[1]何宇,贾琼,罗虎,等.毛冬青汤干预大鼠膝骨关节炎的疗效及作用机制研究[J].中医正骨,2025,37(10):7-14.
　HE Yu,JIA Qiong,LUO Hu,et al.Efficacy and mechanism of Maodongqing Tang(毛冬青汤)against knee osteoarthritis in rats:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(10):7-14.
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毛冬青汤干预大鼠膝骨关节炎的疗效及作用机制研究()

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年10期
页码:
7-14
栏目:
基础研究
出版日期:
2025-10-20

文章信息/Info

Title:
Efficacy and mechanism of Maodongqing Tang(毛冬青汤)against knee osteoarthritis in rats:an experimental study
作者:
何宇; 贾琼; 罗虎; 赵俊淞; 罗凌威; 曹雨林; 张作夫
湖南中医药大学附属省直中医医院,湖南 株洲 412000
Author(s):
HE Yu; JIA Qiong; LUO Hu; ZHAO Junsong; LUO Lingwei; CAO Yulin; ZHANG Zuofu
Hunan Provincial Hospital of Traditional Chinese Medicine Affiliated to Hunan University of Chinese Medicine,Zhuzhou 412000,Hunan,China
关键词:
骨关节炎; 膝;  毛冬青;  类Toll受体4;  髓系分化因子88;  NF-κB;  大鼠;  动物实验
Keywords:
osteoarthritis; knee;  radix ilicis pubescentis;  Toll-like receptor 4;  myeloid differentiation factor 88;  NF-kappa B;  rats;  animal experimentation
摘要:
目的:探讨毛冬青汤干预大鼠膝骨关节炎(knee osteoarthritis,KOA)的疗效及作用机制。方法:将45只雄性SD大鼠随机分为空白对照组、假手术组、模型组、毛冬青汤组和塞来昔布组,每组9只。模型组、毛冬青汤组和塞来昔布组大鼠采用Hulth法建立右侧KOA模型; 假手术组大鼠仅切开右侧膝关节内侧皮肤及皮下组织,暴露关节腔后缝合; 空白对照组大鼠不进行任何处理。造模后4周,毛冬青汤组大鼠按照生药量5.4 g·kg-1·d-1以毛冬青汤药液灌胃,塞来昔布组大鼠按照12 mg·kg-1·d-1以塞来昔布溶液灌胃,空白对照组、假手术组和模型组按照10 mL·kg-1·d-1以生理盐水灌胃。连续干预8周后,采用ELISA试剂盒检测血清白细胞介素(interleukin,IL)-1β、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量,采用Micro-CT分析胫骨平台软骨下骨的骨体积分数、骨矿物质含量、骨小梁数量,采用番红O-固绿染色观察膝关节软骨组织病理改变,采用实时定量PCR检测膝关节软骨组织中Toll样受体(Toll-like receptor,TLR)4、髓系分化初级反应蛋白质88(myeloid differentiation primary response protein 88,MyD88)、核因子κB(nuclear factor-κB,NF-κB)的mRNA表达水平,采用Western Blot法检测膝关节滑膜组织中TLR4、MyD88、NF-κB的蛋白表达水平。结果:①血清促炎性细胞因子含量检测结果。假手术组大鼠血清IL-1β、IL-6、TNF-α含量均高于空白对照组(P=0.000,P=0.000,P=0.000); 模型组大鼠血清IL-1β、IL-6、TNF-α含量均高于假手术组(P=0.000,P=0.000,P=0.000); 毛冬青汤组、塞来昔布组大鼠血清IL-1β、IL-6、TNF-α含量均低于模型组(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); 毛冬青汤组大鼠血清IL-1β、IL-6与塞来昔布组的差异均无统计学意义(P=1.000,P=0.397),TNF-α含量高于塞来昔布组(P=0.001)。②胫骨平台软骨下骨微结构检测结果。5组大鼠胫骨平台软骨下骨骨体积分数比较,组间差异无统计学意义(F=2.642,P=0.097)。模型组大鼠胫骨平台软骨下骨骨矿物质含量低于假手术组(P=0.030),骨小梁数量与假手术组的差异无统计学意义(P=0.592); 毛冬青汤组和塞来昔布组大鼠胫骨平台软骨下骨骨矿物质含量均高于模型组(P=0.008,P=0.007),骨小梁数量均多于模型组(P=0.001,P=0.001); 毛冬青汤组大鼠胫骨平台软骨下骨骨矿物质含量、骨小梁数量与塞来昔布组的差异均无统计学意义(P=1.000,P=1.000)。③膝关节软骨组织病理学观察结果。模型组大鼠表层软骨大面积剥脱,软骨细胞簇集,软骨基质染色较浅; 毛冬青汤组和塞来昔布组大鼠表层软骨缺损较模型组减轻,软骨细胞排列较规律,软骨基质染色较深,其中毛冬青汤组大鼠软骨基质染色深于塞来昔布组。④膝关节软骨组织中TLR4-MyD88-NF-κB信号通路相关基因的mRNA表达水平检测结果。模型组大鼠膝关节软骨组织中TLR4、MyD88、NF-κB的mRNA相对表达量均高于假手术组(P=0.000,P=0.000,P=0.000); 塞来昔布组和毛冬青汤组大鼠膝关节软骨组织中TLR4、MyD88、NF-κB的mRNA相对表达量均低于模型组(P=0.000,P=0.000; P=0.000,P=0.000; P=0.000,P=0.000); 毛冬青汤组大鼠膝关节软骨组织中TLR4的mRNA相对表达量高于塞来昔布组(P=0.011),MyD88、NF-κB的mRNA相对表达量与塞来昔布组的差异均无统计学意义(P=0.975,P=0.264)。⑤膝关节滑膜组织中TLR4-MyD88-NF-κB信号通路相关基因的蛋白表达水平检测结果。模型组大鼠滑膜组织中TLR4、MyD88、NF-κB的蛋白相对表达量均高于假手术组(P=0.000,P=0.000,P=0.017); 毛冬青汤组和塞来昔布组大鼠滑膜组织中TLR4、MyD88、NF-κB的蛋白相对表达量均低于模型组(P=0.000,P=0.000; P=0.000,P=0.003; P=0.006,P=0.000); 毛冬青汤组大鼠膝关节软骨组织中MyD88的蛋白相对表达量低于塞来昔布组(P=0.006),TLR4、NF-κB的蛋白相对表达量与塞来昔布组的差异均无统计学意义(P=0.366,P=1.000)。结论:毛冬青汤干预大鼠KOA,能够抑制炎症反应、修复软骨和软骨下骨,其作用机制与抑制滑膜和软骨细胞中TLR4-MyD88-NF-κB信号通路相关基因的表达有关。
Abstract:
Objective:To investigate the therapeutic efficacy of Maodongqing Tang(毛冬青汤,MDQT)against knee osteoarthritis(KOA)in rats,and to explore its underlying mechanism.Methods:Forty-five male SD rats were selected and randomized into blank control group,sham-operated group,model group,MDQT group,and celecoxib group,with 9 cases in each group.The rats in model group,MDQT group,and celecoxib group were subjected to surgeries on the right knee joints by using Hulth method to build KOA models,while the ones in sham-operated group were merely incised the medial skin and subcutaneous tissues to expose the knee joint cavity at the corresponding sites and then sutured,the ones in blank control group were not given any intervention.Four weeks after successful modeling,the rats in MDQT group were intervened by intragastric administration with MDQT solution at a crude dose of 5.4 g/kg/day,the ones in celecoxib group with celecoxib solution at a dose of 12 mg/kg/day,and the ones in the remaining groups with sterile saline at a dose of 10 mg/kg/day for consecutive 8 weeks.Following the end of intervention,the blood was drawn and the right knee joints were harvested for the following analyses:the serum levels of interleukin(IL)-1β,IL-6,and tumor necrosis factor-α(TNF-α)in the serum samples were measured by ELISA,the knee joint specimens were scanned by Micro-CT for examining the subchondral bone microarchitecture and analyzing the parameters such as bone volume/tissue volume(BV/TV),bone mineral content(BMC),and trabecular number(Tb.N),and the histopathological changes of knee articular cartilage tissues were observed by safranin O-fast green(SO-FG)staining.Furthermore,the mRNA expression le-vels of Toll-like receptor(TLR)4,myeloid differentiation primary response protein 88(MyD88),and nuclear factor-κB(NF-κB)in the knee articular cartilage tissues were determined by real-time quantitative PCR(RT-qPCR),and the protein expression levels of TLR4,MyD88,and NF-κB in knee articular synovial tissues were detected by Western Blot.Results:①Serum pro-inflammatory cytokine levels.The serum levels of IL-1β,IL-6,and TNF-α were higher in sham-operated group compared to blank control group(P=0.000,P=0.000,P=0.000),and these markers were also higher in model group compared to sham-operated group(P=0.000,P=0.000,P=0.000); conversely,these markers reduced in both MDQT group and celecoxib group compared to model group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),with MDQT group showing comparable serum levels of IL-1β and IL-6 to celecoxib group,but higher serum level of TNF-α(P=1.000,P=0.397,P=0.001).②Subchondral bone microstructure of the tibial plateau.The BV/TV showed no significant difference among the 5 groups(F=2.642,P=0.097).The BMC in subchondral bone of tibial plateau reduced in model group compared to the sham-operated group,but Tb.N showed no significant difference between the 2 geoups(P=0.030,P=0.592).Conversely,the BMC and Tb.N were elevated in both MDQT group and celecoxib group compared to model group(P=0.008,P=0.007; P=0.001,P=0.001),with MDQT group showing comparable therapeutic effect to celecoxib group in improving BMC and Tb.N(P=1.000,P=1.000).③Histopathological findings in rat knee articular cartilage.The marked changes,manifesting as large-scale superficial cartilage exfoliation,clustered chondrocytes and lighter stained cartilage matrix,were observed in the knee articular cartilage of rats in model group,whereas,the aforementioned changes were ameliorated,manifesting as reduced superficial cartilage defects,more organized chondrocyte arrangement and deeper stained cartilage matrix,in MDQT group and celecoxib group compared to model group,with the MDQT group showing the deeper stained cartilage matrix compared to the celecoxib group.④The mRNA expression levels of TLR4-MyD88-NF-κB signaling pathway-related genes in knee articular cartilage tissues.The relative mRNA expression levels of TLR4,MyD88,and NF-κB in the knee articular cartilage tissues were all higher in model group compared to sham-operated group(P=0.000,P=0.000,P=0.000),while these markers were all downregulated in celecoxib group and MDQT group compared to model group(P=0.000,P=0.000; P=0.000,P=0.000; P=0.000,P=0.000); furthermore,the relative mRNA expression level of TLR4 was higher in MDQT group compared to celecoxib group(P=0.011),but those of MyD88 and NF-κB showed no significant difference between the 2 groups(P=0.975,P=0.264).⑤The protein expression levels of TLR4-MyD88-NF-κB signaling pathway-related genes in knee articular synovial tissues.The relative protein expression levels of TLR4,MyD88,and NF-κB in rat knee articular synovial tissues were all higher in model group compared to sham-operated group(P=0.000,P=0.000,P=0.017),while these markers were all downregulated in MDQT group and celecoxib group compared to model group(P=0.000,P=0.000; P=0.000,P=0.003; P=0.006,P=0.000); furthermore,the relative protein expression level of MyD88 was lower in MDQT group compared to celecoxib group(P=0.006),but those of TLR4 and NF-κB showed no significant difference between the 2 groups(P=0.366,P=1.000).Conclusion:MDQT can inhibit inflammatory response and repair cartilage and subchondral bone in intervention of KOA in rats.It may exert the effects by inhibiting the expression of TLR4-MyD88-NF-κB signaling pathway-related genes in synoviocytes and chondrocytes.

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备注/Memo

备注/Memo:
基金项目:湖南省中医药科研课题(A2024043); 湖南省“十四五”第二批中医药领军人才、科学带人和骨干人才培养项目(湘中医药〔2024〕3号)
通讯作者:贾琼 E-mail:04170731@163.com
更新日期/Last Update: 1900-01-01