| Objective To observe the effects of sodium aescinate on expression of Beclin-1 protein in spinal cord injury area of rats with hemisection of spinal cord,and explore the mechanism of sodium aescinate in the recovery of nerve function in rats with spinal cord hemisection.Method 89 adult female SD rats were randomly divided into sham operation group(group A),9 rats with laminectomy,saline group(group B)and sodium aescinate group(group C)with 40 rats each.Two groups B and C established stable left hemisection injury models in the L2-3 spinal cord segment.Group A,B and C were injected intraperitoneally with 1.5 ml normal saline and 1.5 ml sodium aescinate solution(6 mg/kg dissolved in normal saline)30 minutes before modeling.Before the establishment of the model,the motor function scores(BBB scores)of the rats in each group were assessed on 6h,1st,3rd,7th and 14 th day.According to the above time points,B and C groups were sampled separately.Regarding the injury point as a center,the injured area of the spinal cord segment 2-3 about 1.5 cm in length was taken out for HE staining to observe the morphology of spinal cord neurons,qualitative and quantitative analysis for immunohistochemistry,and the expression of Beclin-1 protein in the spinal cord injury area of rats was detected by Western-blot immunoblot.All data were analyzed by SPSS 21.0 statistical software.All the measurement datas were expressed by mean ± standard error.Independent sample t-test and one-way analysis of variance(ANOV)were used for statistical test and the difference was statistically significant when P< 0.05.Result 1,Spinal cord neurological function score(BBB score): BBB score of the B and C groups at BBB score of the 3rd,7d and 14 d had significant difference(P< 0.05),indicating that BBB score of the B,7d and 14 d had significant difference(P< 0.05).At the same time,the BBB scores of group B and C,after 7th and 14 th days of modeling,the BBB scores of group C were higher than those of group B(P< 0.05),indicating that sodium aescinate could improve the neurological function of spinal cord hemisection rats and protect neurons.2,HE staining: The morphology of spinal cord tissue in each group was observed under the microscope.The morphology and structure of neurons and glial cells in spinal cord tissue of group A were intact,without edema and necrosis.The biggest difference between group C and group B is that the damage of neurons in group C is lighter than that in group B.Some neurons are preserved intact and glial cells are increased,which indicates that sodium aescinate can protect the integrity of neurons and promote the increase of glial cells.3,Immunohistochemical expression of Beclin-1 positive cells: Beclin-1 expression was mainly localized in the cytoplasm and nuclear membrane of spinal cord injury area,but slightly or not in the interstitium,axon and nucleus.In group A,a small amount of Beclin-1 brown granules were expressed in the spinal cord injury area which was loosely distributed and showed weak positive expression indicating that there was low expression of autophagy in the normal spinal cord area.The expression of Beclin-1 was mainly distributed in the injured area of hSCI,but less in the non-injured area.In the injured group,brown and yellow granules were more expressed and distributed densely showing strong positive expression.The number of Beclin-1 positive cells on the 14 th day was lower than that on the 3rd day(P< 0.01),indicating that the expression of autophagy decreased with time.At the same time,the number of Beclin-1 positive cells in group C was significantly higher than that in group B(P< 0.05).It suggests that sodium aescinate can stimulate the enhancement of autophagy.4,Western blot was used to detect the expression of Beclin-1 protein in hSCI region.The expression of Beclin-1 protein in B and C groups began to increase 6 hours after modeling,peaked on the 3rd day after modeling,decreased on the 7th day and lowest on the 14 th day.Comparisons between groups B and C: There was no significant difference in Beclin-1/beta-actin gray values at 6 hours and 1 day after modeling(P> 0.05).On the third day,Beclin-1/beta-actin gray values peaked and then decreased,and there was significant difference in gray values at each time point(P< 0.05).It showed that the enhancement of autophagy activity was a cumulative process.At the same time,the comparison between groups B and C were as follows: At 6h and 1d,there was no significant difference in Beclin-1/beta-actin gray values between the two groups(P> 0.05).Compared with other time,group C was higher than group B,and the difference was statistically significant(P< 0.05),indicating that sodium aescinate had the effect of stimulating autophagy.Conclusion 1,There was low expression of autophagy in the spinal cord of normal rats and increased expression of autophagy after spinal cord injury.2,In the rat model of hSCI,sodium aescinate could enhance the expression of autophagy-related protein Beclin-1.3,Sodium aescinate can improve the recovery of nerve functions after spinal cord injury by regulating autophagy-related protein Beclin-1. |
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