Study On The Mechanism Of Motor Function Recovery In Rats With Hemisection Spinal Cord Injury By Periplaneta Americana Powder

【Objective】:To observe the recovery of hind limb motor function in rats with spinal cord hemisection injury（h SCI）treated with Periplaneta Americana powder.To investigate the effects of Periplaneta Americana powder on the motor function of hind limbs,autophagy flow and oxidative stress in the injured area of h SCI rats.【Method】:1.Experimental grouping:（1）Part 1:Fifty female SD rats were randomly divided into 5 groups:SD rats were divided into Sham group,normal saline（SCI+NS）group,Periplaneta Americana powder210mg/kg（SCI+210mg/kg）group,Periplaneta Americana powder 420mg/kg（SCI+420mg/kg）group and Periplaneta Americana powder 630mg/kg（SCI+630mg/kg）group.In the Sham group,only the lamina was opened without hemitransection of the spinal cord,and the left hemitransection of the spinal cord was performed in the other groups.The rats were treated by gavage 30min before surgery and daily after surgery,and the volume of each gavage was 5ml,The rats in Sham group and SCI+NS group were given NS,and the other groups were given 5ml of different doses of Periplaneta Americana powder solution.The rats were sacrificed to harvest spinal cord tissue at 14days after surgery.（2）Part II:Sixty female SD rats were randomly divided into normal saline（SCI+NS）group and Periplaneta Americana powder 630mg/kg（SCI+630mg/kg）group.The left half transverse injury of spinal cord was performed in all rats,with 30 rats in each group.Each group was divided into five time points,respectively are 6^th hour,1^st,3^rd,7^th and 14^th day after operation,with 6 rats in each time point.30 minutes before surgery,each rat in the SCI+NS group was given 5ml NS by gavage,and each rat in the SCI+630mg/kg group was given 5ml of 630mg/kg Periplaneta Americana grinding powder solution by gavage,The rats were sacrificed to harvest spinal cord tissue according to the time point.2.Observation indicators:（1）The BBB scores of rats in the first part were observed before operation,6^th hour,1^st,3^rd,7^th,14^th day after operation,and the footprint impression were observed on 14^th day after operation.（2）Hematoxylin-eosin staining（HE staining）:the pathological changes of h SCI area in the first part of rats in each group were observed on the 14^th day after operation.（3）Nissl staining:in the first part rats,the number of neurons in the h SCI area of each group was observed on the 14^th day after operation.（4）ELISA was used to detect the contents of MDA and 8-OHd G in the h SCI area of the rats in the first part on the 14^th day after operation,and the contents of MDA and 8-OHd G in the h SCI area of the rats in the second part on the 6^th hour,1^st,3^rd,7^th and 14^th day after operation.（5）Western blot experiment:The expression of Beclin-1,LC3B and p62 in the h SCI region of the first part of rats was detected on the 14^th day after operation,and the expression of Beclin-1,LC3B-Ⅱ/LC3B-Ⅰand p62 in the h SCI region of the second part of rats was detected on the 6^th hour,1^st,3^rd,7^th and 14^th day after operation.【Result】:1.After 14 days of treatment with Periplaneta Americana powder,compared with Sham group,BBB scores in other groups were lower than those in Sham group from 6^th hour after surgery,and the difference was statistically significant（P<0.05）;Compared with SCI+NS group,BBB scores in SCI+210mg/kg group,SCI+420mg/kg group and SCI+630mg/kg group were higher than those in SCI+NS group on the 7^th and 14^th day after surgery,and the difference was statistically significant（P<0.05）;There were significant differences in BBB scores on day 14 after surgery in SCI+210mg/kg group,SCI+420mg/kg group and SCI+630mg/kg group（P<0.05）,the highest value was found in SCI+630mg/kg group.The footprint results showed that 14 days after treatment with Periplaneta Americana powder,the footprints of rats after h SCI were clear without dragging traces,and the footprint resolution of all dosage groups of Periplaneta Americana powder was better than that of SCI+NS group.2.HE staining:In the SCI+NS group,the tissue structure was loose,the gray matter was obviously damaged,and the neurons showed obvious edema,nuclear fragmentation,and nucleolysis,and no neurons in normal shape were found in the whole field of vision.The neurons in the groups with different doses of Periplaneta Americana powder still showed pathological changes such as edema and nuclear fragmentation,but the neurons were relatively intact.The pathological changes of edema,nuclear fragmentation and nucleolysis were lighter than those of SCI+NS group.All the doses of Periplaneta Americana powder could reduce the degree of neuron damage,and the 630mg/kg group had the least amount of neuron damage.3.Niss staining showed that the number of neurons in Sham group was the highest,followed by SCI+630mg/kg group,SCI+420mg/kg group and SCI+210mg/kg group,and the number of neurons in SCI+NS group was the lowest.4.Content of MDA and 8-OHd G:The part I rats in each group compared with SCI+NS group,MDA and 8-OHd G contents in Sham group,SCI+210mg/kg group,SCI+420mg/kg group and SCI+630mg/kg group were lower than those in SCI+NS group on day 14 after surgery,and the differences were statistically significant（P<0.05）.The SCI+NS group rats of part II compared with SCI+630mg/kg rats at various time points showed that the content of MDA and 8-OHd G in SCI+630mg/kg group was lower than that in SCI+NS group on the 7^th and 14^th day after surgery,and the difference was statistically significant（P<0.05）,MDA and 8-OHd G in SCI+NS group began to increase at 6^th hour after surgery,reached a peak at 7^th day after surgery,and then began to decline.MDA and 8-OHd G in SCI+630mg/kg group began to increase at 6^th day after surgery,reached a peak on the 3^rd day after surgery,and then began to decline.5.Beclin-1,LC3B-Ⅱ/LC3B-Ⅰ,p62 expression:The part I rats in each group compared with SCI+NS group,Beclin-1,LC3B-Ⅱ/LC3B-Ⅰexpression in Sham group,SCI+210mg/kg group,SCI+420mg/kg group and SCI+630mg/kg group were higher than those in SCI+NS group on the 14^th day after surgery.The difference was statistically significant（P<0.05）,p62 expression in SCI+210mg/kg group,SCI+420mg/kg group and SCI+630mg/kg group was lower than that in SCI+NS group,and p62 expression in Sham group was higher than that in SCI+NS group,the difference was statistically significant（P<0.05）.The SCI+NS group rats of part II compared with SCI+630mg/kg rats at various time points,the expressions of Beclin-1,LC3B-Ⅱ/LC3B-Ⅰin the SCI+NS group and the SCI+630mg/kg group were higher than those in the SCI+NS group on the7^th and 14^th after surgery.p62 was lower than SCI+NS group at 6^th hour,1^st,3^rd,7^thand14^th day after surgery,and the difference was statistically significant（P<0.05）.The two groups were respectively compared within the group.In the SCI+NS group,Beclin-1expression increased from 6^th hour,reached a peak on the 1^st day and then began to decline,LC3B-Ⅱ/LC3B-Ⅰexpression increased from 6^th hour,reached a peak on the 3^rd day and then began to decline,and p62 expression decreased from 6^th hour after surgery,reached a low peak on the 3^rd day and then began to increase.The expressions of Beclin-1 and LC3B-Ⅱ/LC3B-Ⅰin SCI+630mg/kg group showed the same trend,both of which increased from 6th hour after surgery and reached a peak on the 3^rd day and then began to decline.The expression of p62 tables decreased from 6^th hour after surgery and reached a low peak on the 3^rd day and then began to rise.【Conclusion】:1.Periplaneta Americana powder can promote the recovery of hind limb motor function in rats with h SCI in a dose-dependent manner,and 630mg/kg has the best effect.2.Periplaneta Americana powder can reduce the content of MDA and 8-OHd G in the h SCI region of rats,and inhibit oxidative stress response.3.Periplaneta Americana powder can up-regulate the autophagy proteins Beclin1 and LC3B-Ⅱ/LC3B-Ⅰin the h SCI region of rats,down-regulate the expression of p62,promote autophagic flow,and enhance autophagy.