摘要
目的：
评估补肾活血复方对老年性膝骨关节炎(Knee Osteoarthritis, KOA)(?)临床疗效和对滑液来源间充质干细胞(Synovial fluid-mesenchymal progenitor cells, SF-MSCs)增殖、抗炎能力的影响,探讨补肾活血法治疗KOA可能的作用机制。
方法：
临床部分：筛选2010.12～2013.1肾虚血瘀型的早期KOA患者进行研究观察,设中药组、中西组、对照组,采用随机对照法,每组观察各34例,中药组每日给予补肾活血复方汤剂口服一次,对照组以塞来昔布0.2g每日两次口服,中西组为每日给予补肾活血复方汤剂口服一次+口服塞来昔布0.2g每日两次,观察治疗6周后WOMAC和中医肾虚血瘀证候评分变化,以ELISA法测定治疗前后患者关节液IL-1β、IL-8、IL-10、TNFα含量的变化。
实验部分：抽取正常人(?)(?)KOA患者膝关节液各5份样本,分离、提纯贴壁细胞,并以流式细胞仪进行鉴定,确定其为SF-MSCs;以低、中、高浓度的补肾活血复方给予SD大鼠灌胃4天取含药血清,设低、中、高浓度及空白对照4组,以MTT法检测不同浓度的含药血清对SF-MSCs增殖活性的影响,以ELISA法测定不同浓度含药血清对SF-MSCs IL-1β、IL-8、IL-10、TNF-α含量的变化。
结果：
临床部分：中药组纳入统计30例,中西组纳入统计29例,对照组纳入统计32例。三组患者在年龄、病程之间的比较(P>0.05),差异无统计学意义；三组患者在X光分级的组间比较存在差异(P<0.05),但治疗前的WOMAC各项评分组间比较、中医证候各项评分组间比较、关节液TNF-α、IL-1β、IL-8和IL-10组间比较(P>0.05),差异无统计学意义,三组具有可比性。
WOMAC评分方面,治疗前,中药组总评分平均18.57±7.35分,其中疼痛6.03±1.50分,僵硬0.27±0.45分,功能12.27±5.98分；中西组总评分平均20.93±4.10分,其中疼痛6.93±1.36分,僵硬0.14±0.35分,功能13.93±3.33分；对照组总评分平均21.25±6.88分,其中疼痛6.13±1.29分,僵硬0.19±0.40分,功能14.94±5.70分；治疗后,三组的WOMAC评分均有明显的下降,其中疼痛、功能项分值降低相对较为明显(P<0.01)；而僵硬项治疗前后三组均无明显变化(P>0.05),治疗后各项WOMAC评分组间比较,疼痛项方面,对照组与中西组得分均较中药组低(P<0.05),中西组与对照组比较差异无统计学意义(P>0.05)；功能项方面,三组比较差异无统计学意义(P>0.05)。
中医证候评分方面,治疗前,中药组中医证候评分5.07±1.14分,其中肾虚1.77±0.73分,血瘀1.87±0.78分,舌脉1.43±0.50分；中西组中医证候评分5.17±1.14分,.其中肾虚1.62±0.56分,血瘀2.00±0.80分,舌脉1.55±0.51分；对照组中医证候评分4.66±1.15分,其中肾虚1.50±0.51分,血瘀1.78±0.91分,舌脉1.38±0.49分。治疗后,三组的中医证候总评分均有明显的下降,差异具有统计学意义(P<0.01),中药组和中西组治疗后在肾虚、血瘀、舌脉项目的得分均有明显下降(P<0.01),而对照组只在血瘀项目与治疗前比较有明显的下降(P<0.01)。
炎症指标方面,治疗6周后,绝大多数患者经治疗后关节液明显减少,无法抽出足够的关节液供ELISA试剂盒检测。中药组仅有10位患者,中西组仅有7位患者,对照组仅有8位患者可以抽出足够的关节液供检测。从可以行前后配对的病例中统计,治疗前,中药组患者关节液中TNF-α35.14±2.42pg/ml,IL-1β89.42±3.71pg/ml, IL-880.60±6.86pg/ml,IL-10131.24±10.42pg/ml；中西组患者关节液中TNF-α36.19±3.54pg/ml,IL-1β88.27±2.10pg/ml,IL-881.32±8.06pg/ml,IL-10133.67±8.77pg/ml；对照组患者关节液中TNF-α35.19±2.40pg/ml,IL-1β85.59±3.42pg/ml,IL-881.44±5.OOpg/ml,IL-10137.53±12.42pg/ml.治疗后,三组的TNF-Q、IL-1β、IL-8含量均有不同程度的下降,治疗前后差异有统计学意义(P<0.05),三组的IL-10治疗后的含量较治疗前明显上升,差异具有统计学意义(P<0.05),三组治疗后的四项指标组间比较差异均无统计学意义(P>0.05)。
实验部分：分离培养的第3代贴壁细胞呈CD90阳性,不表达CD34,可鉴定为SF-MSCs。MTT结果显示,培养第4天后正常人SF-MSCs和KOA患者的SF-MSCs均生长速率增快(P<0.05),培养的6天当中,KOA患者与正常人的SF-MSCs增长速度无明显差异(P>0.05)。补肾活血复方干预KOA患者SF-MSCs6天内,组间OD值比较未见明显差异(P>0.05)。ELISA结果显示,补肾活血复方含药血清能降低KOA患者的SF-MSCs培养液上清炎症因子TNF-α、IL-1β、IL-8的浓度,且降低炎症因子含量的能力与含药血清的浓度呈正相关(P<0.05)；补肾活血复方能提高KOA患者的SF-MSCs培养液上清IL-10抗炎因子的含量(P<0.01),其提高抗炎因子IL-10浓度的能力与含药血清的浓度呈正相关。
结论：
根据补。肾活血法所拟的补肾活血复方安全性好,具有良好的临床疗效,不仅能改善。肾虚血瘀型早期KOA患者的疼痛和功能,而且能改善肾虚血瘀的证候,其改善关节疼痛和功能的作用机制可能通过提高SF-MSCs的抗炎能力,减轻关节内的炎症反应实现。在治疗肾虚血瘀型早期KOA的过程中,配合服用塞来昔布等NSAIDs类药物镇痛效果更佳。
Objective
To Assess Bushenhuoxue compound recipe efficacy of treating eldly osteoarthritis and its affect to SF-MSCs'proliferation and anti-inflammatory ability in vitro, in order to explore the possible mechanisms of treating KOA with Bushenhuoxue way.
Methods
The clinical part:Observating from2010.12to2013.1, kidney insufficiency and blood stasis early KOA patients were selected. Setting TCM group, combined group and control group for randomized controlled trial.34patients were selected into each group for observation. TCM group was given Bushenhuoxue compound recipe once a day, while the control group was given celecoxib0.2g oral twice a day, combined group took both treatment together. After6weeks, WOMAC and kidney deficiency and blood stasis syndrome score of TCM changes were compared. The content changes of the synovial fluid in knee of IL-1β, IL-8, IL-10, TNF-a was measured by ELISA before and after treatment.
The experimental part:Identified SF-MSCs by flow cytometry which was separated and purified from normal and KOA patients in knee; Achieving medical contained serum after gavaged for4days to SD rats with Bushen Huoxue compound recipe. Setting four groups of low, middle, high and control groups to measure the effect on the proliferation of SF-MSCs with Bushen Huoxue compound recipe by MTT and check the culture supernatant content changes of IL-1β, IL-8, IL-10and TNF-α after applied Bushen Huoxue compound recipe contained serum on SF-MSCs by ELISA.
Results
The clinical part:The TCM group included30cases of statistics, combined group included29cases and the control group were32cases. Comparison between three groups in age, course of disease were no significant difference (P>0.05); While comparison between X ray grade of three groups were different(P<0.05), but the score of WOMAC, TCM syndrome score, and the factors of inflammation of TNF-α, IL-1β, IL-8and IL-10of synovial fluid in knee were equal (P>0.05), the three groups were comparable.
In WOMAC score before treatment, TCM group was18.57+7.35, pain item was6.03±1.50, stiff item was0.27±0.45, function was12.27±5.98;The total score of combined group was20.93±4.10, pain item was6.93±1.36, stiff item was0.14±0.35, function was13.93±3.33;Control group was21.25±6.88, pain item was6.13±1.29, stiff item was0.19±0.40, function was14.94±5.70. The total WOMAC score was obviously decline in three groups after treatment, especially in items of pain and function (P<0.01). There was no significant difference between stiff item in three groups after treatment(P>0.05). In pain item, scores of control group and combined group were lower than TCM group in comparison within groups after treatment (P<0.05), while combined group was no significant difference with control group(P>0.05);In function item, there were no significant difference between three groups after treatment(P>0.05).
In TCM syndrome score, TCM group was5.07±1.14, kidney deficiency item was1.77±0.73, blood stasis item was1.87±0.78, tongue and pulse manifestation item was1.43±0.50;The total score of combined group was5.17±1.14, kidney deficiency item was1.62±0.56, blood stasis item was2.00±0.80, tongue and pulse manifestation item was1.55±0.51;The score of control group was4.66±1.15, kidney deficiency item was1.50±0.51, blood stasis item was1.78±0.91, tongue and pulse manifestation was1.38±0.49. The total score of TCM syndrome score were obviously decline after treatment, the difference between before and after treatment was statistical significant in three groups (P<0.01). The score of kidney deficiency item, blood stasis item and tongue and pulse manifestaion item in TCM group and combined group were obviously declined (P<0.01), while it was just declined in blood stasis item of control group.
In inflammatory makers, vast majority of patients were out of joint fluid enough for ELISA detection after treating for6weeks, because of synovial fluid decreased significantly. TCM group remained10cases, combined group remained7cases and control group was8. In matching comparison between before and after treatment, the average concentration of TNF-a was35.14±2.42pg/ml, IL-1β was89.42±3.71pg/ml, IL-8was80.60±6.86pg/ml and IL-10was131.24±10.42pg/ml in knee fluid of TCM group; The average concentration of TNF-a was36.19±3.54pg/ml, IL-1β was88.27±2.10pg/ml, IL-8was81.32±8.06pg/ml, and IL-10was133.67±8.77pg/ml in knee fluid of combined group; The average concentration of TNF-α was35.19±2.40pg/ml, IL-1β was85.59±3.42pg/ml, IL-8was81.44±5. OOpg/ml, IL-10was137.53±12.42pg/ml in knee fluid of control group. After treatment, TNF-α, IL-1β and IL-8content of three groups were decreased in different degree, there are significant differences before and after treatment(P<0.05). The content of IL-10were increase after treatment in three groups, the difference was statistically significant (P<0.05). There were no significant between four makers in three groups after the treatment(P>0.05).
The experimental part:The3rd generation adherented cell was positive in CD90, and did not express CD34, which could be identified as SF-MSCs. The result of MTT showed that normal SF-MSCs and KOA SF-MSCs are grow rapidly after four days of culture (P<0.05). There was no significant difference between growth speed of normal and KOA SF-MSCs in6days of cultivation(P>0.05). There was no significant difference between5groups of the OD value after Bushen Huoxue compound recipe intervention for6days(P>0.05). The result of ELISA showed that Bushen Huoxue compound recipe can reduce the concentration of inflammatory cytokine such as TNF-α, IL-1β, IL-8in KOA SF-MSCs supernatant, and the ability was positively correlated to the concentration of medical contained serum(P<0.05). The Bushen Huoxue compound recipe can increase secretion of anti-inflammatory cytokines IL-10of KOA SF-MSCs(P<0.01), and the ability was positively correlated to the concentration of medical contained serum.
Conelusion
Bushen Huoxue compound recipe is safe and has good clinical curative effect. It is not only release pain and improve function of kidney deficiency and blood stasis type of early KOA patients, but also can improve the deficiency of kidney and blood stasis syndrome. The mechanism may be reducing inflammation in the knee joint by improving the SF-MSCs anti-inflammatory capacity.If treating KOA combined with NSIADs such as celecoxib, the effect of pain release will be better.
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