| Objective:The Chinese medicinal’channel tropism’theory is an important part of the theories of TCM, and is the basis of disease which we know and treat. Under the guidance of the’channel tropism’theory, we formulate prescription to treat osteoarthritis by applying to liver and kidney. In order to further research’channel tropism’nature, and the relevance of’channel tropism’theory and signaling pathways. We select pilos antler of applying to liver and kidney to research its nature as the representative of Chinese traditional medicine, to explore the mechanism of pilos antler regulate TGF beta/Smads signal pathway in the osteoarthritic cartilage.Methods:1Literature resaerchBy means of’The Chinese medical database’,Some medicine cases about Arthralgia Syndrome will be analyzed and imported into database, and using of herbs will be analyzed through mathematics and physics Statistieal methods of SPSS18.0saftware. We will concluded the regulation of different kinds of drugs by analysis frequency of efficacy and’channel tropism’of drugs. At the same time, the system clustering analysis is adopted to clustering statistics on drugs, and using traditional Chinese medicine theory to analyze the results.2Channel tropism researchHulth method is adopted to establish the rat model of osteoarthritis. Twenty four healthy female SD rats were selected and randomly divided into3groups:group of the pilose antler, model group and control group. Animals in each of group after treatment, the expressions of cAMP, cGMP, TGF-βRI, TGF-β RII were measured by ELISA, Quantitative PCR, Western blot to observe the curative effects. 3Explore the different methods to extract total RNA from cartilage tissue of SD ratsSelect9SD rats of3-month-old, respectively Rneasy Lipid Tissue Kit, RNAout kit and classic Trizol method of total RNA was extracted, and Agarose gel electrophoresis was included to detect RNA integrity and quality. In order to explore best extraction scheme of total RNA.4Research mechanism of Hairy Deerhorn regulate TGF-beta/Smad signalling pathwaysOne hundred healthy female SD rats of3months old were selected and subjected to classic Hulth modeling. Knees were harvested to observe the pathologic change of cartilage macroscopically and microscopically, and randomly were divided into5groups:low-dose group of the pilose antler, high-dose group of the pilose antler, saline control group, model group and control group. Animals in each of group at2,4,6weeks after treatment, the expressions of collagen type II, TGF-β1, Smad2, Smad3, Smad4were measured by Immunohistochemistry, Quantitative PCR, Western blot to observe the curative effects.Rusult:1According to the results of frequency analysis, Chinese herb reaches79.2%which are for restoratives Chinese Medicinal Herbs. Diaphoretics or exterior syndrome relieving Chinese Medicinal Herbs, Chinese Medicinal Herbs for invigorating the blood and removing blood stasis, Antirheumatic Chinese Medicinal Herbs and Warming the internal are the main treatment drugs for osteoarthritis. Frequency of kidney is21.4%which is more than others, Frequency of liver and spleen is17.9%which account for second. According to the results of hierarchical cluster analysis, the herbs for treatment could be divided into six cluster,with nourishing liver and kidney, promoting blood circulation to remove meridian obstruction, temperature in the cold,etc as a method for drug combination.2According to the results ELISA method to detect cAMP and cGMP in cartilage tissue, group of Velvet antler of cAMP and cGMP were higher than model group,model group of cAMP and cGMP were lower than normal group. Comparison of three groups had significant difference(cAMP F=10351.408, P=0.000, cGMP F=63032.016, P=0.000, cAMP/cGMP F=6530.879, P=0.000). The expression of TGF-β RImRNA, TGF-β RIImRNA and proteins were increased gradually in low-dose group and high dose group by quantitative PCR and Western blot hybridization assay. By repeated measures analysis of variance, the difference is significant compare to other groups (F=161.672, P=0.000)3Total RNA of Rneasy Lipid Tissue Kit was obtained high purity, integrity and stability, and can successfully reverse transcriptase synthesize double-stranded cDNA. PCR amplification can get gene product what we need. Methods of RNAout kit and classic Trizol can not meet the need.4We prepared successfully osteoarthritis model in the athology. After two week, the surface of chondrocyte were swelling, the flat chondrocyte reduced and clusted through optical microscope. After four week, the surface and middle cell were shrinkage, diffuse, cluster to group. After six week, the most of the surface layer of chondrocyte were disappear, decay defects, chondrocytes disintegration, necrosis, and tidal line disorder or even disappear. According to the results of Mankin’s score of cartilage slice, Mankin’s score of low-dose and high-dose group of the pilose antler was less than the model group and saline group, the difference is significant compare to other groups(P=87.840, P=0.000). The expression of collagen type II by detection of immunohistochemical, chondrocyte layer of low-dose and high-dose group of the pilose antler had a wide range of tan cells, the number was significantly higher than that of saline group and model group, the expression by quantitative PCR and Western blot hybridization assay of TGF-β1was increased gradually in low-dose group and high dose group, the difference is significant compare to other groups by repeated measures analysis ofvariance(F=408.841,P=0.000).Smad2,Smad3gene and proteins were increased gradually after2,4weeks, and reduced after6weeks, the difference is significant compare to other groups by repeated measures analysis of variance(F=240.511, P=0.000,F=1325.174, P=0.000). The expression of Smad4was increased gradually in low-dose group and high dose group, the difference is significant compare to other groups by repeated measures analysis of variance (F=197.035, P=0.000).Conclusion:1The ancients treat’GUBI’under the onset of liver and kidney deficiency as the fundamental principle in clinical, they choose drugs to liver, kidney, spleen. Enriching yin and nourishing kidney, promoting blood circulation to remove blood stasis, etc. is a method for drug combination. In the treatment of liver and kidney deficiency, invigorate the circulation, temperature, Tongluo, dampness to treat the symptoms.2Experimental results demonstrate that pilos antler can influence the changes of cAMP and cGMP, pilos antler can increase the expression of TGF-beta RI,TGF-beta RII on the chondrocyte membrane,so we consider the cartilage may be one of the target tissue which pilos antler select, and TGF-betaRI, TGF-RII of chondrocyte membrane may be one of the targets which pilos antler via selective effect on cartilage.3Based on the specificity of the cartilage, we think Trizol method is not suitable for total RNA extraction of articular cartilage tissue in rat. While using RNeasy Lipid Tissue Mini Kit can guarantee the implementation of the follow-up study.4Pilos antler can influence the expression of TGF-betal, Smad2,3,4genes and proteins in cartilage, and promote chondrocyte proliferation, differentiation, delay the degeneration of cartilage. So we can concluded that pilos antler can regulate the expression of difference molecularof TGF-beta/Smad signaling pathways, thus promotes the signal transduction pathways, play the role of promoting chondrogenesis.And the changes of Smad2,3,4may is one of the important mechanisms of the pathogenesis of osteoarthritis. |
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