| Objective:This study through the method of experimental animals and from cellular and molecular level, the research of the Huo Gu injection on the expression of VEGF, bFGF, IGF-1, OPG, RANKL, Bcl-2, Bax and Caspase-3 and investigate the bone injection treatment of the mechanism of femoral head ischemic necrosis, Huo Gu injection treatment of avascular necrosis of the femoral head provide a theoretical basis.MethodRandomly divided into two groups, namely blank group (30), model group (90). The model group were randomly divided into 3 groups:saline group, control group and experimental group, each of the 30 animals. The blank group were given normal feeding without any drug treatment. Saline group was given 0.9% saline 0.70ml/ times, one time every three days. The control group was given Guanxinning Injection every three days a hip perfusion 0.70ml/. The experimental group was given the active bone injection once every three days, and the dose was 0.70ml/ times. After two weeks of modeling, the rats were sacrificed at 1,3,6,9 and 12 weeks, and each group had 6 rats in each group. Experiment 1, we used three methods of immunohistochemistry, real-time PCR and Western blot detection of rabbit femoral head ischemic necrosis of the repair process of VEGF, bFGF, IGF-1 protein expression; in the second experiment, we used two methods of real time PCR and Western blot detection of rabbit femoral head ischemic necrosis in the healing process of protein and mRNA and expression of OPG/RANKL ratio; Experiment 3, we use two methods of real time PCR and Western blot detection of rabbit femoral head ischemic necrosis repair process in apoptosis regulating genes Bcl-2, Bax and Caspase-3 expression.ResultExperiment 11.Expression of VEGFBlank control group and saline group in various stages of expression had no significant change; control group with the extension of delivery time, expression gradually increased, and there were significant differences (P< 0.05 or P< 0.01); the experimental group with the extension of treatment time, expression can persistently increase and there is a significant difference (P< 0.01), and the expression level compared with the control group increased significantly, but there was no statistical difference.2. Expression of bFGFSustained and stable and blank group at various stages of expression had no obvious change; saline group at each time point and blank groups had no significant difference, and the control group with the administration time prolonged, the expression can continue to rise, and there is significant difference (P< 0.05 or P< 0.01), and the expression amount was increased to 12 weeks, the experimental group with time prolonged, the expression can continue to rise, with the blank and saline group had significant difference (P < 0.01), and the expression level can be sustained and stable increase to 12 weeks, and at each time point relative to the control group increased significantly, and there was difference (P< 0.05 or P< 0.01).3. Expression of IGF-1The blank group in various stages of expression had no obvious change; saline group at third weeks showed high expression, and has significant difference (P< 0.01), then along with the prolongation of time, the expression decreased gradually, and gradually lower than the normal level; the control group with the delivery time is prolonged, and the expression increased gradually. There were significant differences (P< 0.05 or P< 0.01), but sixth weeks later the expression of basic stability without increased significantly; the experimental group with the prolongation of medication, can significantly enhance the expression, there was significant difference with the control and saline group (P< 0.01), and the expression was consistently increased to for Twelfth weeks, and there was significant difference with control group (P< 0.05 or P< 0.01).Experiment 2Ratio of OPG/RANKL, RANKL and OPGThe expression of OPG, RANKL and mRNA in the blank and saline group had no significant change, and there was no significant change in the OPG/RANKL ratio. Extension and blank control group with administration time prolonged, the expression can be significantly increased to 9 weeks significantly increased, and there is a significant difference (P< 0.01), but to the 12 week expression, although a slight increase, but with saline group, but no significant difference (P> 0.05); RANKL protein and mRNA in the 1 week when expression was significantly increased, and there is a significant difference (P< 0.05 or P< 0.01), with the medication time prolonged expression gradually increased to 12 weeks to reach a relatively high, and had significant difference (P< 0.01); OPG/RANKL ratio with the medication can be gradually increased, and there is significant difference (P< 0.01). The experimental group with treatment time prolonged OPG protein and mRNA expression increased gradually and significantly different (P< 0.01), and it can be continuously increased until the 12th week and expression at each time point is higher than that of the control group, and there was significant difference (P< 0.01).; and RANKL mRNA and protein expression although with the extension of treatment time, expression gradually increased, but the next 6 weeks has with the blank and saline group compared with significant difference (P< 0.01) and can be sustained and stable high expression to the end of the 12 week, but the expression amount had no significant change; the modulation of RANKL ratio can also gradually increased and at each time point were always higher than that of the blank, saline treated and control group and statistical difference (P< 0.05 or P< 0.01).Experiment 31.The expression of Bax, Bcl-2 and the ratio of bcl-2/baxBcl-2 in blank group at all stages of expression did not change; saline group at each time point with the blank group there is no difference (P> 0.05); control group with time prolonged expression can continue to rise, with the blank and saline group compared with significant difference (P< 0.01); experimental group with time prolonged expression can continue to rise, with the blank and saline group compared with significant difference (P< 0.01) and each time point expression than the control group. Blank group Bax in various stages of expression did not change; saline group at each time point with the blank group there is no difference (P> 0.05); the medicine time prolonged expression continued to decline, with the blank and saline group compared with significant difference (P< 0.05 or P< 0.01) in the control group; experimental group can also with the extension of time, the expression continued to decline, with the blank and saline group compared with significant difference (P< 0.01) and in each time point expression, compared with the control group to be low, and there was significant difference (P< 0.05). There has been no change in the blank and saline group Bcl-2/ Bax ratio; control and the ratio of Bcl-2/ Bax in experimental group at each time point is gradually increasing and blank and saline group there were significant differences (P< 0.05 or P< 0.01), experimental group Bcl-2/ Bax ratio in each time point compared to that of the control group significantly increased and statistically significant difference (P< 0.05).2.The expression of Caspase-3The blank group in each stage was no change; saline group at 3 weeks and compared with the control group there was significant difference in drug first, (P< 0.05 or P< 0.01), with the prolongation of medication, the expression gradually reduced to twelfth weeks, but no difference compared with the control group (P> 0.05); the control group with the prolongation of medication, the expression is going down, and there is a significant difference (P< 0.01), the expression also showed decreased gradually; the experimental group also showed high expression in after first and 3 weeks, there was significant difference compared with the control group (P< 0.01). The expression decreased gradually after sixth weeks and gradually lower than the normal level, and compared with the blank control group were significantly decreased (P< 0.01), along with the prolongation of time, the expression can be sustained significantly reduced to twelfth weeks, and was significantly lower than the normal level, and with the blank and saline Group with significant difference (P< 0.01), at the same time was significantly lower than the control group.ConclusionHuo Gu injection can enhance the expression of rabbit femoral head necrosis, bFGF and local VEGF IGF-1 to enhance angiogenesis, to repair ischemic necrosis of femoral headHuo Gu injection can increase the expression of rabbit femoral head necrosis OPG, also upregulate the expression of RANKL, and increase the ratio of OPG/RANKL It is beneficial to promote the absorption of necrotic bone, improve the bone metabolism, and restore a dynamic balance of bone metabolism, promote bone repair and new bone formation. OPG/RANKL ratio may be one of the targets of drug action in the active bone injection.Huo Gu injection through the upregulation of rabbit femoral head necrosis of bcl-2 expression downregulation of Bax and Caspase-3 expression of Bcl-2/ Bax ratio increased, to local holding head necrosis cell apoptosis and bone cells prolong the survival time, and can inhibit osteoclast activity and function, promote the repair and regeneration of bone. |
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