| ObjectiveSpinal cord injury(SCI)is caused by spinal fracture or dislocation,such as traumatic or non-traumatic events.The long-term disability of patients will bring serious burden to individuals,families and society.A series of secondary pathophysiological changes,including apoptosis,inflammation and neurodegeneration,are the main obstacles to the regeneration and recovery of nerve after SCI.Phosphoinosmde-3-kinase/protein kinase B/the mammalian target of rapamycin(PI3K/AKT/mTOR)signaling pathway is one of the signaling pathways that regulates cell apoptosis,autophagy,and participates in immune,metabolism and other important body activities.It is very important for the development,treatment and prognosis of SCI.Electroacupuncture at Dazhui and Mingmen has a clear clinical effect in the treatment of SCI,but the relevant mechanism is not clear.The purpose of this study is to clarify the effect of electroacupuncture at Dazhui and Mingmen after SCI by modulating PI3K/AKT/mTOR signaling pathway.MethodAll 13-week-aged male Sprague-Dawley rats were randomly divided into four groups:Control Group(n=18)without any intervention,Sham-operation Group(n=18)received only a laminectomy,Model Group(n=54)received SCI induction at the T10 spinal segment,and Electroacupuncture(EA)Group(n=54)received SCI induction at the T10 spinal segment and EA intervention at Dazhui(GV14)and Mingmen(GV4).Rats in the Model Group and EA Group were randomly divided into the following subgroups;1-day Subgroup(n=18),14-day Subgroup(n=18)and 28-day Subgroup(n=18).The limb motor function of rats was evaluated by Basso,Beattie&Bresnahan locomotor rating scale(BBB scale),which showed the effect of electroacupuncture at Dazhui and Mingmen 1 day,14 days and 28 days on motor function recovery after SCI.The spinal cord tissue of rats was observed by Magnetic Resonance Imaging(MRI)and Hematoxylin-eosin(HE)staining,which showed the effect of electroacupuncture at Dazhui and Mingmen 1 day,14 days and 28 days on histomorphology of injured spinal cord.The PI3K/AKT/mTOR signaling pathway was detected by Immunohistochemistry and Western blotting(WB),including PI3K,p-PI3K,AKT,mTOR,p-mTOR,p70 Ribosomal Protein S6 Kinase(p70S6K),p-p70S6K,phosphatase and tensinhomology deleted on chromosome ten(PTEN)and cysteinyl aspartate specific proteinase 3(caspase 3),which showed the effect of electroacupuncture at Dazhui and Mingmen on PI3K/AKT/mTOR signaling pathway after SCI.ResultThe result of BBB scale showed that compared with the Control Group and Sham-operation Group,BBB scale scores in the Model Group and EA Group were significantly lesser at all time points(P<0.01).Compared with the Model Group,BBB scale scores in the EA Group were high on 1-day,14-day and 28-day Subgroups(P<0.05).There was significant difference in the BBB scale scores on the 1st,14th and 28th day in EA group(P<0.01).MRI showed morphology of thoracic segment of spinal cord in all rats,especially T10 segment.The spinal cords of rats from Control Group and Sham-operation Group have no obvious compression,which MRI shows no abnormal signal.The spinal cords of rats from Model Group and EA Group have compressive deformation forming necrosis cavity and reactive proliferative tissue,which MRI shows that on inflammation was in hyperintense region on T2WI.which progressive decrease as time goes on.And the inflammatory area of spinal cord in the Model Group and EA Group decreased gradually as time goes on(1 day,14 days,28 days)(P<0.01).Compare with Model Group,MRI in EA Group shows the more complete organizational structure,smaller volume of the T2 hyperintense region and lower signal on 1day,14day and 28day,which means that rats in EA Group has less damage tissue and inflammation(P<0.05).HE staining showed that the spinal cord tissues are normal in Control Group and Sham-operation Group,which had complete structure,normal gray and white matter and regular cells.In contrast,the injured regions in the spinal cord in Model Group and EA Group displayed disruption of structures.Compared with Model Group at the same time,the injured region in the spinal cord in EA Group displayed improved tissue repair and recovery with less pathologic injury in necrosis cavity,which had less bleeding,lighter neurons cell edema,more normal neurons cells and more dense tissue.The result of Immunohistochemistry showed that Compared with Control Group and Sham-operation Group,the numbers of caspase 3-immunoreacti ve cells and p-PI3K-immunoreactive cells were increased in Model Group and EA Group on 1-day,14-day and 28-day Subgroups(P<0.01).Compared with Model Group,the numbers of caspase 3-immunoreactive cells were decreased in EA Group on 1-day,14-day and 28-day Subgroups(P<0.01),p-PI3K-immunoreactive cells and p-mTOR-immunoreactive cells were increased in EA Group on 1-day,14-day and 28-day Subgroups(P<0.01).The result of WB showed that the levels of p-mTOR protein expression in all groups were significantly increased compared with the rats received intervention of rapamycin(P<0.01),whereas the level of p-mTOR protein expression in EA Group was still significantly increased than other groups(P<0.01).Compared with Control Group and Sham-operation Group,the expressions of p-PI3K/PI3K,p-mTOR/mTOR,p-p70S6/p70S6 and AKT were significantly decreased in Model Group(P<0.01),and the expressions of caspase 3 and PTEN were significantly increased in Model Group(P<0.01).Compared with Model Group,the expressions of p-PI3K/PI3K,p-mTOR/mTOR,p-p70S6/p70S6 and AKT were significantly increased in EA Group(P<0.01),and the expressions of caspase 3 and PTEN were significantly decreased in EA Group(P<0.01).Compared with the 1-day Subgroup,the difference of each index was statistically significant(P<0.01).ConclusionElectroacupuncture at Dazhui and Mingmen has an excellent therapeutic effect on SCI,which the results suggest that electroacupuncture at Dazhui and Mingmen could improve the recovery of locomotor function and histological morphology change after SCI in rats,through PI3K/AKT/mTOR signaling pathway on cell growth,apoptosis and autophagy.And the intervention time of electroacupuncture at Dazhui and Mingmen is longer,the advantage of treatm is the more obvious. |
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