Mechanisms Research Of Protective Effect Of Lithium-Mediated The Activity Of GSK3? In Neurons After Spinal Cord Injury

Objective: Spinal cord injury is a common clinical traumatic disease which mainly caused by falling accident and traffic injuries.It has the characteristics of disability and high mortality.The pathophysiological processes of SCI include two stages: primary injury and secondary injury.Primary injury is caused by transient violence directly causing destruction of spinal cord tissue,which in turn causes nerve loss or vascular injury.Secondary injury caused by factors such as ischemia and hypoxia after primary injury is caused by large-scale death of nerve cells,which leads to dysfunction.The main reason for cell survival is that it retains the necessary anatomy for the spinal cord and is the basis for functional recovery after injury.Lithium have been clinically treated for bipolar disorder for more than 60 years.Recently,lithium have been found to be effective in the treatment of other diseases of the central nervous system.With the deepening of research,it is found that lithium can promote the nervous system of the central nervous system,inhibit the expression of pro-apoptotic genes,and play a protective role in ischemic and traumatic brain injury,suggesting that lithium may have similar protective effects after spinal cord injury.Recent studies have confirmed that lithium promote the proliferation of neural stem cells,differentiation into neurons,and synthesis and secretion brain-derived neurotrophic factors.The inhibitory effect of lithium on GSK-3? has been accepted,and this effect is also likely to be closely related to its therapeutic effect.However,the molecular mechanism of lithium that triggers this inhibition is not fully explored.Methods: 1.Primary culture of spinal cord neurons obtained from five or six C57BL/6J mice embryos(E13–14 days).Mature neurons were obtained after culture.2.7-day-old neurons were incubated in DMEM without serum.A transfection solution containing siRNA(specifically on NKA ?1 or SGK1)was added to the cultures.3.The Lowery method was used to determine the protein concentration of protein samples collected using protein lysates.4.To determine the effect of LiClon the level of the phosphrylation of GSK3? and AKT??-Catenin ?SGK 1 and Na+-K+-ATPase ?1 subtype(NKA ?1)by Western blotting after treating for 1 hour ?2 hours?4 hours?8 hours?12 hours?24 hours and 48 hours;we selectively inhibited the expression of NKA ?1 or SGK1 using RNAinterference in the cultured spinal cord neurons to detect the NKA ?1 or SGK1 on the level of p-GSK3? and ?-catenin regulated by lithium.5.The treatment of LiCl(dissolved in PBS intraperionrally injected at 20mg/kg/day for 3 days)or Ouabain(1mM dissolved in 5?l ACSF intrathecally injected)on transgenic mice Thy1-YFP.6.Using Western blotting method to detect the effect of lithium or ouabain on the level of regulate SGK1?p-GSK3? and ?-catenin.7.The results were analysed with one-way ANOVA by SPSS22.0 software.P<0.05 indicates the statically significant difference.Results: 1.The administration of 1 mM LiCl significantly increased the phosphorylation of GSK3? at the serine-9(Ser9)site and inhibited its activity from hours 1 to 48.The effects of lithium on ?-catenin was similar to that of the effects on the phosphorylation of GSK3?.Lithium induced the phosphorylation of AKT by approximately 70% from hours 1 to 48.However lithium suddenly increased the level of p-GSK3? and ?-catenin after 8 hours.2.The effect of NKA ?1 and SGK1 induced by lithium.LiCl significantly increased the expression of NKA ?1 and SGK1 at hours 12.We selectively inhibited the expression of NKA ?1 and SGK1 in the cultured spinal cord neurons.LiCl significantly increased the expression of SGK1 in the neurons without interference treatment.After deleting the expression of NKA ?1,the expression of SGK1,the increased tendency of p-GSK3? and ?-catenin by lithium were clearly supressed.Similar trends were present in the neurons pretreated with the siRNA duplex of SGK1.3 The effect of lithium in vivo.After the treatment of LiCl on transgenic mice Thy1-YFP,the expression of SGK1?the level of ?-catenin and the phosphorylation of GSK3? significantly increased.Ouabain obviously decreased the level of the protein above.Pretreatment with ouabain,then LiCl was injected.Ouabain obviously decreased the stimulation of lithium.Conclusions:1.Lithium downregulates GSK3? activity via two different singaling pathways in vivo and in vitro.2.In the acute phase,lithium inhibited GSK3? activity by stimulating phosphorylation of AKT.3.In the chronic phase,lithium upregulates the expression of NKA ?1,which can in turn supress GSK3? activity by increasing the expression of SGK1.