| ObjectiveTo 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.MethodsThe 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. ResultsThe 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.ConelusionBushen 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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