Mechanism Of BMP2 / BMP4 Signaling Pathway In Prevention And Treatment Of Articular Cartilage Degeneration By Bone - Tonifying Traditional Chinese Medicine

Objective: 1. Thoroughly analyzing the conception of kidney, essence, marrow, bone and articular cartelige in traditional Chinese medicine to establish the “kidney-essense-marrow-bonearticular cartilage” system and eventually draw the hypothesis that TCM kidney has influence in articular cartilage metabolism. 2. To observe the phenotype of deleting BMP 2 and BMP 4 gene in chondrocytes in the development and maintaining of articular cartilage in mice. 3. Try to demonstrate the molecular mechanism of articular cartilage development and degradation through BMP 2 and BMP 4 gene related signaling. 4. To evaluate the effect of Icariin, Psoralen, Oleanolic and 2,3,5,4-tetrahydroxyl diphenylethylene-2-o-glucoside in regulating the expression of BMP 2 and BMP 4 during the process of mesenchymal cell differentiation into osteoblast. 5. To detect the function of benefiting kidney herbs in improving articular cartilage self-maintaining and approach the molecular mechanism, by which means to demonstrate the relation between kidney and articular cartilage in Chinese medicine theory, and more comprehensively to extend the conception of ‘kidney governs bone’ theory. Material and method: Theoretical researchEstablish the systematical connection in physics and ‘kidney deficiency-essence shortage-marrow deduction-bone withered-AC degradation’ pathologically, by explaining comprehensively the conception of kidney, essence, marrow, bone and articular cartilage.Hypothesis is ‘Articular cartilage is regulated by kidney system’. In vivo experiment Mice and grouping:BMP2/BMP4 double conditional knockout mice were generated by crossing BMP 2 fx/fx and BMP 4 fx/fx mice with Col2 Cre ER transgenic mice, Tamoxifen was inducted when mice were 2 weeks old(1mg/10 g bodyweight, i.p., for 5days). The Cre-positive mice were used as cKO mice, grouping into(BMP2, BMP4)Col 2 ER, and the Cre-negative littermates were used as controls, gender randomly. Methods:Knee joints were collected from 3 and 6 months old mice, fixed and decalcified, embedded in paraffin and sliced into 3um slides. Alcein Blue/orange G staining was performed to evaluate the condition articular cartilage, images were shot and the area of AC of both tibial plateau and femoral condyle was calculated with OsteoMeasure software. Immunohistochemistry was performed to detect the protein expression of Mmp13, Adamts4 and Adamts 5 in articular cartilage. In vitro experiment Cells culture:Mesenchymal cells were isolated from 1 months old wild type mice(C57BL/6) and cultured in dish. Icariin, Psoralen, Oleanolic acid and 2,3,5,4-tetrahydroxyl diphenylethylene-2-o-glucoside was added into the culture medium diversely. Methods:Cells were harvested for RNA extraction on 48 h time points to detect the concentration of BMP2, BMP4, and RUNX2 genes. Western-blotting was performed to examine the protein expression of BMP2, BMP4, and RUNX2 on Day 5. ALP staining was performed on Day 7. Result 1. Articular cartilage is considered in conception of the bone in TCM theory. Kidney-essence-marrow-bone-articular cartilage is an interacting system. Analyzing the mechanism of benefitting kidney herbs in treating OA can establish the hypothesis that kidney regulates AC development. 2. Deletion of BMP2 and BMP4 induced articular cartilage phenotype, such as narrowed AC area, shortened AC depth, and massive subchondral bone, and the osteometric data of calculated AC area is consistent to the dramatically morphology changes in both 3- and 6-month old mice.(P<0.05) 3. The protein expression of MMP 13 in superfacial zone of articular cartilage tissue is significant increased in(BMP2, BMP4)Col 2 ER Group in 3-month old mice(P<0.05). While in the 6-month old mice there showed the same tendency but the data is not stastatic. 4. The protein expression of Adamts5 in superfacial zone of articular cartilage tissue is dramatically stronger in(BMP2, BMP4)Col 2 ER Group in both 3- and 6- month old mice, and the osteometric data is consistent to the morphologic result, significantly increased(P<0.05). 5. The protein expression of Adamts5 in superfacial zone of articular cartilage tissue is dramatically stronger in(BMP2, BMP4)Col 2 ER Group in both 3- and 6- month old mice, and the osteometric data is consistent to the morphologic result, significantly increased(P<0.05). 6. Icariin, Psoralen, Oleanolic acid and 2,3,5,4-tetrahydroxyl diphenylethylene-2-o-glucoside, which is extracted from benefiting kidney herbs, promote the differentiation of mesenchymal cells into osteoblasts through upregulating the expression of BMP2 and BMP4. 7. Icariin, Psoralen, Oleanolic acid and 2,3,5,4-tetrahydroxyl diphenylethylene-2-o-glucoside, which is extracted from benefiting kidney herbs, can upregulate the gene expression of BMP2, BMP4, RUNX2 and ALP. 8. Icariin, Psoralen, Oleanolic acid and 2,3,5,4-tetrahydroxyl diphenylethylene-2-o-glucoside, which is extracted from benefiting kidney herbs, can upregulate the protein expression of BMP2, BMP4, RUNX2 and ALP. Conclusion 1. Comprehensively explaining the conception of kidney, essence, marrow, bone and articular cartilage to establish the systematical connection in physics and ‘kidney deficiency-essence shortage-marrow deduction-bone withered-AC degradation’ pathologically. 2. Articular cartilageoriginates from congenital essence stored in kidney and nourished by body fluid adjusted by kidney, soarticular cartilage is connected to kidney system theroreticlly. 3. Deletion of BMP2/BMP4 gene in chondrocytesinduce AC degradation phenotype and over expression of OA related protein MMP13, ADAMTS4 and ADAMTS5 in surperficial zone, which indicates BMP2 and BMP4 are essential to the development and self-maintaining of articular cartilage. 4. Icariin, Psoralen, Oleanolic acid and TSG can upregulate the gene and protein expression of BMP2 and BMP4 in mesenchymal cells, which might be the melacuar mechanism of benifiting kidney herbs in treating and preventing OA and slow down the AC degration. 5. ‘Kidney governs bone’ theory also involves the relation between kidney and articular cartilage, which is worth more endeavor to search and analyze.