| Objective:Through experimental study,the expressing characteristics of autophagy after SCI are determined,and to speculate the repairment mechanism of Jiaji EA of SCI rats is achieved by affecting autophagy process and inhibiting apoptosis.To reveal the regulation mechanism of autophagy in spinal cord tissue after SCI.Methods:In this study,Allen’s modified weight falling method wad used to replicate the model of SCI in rats.Autophagy inhibitor 3-MA is applicated to interfere with autophagy.192 SPF adult SD rats,with weight 200±20g were randomly divided into 4 groups:Sham-operation Group(Sham Group)、Model Group、3-MA+Jiaji EA Group(3-MA+EA Group)、Jiaji EA Group(EA Group).Each team included 48 rats and these rats were divided into 4h,Id,3d,7d,four sub-groups based on the treatment time after the surgery.Using BBB score to evaluate the movement function for each group after SCI;histopathological changes are observed by HE staining;using immunohistochemical staining to detect the change of Beclin1、LC3 positive cells in the injured segment of the spinal cord tissue.Using the Transmission Electron Microscope to observe the ultrastructure of spinal cord tissue in order to find the autophagosome.The proteinic of Beclinl and LC3 in each group evaluated by the fluorescence immunohistochemistry.The expression of Beclin1、LC3、P62、Bcl-2、Bax tasted by the Western-Blot.Using qRT-PCR to analyze the change of Beclin1、LC3、Bcl-2、BaxmRNA expression in the injured spinal cord.Results:(1)BBB score:To compared with the Sham Group,at 4h,1d、3d、7d post-SCI,the scores in the Model Group、the 3-MA+EA Group and the EA Group are lower,there is significant difference(p<0.01);to compared with the EA Group,the 3-MA+EA Group has lower score but no difference(p>0.05).After the treatment of Jiaji EA,the score at 1d,3d and 7d post-SCI showed a gradually increasing and higher than the Model Group(p<0.05).(2)HE dyeing observation results:The spinal cord tissue in the Sham Group is close to normal.Significantly vacuoles are found after 4h post-SCI in segment of spinal cord,and there are fragments of necrotic tissue in it,and haemorrhage can also be found.The tissue of the EA Group have a better recovered than the Model Group and the 3-MA+EA Group since 3d post-SCI.(3)The activity of Beclin1、LC3 positive cells:The protein’s expression was weak in the Sham Group,and in the Model Group、the 3-MA+EA Group、the EA Group they’re increased after 4h post-SCI in neuron cell and glial cell,show as Yellow and Tan.Both of them reached the peak of the activity after 1d post-SCI.The EA Group has the most expression of Beclinl、LC3 positive cells.Compared with the Sham Group,the Model Group.the 3-MA+EA Group、the EA Group have a higher expression on Beclin1 protein’s at 4h post-SCI(p<0.01);Compared with the Sham Group,the Model Group、the EA Group have a higher expression on LC3 protein’s at 4h post-SCI(p<0.01),the 3-MA+EA Group have a higher expression on LC3 protein’s at 4h post-SCI,but there is no difference(p>0.05).Compared with the Sham Group,the Model Group、the 3-MA+EA Group、the EA Group have a higher expression on both protein’s at 1d、3d、7d post-SCI,there are significant difference(p<0.01).Compared with the EA Group,the Model Group、the 3-MA+EA Group have a lower expression on both protein’s and there is significant difference at 4h、Id、3d、7d post-SCI(p<0.01).(4)Through Transmission Electron Microscope:After SCI,there was cytoplasmic edema in the spinal cord tissue,and the mitochondrial swelling was obvious,and the myelin sheath became thin,broken and demyelinating.At 4h post-SCI,there were demyelination occurred,the arrangement of interlayer happened disorder,appearred a large number vacuoles.Since Id post-SCI,the number of autophagic lysosomes and autophagy in the spinal cord are increased in the EA Group,compare with the Model Group and the 3-MA+EA Group.At 7d post-SCI,the autophagy was still visible,but the numbers were reduced and the new myelin sheath can be seen.(5)Fluorescence Immunohistochemistry:The Beclinl-positive and LC3-positive cells in the Sham Group uniformed low expression after SCI.The staining is gradually deepened since 4h post-SCI,and both of them’s positive cells are increased until 7d post-SCI.The Beclin1-positive and LC3-positive cells in the EA Group are significantly increased compared to the Model Group and the 3-MA+EA Group.(6)Western Blot:The expression level of five kinds of proteins have no obvious difference at 4h、Id、3d、7d post-SCI in Sham Group(p>0.05).Compare with the Sham Group,in the Model Group、the 3-MA+EA Group and the EA Group Beclinl、LC3 protein expressions are higher,and there were significant difference(p<0.01);and P62 protein expression was lower,which also has significant difference(p<0.01);Compare with the EA Group,the Model Group has lower protein expression level of Beclin1,but no differences(p>0.05);the protein expression level of LC3 are lower,and the protein expression level of P62 are higher,both have difference(p<0.05)at 4h post-SCI.Compare with the Sham Group,in the Model Group、the 3-MA+EA Group and the EA Group the Beclinl、LC3 protein expressions were higher,and there were significant difference(p<0.01);and the P62 protein expression is lower,which also has significant difference(p<0.01);Compare with the EA Group,the Model Group,the 3-MA+EA Group have lower protein expression level of the Beclinl and LC3,and there are difference(p<0.01)at 1d、3d、7d post-SCI.The Beclinl and LC3 protein expressions increased from 4h post-SCI,and gradually decreased after the peak at Id post-SCI.Compare with the Sham Group,the Model Group、the 3-MA+EA Group and the EA Group the Bcl-2 and Bax protein expressions are higher,and there was significant difference(p<0.01).Compare with the EA Group,the Model Group and the 3-MA+EA Group have lower protein expression level of the Bcl-2,and there was difference(p<0.05)at 4h、Id、3d、7d post-SCI.Since 4h post-SCI,the protein expression level of the Bcl-2 has gradually increased,and the expression still high at 7d post-SCI.Compare with the EA Group,the Model Group and the 3-MA+EA Group have higher protein expression level of Bax,and there was difference(p<0.05)at 4h、1d、3d、7d post-SCI.The protein expression level of Bax has increased from 4h post-SCI and decreased after the peak of 3d post-SCI in the Model Group.The protein expression level of Bax was at its lowest point at Id post-SCI and then gradually increased.(7)qRT-PCR:There is no significant difference of all kinds of mRNA in expression of the Sham Group at different time points(p>0.05).Compare with the Sham Group,in the Model Group、the 3-MA+EA Group and the EA Group the BeclinlmRNA and LC3 mRNA are higher,and there was significant difference(p<0.05);LC3mRNA and BaxmRNA are higher,and there was significant difference(p<0.01);and the 3-MA+EA Group has lower Bcl-2mRNA which is no difference to compare with the Sham Group(p>0.05).Compared with the EA Group,the Model Group and the 3-MA+EA Group have lower expression in BeclinlmRNA,LC3mRNA,Bcl-2mRNA,and higher expression in the BaxmRNA,and there is difference(p<0.05)at 4h post-SCI.the Model Group、the 3-MA+EA Group and the EA Group have a higher expression in Beclin1mRNA,LC3mRNA Bcl-2mRNA and BaxmRNA to compared with the Sham Group at Id、3d、7d post-SCI,and there is difference(p<0.05).Compare with the EA Group,the Model Group and the 3-MA+EA Group have lower expression in BeclinlmRNA、LC3mRNA、Bcl-2mRNA,and higher expression in BaxmRNA,and there are both have difference(p<0.05).Conclusion:1.Jiaji EA can improve the hind limb motor dysfunction of SCI in rats.2.Jiaji EA can promote the pathological morphological recovery of spinal cord after SCI,3.The number of Beclinl.LC3 protein positive cells are increased in neuron cell and glial cell after SCI.The autophagy was activated by increased the protein ’ s expression level of Beclinl and LC3,and the dynamic process of autophagy can be observed after SCI by decreased the P62-protein expression.4.Jiaji EA can enhance autophagy and inhibit apoptosis after SCI. |
PDF Full Text Request