[1]廖太阳,马振源,康俊峰,等.基于“柔肝养筋”理论探讨小鼠肝脏来源外泌体对膝骨关节炎模型小鼠膝关节软骨的影响[J].中医正骨,2024,36(03):7-14,22.
　LIAO Taiyang,MA Zhenyuan,KANG Junfeng,et al.Effects of hepatocyte-derived exosomes from mice on knee cartilage of model mice with knee osteoarthritis:a softening liver and nourishing tendons theory-based experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(03):7-14,22.
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基于“柔肝养筋”理论探讨小鼠肝脏来源外泌体对膝骨关节炎模型小鼠膝关节软骨的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期数:
2024年03期
页码:
7-14,22
栏目:
基础研究
出版日期:
2024-03-20

文章信息/Info

Title:
Effects of hepatocyte-derived exosomes from mice on knee cartilage of model mice with knee osteoarthritis:a softening liver and nourishing tendons theory-based experimental study
作者:
廖太阳; 马振源; 康俊峰; 杨楠; 王培民; 邢润麟
江苏省中医院,江苏 南京 210029
Author(s):
LIAO Taiyang; MA Zhenyuan; KANG Junfeng; YANG Nan; WANG Peimin; XING Runlin
Jiangsu Province Hospital of Chinese Medicine,Nanjing 210029,Jiangsu,China
关键词:
外泌体;  软骨;  骨关节炎; 膝;  小鼠;  动物实验
Keywords:
exosomes;  cartilage;  osteoarthritis; knee;  mice;  animal experimentation
摘要:
目的:基于“柔肝养筋”理论探讨小鼠肝脏来源外泌体对膝骨关节炎(knee osteoarthritis,KOA)模型小鼠膝关节软骨的影响。方法:以超速离心法从3月龄(年轻)和22月龄(老年)C57BL/6雄性小鼠肝脏提取外泌体。将48只7周龄SPF级C57BL/6J雄性小鼠随机分为4组,每组12只。KOA组、Young-EVs组及Aged-EVs组小鼠采用前交叉韧带横断术进行KOA造模,Sham组小鼠接受手术但不离断前交叉韧带。术后14 d开始,向Young-EVs组和Aged-EVs组小鼠膝关节腔分别注射10 μL年轻和老年小鼠肝脏来源外泌体,每周2次,连续注射4周; Sham组和KOA组用同样方法向膝关节腔注射10 μL PBS溶液。外泌体干预结束后,对各组小鼠造模侧膝关节进行X线检查; 苏木精-伊红染色和番红O-固绿染色后进行组织病理学观察,并采用国际骨关节炎研究协会(Osteoarthritis Research Society International,OARSI)骨关节炎软骨组织病理学评价系统及Mankin病理评分系统评价小鼠软骨退变程度; 免疫组化法检测软骨组织中Ⅱ型胶原蛋白(collagenⅡ,COLⅡ)、聚集蛋白聚糖(aggrecan,ACAN)、基质金属蛋白酶(matrix metalloproteinase,MMP)13和一种具有血小板反应蛋白基序的去整合素和金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs,ADAMTS)5表达量。结果:①小鼠膝关节X线检查结果。Sham组小鼠膝关节关节间隙正常,关节面平整光滑,软骨完整,关节边缘无骨赘形成,Kellgren-Lawrence分级为0级; KOA组小鼠膝关节间隙未见明显狭窄,关节面粗糙,软骨出现部分缺损,关节边缘可见明显骨赘形成,Kellgren-Lawrence分级为Ⅱ级; Young-EVs组小鼠膝关节间隙轻微变窄,关节表面稍有粗糙表现,软骨缺损较KOA组减少,关节边缘可见轻微骨赘形成,Kellgren-Lawrence分级为Ⅰ级; Aged-EVs组小鼠膝关节间隙、关节表面粗糙程度、软骨缺损情况、关节内游离体数量较KOA组更为严重,Kellgren-Lawrence分级为Ⅲ级。②小鼠膝关节软骨组织病理学观察结果。4组小鼠膝关节软骨OARSI评分和Mankin评分组间总体比较,差异均有统计学意义[OARSI评分:(0.417±0.376)分,(4.500±0.632)分,(2.417±0.492)分,(5.417±0.492)分,F=116.800,P=0.000; Mankin评分:(1.083±0.665)分,(7.250±0.880)分,(3.583±0.917)分,(8.833±0.683)分,F=117.100,P=0.000]。KOA组、Young-EVs组及Aged-EVs组的OARSI评分和Mankin评分均高于Sham组(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); Young-EVs组的OARSI评分和Mankin评分均低于KOA组(P=0.000,P=0.000); Aged-EVs组的OARSI评分和Mankin评分均高于KOA组、Young-EVs组(P=0.025,P=0.000; P=0.015,P=0.000)。③小鼠膝关节软骨组织免疫组织化学检测结果。4组小鼠膝关节软骨中COLⅡ和ACAN阳性表达面积比组间总体比较,差异均有统计学意义[COLⅡ:(40.050±2.324)%,(26.083±1.119)%,(23.317±2.599)%,(16.717±2.479)%,F=118.500,P=0.000; ACAN:(51.500±3.191)%,(17.700±3.415)%,(19.383±3.924)%,(12.300±1.908)%,F=185.500,P=0.000]。KOA组、Young-EVs组及Aged-EVs组的COLⅡ和ACAN阳性表达面积比均低于Sham组(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); Young-EVs组与KOA组COLⅡ、ACAN阳性表达面积比的组间差异均无统计学意义(P=0.167,P=0.799); Aged-EVs组的COLⅡ和ACAN阳性表达面积比均低于KOA组、Young-EVs组(P=0.000,P=0.000; P=0.039,P=0.005)。4组MMP13和ADAMTS5阳性表达面积比组间总体比较,差异均有统计学意义[MMP13:(11.733±2.191)%,(46.150±6.237)%,(34.417±5.027)%,(57.233±4.049)%,F=106.600,P=0.000; ADAMTS5:(7.950±1.344)%,(30.533±6.184)%,(22.083±3.418)%,(39.317±5.606)%,F=51.450,P=0.000]。KOA组、Young-EVs组及Aged-EVs组的MMP13和ADAMTS5阳性表达面积比均高于Sham组(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); Young-EVs组的MMP13和ADAMTS5阳性表达面积比均低于KOA组(P=0.002,P=0.021); Aged-EVs组的MMP13和ADAMTS5阳性表达面积比均高于KOA组、Young-EVs组(P=0.003,P=0.000; P=0.016,P=0.000)。结论:小鼠肝脏来源外泌体可能参与软骨细胞合成及分解代谢反应; 其中老年小鼠肝脏来源外泌体可加速细胞外基质降解、减少聚集蛋白聚糖合成,加剧KOA小鼠软骨退变; 而年轻小鼠肝脏来源外泌体可抑制软骨细胞外基质降解,保护KOA小鼠软骨。
Abstract:
Objective:To explore the effects of hepatocyte-derived exosomes(HDEs)from mice on knee cartilage of model mice with knee osteoarthritis(KOA)based on softening liver and nourishing tendons theory.Methods:The exosomes were isolated and extracted from the livers of young(3-month-old)and aged(22-month-old)male C57BL/6 mice by using ultracentrifugation.Forty-eight 7-week-old specific pathogen free(SPF)-grade male C57BL/6J mice were randomized into KOA group,Young-EVs group,Aged-EVs group and Sham group,12 ones in each group.The mice in KOA group,Young-EVs group and Aged-EVs group were subjected to anterior cruciate ligament(ACL)transection for inducing KOA; while the ones in Sham group only underwent surgery,but without transecting ACL.At postoperative day 14 after the modeling,the mice in Young-EVs group and Aged-EVs group were intervened by knee intra-articular injection of 10 μL HDEs extracted from young and aged mice,respectively,two times a week for consecutive 4 weeks; while the ones in Sham group and KOA group with 10 μL phosphate buffered saline(PBS)solution.After the end of intervention,the knee joints of the modeling side in mice of each group were checked by X-ray scanning,and were evaluated by Kellgren-Lawrence(K-L)grading system.After the end of X-ray examination,the knees were harvested from mice of each group,and the knee tissue sections were stained with hematoxylin-eosin(HE)and safranin O-fast green(SO-FG)for observing the histopathological changes; meanwhile,the degree of knee articular cartilage degeneration was evaluated by using Osteoarthritis Research Society International(OARSI)osteoarthritis cartilage histopathology assessment system and Mankin scoring system.Furthermore,the expression levels of collagenⅡ(COLⅡ),aggrecan(ACAN),matrix metalloproteinase(MMP)13 and a disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS)5 in knee cartilage tissues were detected by using immunohistochemistry(IHC).Results:①The results of X-ray examination showed that the mice in Sham group presented with normal knee joint space,smooth articular surface,and intact cartilage,and no osteophyte was observed at the margin of knee joint.The K-L classification belonged to grade 0.The changes,manifesting as rough articular surface,partially defective cartilage and definite osteophytes at the margin of knee joint,were observed,while,the marked knee joint space narrowing(JSN)were unobserved in mice of KOA group.The K-L classification belonged to gradeⅡ.The slight JSN,slightly rough articular surface,defective cartilage(decreased compared to KOA group),and small osteophytes at the margin of knee joint were observed in mice of Young-EVs group.The K-L classification belonged to gradeⅠ.Compared to KOA group,the changes,including knee joint space,articular surface roughness,cartilage defects,and the number of free bodies within the joint,were more severe in mice of Aged-EVs group.The K-L classification belonged to gradeⅢ.②There was statistical difference in OARSI score and Mankin score between the 4 groups(OARSI score:0.417±0.376,4.500±0.632,2.417±0.492,5.417±0.492 points,F=116.800,P=0.000; Mankin score:1.083±0.665,7.250±0.880,3.583±0.917,8.833±0.683 points,F=117.100,P=0.000).The OARSI score and Mankin score were higher in KOA group,Young-EVs group and Aged-EVs group compared to Sham group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),and were higher in KOA group compared to Young-EVs group(P=0.000,P=0.000),and were highest in Aged-EVs group(P=0.025,P=0.000; P=0.015,P=0.000).③There was statistical difference in the positive expression area ratios of COLⅡand ACAN in knee cartilage between the 4 groups(COLⅡ:40.050±2.324,26.083±1.119,23.317±2.599,16.717±2.479%,F=118.500,P=0.000; ACAN:51.500±3.191,17.700±3.415,19.383±3.924,12.300±1.908%,F=185.500,P=0.000).The positive expression area ratios of COLⅡand ACAN were lower in KOA group,Young-EVs group and Aged-EVs group compared to Sham group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),and were lower in Aged-EVs group compared to KOA group and Young-EVs group(P=0.000,P=0.000; P=0.039,P=0.005),while,the comparison between Young-EVs group and KOA group revealed no significant differences(P=0.167,P=0.799).There was statistical difference in the positive expression area ratios of MMP13 and ADAMTS5 in knee cartilage between the 4 groups(MMP13:11.733±2.191,46.150±6.237,34.417±5.027,57.233±4.049%,F=106.600,P=0.000; ADAMTS5:7.950±1.344,30.533±6.184,22.083±3.418,39.317±5.606%,F=51.450,P=0.000).The positive expression area ratios of MMP13 and ADAMTS5 were higher in KOA group,Young-EVs group and Aged-EVs group compared to Sham group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),and were higher in KOA group compared to Young-EVs group(P=0.002,P=0.021),and were highest in Aged-EVs group(P=0.003,P=0.000; P=0.016,P=0.000).Conclusion:The mice HDEs may be involved in the synthesis and catabolic reactions of chondrocytes,among which HDEs from the aged mice can accelerate the degradation of extracellular matrix and reduce the synthesis of ACAN,thereby exacerbating the cartilage degeneration in KOA mice; whereas the ones from the young mice can inhibit the degradation of cartilage extracellular matrix,thus exerting the effect of protecting cartilage in KOA mice.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(81804123); 江苏省第六期“333人才”培养资助项目(第三层次青年人才项目); 江苏省中医药科技发展计划青年人才项目(QN202210); 南京中医药大学自然科学基金项目(XZR2020005); 江苏省中医院高峰学术人才项目(Y2021rc39); 江苏省医学重点学科和医学重点实验室建设项目(JSDW202252)
通讯作者:邢润麟 E-mail:xingrunlin@126.com
更新日期/Last Update: 1900-01-01