| ObjectiveTo observe the effect of DuHuoJiSheng decoction on the serum interleukin-1 and TNF-a in the serum and articular cavity effusion in the rabbit model with osteoarthritis, We try to obtain the difference of protein expression following DuHuoJiSheng decoction treatment through choosing the different proteins in the every group rabbits by proteomics in order to definite the protective mechanism and target. Finally, we observe the effect of DuHuoJiSheng decoction on the protein expression of Wnt3a, beta-catenin, MMP13 in the rabbit model with osteoarthritis, proving the possible protective mechanism and target.Methods1. The 30 big white rabbits was randomly divided into normal group (n=10), model group (n=10), and DuHuoJiSheng decoction treatment group (n=10), and the rabbit osteoarthritis model was established by means of fixing right leg in plaster for eight weeks. The rabbits with normal feed in the normal group have not experienced the establishment of model, and received the vehicle NS with an equivalent volume by intragastric administration per day after 8 week. However, The rabbits in the model group have experienced the establishment of model, and received the vehicle NS with an equivalent volume by intragastric administration per day after 8 week.As the same, The rabbits in the model group have experienced the establishment of model, but received DuHuoJiSheng decoction by intragastric administration per day after 8 week, All the rabbits following 4 weeks were sacrificed, and the samples including the serum, articular cavity effusion, the knee joint cartilage were collected.2. The histological examinations of rabbits knee joint cartilages were performed by HE staining under Light microscopy, and the semi-quantitative score of knee joint cartilages was obtained according the modified version of the Mankin scale. And both IL-1 and TNF-a level in the serum and articular cavity effusion were detected by Enzyme-linked immunosorbent method.3. The extracted proteins of the knee joint cartilage in the rabbits was differed by means of two-dimensional gel electrophoresis,next scanned with Imagescannerll scanner. The dates of gelatin images finally were analyzed by ImageMaster2-DE Platinum6 software.The point of protein was detected with ImageMaster in the GE company. following, proteins was zymolysised according the standard accomplished by Gharahdaghi. After Peptide samples DuHuoJiSheng decoction group (382±31) is less than that in the model group, the difference was statistically significant (P< 0.01).Comparison study found that,the protein point with a difference of 2 times or more is 12,and 10 protein points are identified.when compared with normal group, there are six differences in the model group, and 4 protein expression level are up-regulation,2 protein expression is down-regulation.When compared with model group, there are five differences in the DuHuoJiSheng decoction group,2 protein expression level are up-regulation,3 protein expression is down-regulation.The physiological functions of proteins identified on the basis of the existence of which can be divided into five types:(1) metabolism related enzymes (triose phosphate isomerase,beta 1-heat shock protein); (2) signaling (wnt3a, beta serial protein); (3) oxidoreductase (thiol-specific antioxidant protein); (4) the cartilage matrix proteins (type I collagen a2 precursor, II collagen, metal matrix protein 13); (5) the cytoskeleton protein (beta tubulin, cytoskeleton type I (10).4. Western blot results showed, there is little of wnt3a, beta serial protein, MMP13 protein expression in articular cartilage in the normal group.The protein expression of wnt3a, beta serial protein, MMP13 in the model group are up-regulated, when compared with normal group, the difference was statistically significant (P< 0.01). However, after DuHuoJiSheng decoction treatment,The protein expression of wnt3a, beta serial protein, MMP13 in the DuHuoJiSheng decoction group are down-regulated, when compared with model group,the difference was statistically significant (P< 0.01).Conclusion1.There is pathology of osteoarthritis on the rabbits in the model group, and the IL-1 and TNF alpha level both in the serum and articular cavity effusion in the model group are increased,suggesting a succeed osteoarthritis model.Inflammation mechanism may be associated with the devolepment of osteoarthritis.2.DuHuoJiSheng decoction treatment could improve the pathology of osteoarthritis,reduce the Mankin semi-quantitative score, decrease the IL-1 and TNF-a level both in the serum and articular cavity effusion, which proves that DuHuoJiSheng decoction could provide a protective role of articular cartilage,its mechanism may involved with anti-inflammation.3.There are up-regulation of protein expression of Wnt3a,beta serial protein,MMP13 in the rabbits knee articular cartilage with osteoarthritis,which suggesting that Wnt/p-catenin pathway activation is associated with the devolopment and progress of osteoarthritis by the... |
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