独一味对兔骨关节炎模型关节软骨影响的实验研究
摘要
目的探讨藏药独一味对兔KOA模型软骨退变的保护或治疗机制,为临床独一味防治骨关节炎的推广应用提供理论支持。
方法将40只新西兰大白兔随机编号,分为五组,即,(A)空白对照组、(B)模型对照组、(C)小剂量组治疗组、(D)中剂量组治疗组、(E)大剂量组治疗组,每组8只。A组作为空白对照组,行模拟手术。B、C、D、E组按Hulth方法,制造兔膝关节骨关节炎模型。术后1周开始全部实验动物每天驱赶行走30min,于造模1周后开始灌胃,各治疗组给药剂量根据Meeh-Rubner公式计算,将按体表面积折算的临床常用量等效剂量,溶于10mL蒸馏水中,独一味小剂量治疗组、中剂量治疗组和大剂量治疗组分别灌胃给予独一味胶囊60mg/kg、120mg/kg、240mg/kg。每天灌胃给药一次；空白对照组、模型对照组于同一时间点给予等量生理盐水灌胃。治疗6周,末次给药后禁食12h。处死动物。再用组织学、免疫组化、RT-PCR学等技术对兔KOA模型关节软骨、关节液等部位进行检测和评估。
切开实验侧兔膝关节,对兔膝关节关节软骨行大体观察并参照文献标准进行评分,比较各组间的差异；将关节软骨制作成普通光学显微镜切片进行观察,并按照Mankin's关节软骨组织学评分系统进行评分,比较各组间的差异；制作透射电子显微镜切片,观察其超微病理变化。
在切开膝关节之前抽取关节液,检测关节液中IL-1β、NO的含量变化,比较各组间的差异；
应用RT-PCR技术检测药物治疗后,关节软骨MMP-3, MMP-13, TIMP-1mRNA表达的变化,比较各组间的差异；
用免疫组化法分别测量关节软骨组织GAG含量、II型胶原阳性染色面积的变化,并比较各组间的差异；
结果给药前后体重变化：与给药前动物体重相比较,各剂量治疗组在实验结束时体重均有不同程度的增加,但无统计学差异(均为P>0.05)。关节活动度：实验动物关节被动活动度测定结果显示,实验6周时,模型对照组与小剂量治疗组相比,P<0.05。各治疗组关节活动度均有不同程度的恢复,独一味中剂量、大剂量治疗组恢复程度尤为明显(P<0.01)。
大体观察,中、大剂量治疗组与模型对照组比较,软骨磨损、退变程度明显减轻(P<0.01),光镜下观察,治疗组与模型对照组比较,Mankin's评分明显降低,小剂量治疗组与模型对照组比较,P<0.05；中剂量、大剂量治疗组与模型对照组比较,P<0.01；电镜观察发现,药物治疗后中、大剂量治疗组关节软骨细胞的形态、结构、排列等均优于模型对照组。
经不同剂量独一味治疗后,各组关节液中的NO、IL-1β含量均值相对于模型对照组有所降低,各治疗组间进行比较,中、大剂量治疗组关节液中NO、IL-1β含量与模型对照组比较明显降低,比较有显著性差异(P<0.01)。小剂量治疗组与模型对照组比较,经检验,P<0.05；提示随着独一味剂量的增大,关节液中NO、IL-1β含量进一步降低。
治疗后,中、大剂量治疗组与模型对照组比较,中、大剂量治疗组MMP-3,MMP-13的mRNA表达较低、而TIMP-1的mRNA表达较高；模型对照组关节软骨中MMP-3,MMP-13的mRNA表达明显增高,而TIMP-1表达明显降低。组间比较有显著性差异(P<0.01)。
II型胶原阳性染色后,模型对照组,阳性染色显著减少,分布不均匀,且表浅层出现缺损；经独一味胶囊治疗后,中、大剂量治疗组与模型对照组比较,中、大剂量治疗组II型胶原阳性染色面积明显增大,比较有显著性差异(P<0.01)。检测软骨基质中GAG含量,模型组与小剂量治疗组比较,软骨基质中GAG含量有显著性差异,P<0.05；中剂量组治疗组、高剂量组治疗组与模型组分别比较,中、高剂量治疗组软骨基质中GAG含量有显著性差异,P<0.01；
结论
1. Hulth手术方法建立家兔膝OA模型,稳定性好,成功率高,诱导周期较短,重复性好。
2.独一味通过减少兔膝OA模型关节液中NO、IL-1β含量,抑制MMPs表达,增加TIMP-1表达,提高ECM中GAG含量,促进II型胶原的合成,延缓或一定程度上能够阻止兔膝OA病变过程的发展,进而改善兔膝OA关节功能。
3.独一味胶囊对骨性关节炎的治疗机制可能与这几方面有关：
在兔膝关节骨性关节炎模型上,经不同剂量独一味治疗后,能增加模型OA关节的关节活动度,减轻或延缓软骨退变、磨损程度和减少关节周围软骨性骨赘的增生,降低关节软骨光镜下的Mankin's评分,中、高剂量治疗组表现尤为明显。
应用独一味干预OA关节模型后,经检测关节液中的NO、IL-1β含量明显降低,表明独一味胶囊通过降低OA关节局部IL-1β、NO的水平,减轻关节滑液和滑膜的炎性反应,并呈现出一定的剂量依赖性。通过降低关节液中NO的含量,减少或延缓NO对关节软骨基质的破坏作用,减少软骨细胞的破坏,促进软骨细胞合成软骨基质,抑制或减少软骨细胞凋亡,使关节软骨的破坏减少。通过降低关节液中IL-1β的含量,调节软骨细胞的合成代谢,抵抗强大的促炎症作用,调节关节软骨分解代谢的影响；抑制或延缓骨关节炎进程。
实验结果显示,空白对照组MMP-3、MMP-13的mRNA表达低于模型对照组,而治疗组中TIMP-1的mRNA表达高于模型对照组,进一步验证了OA与基质金属蛋白酶、金属蛋白酶抑制剂的调控密切相关。经不同剂量独一味治疗后的各组MMP-3、MMP-13的mRNA表达均值均明显少于模型对照组,提示独一味能抑制MMP-3、MMP-13在关节软骨组织中的表达,并呈现出一定的剂量依赖性,剂量越大,抑制作用越强。证明独一味胶囊能延缓骨性关节炎进程,起到延缓关节软骨基质的破坏、保护关节软骨的作用。
藏药独一味可能是通过降低IL-1β等炎性细胞因子对软骨细胞的作用,减少MMPs的分泌,进而有效地促进了软骨细胞II型前胶原mRNA的表达,促进合成蛋白多糖和II型胶原,有效地防止了基质胶原的降解,一定程度上缓解了骨关节炎关节软骨的破坏进程,起到了一定的保护和修复作用。
Objective To explore the protection and treatment mechanism of Duyiwei capsule on rabbit KOA model of cartilage degeneration and to provide theoretical support for the clinical application of the Duyiwei capsule on osteoarthritis.
Methods40New Zealand rabbits were numbered and divided into five groups at random:the control group (A), model control group (B), the low dose group(C), the middle dose group(D) and the high dose group (E),8for each group. Group A was simulated operation as sham operation group. Group B, C, D, and E were duplicated into the rabbit model of knee osteoarthritis by Hulth's technique. All the rabbits moved with goad for30min evryday after1week. A week after the Modeling, the drug was given to the rabbits through stomach by Meeh-Rubner. The dosage of administration was converted through the surface area of human and the rabbit. Duyiwei capsule was dissolved in the2ml distilled water. The low dose group, the middle dose group and the high dose group were respectively given the drug (60mg/kg、120mg/kg、240mg/kg),one time a day.At each time point, the control group and model control group were respectively given the equivalent normal saline. After6weeks of treatment the animals were given the last dose of the drugs before fasting for24h, Then the rabbits were sacrificed. The articular cartilage, synovial fluid of rabbit KOA model were measured and evaluated by histological techniques, immunohistochemistry, RT-PCR and other techniques.
After opening the rabbit knee joint in experimental side, observed and scorded the articular cartilage of the rabbit' s knee joint according to previous literature and compared the differences between each two groups. The articular cartilage was prepared for optical microscope sections. Observed and scorded the optical microscope sections by Mankin scoring system and compared the differences between each two groups. The the articular cartilage was determined with transmit electron microscope to observe the ultrastructural pathologic changes.
Synovial fluid was taken before opening the rabbit knee joint. The IL-1β and NO levels in synovial fluid were measured and compared the differences between each two groups.
After therapy, the MMP-3, MMP-13and TIMP-1mRNA expression levels were measured of articular cartilage and compared the differences between each two groups.
The GAG levels, changes in the positive staining area of type Ⅱ collagen in articular cartilage were measured by immunohistochemical method and compared the differences between each two groups.
Results
The rabbit body weight changed before and after admini-stration:Compared with that before administration, all the body weight of treatment groups increased at different degrees, but there is no significant differences compared with normal group (P>0.05). The range of motion:The results of the knee joint mobility determination showed that:All the knee joint mobility of treatment groups recovered in varying degrees, especial ly the middle dose group and the high dose group (P<0.01)
General observation, compared with model control group, the cartilage abrasion, degeneration of the middle dose group and the high dose group were less obvious (P<0.01).Light microscopy observation, compared with model control group, the scores of the low dose group by Mankin scoring system were less obvious (P<0.05), so did the middle dose group and the high dose group (P<0.01),Electron microscopy observation, Cells morphology, structure, arrangement of the middle dose group and the high dose groupwere better than the model control group.
After Duyiwei capsule therapy, compared with the model control group, the IL-1β and NO levels in synovial fluid of each group was decreased. Compared in each group, the IL-1β and NO levels in synovial fluid of the middle dose group and the high dose group were less obvious, and there was significant difference in the comparison between the groups (P<0.01). Compared with the model control group, the IL-1β and NO levels of the low dose group were less (P<0.05). These results suggested that the IL-1(3and NO levels in synovial fluid decreased with the elevation of the dose.
After therapy, compared with the model control group, the MMP-3and MMP-13mRNA expression levels of the middle dose group and the high dose group were less obvious, whi le the TIMP-1mRNA expression levels were obvious. The MMP-3and MMP-13mRNA expression levels in synovial fluid of the model control group were significantly increased, while the TIMP-1mRNA expression levels were decreased significantly. There was significant difference in the comparison between the groups (P<0.01).
After positive staining of type Ⅱ collagen, positive staining aere was increased significantly with uneven distribution and a breach appeared in the superficial zone. After Duyiwei capsule therapy, compared with the model control group, the positive staining area of type Ⅱ collagen of the middle dose group and the high dose group were increased significantly, and there was significant difference in the comparison between the groups (P<0.05). Compared with the model control group, the GAG levels of type Ⅱ collagen of the low dose group was increased significantly (P<0.05), the GAG levels of type Ⅱ collagen of the middle dose group and the high dose group were obvious increased (P<0.01).
Conclusions
1. Build the model of rabbit knee osteoarthritis by Hulth's technique with favorable stability, high success rate, shorter inducement duration and good reproducibility.
2. Duyiwei capsule inhibits the expression of MMPs, promotes the TIMPs expression, advance the GAG content in ECM, promote synthesis of type Ⅱ collagen, delays and prevents the development of rabbit knee osteoarthritis to some extent, improves osteoarthritis of rabbit knee by decreasing the IL-1β and NO levels in synovial fluid of rabbit KOA model.
3. There Maybe some factors Duyiwei capsule correlates to the therapeutic mechanism on OA as follow:
After different doses of Duyiwei capsule therapy, there are same changes on KOA model. experimental models, especially the middle dose group and the high dose group. The knee joint mobility was improved. Cartilage degeneration wear degree were reduced or delayed.The formation of the cartilaginous osteophyte was decreased and the scores by Mankin was reduced.
After the Intervention Effect of Duyiwei capsule on KOA model, the IL-1β and NO levels in synovial fluid of each group were decreased. It showed that The synovial fluid and inflammatory response of synovial were reduced by Duyiwei capsule reducing the IL-1β and NO levels inside the lesion joints, and it showed a relationship with dose-dependent manner. Duyiwei capsule reduced destruction of articular cartilage by reducing the NO levels in synovial fluid, reducing or delaying the destructive action of NO on articular cartilage. decreasing the chondrocyte destruction, promoting the synthesis of extracellular matrix, inhibiting apoptosis of rabbit chondrocytes. Duyiwei capsule delayed the process of OA by the IL-1β levels in synovial fluid, controlling the anabolism and catabolism of chondrocyte and the powerful resistance to inflammation.
The experimental results show that the MMP-3and MMP-13mRNA expression levels of the control group lower than the model control group, while the TIMP-1mRNA expression levels of treatment groups were higher than the model control group. This conclusion was further verified that MMPs and TIMPs were related to OA. After Duyiwei capsule therapy, the MMP-3and MMP-13mRNA expression levels of each group lower than the model control group obviously. It showed that Duyiwei capsule could inhibit the MMP-3and MMP-13mRNA expression with dose-dependent manner. It proved that Duyiwei capsule could delay the process of OA and the destruction of cartilage matrix and protect articular cartilage.
Duyiwei capsule may delay the process of OA and the destruction of articular cartilage to some extent and has played a certain action of protection and repair by decreasing the effect of IL-1β and other inflammatory cytokines on chondrocytes, reducing the secretion of MMPs, promoting the synthesis of type Ⅱ collagen and proteoglycan and the ex-pression of procollagen type Ⅱ mRNA, preventing degradation of matrix collagen.
方法将40只新西兰大白兔随机编号,分为五组,即,(A)空白对照组、(B)模型对照组、(C)小剂量组治疗组、(D)中剂量组治疗组、(E)大剂量组治疗组,每组8只。A组作为空白对照组,行模拟手术。B、C、D、E组按Hulth方法,制造兔膝关节骨关节炎模型。术后1周开始全部实验动物每天驱赶行走30min,于造模1周后开始灌胃,各治疗组给药剂量根据Meeh-Rubner公式计算,将按体表面积折算的临床常用量等效剂量,溶于10mL蒸馏水中,独一味小剂量治疗组、中剂量治疗组和大剂量治疗组分别灌胃给予独一味胶囊60mg/kg、120mg/kg、240mg/kg。每天灌胃给药一次；空白对照组、模型对照组于同一时间点给予等量生理盐水灌胃。治疗6周,末次给药后禁食12h。处死动物。再用组织学、免疫组化、RT-PCR学等技术对兔KOA模型关节软骨、关节液等部位进行检测和评估。
切开实验侧兔膝关节,对兔膝关节关节软骨行大体观察并参照文献标准进行评分,比较各组间的差异；将关节软骨制作成普通光学显微镜切片进行观察,并按照Mankin's关节软骨组织学评分系统进行评分,比较各组间的差异；制作透射电子显微镜切片,观察其超微病理变化。
在切开膝关节之前抽取关节液,检测关节液中IL-1β、NO的含量变化,比较各组间的差异；
应用RT-PCR技术检测药物治疗后,关节软骨MMP-3, MMP-13, TIMP-1mRNA表达的变化,比较各组间的差异；
用免疫组化法分别测量关节软骨组织GAG含量、II型胶原阳性染色面积的变化,并比较各组间的差异；
结果给药前后体重变化：与给药前动物体重相比较,各剂量治疗组在实验结束时体重均有不同程度的增加,但无统计学差异(均为P>0.05)。关节活动度：实验动物关节被动活动度测定结果显示,实验6周时,模型对照组与小剂量治疗组相比,P<0.05。各治疗组关节活动度均有不同程度的恢复,独一味中剂量、大剂量治疗组恢复程度尤为明显(P<0.01)。
大体观察,中、大剂量治疗组与模型对照组比较,软骨磨损、退变程度明显减轻(P<0.01),光镜下观察,治疗组与模型对照组比较,Mankin's评分明显降低,小剂量治疗组与模型对照组比较,P<0.05；中剂量、大剂量治疗组与模型对照组比较,P<0.01；电镜观察发现,药物治疗后中、大剂量治疗组关节软骨细胞的形态、结构、排列等均优于模型对照组。
经不同剂量独一味治疗后,各组关节液中的NO、IL-1β含量均值相对于模型对照组有所降低,各治疗组间进行比较,中、大剂量治疗组关节液中NO、IL-1β含量与模型对照组比较明显降低,比较有显著性差异(P<0.01)。小剂量治疗组与模型对照组比较,经检验,P<0.05；提示随着独一味剂量的增大,关节液中NO、IL-1β含量进一步降低。
治疗后,中、大剂量治疗组与模型对照组比较,中、大剂量治疗组MMP-3,MMP-13的mRNA表达较低、而TIMP-1的mRNA表达较高；模型对照组关节软骨中MMP-3,MMP-13的mRNA表达明显增高,而TIMP-1表达明显降低。组间比较有显著性差异(P<0.01)。
II型胶原阳性染色后,模型对照组,阳性染色显著减少,分布不均匀,且表浅层出现缺损；经独一味胶囊治疗后,中、大剂量治疗组与模型对照组比较,中、大剂量治疗组II型胶原阳性染色面积明显增大,比较有显著性差异(P<0.01)。检测软骨基质中GAG含量,模型组与小剂量治疗组比较,软骨基质中GAG含量有显著性差异,P<0.05；中剂量组治疗组、高剂量组治疗组与模型组分别比较,中、高剂量治疗组软骨基质中GAG含量有显著性差异,P<0.01；
结论
1. Hulth手术方法建立家兔膝OA模型,稳定性好,成功率高,诱导周期较短,重复性好。
2.独一味通过减少兔膝OA模型关节液中NO、IL-1β含量,抑制MMPs表达,增加TIMP-1表达,提高ECM中GAG含量,促进II型胶原的合成,延缓或一定程度上能够阻止兔膝OA病变过程的发展,进而改善兔膝OA关节功能。
3.独一味胶囊对骨性关节炎的治疗机制可能与这几方面有关：
在兔膝关节骨性关节炎模型上,经不同剂量独一味治疗后,能增加模型OA关节的关节活动度,减轻或延缓软骨退变、磨损程度和减少关节周围软骨性骨赘的增生,降低关节软骨光镜下的Mankin's评分,中、高剂量治疗组表现尤为明显。
应用独一味干预OA关节模型后,经检测关节液中的NO、IL-1β含量明显降低,表明独一味胶囊通过降低OA关节局部IL-1β、NO的水平,减轻关节滑液和滑膜的炎性反应,并呈现出一定的剂量依赖性。通过降低关节液中NO的含量,减少或延缓NO对关节软骨基质的破坏作用,减少软骨细胞的破坏,促进软骨细胞合成软骨基质,抑制或减少软骨细胞凋亡,使关节软骨的破坏减少。通过降低关节液中IL-1β的含量,调节软骨细胞的合成代谢,抵抗强大的促炎症作用,调节关节软骨分解代谢的影响；抑制或延缓骨关节炎进程。
实验结果显示,空白对照组MMP-3、MMP-13的mRNA表达低于模型对照组,而治疗组中TIMP-1的mRNA表达高于模型对照组,进一步验证了OA与基质金属蛋白酶、金属蛋白酶抑制剂的调控密切相关。经不同剂量独一味治疗后的各组MMP-3、MMP-13的mRNA表达均值均明显少于模型对照组,提示独一味能抑制MMP-3、MMP-13在关节软骨组织中的表达,并呈现出一定的剂量依赖性,剂量越大,抑制作用越强。证明独一味胶囊能延缓骨性关节炎进程,起到延缓关节软骨基质的破坏、保护关节软骨的作用。
藏药独一味可能是通过降低IL-1β等炎性细胞因子对软骨细胞的作用,减少MMPs的分泌,进而有效地促进了软骨细胞II型前胶原mRNA的表达,促进合成蛋白多糖和II型胶原,有效地防止了基质胶原的降解,一定程度上缓解了骨关节炎关节软骨的破坏进程,起到了一定的保护和修复作用。
Objective To explore the protection and treatment mechanism of Duyiwei capsule on rabbit KOA model of cartilage degeneration and to provide theoretical support for the clinical application of the Duyiwei capsule on osteoarthritis.
Methods40New Zealand rabbits were numbered and divided into five groups at random:the control group (A), model control group (B), the low dose group(C), the middle dose group(D) and the high dose group (E),8for each group. Group A was simulated operation as sham operation group. Group B, C, D, and E were duplicated into the rabbit model of knee osteoarthritis by Hulth's technique. All the rabbits moved with goad for30min evryday after1week. A week after the Modeling, the drug was given to the rabbits through stomach by Meeh-Rubner. The dosage of administration was converted through the surface area of human and the rabbit. Duyiwei capsule was dissolved in the2ml distilled water. The low dose group, the middle dose group and the high dose group were respectively given the drug (60mg/kg、120mg/kg、240mg/kg),one time a day.At each time point, the control group and model control group were respectively given the equivalent normal saline. After6weeks of treatment the animals were given the last dose of the drugs before fasting for24h, Then the rabbits were sacrificed. The articular cartilage, synovial fluid of rabbit KOA model were measured and evaluated by histological techniques, immunohistochemistry, RT-PCR and other techniques.
After opening the rabbit knee joint in experimental side, observed and scorded the articular cartilage of the rabbit' s knee joint according to previous literature and compared the differences between each two groups. The articular cartilage was prepared for optical microscope sections. Observed and scorded the optical microscope sections by Mankin scoring system and compared the differences between each two groups. The the articular cartilage was determined with transmit electron microscope to observe the ultrastructural pathologic changes.
Synovial fluid was taken before opening the rabbit knee joint. The IL-1β and NO levels in synovial fluid were measured and compared the differences between each two groups.
After therapy, the MMP-3, MMP-13and TIMP-1mRNA expression levels were measured of articular cartilage and compared the differences between each two groups.
The GAG levels, changes in the positive staining area of type Ⅱ collagen in articular cartilage were measured by immunohistochemical method and compared the differences between each two groups.
Results
The rabbit body weight changed before and after admini-stration:Compared with that before administration, all the body weight of treatment groups increased at different degrees, but there is no significant differences compared with normal group (P>0.05). The range of motion:The results of the knee joint mobility determination showed that:All the knee joint mobility of treatment groups recovered in varying degrees, especial ly the middle dose group and the high dose group (P<0.01)
General observation, compared with model control group, the cartilage abrasion, degeneration of the middle dose group and the high dose group were less obvious (P<0.01).Light microscopy observation, compared with model control group, the scores of the low dose group by Mankin scoring system were less obvious (P<0.05), so did the middle dose group and the high dose group (P<0.01),Electron microscopy observation, Cells morphology, structure, arrangement of the middle dose group and the high dose groupwere better than the model control group.
After Duyiwei capsule therapy, compared with the model control group, the IL-1β and NO levels in synovial fluid of each group was decreased. Compared in each group, the IL-1β and NO levels in synovial fluid of the middle dose group and the high dose group were less obvious, and there was significant difference in the comparison between the groups (P<0.01). Compared with the model control group, the IL-1β and NO levels of the low dose group were less (P<0.05). These results suggested that the IL-1(3and NO levels in synovial fluid decreased with the elevation of the dose.
After therapy, compared with the model control group, the MMP-3and MMP-13mRNA expression levels of the middle dose group and the high dose group were less obvious, whi le the TIMP-1mRNA expression levels were obvious. The MMP-3and MMP-13mRNA expression levels in synovial fluid of the model control group were significantly increased, while the TIMP-1mRNA expression levels were decreased significantly. There was significant difference in the comparison between the groups (P<0.01).
After positive staining of type Ⅱ collagen, positive staining aere was increased significantly with uneven distribution and a breach appeared in the superficial zone. After Duyiwei capsule therapy, compared with the model control group, the positive staining area of type Ⅱ collagen of the middle dose group and the high dose group were increased significantly, and there was significant difference in the comparison between the groups (P<0.05). Compared with the model control group, the GAG levels of type Ⅱ collagen of the low dose group was increased significantly (P<0.05), the GAG levels of type Ⅱ collagen of the middle dose group and the high dose group were obvious increased (P<0.01).
Conclusions
1. Build the model of rabbit knee osteoarthritis by Hulth's technique with favorable stability, high success rate, shorter inducement duration and good reproducibility.
2. Duyiwei capsule inhibits the expression of MMPs, promotes the TIMPs expression, advance the GAG content in ECM, promote synthesis of type Ⅱ collagen, delays and prevents the development of rabbit knee osteoarthritis to some extent, improves osteoarthritis of rabbit knee by decreasing the IL-1β and NO levels in synovial fluid of rabbit KOA model.
3. There Maybe some factors Duyiwei capsule correlates to the therapeutic mechanism on OA as follow:
After different doses of Duyiwei capsule therapy, there are same changes on KOA model. experimental models, especially the middle dose group and the high dose group. The knee joint mobility was improved. Cartilage degeneration wear degree were reduced or delayed.The formation of the cartilaginous osteophyte was decreased and the scores by Mankin was reduced.
After the Intervention Effect of Duyiwei capsule on KOA model, the IL-1β and NO levels in synovial fluid of each group were decreased. It showed that The synovial fluid and inflammatory response of synovial were reduced by Duyiwei capsule reducing the IL-1β and NO levels inside the lesion joints, and it showed a relationship with dose-dependent manner. Duyiwei capsule reduced destruction of articular cartilage by reducing the NO levels in synovial fluid, reducing or delaying the destructive action of NO on articular cartilage. decreasing the chondrocyte destruction, promoting the synthesis of extracellular matrix, inhibiting apoptosis of rabbit chondrocytes. Duyiwei capsule delayed the process of OA by the IL-1β levels in synovial fluid, controlling the anabolism and catabolism of chondrocyte and the powerful resistance to inflammation.
The experimental results show that the MMP-3and MMP-13mRNA expression levels of the control group lower than the model control group, while the TIMP-1mRNA expression levels of treatment groups were higher than the model control group. This conclusion was further verified that MMPs and TIMPs were related to OA. After Duyiwei capsule therapy, the MMP-3and MMP-13mRNA expression levels of each group lower than the model control group obviously. It showed that Duyiwei capsule could inhibit the MMP-3and MMP-13mRNA expression with dose-dependent manner. It proved that Duyiwei capsule could delay the process of OA and the destruction of cartilage matrix and protect articular cartilage.
Duyiwei capsule may delay the process of OA and the destruction of articular cartilage to some extent and has played a certain action of protection and repair by decreasing the effect of IL-1β and other inflammatory cytokines on chondrocytes, reducing the secretion of MMPs, promoting the synthesis of type Ⅱ collagen and proteoglycan and the ex-pression of procollagen type Ⅱ mRNA, preventing degradation of matrix collagen.
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