The Expression Of Cytokines And Regulatory Gene Of Apoptosis And The Effect Of Combination Of MP/AG To Its Following Spinal Cord Injury

Spinal cord injury (spinal cord injury, SCI) can be induced of various primary factors. After injury, the body quickly made a series of pathophysiological processes and reactions of self-defense, including electrolyte imbalance, local edema, apoptosis, production of free radicals, induced immune reaction caused by injury and so on. The intervention to the different types of damage is a hot issue in the clinical treatment of spinal cord injury.Treatment of spinal cord injury mainly includes the operation and non-operationtreatment. The operation treatment to spinal cord injury is mainly to alleviate spinal cord compression or to maintain spinal stability by in vivo fixation; non operation treatment, which includes drug, hyperbaric oxygen therapy, cell transplantation, gene therapy and so on, is mainly directed against the secondary injury after spinal cord injury. The hormone is convenient use and wide application of the drug treatment to spinal cord injury. Methylprednisolone (Methylprednisolone, MP) is the most representative of hormone.Methylprednisolone (MP) is a synthetic glucocorticoid and the only therapeutic agent approved by the Food and Drug Administration for reducing the extent of the post-traumatic inflammatory reaction following acute SCI. MP is neuroprotective in animal models and the general use of MP in the clinic, Although the application of MP after SCI is associated with a wide array of anti-inflammatory effects, the long-term administration of this therapeutic steroid results in a variety of side-effects. There is need for additional and improved treatments for SCI, so a combination represents a promising strategy of clinically applicable pharmacological therapy for the rapid initiation of neuroprotection following SCI.Aminoguanidine (AG), a small water-soluble compound, is be widely used for the prevention of the chronic tissue complications of diabetes mellitus in humans. Previous studies have shown that AG reduces the extent of brain edema in animal models of surgical brain injury, stroke and post-traumatic brain injury. Notably, some studies demonstrate that AG improve the motor functions of injured spinal cords in rats and may have a potential role in the treatment of acute SCI. However, high doses of AG lead to toxic effects, which limit its usefulness clinically. In a word, treating a single target is unlikely to achieve complete inhibition of the inflammation due to the complexity and redundancy of the inflammatory response associated with SCI. It is being shown that the strategy of targeting multiple proinflammatory pathways may be more effective than targeting a single effector molecule. Our study provides convincing evidence that the combination of MP with AG significantly proves the damaged motor function caused by SCI in ratby reducing the levels of spinal cord edema and inhibiting the apoptosis, whereas a single treatment did not significantly improve them.In word, our study could contribute to a better understanding of the combination of MP with AG and provide theoretical and experimental basis for the clinical treatment of spinal cord injuryThe study consists of three sections as follows:1.Theeffect of the combination of MP with AG to spinal cord edema and function recovery after spinal cord injury in rats.1) Combination treatment with MP and AG results in functional recovery following SCI.BBB score and the Grid-walking lattice are used to determinate the functional recovery of SCI rats. The BBB scores in the AG and MP combination treatment group are consistently higher than those in the other groups and the differences between BBB scores of the AG and MP combination group and those of the other three groups are statistically significant starting from the third week and continuing until the eight week. The score of the AG and MP combination group at56d is16.55+2.01, and the damaged rats appears fine activity of the distal limbs such as dragging the toe and tail, instability of trunk, alternating rotation of claws. Our result further suggests that recovery of motor function of the hind limb is well. A minor but not statistically significant increase of the BBB scores is observed in the AG and MP groups from the fifth week after SCI. Results from the grid walking test also show that the percentage of missteps of hind paws is markedly reduced in the combination-treated rats compared with that in the other groups. The differences are statistically significant (p <0.05).Our results suggest that combined treatment is beneficial to the functional recovery after spinal cord injury.2) The combination MP with AG therapy has significant anti-edematous activity.The combination therapy had significant anti-edematous activity compared with that observed in the control group, whereas in the single treatment groups the levels of cerebral edema did not significantly change compared with those of the control group by measuring the water content of each segment of spinal cord. However, the levels of cerebral edema in the single treatment groups were significantly increased compared with those of the sham group. These data showed that the combination therapy with AG and MP significantly ameliorated the increased water content of the injured spinal cords.2. The effect of the combination of MP with AG to inflammatory cytokines including in MPO after spinal cord injury in rats.1) The combination MP with AG therapy inhibits neutrophil infiltration.MPO activity is significantly elevated in the rats subjected to SCI when compared with those of the rats in the sham surgery group by the ELAISA methods and the differences are statistically significant. The result suggests that a large number of neutrophil infiltrates in the lesion site. The levels of MPO activity were significantly reduced by the combination therapy with AG and MP compared with those of the rats in the control group. However, administering either of the compounds as a single treatment did not reduce the levels of neutrophil infiltration in the injured spinal cord compared with those of the rats in the control group and the differences are not statistically significant.2) The effect of the combination therapy on the expression levels of TNF-a and IL-1β following SCI.The combination therapy significantly reduced the spinal cord levels of TNF-a and IL-1β compared with those of the control group by the ELAISA methods. Furthermore, the MP treatment group exhibited markedly reduced spinal cord levels of TNF-a and IL-1β compared with those of the control group, whereas the AG treatment group did not show clearly reduced TNF-α and IL-1β levels compared with those of the control group. The results show that combined therapy can inhibit the expression of proinflammatory cytokines and obviously reduce the secondary reactions. 3. The effect of the combination of MP with AG to apoptosis after spinal cord injury in rats.1) Western blot analysis of Bax and Bcl-2.The Bax expression levels were appreciably increased in the spinal cords from the rats subjected to SCI, compared with those in the spinal cord from the sham rats by western blot. However, the combination therapy with MP and AG significantly reduced Bax expression levels compared with those in the control. Furthermore, whole extracts from the spinal cord of each rat were also analyzed to detect Bcl-2expression levels. Combination treatment of the rats with MP and AG significantly increased the SCI induced inhibition of Bcl-2expression. These results indicate that combination therapy with MP and AG inhibits apoptosis following SCI.2) Effect of the combination therapy on the expression levels of caspase3mRNA following SCI.The expressionlevel of caspase3mRNA was appreciably increased in the spinal cords from the rats subjected to SCI, compared with those in the spinal cord from the sham rats. However, the combination therapy with MP and AG significantly reduced the expression level of caspase3mRNA compared with those in the control, p<0.05.The experimental results suggest that the expression of combined therapy can significantly reduce the mRNA level of caspase3.In brief, the present study provides convincing evidence that the combination of MP with AG significantly reduce the levels of spinal cord edema, reduce the levels of neutrophil infiltration, inhibit TNF-α and IL-1β levels, reduce Bax expression levels, increase the Bcl-2protein expression,and improve the damaged motor function caused by SCI in rats compared with those of the AG group and MP group based on rat model of spinal cord injury. In conclusion, the data imply that strategies targeting multiple proinflammatory pathways may be more effective than those targeting a single effector molecule. Our data supply to substantial theoretical and experimental basis for the treatment of spinal cord injury.