| Objectives: Spinal cord injury is a CNS injury disease with high morbidity.Spinal cord injury in time sequence can be divided into primary injury and secondary injury.Primary injury is an irreversible physical injury,and secondary damage(such as inflammation,edema,apoptosis,oxidative stress,etc.)caused by changes in the microenvironment plays an important role in spinal nerve tissue injury.Secondary injury is regulatable,and human intervention can improve the adverse microenvironment caused by secondary injury,thereby reducing the damage degree of nervous function after spinal cord injury.Currently,in addition to traditional methods such as surgical decompression,hormone drugs,and hypothermia therapy,the treatment of traditional Chinese medicine is an important therapeutic intervention.But traditional Chinese medicine in the treatment of diseases of nervous system damage specific nerve protection mechanism needs further research.This study in order to maximize the simulation environment change of spinal cord injury.The saikosaponin a(SSa),a Chinese herbal medicine extract of different concentration,was injected into rats.In order to explore the possible mechanism of neuroprotection and the choice of optimal therapeutic drug concentration after acute spinal cord injury.The BBB score and the oblique plate test at 4 weeks after operation were used to evaluate the neurological function of rats.Methods:1.Intraperitoneal injection of 5mg/kg,10mg/kg,20mg/kg SSa in different drug concentration groups,using Allen,s weight hitting method to establish acute spinal cord injury model in rats,observe the efficacy of different drug concentration group,to obtain optimal drug concentration selection in this experiments.2.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of inflammatory cytokines in spinal cord tissues and serum of rats in each group.The effect of SSa on the immune microenvironment after acute spinal cord injury in rats was observed.3.Hematoxylin-eosin staining(HE)method was used to observe the morphological changes in the gray matter area of spinal cord injury under inverted phase contrast microscope.4.Use Western Blot method,GAPDH as an internal reference,and Image J software to measure protein gray value,calculate the ratio of target protein NF-κB P65,NF-κB PP65 and AQP4 protein to GAPDH gray value,observe the SSa effect of apoptotics signaling pathway in NF-κB after acute spinal cord injury in rats.5.Using Western Blot method,GAPDH as an internal reference,Image J software to calculate the protein gray value,calculate the ratio of the target protein BCL-2,Caspase-3 and GAPDH gray value,observe the SSa effect of apoptotics signaling pathway in BCL-2/Caspase-3 after acute spinal cord injury in rats.6.Using immunohistochemistry,microscopy and image acquisition software were used to analyze the cumulative absorbance values(IOD)of BCL-2 and Caspase-3 positive cells.Observe the effect of SSa on acute spinal cord injury in rats BCL-2 and Caspase-3 activity in neurons.7.Using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)method to detect the status of apoptotic cells in the spinal cord tissue,BBB score and sloping plate test to evaluate the function of the lower limbs of each group of rats,observed the SSa protective effect of neuronal cells and recovery of neurological function after acute spinal cord injury in rats.Results:1.Rats model of acute spinal cord injury was established successfully,after hitting the rat double hind limb twitching,tail flick,and then completely paralyzed.All animals survived after modeling,no wound infection,paralysis of both hind limbs in rats.2.ELISA showed the levels of that TNF-α and IL-6 in the spinal cord tissue and serum of the rats in the SCI injury group were higher than those in the sham group.The levels of TNF-α and IL-6 in spinal cord tissue and serum of rats treated with saikosaponin a 5mg / kg,10 mg / kg and 20 mg / kg were lower than those of the injury group.The expression of TNF-α and IL-6 in the spinal cord tissue of rats treated with 10 mg / group and 20 mg / kg group the difference was not statistically significant(P> 0.05).The levels of TNF-α and IL-6 in serum of rats treated with 10 mg / kg and 20mg/kg were lower than those of 5mg/kg group(P> 0.05).3.HE staining demonstrated normal neurons of the spinal cord and no obvious lesion in group Sham;neuronal cells were observed in the injured area of group SCI,with hemorrhage,neutrophil infiltration,and nerve cell edema in the injured area;the neuronal cells were visible in the spinal cord of group SCI+SSa,with microglia mild hyperplasia,and the pathological changes were improved when compared with group SCI.4.Western blot results showed that the protein expression levels of NF-κB P65,NF-κB P-P65,Caspase-3 and AQP4 were significantly lower in group Sham than groups SCI and SCI+SSa,and in group SCI+SSa than group SCI.Meanwhile,protein expression levels of BCL-2 were significantly lower in group Sham than groups SCI,and in group SCI+SSa than group SCI.5.Immunohistochemistry results showed that the expression of Bcl-2 were significantly lower in groups SCI and SCI+SSa than Sham group,in SCI+SSa group was higher than that in the SCI group.Meanwhile,the expression of Caspase 3 in SCI+SSa group was lower than that in SCI group.6.TUNEL results showed that the positive cells were significantly higher in group SCI than Sham and SCI+SSa groups,and the number of TUNEL-positive cells in SCI+SSa group is lower than that in SCI group.7.The BBB score and tiltboard experiment maximum angle were significantly higher in Sham group than groups SCI and SCI+SSa at each time point and in group SCI+SSa than group SCI at 14,21,and 28 days after operation.Conclusions:1.Saikosaponin a can reduce the early expression of inflammatory factors in acute spinal cord injury,improve immune function and reduce secondary injury after spinal cord injury immune response.2.In this study,Saikosaponin a 10mg/kg group was superior to other groups in inhibiting the inflammatory reaction.After acute spinal cord injury,the spinal blood barrier may affect drug absorption,the drug may partially pass through the spinal blood barrier.3.Saikosaponin a by inhibiting the NF-kappa B signaling pathway and the expression of AQP4 protein,reduce the body inflammatory reaction and tissue edema after acute spinal cord injury,which could regulate the BCL-2 / Caspase–3 signaling pathways,alleviate nerve cell apoptosis after acute spinal cord injury,which has a certain ability to protect the nerve. |
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