney in the mice of the 3 groups.Conclusion:SSAKY can effectively alleviate the pain sensitization and cartilage degeneration in KOA mice with high safety.It may work by inhibiting the expression of NGF and regulating the cartilage extracellular matrix metabolism.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(82274280); 浙江省基础公益研究计划项目(LR23H270001)
通讯作者:金红婷 E-mail:hongtingjin@163.com
更新日期/Last Update: 1900-01-01