[1]张毓杰,田飚,闻胜月,等.石氏薰洗方药浴干预兔膝骨关节炎的效果及作用机制研究[J].中医正骨,2025,37(07):4-14.
　ZHANG Yujie,TIAN Biao,WEN Shengyue,et al.Efficacy and mechanism of Shi's Xunxi Fang(石氏薰洗方)medicated bath therapy against knee osteoarthritis in rabbits:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(07):4-14.
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石氏薰洗方药浴干预兔膝骨关节炎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07期
页码:
4-14
栏目:
基础研究
出版日期:
2025-07-20

文章信息/Info

Title:
Efficacy and mechanism of Shi's Xunxi Fang(石氏薰洗方)medicated bath therapy against knee osteoarthritis in rabbits:an experimental study
作者:
张毓杰; 田飚; 闻胜月; 张旻; 庞坚; 詹红生; 陈博
上海中医药大学附属曙光医院,上海 201203
Author(s):
ZHANG Yujie; TIAN Biao; WEN Shengyue; ZHANG Min; PANG Jian; ZHAN Hongsheng; CHEN Bo
Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China
关键词:
骨关节炎; 膝;  兔;  石氏薰洗方;  沐浴疗法;  白细胞介素-1β;  肿瘤坏死因子-α;  核因子-κB;  环氧化酶-2;  基质金属蛋白酶;  一氧化氮合酶
Keywords:
osteoarthritis; knee;  rabbits;  Shi's Xunxi Fang;  bath therapy;  interleukin-1beta;  tumor necrosis factor-alpha;  NF-kappa B;  cyclooxygenase 2;  matrix metalloproteinases;  nitric oxide synthase
摘要:
目的:探讨石氏薰洗方药浴干预兔膝骨关节炎(knee osteoarthritis,KOA)的效果及作用机制。方法:将50只新西兰兔随机分为假手术组(6只)、模型组(6只)、石氏薰洗方浸泡组(16只)、纯净水浸泡组(16只)和扶他林组(6只)。模型组、石氏薰洗方浸泡组、纯净水浸泡组及扶他林组在兔右膝关节腔内注射0.3 mL含0.03 mol·L-1左旋半胱氨酸和4%木瓜蛋白酶的生理盐水; 假手术组仅穿刺右膝关节腔,不注射药物。分别从各组随机选取1只兔,验证造模成功后,将石氏薰洗方浸泡组剩余的15只兔随机分为药浴45 ℃组、40 ℃组和35 ℃组,每组5只,分别用相应温度的石氏薰洗方药液浸泡右膝关节,每次30 min,每天1次; 将纯净水浸泡组剩余的15只兔随机分为水浴45 ℃组、40 ℃组和35 ℃组,每组5只,分别用相应温度的纯净水浸泡右膝关节,每次30 min,每天1次; 扶他林组兔于右膝关节处外涂双氯芬酸二乙胺乳胶剂,每次涂抹5 min,每天2次; 各组均连续干预4周。模型组与假手术组兔不做任何处理。干预结束后,行右膝关节X线检查,观察关节间隙及骨赘形成情况; 解剖分离右膝关节,观察软骨表面及软骨下骨暴露情况,评估膝关节软骨退变情况; 采用HE染色观察右膝关节滑膜组织病理学变化,评估滑膜炎严重程度; 采用ELISA法检测膝关节液中白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、核因子-κB(nuclear factor-κB,NF-κB)、环氧化酶-2(cyclooxygenase-2,COX-2)、一氧化氮合酶(nitric oxide synthase,NOS)、基质金属蛋白酶(matrix metalloproteinases,MMP)-3、MMP-13的蛋白表达水平; 采用实时定量逆转录PCR检测IL-1β、TNF-α、NF-κB、COX-2、NOS、MMP-3、MMP-13的mRNA表达水平。结果:①膝关节影像学观察结果。干预结束后,假手术组膝关节间隙正常、无骨赘增生; 模型组膝关节间隙明显变窄,有明显骨赘形成; 药浴各温度组(45 ℃、40 ℃、35 ℃)相较于同温度水浴组,关节间隙略宽,骨赘体积减小,骨质密度异常区域缩小,且影像灰度层次更清晰; 药浴40 ℃组相较于药浴45 ℃组和35 ℃组,关节间隙更宽,骨赘数量更少、体积更小; 水浴40 ℃组相较于水浴45 ℃组和35 ℃组,关节间隙略宽,骨赘数量减少、体积减小; 扶他林组关节间隙接近正常,骨赘较少,形态接近正常。②膝关节软骨大体观察结果。药浴40 ℃组关节软骨退变评分低于药浴45 ℃组和35 ℃组(P=0.047,P=0.028),水浴40 ℃组关节软骨退变评分低于水浴45 ℃组和35 ℃组(P=0.049,P=0.033),药浴40 ℃组关节软骨退变评分低于水浴45 ℃组和35 ℃组(P=0.007,P=0.015)、与水浴40 ℃组的差异无统计学意义(P=0.330),药浴40 ℃组关节软骨退变评分与扶他林组的差异无统计学意义(P=0.083),水浴40 ℃组关节软骨退变评分高于扶他林组(P=0.021)。③膝关节滑膜组织病理学观察结果。药浴40 ℃组滑膜病理学评分低于药浴45 ℃组和35 ℃组(P=0.047,P=0.020),水浴40 ℃组滑膜病理学评分低于水浴45 ℃组和35 ℃组(P=0.044,P=0.002),药浴40 ℃组滑膜病理学评分低于水浴45 ℃组、40 ℃组和35 ℃组(P=0.004,P=0.037,P=0.000),药浴40 ℃组滑膜病理学评分与扶他林组的差异无统计学意义(P=0.679),水浴40 ℃组滑膜病理学评分高于扶他林组(P=0.017)。④膝关节液中炎症相关因子及基质降解酶的蛋白表达水平检测结果。药浴各温度组(45 ℃、40 ℃、35 ℃)、水浴各温度组(45 ℃、40 ℃、35 ℃)及扶他林组膝关节液中IL-1β、TNF-α、NF-κB、COX-2、NOS、MMP-3、MMP-13的蛋白表达水平均低于模型组(均P=0.000),药浴45 ℃组膝关节液中IL-1β、NF-κB、NOS、MMP-3、MMP-13的蛋白表达水平均低于药浴40 ℃组和35 ℃组(IL-1β:P=0.001,P=0.000; NF-κB:P=0.017,P=0.027; NOS:P=0.029,P=0.002; MMP-3:P=0.035,P=0.001; MMP-13:P=0.045,P=0.000),药浴40 ℃组膝关节液中TNF-α、COX-2的蛋白表达水平均低于药浴45 ℃组和35 ℃组(TNF-α:P=0.011,P=0.017; COX-2:P=0.009,P=0.002); 药浴40 ℃组膝关节液中IL-1β、TNF-α、NF-κB、COX-2、NOS、MMP-3、MMP-13的蛋白表达水平均低于水浴40 ℃组(P=0.002,P=0.035,P=0.000,P=0.001,P=0.026,P=0.005,P=0.004)。⑤膝关节液中炎症相关因子及基质降解酶的mRNA表达水平检测结果。药浴各温度组(45 ℃、40 ℃、35 ℃)、水浴各温度组(45 ℃、40 ℃、35 ℃)及扶他林组膝关节液中IL-1β、TNF-α、NF-κB、COX-2、NOS、MMP-3、MMP-13的mRNA表达水平均低于模型组(均P=0.000),药浴40 ℃组膝关节液中IL-1β、TNF-α、NF-κB、COX-2、NOS、MMP-3、MMP-13的mRNA表达水平均低于药浴45 ℃和35 ℃组(IL-1β:P=0.000,P=0.000; TNF-α:P=0.010,P=0.000; NF-κB:P=0.013,P=0.002; COX-2:P=0.000,P=0.013; NOS:P=0.024,P=0.018; MMP-3:P=0.038,P=0.014; MMP-13:P=0.001,P=0.000),药浴40 ℃组膝关节液中IL-1β、TNF-α、NF-κB、COX-2、NOS、MMP-3、MMP-13的mRNA表达水平均低于水浴40 ℃组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.001,P=0.000,P=0.000)。结论:石氏薰洗方药浴能减轻KOA模型兔滑膜炎症和延缓关节软骨退变,且在40 ℃时疗效最佳,其效果与双氯芬酸二乙胺乳胶剂外涂相当; 其作用机制可能与其能够抑制IL-1β、TNF-α等促炎性细胞因子,阻断NF-κB信号通路,降低COX-2、NOS、MMPs的表达有关。
Abstract:
Objective:To investigate the therapeutic efficacy of Shi's Xunxi Fang(石氏薰洗方,SXXF)medicated bath therapy against knee osteoarthritis(KOA)in rabbits,and to explore its underlying mechanism.Methods:Fifty New Zealand rabbits were randomly assigned into sham-operated group(6 ones),model group(6 ones),SXXF immersion group(16 ones),purified water immersion group(16 ones),and voltaren group(6 ones).All rabbits but the ones in sham-operated group were treated with intra-articular injection of 0.3 mL normal saline containing 0.03 mol/L L-cysteine and 4% papain into the right knee joints for inducing KOA model,while the ones in sham-operated group only underwent puncture of the right knee joint cavity,without any drug injection.After that,one rabbit was randomly selected from each group to verify whether the KOA models were built successfully.After successful modeling,the remaining 15 rabbits in the SXXF immersion group and 15 ones in the purified water immersion group were further randomized into 45,40,and 35 ℃ subgroups,respectively,with 5 ones in each group.The right knee joints of rabbits in the 6 subgroups were immersed in SXXF decoction and purified water,respectively,at their corresponding temperatures,once a day,30 minutes at a time for consecutive 4 weeks.The rabbits in the voltaren group received external application of diclofenac diethylamine emulgel to the right knee joints,twice a day,5 minutes at a time for consecutive 4 weeks.The ones in model group and sham-operated group were not given any intervention.After the end of intervention,the joint space and osteophyte formation were observed on the right knee joints by X-ray examination,and the right knee joints were dissected and separated to examine the articular cartilage surface and subchondral bone exposure and evaluate the degree of knee joint cartilage degeneration.Besides,the right knee synovial tissues were harvested and stained with HE staining kit to observe the histopathological changes and evaluate the severity of synovitis.Furthermore,the protein expression levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),nuclear factor-κB(NF-κB),cyclooxygenase-2(COX-2),nitric oxide synthase(NOS),matrix metalloproteinase(MMP)-3,and MMP-13 in the knee joint synovial fluid were quantified using enzyme-linked immunosorbent assay(ELISA),and the mRNA expression levels of IL-1β,TNF-α,NF-κB,COX-2,NOS,MMP-3,and MMP-13 were detected by real-time quantitative reverse transcription PCR(qRT-PCR).Results:①Post-intervention radiographic observations on knee joint.After the end of the intervention,the rabbits in sham-operated group exhibited normal knee joint space without osteophyte formation.In contrast,the rabbits in model group displayed a significantly narrowed knee joint space,with prominent osteophyte formation.Moreover,compared to the 45,40,and 35 ℃ purified water immersion subgroups,the marked changes,manifesting as as slightly widened knee joint space,reduced osteophyte volume,diminished areas of abnormal bone density,and improved grayscale resolution on radiographic images,were observed in the rabbits of SXXF immersion subgroups at the corresponding temperatures.Specifically,compared to the 45 and 35 ℃ SXXF immersion subgroups,wider joint space,fewer osteophytes with smaller volume were observed in rabbits of 40 ℃ SXXF immersion subgroup.Similarly,compared to the 45 and 35 ℃ purified water immersion subgroups,the slightly wider joint space and decreased osteophytes with reduced volume were observed in rabbits of 40 ℃ purified water immersion subgroup.The rabbits in voltaren group presented a joint space approaching normal,with fewer osteophytes and basically normal morphology.②Gross morphological observation on knee joint cartilage.The degenerative score of joint cartilage was lower in 40 ℃ SXXF immersion subgroup compared to 45 and 35 ℃ SXXF immersion subgroups(P=0.047,P=0.028).Similarly,the degenerative score of joint cartilage was lower in 40 ℃ purified water immersion subgroup compared to 45 and 35 ℃ purified water immersion subgroups(P=0.049,P=0.033),and it was lower in 40 ℃ SXXF immersion subgroup compared to 45 and 35 ℃ purified water immersion subgroups(P=0.007,P=0.015),but the comparison between 40 ℃ SXXF immersion subgroup and 40 ℃ purified water immersion subgroup or between 40 ℃ SXXF immersion subgroup and voltaren group revealed no significant differences(P=0.330,P=0.083),however,the degenerative score was higher in 40 ℃ purified water immersion subgroup compared to voltaren group(P=0.021).③Pathological observation on knee joint synovial tissues.The pathological score of synovium was lower in 40 ℃ SXXF immersion subgroup compared to 45 and 35 ℃ SXXF immersion subgroups(P=0.047,P=0.020).Similarly,the pathological score of synovium was lower in 40 ℃ purified water immersion subgroup compared to 45 and 35 ℃ purified water immersion subgroups(P=0.044,P=0.002),and it was lower in 40 ℃ SXXF immersion subgroup compared to 45,40,and 35 ℃ purified water immersion subgroups(P=0.004,P=0.037,P=0.000).The comparison between 40 ℃ SXXF immersion subgroup and voltaren group revealed no significant differences(P=0.679),however,the pathological score was higher in 40 ℃ purified water immersion subgroup compared to voltaren group(P=0.017).④The protein expression levels of inflammation-related factors and matrix-degrading enzymes in the knee joint synovial fluid.The protein expression levels of IL-1β,TNF-α,NF-κB,COX-2,NOS,MMP-3,and MMP-13 in the knee joint synovial fluid were lower in 45,40,and 35 ℃ SXXF immersion subgroups,45,40,and 35 ℃ purified water immersion subgroups,and voltaren group compared to model group(all P=0.000).Furthermore,the protein expression levels of IL-1β,NF-κB,NOS,MMP-3,and MMP-13 in the knee joint synovial fluid were lower in 45 ℃ SXXF immersion subgroup compared to 40 and 35 ℃ SXXF immersion subgroups(IL-1β:P=0.001,P=0.000; NF-κB:P=0.017,P=0.027; NOS:P=0.029,P=0.002; MMP-3:P=0.035,P=0.001; MMP-13:P=0.045,P=0.000),while that of TNF-α and COX-2 were lower in 40 ℃ SXXF immersion subgroup compared to 45 and 35 ℃ SXXF immersion subgroups(TNF-α:P=0.011,P=0.017; COX-2:P=0.009,P=0.002).Additionally,the protein expression levels of IL-1β,TNF-α,NF-κB,COX-2,NOS,MMP-3,and MMP-13 in the knee joint synovial fluid were lower in 40 ℃ SXXF immersion subgroup compared to 40 ℃ purified water immersion subgroup(P=0.002,P=0.035,P=0.000,P=0.001,P=0.026,P=0.005,P=0.004).⑤The mRNA expression levels of inflammation-related factors and matrix-degrading enzymes in the knee joint synovial fluid.The mRNA expression levels of IL-1β,TNF-α,NF-κB,COX-2,NOS,MMP-3,and MMP-13 in the knee joint synovial fluid were lower in 45,40,and 35 ℃ SXXF immersion subgroups,45,40,and 35 ℃ purified water immersion subgroups,and voltaren group compared to model group(all P=0.000),and they were lower in 40 ℃ SXXF immersion subgroup compared to 45 and 35 ℃ SXXF immersion subgroups(IL-1β:P=0.000,P=0.000; TNF-α:P=0.010,P=0.000; NF-κB:P=0.013,P=0.002; COX-2:P=0.000,P=0.013; NOS:P=0.024,P=0.018; MMP-3:P=0.038,P=0.014; MMP-13:P=0.001,P=0.000),and were lower in 40 ℃ SXXF immersion subgroup compared to 40 ℃ purified water immersion subgroup(P=0.000,P=0.000,P=0.000,P=0.000,P=0.001,P=0.000,P=0.000).Conclusion:SXXF medicated bath therapy can mitigate the synovial inflammation and delay the articular cartilage degeneration in KOA model rabbits,with optimal efficacy observed at 40 ℃,and its therapeutic effect is comparable to that of external application of diclofenac diethylamine emulgel.It may work by inhibiting the pro-inflammatory cytokines such as IL-1β and TNF-α,blocking the NF-κB signaling pathway,and down-regulating the expression of COX-2,NOS,and MMPs.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(81774340); 第七批全国老中医药专家学术经验继承工作指导老师及继承人项目(国中医药人教函〔2022〕76号); 上海市科技计划项目(21Y11921600,20MC1920600); 上海市名老中医学术经验研究工作室建设项目(SHGZS-202238)
通讯作者:陈博 E-mail:cbm818@126.com
更新日期/Last Update: 1900-01-01