| Background:Several studies found that the lack of neurotrophic factors(NTFs) and the product of Myelin associated inhibitor factors(MAIFs) after SCI are two important factors which affect the axon regeneration microenvironment, and seriously prevent the neural axon regeneration after SCI. Traditional Chinese medicine, with the advantages of multiple factors and multiple targets, has been gradually become the focus in the improvement of nerve regeneration microenvironment."Jisuikang", the prescription based on the theory of "treat both Du meridian and kidney" and years of our clinical practice, could remove blood stasis and smooth Du meridian, nourish liver and kidney, and harmonize Qi and blood". Previous study showed that,"Jisuikang" could promote nerve functional recovery, inhibit the lipid peroxidation, remove oxygen free radicals, and effectively improve the local microenvironment after SCI. Therefore, we speculated that such effect may be related to its function of promoting NTFs and inhibiting MAIFs.Objective:To further clarify the effect of "Jisuikang" on neural functional recovery and spinal cord pathological changes after SCI, explore its effect on the expression of BDNF, NGF and NgR, and analysis its probable mechanism to improve the axon regeneration microenvironment.Methods:210clean grade SD rats, female, weight180～220g,30were only used to evaluate the SCI animal models, other180only for formal experiment. The rat models of spinal injury were established by modified Allen’s method in the experiment. The vertebral plates and spines of pseudo surgery group were bitten away. The BBB evaluation, oblique board test and histopathology were applied to test the availability of the SCI rat models.30rats were randomly extracted into pseudo surgery group (group A), other150rats were randomly divided into model group (group B), prednisolone group (group C), high, middle and low doses jisuikang group (group D～F). The rats were intervened30min after anesthesia recovery. The rats in group C were given0.06g/(kg.d) prednisolone, in group D-F were given respectively50,25, and12.5g/(kg.d)"Jisuikang". Each group were given20ml/(kg.d) volume by intragastric administration. The rats in group A and B was given the same dose of normal saline (NS).The BBB evaluation, oblique board test and histopathology were applied to test the postoperative results24hours,3,7, and14days after SCI, and executed the rats and extracted the spinal cord tissue3,7, and14days after SCI. Observed the change of the spinal cord tissues for the HE staining, transmission electron microscope (TEM), analyzed the expression of serum BDNF, NGF after SCI. Immunohistochemistry, western blot and RT-PCR were applied to test the expression of protein and mRNA of BDNF, NGF, NgR.Results:(1)BBB score and oblique board test:group B were significantly lower than group A and control group (P<0.01). HE staining:In control group and group A, the spinal cord structures were clear, no neurons swelling, nucleus pycnosis, necrosis or inflammatory in the cell body, while in group B, the structure of spinal cord was damaged, nerve fibers arranged disorder, with neuronal pyknosis, necrosis, dissolved, and flake bleeding area.(2)BBB score and the angle of oblique board test of each group were significantly lower than group A24h after injury (P<0.01). In group C and E, ethology score were both higher than group B3～14days after injury (P<0.05), with no significant difference between the two groups (P>0.05). The activity of rats’hind legs in treatment groups were improved7days after injury, with no significant difference between group C and group E(P>0.05), BBB score of group E was significantly higher than other groups14days after injury, with significant difference from group C(P<0.01).(3)HE staining:In Group B, the structure of spinal cord tissues was damaged, with severe gap in nerve fibers, neuronal pyknosis, necrosis, dissolve could be seen, with progressive aggravation. In group C-F also could see spinal cord damage, but as the intervention time extended, the neuron survival were more obvious compared with group B, especially in group C and E; the result of SEM showed that3-7days after injury, group B had a progressive aggravation, with some irreversible damage, while group C～F had more clear structure of glial cells.(4)ELISA:BDNF and NGF had different degree rise in rats serum after SCI, but the maintain time is short.3days after injury, BDNF and NGF were significantly increased in group B～F compared with group A,7～14days after injury, BDNF remained at a high level, while the expression of NGF fell slightly. Statistics showed that, at each time point, prednisone and "Jisuikang" both could effectively promote the expression of BDNF and NGF in serum after SCI, and maintain at a high level, with no obvious difference between group C and E7-14days after injury(P>0.05).(5)Immunohistochemistry and western blot:the expression of BDNF, NGF rose at different degree, similar result to the ELISA. The different time points expression of BDNF, NGF of group C-F were significantly higher than group B, the postoperative expression of BDNF remained at high levels after7～14days, while the expression of NGF was slightly down7days after injury. The spinal cord injury area highly expressed NgR after SCI, but prednisolone and "Jisuikang" could restrain the expression of NgR, and both significantly reduced at different time points, compared with group B (P<0.05). (6)RT-PCR:Prednisone and "Jisuikang" could promote the expression of BDNF and NGF mRNA, inhibit the expression of NgR mRNA. Early after SCI, prednisone had a more obvious effect, group C～F were also better than group B, especially in group C, but compared with prednisone group, the action is slower. But14days after injury, group C also maintained a higher level of BDNF, NGF mRNA and a lower level of NgR mRNA.Conclusion:(1)The modified Allen’s method built in this research is a reliable method with good stability, and easy to repeat.(2)Both prednisolone and "Jisuikang" can promote the recovery of the motor function of hind limb of the SCI rats, and "Jisuikang" has a better effect7days after injury.(3)Both prednisolone and "Jisuikang" can reduce and delay the pathological damage, prevent the occurrence and development of irreversible deformation phenomenon after SCI.(4)Both prednisolone and "Jisuikang" can promote the expression of BDNF, NGF and inhibit the expression of NgR after SCI, and the prednisolone has a more obvious effect early after SCI, while "Jisuikang" has a lasting effect. It reminders that "Jisuikang" has obvious advantages in the middle and late period after SCI. Maybe it is one of the factors of "Jisuikang" to promote neural functional recovery and improve the axon regeneration microenviromnent. |
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