| 1 ObjectiveSpinal Cord injury(SCI) is a serious traumatic disease of the central nervous system, with a high rate of disability, and the incidence rate is rising year by year, bring about heavy burden to patients, families and society.Du meridian electroacupuncture therapy, achieved good clinical effect on restoration of nerve function after SCI, but its mechanism is still unclear. The repair mechanism of nerve function after SCI:to complementary and alternative apoptosis of nerve cells, to promote the regeneration and remyelination of injured axons, to improve the local microenvironment of injuried spinal cord.Based on the former research found of our research group, Du meridian electroacupuncture therapy can inhibit the apoptosis of nerve cells, promote the regeneration and repair of nerve cells, and promote the recovery of nerve function after SCI effectively. In this research, with the intervention of Du meridian electroacupuncture, we will observation the influence of Du meridian electroacupuncture on neural stem cells, neurons, oligodendrocytes, and astrocytes in the injured site of SCI systematically. With Wnt and Notch signal pathway as the breakthrough point,which are closely related to the proliferation and differentiation of neural stem cell, we will study whether we can adjusting the Wnt and Notch signaling pathway to regulate the proliferation and differentiation of endogenous neural stem cell in the injured site of SCI by Du meridian electroacupuncture,and then promote the repair of nerve function after SCI, to provide a scientific experimental basis for its clinical application of Du meridian electroacupuncture treatment of SCI.2 MethodsThis research is animal experiment. Animal model of SCI is replicated by modified Allen’s. The research is divided into four batches,1d,7d,14d,28d. SD rats were selected, randomly divided into blank control group (B), blank+ Electroacupuncture treatment control group (BE), model group (M), sham group (S), Electroacupuncture treatment group (EA). Group EA is treated by electroacupuncture therapy, selected "Da Zhui", "Ming Men" acupoints,20min/d, treatment to the corresponding number of days; Group BE, treated as Group EA; Group B, Group S, Group M, given the same strength, grasping, and fixed as Group EA, but not treated by electroacupuncture. For the rats of the 28d batch, detect the body weight and evaluate nerve function with BBB in day1,day7,day14,day28, so as to compare the effects of Du meridian electroacupuncture therapy on body weight changes and the recovery of nerve function of the rats in each group; After the completion of the treatment of each batch, HE staining was used to observe the pathological changes in the injured site of the rats in each group, so as to compare the effects of Du meridian electroacupuneture therapy on the pathological changes in the injured site of the rats in each group; Immunohistochemistry was used to detect the expression of Nestin, NSE, APC, and GFAP in the injured site of the rats in each group, so as to compare the effects of Du meridian electroacupuncture therapy on the proliferation and differentiation of endogenous neural stem cells in the injured site of the rats in each group; Immunohistochemical and Western Blot were used to detect the expression of Wntl, Wnt3a,β-catenin in the injured site of the rats in each group, so as to compare the effects of Du meridian electroacupuncture on the Wnt signaling pathway in the injured site of the rats in each group;Immunohistochemical and Western Blot were used to detect the expression of Deltal, Presenilin1, Hes1, Hes5 in the injured site of the rats in each group, so as to compare the effects of Du meridian electroacupuncture on the Notch signaling pathway in the injured site of the rats in each group.3 Results3.1 The results of body weight changes:Day1, Day7, Day 14, Day28, compared with group B at the same time point, the body weight of rats in group BE and S were not changed significantly (P>0.05);Day7, Day14, Day28, compared with group B at the same time point, the body weight of rats in group M significantly reduced (P<0.01);After treatment with Du meridian electroacupuncture, the body weight of rats in group EA significantly increased than M group in Day7, Day14, Day28, the difference was statistically significant (P<0.01). In all, after SCI, compared with group B, the growth trends of the body weight of the rats in group M and group EA were reduced significantly, but after the treatment with Du meridian electroacupuncture, the growth trend of the body weight of the rats in group EA are significantly increased than group M.3.2 The results of BBB:The BBB score of group BE and S are the same as group B in Day1, Day7, Day14, Day28, all get full marks (21); In Day1, Day7, Day14, Day28, The BBB score of group M are significantly lower than group B (P<0.01), while the BBB score of group EA increased significantly in Day7, Day 14, Day28, compared with group M, the difference was statistically significant (P<0.01). In all, the general trend of the BBB score of group M and group EA increased with the change of time, and the growth trend of the BBB score of group EA is significantly higher than group M.3.3 The results of HE staining:in Day1, Day7, Day14, Day28, group B, BE and S,in the spinal cord tissue structure, the gray and white matter are complete, and have clear boundary, the neurons of anterior horn are large and complete, clear and round nucleus are visible, nissl body are rich, and gathered around the nucleus; About group M, 1d after SCI, a large area of red blood cells exudation in gray matter, neurons structure damaged, nucleus are ambiguous, and accompanied by mild edema, the number of neuron is reduced,7d after SCI, the boundaries of gray matter lost, liquefactive necrosis and cavities can be seen in the centres of injury, and a number of macrophages, foam cells, and glial cells, neurons disappeared,14d after SCI, boundaries of grey matter are faintly visible, with a small number of neurons, but edema, and bubble formation,28d after SCI, boundaries of gray matter is relatively clear, there are still a lot of foam cells and inflammatory cells infiltration, neurons edema, and glial scar formed;but Du meridian electroacupuncture therapy can obviously reduce the number of neurons apoptosis, reduce inflammation infiltration, inhibit the formation of glial scar, promote the repair of spinal cord tissue in the injury.3.4 The results of the proliferation and differentiation of endogenous neural stem cells:Day1, Day7, Day 14,and Day28, compared with group B, the expression of Nestin, NSE, APC, GFAP of group BE and S had no significant differences; Day1, Day7, Day14,and Day28, compared with group B, the expression of Nestin of group M were significantly inceased(P<0.01),while the expression of NSE, APC were significantly decreased (P<0.01), Day1, Day7, and Day14, compared with group B, the expression of GFAP of group M were significantly inceased(P<0.01 or P<0.05); Day1, Day7, Day 14,and Day28, compared with group M, the expression of Nestin, NSE of group EA were significantly increased (P<0.01);Compared with group M, the expression of APC of group EA increased, but had significant difference only in the day28 (P<0.05); Day1, Day7, and Day14, compared with group M, the expression of GFAP of group EA is significantly reduced (P<0.01 or P<0.05).3.5 The result of the related factors of Wnt signaling pathway:Day1, Day7, Day 14,and Day28, compared with group B, BE group, the expression of Wnt1, Wnt3a, β-catenin of group BE and S had no significant differences; Day1, Day7, Dayl4,and Day28,compared with group B, the expression of Wntl, Wnt3a, β-catenin of group M increased significantly (P<0.01 or P<0.05); Day1, Day7, Day14,and Day28, compared with group M, the expression of Wntl, Wnt3a, β-catenin of group EA increased significantly (P<0.01 or P<0.05).3.6 The result of the related factors of Notch signaling pathway:Day1, Day7, Day14,and Day28, compared with group B, BE group, the expression of Delta1,Presenilin1,Hes1,Hes5 of group BE and S had no significant differences; Day1, Day7, Day14,and Day28,compared with group B, the expression of Delta1,Presenilin1,Hes1,Hes5 of group M increased significantly (P<0.01 or P<0.05); Day1, Day7, Day14,and Day28, compared with group M, the expression of Delta1,Presenilin1,Hes1,Hes5of group EA decreased significantly (P<0.01 or P<0.05).4 Conclusions4.1 Spinal cord injury can cause body weight loss and nerve function loss of the rats, while Du meridian electroacupuncture therapy can promote the recovery of body weight and neural functional of the rats after SCI.4.2 Du meridian electroacupuncture therapy can markedly reduce apoptosis and inflammatory infiltration in the injury site of the SCI rats, inhibit the formation of glial scar in the injury site, and promote the repair of spinal cord tissue of the SCI rats.4.3 Du meridian electroacupuncture therapy can promote the proliferation of endogenous neural stem cells in the injury site of the SCI rats, and promote endogenous neural stem cells differentiate into neurons and oligodendrocytes, while inhibit endogenous neural stem cells differentiate into astrocytes excessively.4.4 Du meridian electroacupuncture therapy can promote the expression of Wnt1, Wnt3a in the injury site of the SCI rats, and thus inhibit the phosphorylation and ubiquitination of β-catenin in the cytoplasm, promote the accumulation of β-catenin, so as to upregulate the activation of Wnt signaling pathway, this may be one of the mechanisms of that Du meridian electroacupuncture therapy can promote the proliferation of endogenous neural stem cells, and promote endogenous neural stem cells differentiate into neurons in the injury site of the SCI rats.4.5 Du meridian electroacupuncture therapy can inhibit the expression of Deltal,one of the ligands of Notch, in the injury site of the SCI rats, and thus inhibit the expression of Presenilinl in the cytoplasm, and then inhibit the excessive activation of Notch signaling pathway, and then reduce the expression of Hes1 and Hes5,which are the downstream genes of Notch signaling pathway, this may be one of the mechanisms of that Du meridian electroacupuncture therapy can promote endogenous neural stem cells differentiate into neurons and oligodendrocytes, and inhibit endogenous neural stem cells differentiate into astrocytes excessively in the injury site of the SCI rats.
|
PDF Full Text Request