Research The Mechanism Of Hydroxyl Safflor Yellow A Promote The Rabbit Steroid-induced Avascular Necrosis Of Femoral Head Restoration

Purpose:Observe the Restoration Mechanism of traditional Chinese medicine saffron main active ingredient HSYA to steroid-induced avascular necrosis of the femoral head, investigate the mechanism from the MAPK signaling pathway, provide a theoretical basis for the prevention and treatment of steroid-induced avascular necrosis.Method:1. Using the modeling method of He, by gluteus injection of prednisolone acetate to New Zealand rabbits, to establishment of a rabbit steroid-induced femoral head avascular necrosis of the model, by transmission electron microscope observation, histological and morphological studies,rate calculation of empty lacunae and other aspects identification.2. After the success of modeling, the experimental animals were randomly divided into three groups, and use the different methods to intervene. The blank group: the normal diet feeding; The control group:A:Core decompression and normal saline50μl local injection; B: Auricular Vein injection of normal saline2ml/d; The treatment group:A:Core decompression and HSYA50μl local injection; B:ear vein injection HSYA2ml/d.3. The three groups of animals cut out of the femoral head at two weeks after the intervention, by transmission electron microscope observation, histological and morphological studies,rate calculation of empty lacunae, ALP detection,use PCR to detecting the expression of type I collagen, by immunohistochemical method to detect the expression of JNK in MAPK signaling pathway and the MAPK signaling pathway proteins ERK1, ERK2, P38expression was analyzed by Western blot to detect.Results:1. Observed under light microscope:Blank group femoral cartilage layer is thicker, Osteoblast activity, relatively regular are arranged of trabecular bone, bone cells are clearly visiblethe nuclear larger and centrally located, and occasionally of emptiness bone lacuna, the bone marrow tissue proliferated actively seen in Marrow cavity. The control group of the femoral head cartilage layer is thinner, subchondral trabecular bone thinning and structure disorder, part of them has fracture phenomena, the emptiness bone lacuna phenomenon significantly increased bone cells decreased, increased adipocytes, marrow cavity almost entirely replaced by adipose tissue. The treatment group was significantly thicker layer of the femoral head cartilage, and subchondral trabecular bone thicker, relatively regular are arranged, bone cell hyperplasia activity, nuclear larger and centrally located but the fat cells to reduce and smaller. The emptiness bone lacuna phenomenon significantly reduced, showing active bone Marrow cavity hyperplasia within the organization gradually replace fat tissue.2. TEM to observe the ultrastructure:Blank group the bone cell morphology were normal, intracellular of organelles more complete, plump cytoplasm, no significant of lipid droplets; Control group the bone cellular structures vague and reducing quantity, and more degeneration and necrosis, the nucleus pyknosis, bone cells have varying sizes, varying the number of of lipid droplets appear, nucleus pushed to one side; The treatment group, surface of bone were covered with a layer bobbin osteoblasts, Osteoblasts small, nucleoli visible and less pulp, a small amount of mitochondria and the rough endoplasmic reticulum, lysosomes, individual bone cellular structures vague and intracellular of lipid droplets decreased or disappeared.3. Empty bone lacuna rates:Blank group rate of empty lacunae low of14.38%; Empty lacunae in the control group was significantly higher, A high as26.43%, B up to25.47%; Compared the treatment group with the control group rate of empty lacunae was significantly lower rates, A is14.63%, B is16.32%.4. ALP detection:The control group ALP content was significantly lower than the control group, but the content of the HSYA treatment group was significantly higher than the control group, and the treatment group A and B are different. Compared with the control group A, B, The treatment group A, B,ALP levels were significantly elevated.5. RT-PCR analysis:In expression of collagen type Ⅰ the control group were significantly lower than the control group, but the HSYA treatment group were significantly higher than the control group,and treatment groups A and B were no significant differences. Compared with the control group A, B, treatment group A, B, type Ⅰ collagen expression were significantly increased.6. Immunohistochemistry:JNK expression in control group and treatment group were raised than blank group,and more pronounced increase in the control group. The treatment group compared with the control group JNK expression was significantly reduced.7. Expression of MAPK pathaway associated proteins:MAPK signaling pathway proteins ERK1, ERK2, P38expression was analyzed by Western blot to detect that the control group compared with the blank group, P-ERK1, P-ERK2expression was significantly reduced, whereas the expression of P-P38is significantly increased. The treatment group compared with the control group, P-ERK1, P-ERK2expression were significantly increased, whereas the expression of P-P38is significantly decreased. While P-ERK1, P-ERK2expression between The treatment group A, B have significant difference, while P-P38protein expression was not significantly different.Conclusion:1. Through the femoral head were observed by TEM, histological and morphological studies, rate of empty lacunae, ALP detection, and to detect the expression of type I collagen, showed that HSYA can promote osteogenic differentiation of BMSCs conducive repair of femoral head necrosis of bone tissue.2. Experimentally found that Pith Decompression combined with local injection HSYA rehabilitation effects to femoral head necrosis is better than Simply ear vein injection HSYA, this indicates that the combined treatment of SANFH may be related to by reducing the internal pressure of the Bone, improving partial blood circulation and meanwhile through promoting blood circulation, promoting the osteogenic differentiation of BMSCs to accelerate in tissue repairof the damaged bone.3. The mechanism of HSYA prevention of steroid-induced avascular necrosis, not only is the anti-oxidation, affect platelet activity and aggregation, promote vascular endothelial cell proliferation and improve the microcirculation of the femoral head, but also related to the inhibition of bone marrow mesenchymal stem cells into fat cells and promote its transformation into osteoblasts.