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[1]李雪楓,王玉杭,鄒凱奧,等.腎髓安康飲干預(yù)小鼠膝骨關(guān)節(jié)炎的效果和安全性及作用機(jī)制研究[J].中醫(yī)正骨,2025,37(02):4-12.
　LI Xuefeng,WANG Yuhang,ZOU Kaiao,et al.Efficacy,safety,and mechanism of Shensui Ankang Yin(腎髓安康飲)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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腎髓安康飲干預(yù)小鼠膝骨關(guān)節(jié)炎的效果和安全性及作用機(jī)制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期數(shù):
2025年02期
頁碼:
4-12
欄目:
基礎(chǔ)研究
出版日期:
2025-02-20

文章信息/Info

Title:
Efficacy,safety,and mechanism of Shensui Ankang Yin(腎髓安康飲)against knee osteoarthritis in mice:an experimental study
作者:
李雪楓1; 王玉杭1; 鄒凱奧1; 溫經(jīng)淵1; 陳文哲1; 袁文華2; 肖魯偉2; 金紅婷2
(1.浙江中醫(yī)藥大學(xué)第一臨床醫(yī)學(xué)院,浙江 杭州 310053; 2.浙江省中醫(yī)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
關(guān)鍵詞:
骨關(guān)節(jié)炎; 膝;  腎髓安康飲;  疼痛;  敏化;  軟骨細(xì)胞;  神經(jīng)生長(zhǎng)因子;  小鼠
Keywords:
osteoarthritis; knee;  Shensui Ankang Yin;  pain;  sensitization;  chondrocytes;  nerve growth factor;  mice
摘要:
目的:探討腎髓安康飲干預(yù)小鼠膝骨關(guān)節(jié)炎(knee osteoarthritis,KOA)的效果、安全性及作用機(jī)制。方法:將30只C57BL/6J小鼠隨機(jī)分為假手術(shù)組、模型組和腎髓安康飲組。模型組和腎髓安康飲組小鼠采用內(nèi)側(cè)半月板失穩(wěn)術(shù)在右后肢構(gòu)建KOA模型,假手術(shù)組小鼠切開右后肢皮膚、暴露膝關(guān)節(jié)后縫合。自造模手術(shù)后第2天起,腎髓安康飲組小鼠以腎髓安康飲濃縮液(生藥濃度1.6 g·mL-1)灌胃,每次0.5 mL,每天1次,連續(xù)灌胃8周; 模型組和假手術(shù)組小鼠以等量生理鹽水灌胃。干預(yù)8周后,分別采用Up-down法和熱板實(shí)驗(yàn)測(cè)定小鼠機(jī)械痛閾值和熱痛閾值,采用DigiGait步態(tài)分析系統(tǒng)分析小鼠步態(tài); 采用Micro-CT掃描儀掃描小鼠右膝關(guān)節(jié),測(cè)定脛骨平臺(tái)軟骨下骨的骨體積分?jǐn)?shù); 制備小鼠膝關(guān)節(jié)軟骨組織切片,采用阿爾辛藍(lán)-蘇木素/橙黃G染色觀察膝關(guān)節(jié)軟骨退變情況,采用國(guó)際骨關(guān)節(jié)炎研究學(xué)會(huì)(Osteoarthritis Research Society International,OARSI)評(píng)分標(biāo)準(zhǔn)評(píng)價(jià)膝關(guān)節(jié)軟骨退變情況,采用免疫組織化學(xué)染色法檢測(cè)膝關(guān)節(jié)軟骨中Ⅱ型膠原、基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP)-13的表達(dá)情況; 制備小鼠膝關(guān)節(jié)軟骨下骨和L4右側(cè)背根神經(jīng)節(jié)組織切片,采用免疫熒光染色法檢測(cè)小鼠膝關(guān)節(jié)軟骨下骨和L4右側(cè)背根神經(jīng)節(jié)內(nèi)神經(jīng)生長(zhǎng)因子(nerve growth factor,NGF)的表達(dá)情況; 取小鼠心臟、肝臟、脾臟、肺臟、腎臟,制備組織切片,采用HE染色法觀察小鼠內(nèi)臟組織病變情況。結(jié)果:①疼痛閾值測(cè)定結(jié)果。模型組小鼠50%縮足閾值低于假手術(shù)組(P=0.000),右后足反應(yīng)時(shí)間短于假手術(shù)組(P=0.000); 腎髓安康飲組小鼠50%縮足閾值高于模型組(P=0.000),右后足反應(yīng)時(shí)間長(zhǎng)于模型組(P=0.000)。②步態(tài)分析結(jié)果。模型組小鼠右后肢的擺動(dòng)相時(shí)間長(zhǎng)于假手術(shù)組(P=0.001),支撐相時(shí)間短于假手術(shù)組(P=0.000),步幅和足底接觸面積均小于假手術(shù)組(P=0.000,P=0.000); 腎髓安康飲組小鼠右后肢的擺動(dòng)相時(shí)間短于模型組(P=0.001),支撐相時(shí)間長(zhǎng)于模型組(P=0.000),步幅和足底接觸面積均大于模型組(P=0.000,P=0.000)。③膝關(guān)節(jié)Micro-CT分析結(jié)果。模型組小鼠脛骨平臺(tái)軟骨下骨異常骨重塑明顯,腎髓安康飲組小鼠脛骨平臺(tái)軟骨下骨異常骨重塑較模型組明顯減少。模型組小鼠脛骨平臺(tái)軟骨下骨骨體積分?jǐn)?shù)大于假手術(shù)組(P=0.000),腎髓安康飲組小鼠脛骨平臺(tái)軟骨下骨骨體積分?jǐn)?shù)小于模型組(P=0.000)。④膝關(guān)節(jié)軟骨組織病理學(xué)觀察結(jié)果。模型組小鼠膝關(guān)節(jié)軟骨磨損嚴(yán)重,軟骨層變薄、缺失,裂隙深達(dá)軟骨下骨; 腎髓安康飲組小鼠膝關(guān)節(jié)軟骨損傷較模型組有明顯好轉(zhuǎn),軟骨表面部分損傷、基質(zhì)結(jié)構(gòu)較完整。模型組小鼠OARSI評(píng)分高于假手術(shù)組(P=0.000),腎髓安康飲組小鼠OARSI評(píng)分低于模型組(P=0.000)。⑤膝關(guān)節(jié)軟骨Ⅱ型膠原和MMP-13表達(dá)的檢測(cè)結(jié)果。模型組小鼠膝關(guān)節(jié)軟骨Ⅱ型膠原陽性表達(dá)面積比小于假手術(shù)組(P=0.000),MMP-13陽性表達(dá)面積比大于假手術(shù)組(P=0.000); 腎髓安康飲組小鼠膝關(guān)節(jié)軟骨Ⅱ型膠原陽性表達(dá)面積比大于模型組(P=0.000),MMP-13陽性表達(dá)面積比小于模型組(P=0.000)。⑥膝關(guān)節(jié)軟骨下骨和L4右側(cè)背根神經(jīng)節(jié)內(nèi)NGF表達(dá)的檢測(cè)結(jié)果。模型組小鼠膝關(guān)節(jié)軟骨下骨和L4右側(cè)背根神經(jīng)節(jié)內(nèi)NGF陽性表達(dá)面積比均大于假手術(shù)組(P=0.000,P=0.000),腎髓安康飲組小鼠膝關(guān)節(jié)軟骨下骨和L4右側(cè)背根神經(jīng)節(jié)內(nèi)NGF陽性表達(dá)面積比均小于模型組(P=0.000,P=0.000)。⑦小鼠內(nèi)臟組織病理學(xué)觀察結(jié)果。3組小鼠心臟、肝臟、脾臟、肺臟、腎臟均未見病理性改變。結(jié)論:腎髓安康飲能夠有效減輕KOA小鼠疼痛敏化和軟骨退化,且安全性高,其作用機(jī)制可能與其能夠抑制NGF表達(dá)、調(diào)節(jié)軟骨細(xì)胞外基質(zhì)代謝有關(guān)。
Abstract:
Objective:To observe the outcomes and safety of Shensui Ankang Yin(腎髓安康飲,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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備注/Memo

備注/Memo:
基金項(xiàng)目:國(guó)家自然科學(xué)基金項(xiàng)目(82274280); 浙江省基礎(chǔ)公益研究計(jì)劃項(xiàng)目(LR23H270001)
通訊作者:金紅婷 E-mail:[email protected]
更新日期/Last Update: 1900-01-01