| Objective:By observing the clip ridge electroacupuncture combined with nerve mobilization treatment of rabbit sciatic nerve injury,nerve conduction velocity and Netrin-1,Slit2 and Rho GTPases expression,the effect of clip ridge electroacupuncture combined with nerve mobilization therapy through regulating the Netrin-1,the expression of Slit2,inhibition of Rho GTPases imbalances,promote axon regeneration mechanism,provide theory support for the clinical treatment of peripheral nerve injury.Methods:A total of 216 New Zealand rabbits were randomly divided into 6 groups,including normal control group,virus no-load group,electroacupuncture combined with nerve mobilization group,Netrin-1 group,Slit2 group,Netrin-1+Slit2 adenovirus binding group,and then divided into 3 subgroups at 3 time points 1,2 and 4 weeks postoperatively,with 12 rabbits in each subgroup.Clamps method for rabbit left sciatic nerve Sunderland I damage model.The normal control group grabbed on time without doing any processing;Virus injection was completed in each virus group at the time of model preparation.Target virus injection was carried out at the upper and lower 3mm of the injured nerve,respectively,and 5uL was injected at each injection point.Virus no-load group was injected with no-load virus.Netrin-1 group was injected with Netrin-1 adenovirus(titer:1.2×108 virions ·mL-1).Slit2 group was injected with Slit2 adenovirus(titer:was 1.6×108 virus particles ·mL-1).The Netrin-1+Slit2 adenovirus binding group was injected with adenovirus binding virus(titer was the same as that of the monovirus-injected group).electroacupuncture combined with nerve mobilization group:the treatment was performed on the third day after surgery.Electroacupuncture was performed on the spine,30min/time,once a day,6 days/week;After that,nerve mobilization was performed,stretching for 1s and relaxing for 5s,10/group,lgroup/day,6days/week.The nerve electrophysiological level of rabbits was assessed by nerve conduction velocity.The expressions of nerve growth guiding factors Netrin-1 and Slit2 were detected by real-time quantitative PCR and immunofluorescence double staining.The expressions of Racl,Cdc42 and RhoA proteins in Rho GTPases were detected by Western Blot.Result:1.Neuroelectrophysiological assessmentThe nerve conduction velocity of all treatment groups was lower than that of normal control group at 1,2 and 4 weeks,and the difference was statistically significant(P<0.05).The nerve conduction velocity of electroacupuncture combined with nerve mobilization group and Netrin-1+Slit2 adenovirus binding group was higher than that of Netrin-1 and Slit2 group,the difference was statistically significant(P<0.05).There was no significant difference in nerve conduction velocity between the electroacupuncture combined with nerve mobilization group and the Netrin-1+Slit2 adenovirus binding group(P<0.05).There was no significant difference in nerve conduction velocity between Netrin-1 group and Slit2 group(P<0.05).At 1 week,the nerve conduction velocity of virus no-load group was higher than that of other treatment groups,and the difference was statistically significant(P<0.05).At 2 and 4 weeks,the nerve conduction velocity of the virus no-load group was lower than that of other treatment groups,and the difference was statistically significant(P<0.05).From the general trend,except for the normal control group and the virus no-load group,the nerve conduction velocity of the other treatment groups was the highest in the 4-week group.2.Netrin-1 expressionAt the time points of 1,2 and 4 weeks,the expression of Netrin-1 mRNA in sciatic nerve and Spinal cord segments corresponding to L4-L6 spinal segments in treatment groups was higher than that in normal control group,and the difference was statistically significant(P<0.05).The expression of Netrin-1 mRNA in Netrin-1 group and Netrin-1+Slit2 adenovirus binding group was higher than that in the no-load group and Slit2 group,and the difference was statistically significant(P<0.05).There was no significant difference in the expression of Netrin-1 mRNA between virus no-load group and Slit2 group(P>0.05).There was no significant difference in the expression of Netrin-1mRNA between Netrin-1 group and Netrin-1+Slit2 adenovirus binding group(P>0.05).The expression of Netrin-1 mRNA in the electroacupuncture combined with nerve mobilization group was consistent with that in the Netrin-1 group and the Netrin-1+Slit2 adenovirus binding group,and was higher than that in the no-load group,with statistical significance(P<0.05).The expression of Netrin-1 mRNA was up-regulated gradually with time,and the best expression was found in 4-week group.3.Slit2 expressionAt the time points of 1,2 and 4 weeks,the expression of Slit2 mRNA in sciatic nerve and Spinal cord segments corresponding to L4-L6 spinal segments in treatment groups was higher than that in normal control group,and the difference was statistically significant(P<0.05).The expression of Slit2 mRNA in Slit2 group and Netrin-1+Slit2 adenovirus binding group was higher than that in Netrin-1 group and no-load group,and the difference was statistically significant(P<0.05).There was no significant difference in Slit2 mRNA expression between Netrin-1 group and virus no-load group(P>0.05).There was no significant difference in Slit2 mRNA expression between the Slit2 group and the Netrin-1+Slit2 adenovirus binding group(P>0.05).The expression of Slit2 mRNA in the electroacupuncture combined with nerve mobilization group was consistent with that in the Slit2 group and the Netrin-1+Slit2 adenovirus binding group,both of which were higher than that in the no-load group,with statistical significance(P<0.05).The expression of Slit2 mRNA was up-regulated gradually with time,and the best expression was found in the 4-week group.4.Racl expressionAt each time point of 1,2 and 4 weeks,the expression of Racl in sciatic nerve and Spinal cord segments corresponding to L4-L6 spinal segments in normal control group was higher than that in treatment groups,and the difference was statistically significant(P<0.05).The expression of Racl in electroacupuncture combined with nerve mobilization and Netrin-1+Slit2 adenovirus binding group was higher than that in no-load group,Netrin-1 group and Slit2 group,and the difference was statistically significant(P<0.05).The expression of Racl in Netrin-1 group was higher than that in no-load group and Slit2 group,and the difference was statistically significant(P<0.05).There was no significant difference in Racl expression between virus no-load group and Slit2 group(P>0.05).Racl expression in the sciatic nerve:At 1 week,the Racl expression in the electroacupuncture combined with nerve mobilization group was higher than that in the Netrin-1+Slit2 adenovirus binding group,with statistical significance(P<0.05).At 2 and 4 weeks,there was no significant difference in Racl expression between the electroacupuncture combined with nerve mobilization group and the Netrin-1+Slit2 adenovirus binding group(P>0.05).The expression of Racl in the Spinal cord segments corresponding to L4-L6 spinal segments levels:At 1 and 4 weeks,the expression of Racl in the electroacupuncture combined with nerve mobilization group was higher than that in the Netrin-1+Slit2 adenovirus binding group,and the difference was statistically significant(P<0.05).At 2 weeks,there was no significant difference in Racl expression between the electroacupuncture combined with nerve mobilization group and the Netrin-1+Slit2 adenovirus binding group(P>0.05).Overall,the 4-week group had the best expression effect.5.Cdc42 expressionAt each time point of 1,2 and 4 weeks,the expression of Cdc42 in sciatic nerve and Spinal cord segments corresponding to L4-L6 spinal segments in normal control group was higher than that in treatment groups,and the difference was statistically significant(P<0.05).The expression of Cdc42 in electroacupuncture combined with nerve mobilization and Netrin-1+Slit2 adenovirus binding group was higher than that in no-load group,Netrin-1 group and Slit2 group,and the difference was statistically significant(P<0.05).The expression of Cdc42 in Netrin-1 group was higher than that in virus no-load group and Slit2 group,and the difference was statistically significant(P<0.05).There was no significant difference in Cdc42 expression between virus no-load group and Slit2 group(P>0.05).Expression of Cdc42 in sciatic nerve:At 1 week,the expression of Cdc42 in electroacupuncture combined with nerve mobilization group was higher than that in Netrin-1+Slit2 adenovirus binding group,with statistical significance(P<0.05).At 2 and 4 weeks,the expression of Cdc42 in Netrin-1+Slit2 adenovirus binding group was higher than that in electroacupuncture combined with nerve mobilization group,and the difference was statistically significant(P<0.05).The expression of Cdc42 in the Spinal cord segments corresponding to L4-L6 spinal segments levels:At 1 and 4 weeks,the expression of Cdc42 in the electroacupuncture combined with nerve mobilization group was higher than that in the Netrin-1+Slit2 adenovirus binding group,with statistical significance(P<0.05).At 2 weeks,the expression of Cdc42 in Netrin-1+Slit2 adenovirus binding group was higher than that in electroacupuncture combined with nerve mobilization group,and the difference was statistically significant(P<0.05).Overall,the 4-week group had the best expression effect.6.RhoA expressionAt each time point of 1,2 and 4 weeks,the expression of RhoA in sciatic nerve and Spinal cord segments corresponding to L4-L6 spinal segments in normal control group was lower than that in the treatment group,with statistical significance,and the difference was statistically significant(P<0.05).The expression of RhoA in electroacupuncture combined with nerve mobilization group and Netrin-1+Slit2 adenovirus binding group was lower than that in no-load group,Netrin-1 group and Slit2 group,and the difference was statistically significant(P<0.05).The RhoA expression in Slit2 group was lower than that in the virus no-load group and Netrin-1 group,and the difference was statistically significant(P<0.05).There was no significant difference in RhoA expression between the no-load group and the Netrin-1 group(P>0.05).Expression of RhoA in sciatic nerve:At 1 and 4 weeks,the expression of RhoA in Netrin-1+Slit2 adenovirus binding group was lower than that in electroacupuncture combined with nerve mobilization group,the difference was statistically significant(P<0.05).At 2 weeks,RhoA expression in electroacupuncture combined with nerve mobilization group was lower than that in Netrin-1+Slit2 adenovirus binding group,and the difference was statistically significant(P<0.05).RhoA expression in the Spinal cord segments corresponding to L4-L6 spinal segments levels:At 1 and 4 weeks,RhoA expression in the Netrin-1+Slit2 adenovirus binding group was lower than that in the electroacupuncture combined with nerve mobilization group,and the difference was statistically significant(P<0.05).At 2 weeks,RhoA expression in electroacupuncture combined with nerve mobilization group was lower than that in Netrin-1+Slit2 adenovirus binding group,with statistical significance(P<0.05).Overall,the 4-week group had the best expression effect.Conclusion:The possible mechanism of Jiaji electroacupuncture combined with nerve mobilization in promoting axonal regeneration is to regulate the expression of Netrin-1 and Slit2,up regulate the expression of downstream molecules Racl and Cdc42,down regulate the expression of RhoA,adjust the imbalance of Rho GTPases enzyme system,promote the reorganization of cytoskeleton,and promote the regeneration of injured peripheral nerves. |
