| ObjectiveTo explore the Wnt/β-catenin signal transduction mechanism by Bushen Huoxue decoction intervening the adipogenic and osteogenic induced differentiation of BMSCs.Methods1 In vivo experiment:90 New Zealand white rabbits were randomly divided into blank group 30, model group 30, treatment group 30. After the establishment of hormone femoral bone ischemia and necrosis in rabbit model of success by the HeShi molding gluteal muscle injection of prednisolone acetate modeling, in the treatment group, the treatment group was treated with the Bushen Huoxue decoction and the other two groups were treated with physiological saline, the morphology of the tissues was observed by HE staining, and the serum alkaline phosphatase and glycerol three were detected after 8 weeks, observe the effect of Bushen Huoxue decoction on the ischemic necrosis of the femoral head.2 In vitro experiment:The New Zealand white rabbits for 5 monthswere used to isolate and identify the BMSCs,and then induce the osteogenic and adipogenic differentiation of rabbit BMSCs and interve them by Bushen Huoxue decoction and Tuoluo Shenggu capsule. Oil red O and Alkaline phosphatase staining,and then detect TG and ALP content. RT-PCR and Western Blotting were used to detect the gene and protein expression changes of the related factors on the Wnt/β-catenin signaling pathway in cells.Results1 In vivo experimental results:(1) The detection results of alkaline phosphatase and the fat content of the markers of the osteogenic markers in the serum of the blood serum:the content of alkaline phosphatase in serum of the prognosis of Bushen Huoxue decoction was high. Results of ALP content in serum:compared with the blank group, the model group and the treatment group were significantly lower than the blank group(P= 000, P=0.015); Compared with model group, the treatment group was significantly higher than the model group(P=0.006); But the serum triglyceride levels in the serum of the prognosis of Bushen Huoxue decoction were low. Serum triglyceride content results:compared with the blank group, model group, treatment group was significantly higher than the blank group (P=0.000, P=0.005), and the model group, the treatment group was significantly lower than the model group (P=0.003).(2)The morphological observation of HE staining:In the blank group, the trabecular bone was compact and orderly, and the bone cells were clear and the nucleus was located in the center of the central and large. Model group, the cartilage layer became thin, structural disorder, trabecular bone is sparse, part of a fracture phenomenon, trabecular bone necrosis, bone lacunae were empty phenomenon compared with the blank group increased significantly, subchondral marrow fat cells increased, the number of hematopoietic cells reduce. In the treatment group, the bone cell structure was clear, there were more trabecular bone, and the necrotic bone cells were less than the model control group.2 In vitro experimental results:(1)Comparison of Alkaline phosphatase staining:Blank control group, model group and blank serum alkaline phosphatase staining was negative. High, medium and low dose group of Bushen Huoxue decoction were positive, positive result was gradually increased along with the increase of the dose. Positive drug group:alkaline phosphatase staining strongly positive, and stronger than the other groups (P<0.05)(2)Comparison of Oil red O staining:The red stained fat particles does not appear in the control group, and other groups obviously is dyed red fat particles; High Bushen Huoxue Decoction, distribution, low dose group were stained red fat particles, but the three groups of red dye fat granule was significantly less than that in the model group, the high dose group and positive drug group red staining results approximate, was less than that of the middle and low dose (P<0.05).(3)Intracellular triglyceride (TG), alkaline phosphatase (ALP) test results:Bushen Huoxue Decoction group and positive drug group and model group TG content were significantly decreased compared and showed a decreasing trend with increase of the dose; and Bushen Huoxue Decoction and positive drug group and model group were ALP levels increased significantly compared and increased with the dose increased. (P<0.01).(4)Transmission electron microscope was used to observe the stem of prognosis of BMSCs ultrastructure:cells in the control group were oval or irregular in shape, less cytoplasm, nuclear and cell surface without microvilli, mitochondria had mostly, crest no obvious fracture or disappear; part of the rough endoplasmic reticulum showed no obvious expansion of. No obvious removal particle phenomenon. Model group, cells were long spindle shape, no obvious surface protrusions and the nucleus was oval, no obvious nucleolus, more mitochondria organelles and more vacuoles were, the mitochondrial fraction showed a high electron density changes, visible secondary lysosomes and lipid droplets and some visible smooth shape, a large section of the expansion of the rough endoplasmic reticulum. Low dose group showed long fusiform, many cell processes, compared with model group, the cavity surface is less, the part of the mitochondrial fine has been restored, the number of mitochondria damage, dilated rough endoplasmic reticulum is less. In the middle dose group, there was a long spindle shape, and the number of mitochondria was increased, and the crest was also increased, and there was no obvious lipid droplet. High dose group and positive drug group were long fusiform, vacuolar degeneration significantly reduced, significantly reduced the secondary lysosomes, dilation of rough endoplasmic reticulum is not too bad, no obvious lipid drops.(5) BMSCs osteogenic and adipogenic factor mRNA expression:(1) Compared by the model group, positive drug group and Bushen Huoxue Decoction group in osteogenesis related factors of WntlOb, Wntl, beta catenin Ror2 gene showed a increasing trend, and the positive drug Tongluo Shenggu Capsule on the osteogenic differentiation related gene expression enhanced effect was superior to other groups; Bushen Huoxue Decoction group of osteogenic gene expression is enhanced, and with the dose enhancement effects showed a gradual upward trend, and Bushen Huoxue Decoction high dose group of each gene expression enhanced effect and positive drug group compared with no significant difference (P>0.05). (2) The model group compared with positive drug group, and Bushen Huoxue Decoction group into lipid related factor DDK1, SFRP4 gene expression showed decreasing trends, positive drug Tongluo Shenggu Capsule on DDK1, SFRP4 into adipogenic gene expression decreased, significantly higher than the other groups; Bu Shen Huo Xue Decoction group the expression of osteogenic genes also has a limiting effect and with increasing doses of limiting effect is gradually rising trend and Bushen Huoxue Decoction high dose group of each gene expression effect is weakened and positive drug group compared with no significant difference (P>0.05).(6)BMSCs in the Wnt/β-catenin catenin signaling pathway related factors of protein expression changes:(1) Compared with model group:WntlOb protein. Wntl. beta catenin,FZD6 Ror2 which to promote signaling pathway open showed upward trend, positive drug Tongluo students bone capsule on the osteogenic differentiation protein table of enhancement effect, significantly better than the other groups; Bushen Huoxue Decoction on osteogenic protein expression also has strengthening effect, and compared with increasing dose enhancement effect was gradually rising trend, and Bushen Huoxue Decoction high dose group of each protein expression enhanced effect and positive drug group, no significant difference (P>0.05). (2) Compared with model group:DDK1. SFRP4 protein expression showed a decreasing trend, positive medicine Tongluoshenggu Capsule on DDK1, SFRP4 into fat differentiation protein expression decreased, significantly higher than the other groups; Bushen Huoxue Decoction on expression of bone protein also has a limited role and with increasing doses of limiting effect is gradually rising trend and Bushen Huoxue Decoction high dose group of each protein expression weakened the effect of positive drug group and compared with no significant difference (P>0.05).Conclusion:1 Bushen Huoxue Decoction can treat the steroid induced avascular necrosis of the femoral head in rabbits and it can promote BMSCs osteogenic differentiation, and inhibit their differentiation into adipocytes.2 Bushen Huoxue Decoction for regulating BMSCs bone into adipogenic differentiation mechanism is mainly by enhancing the gene and protein expression of of Wnt/p-catenin signaling pathway into bone factor WntlOb, Wntl, beta catenin, FZD6 Ror2, while limiting into lipid factors DDK1, SFRP4 expression to achieve. |
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