Effect Of PI3K/Akt Signal Pathway On The Expression Of SREBP-1 In Schwann Cells Of Diabetic Peripheral Neuropathy

Objective:Diabetic peripheral neuropahty(DPN)is one of most common complications of Diabetic mellitus(DM).Abnormal lipid metabolism is regarded as an important factor that may be accounted for the pathogenesis and development of DPN.SREBP-1 is a vital transcription factor which regulates lipid metabolism in various tissues including liver,kidney,and adipose tissue.PI3K/Akt pathway is one of the cell signal pathways and was reported to play a key role in regulating axon wrapping and myelin sheath thickness of peripheral nerve system(PNS).It was suggested that PI3K/Akt pathway was inhibited in DPN.However,it has not been clarified whether PI3K/Akt inhibition is associated with downregulation of SREBP-1 in diabetic peripheral neuropathy Therefore,in the current study,diabetic mice and high glucose-stimulated cultured RSC96 cells were chosen to detect the expression of phospho-Akt and SREBP-1.Importantly,our research explored the relationship between inhibition of the PI3K/Akt signaling pathway and SREBP-1expression in DPN.Methods:1.Establish the diabetic animal model and detect the expression of Akt and SREBP-1 in saciatic nervesDiabetic mice were constructed by intraperitoneally injecting streptozocin(STZ,150mg/kg body weight)and the corresponding control mice were only injected with sodium citrate solution.Mice with blood glucose greater than 11.1 mmol/L were regarded as diabetic models.Sixteen male CD1 mice were randomly divided into two groups,the normal control group and diabetes group.Sixteen weeks later,all mice were killed and the sciatic nerves were collected for the Akt and SREBP-1 detections through westernblot and immunohistochemistry.2.Pretreat the RSC96 cells with high glucose and insulin and detect the expression of Akt and SREBP-1 in RSC96 cellsRSC96 is divided into four groups,the normal concentration of glucose group(N),the high concentration of glucose group(H),the normal concentration of glucose & insulin group(N+INS),the high concentration of glucose & insulin group(H+INS).After three days' different stimulation,phospho-Akt(Ser473,Thr308),Akt and SREBP-1 expression were detected through western blot and immunohistochemistry.Results:1.The p-Akt(SER 473),p-Akt(THR 308),and SREBP-1 expression decreased in the sciatic nerves of diabetic miceChanges in p-Akt(Ser 473),p-Akt(Thr308),and SREBP-1 expression were detected by the method of Western blot in the sciatic nerves of normal mice and diabetic mice fed for 4 months.The abundance of p-Akt(Ser 473)and p-Akt(Thr 308)were decreased in diabetic mice compared with normal mice(P< 0.05,P< 0.05),without change in the expression of total Akt(P> 0.05).In parallel,decreases in precursor segment of SREBP-1 and mature segment of SREBP1 were both consistently revealed in the sciatic nerves of diabetic mice versus those of normal mice(P< 0.05,P< 0.05).Immunohistochemistry was performed for SREBP-1 detection and showed a weak positive expression in the sciatic nerves.2.The time-dependent effect of high glucose on PI3K/AKT signaling pathway and SREBP-1 protein expression in RSC96 cellsRSC96 cells were respectively treated with glucose at different concentrations(25mmol/L,50mmol/L,and 100mmol/L)for 3 days and 5 days.The results revealed that at 3 days and 5 days after high?glucose stimulation as depicted,RSC96 cells showed evident decreases in the expression of p-Akt of SREBP-1 in comparison with normal glucose or corresponding mannitol-treated cells(P< 0.05,P< 0.05).These data suggested that the 25mmol/L glucose was enough to inactivate Akt and decrease SREBP-1 expression.Statistical analysis revealed that 25mmol/L glucose respectively decreased p-Akt(Ser 473),p-Akt(Thr 308),and mature segment of SREBP-1 by 89.9%,78.6%,and 43.3% after 3 days?treatment compared with the mannitol control group.As well,5 days?treatment of 25mmol/L glucose reduced p-Akt(Ser 473),p-Akt(Thr 308),and mature segment of SREBP-1 expression by 87.8%,82.0%,and 24.8% relative to the mannitol group(P < 0.05).The expression and localization of SREBP-1 in RSC96 cells treated was also investigated by immunocytochemistry.The results shown in that SREBP-1 was localized in the nucleus and cytoplasm of RSC96 cells.High?glucose treatment of 3 days caused an evident decrease in SREBP-1 expression compared with normal glucose or mannitol treatment.3.Insulin-induced activated PI3K/AKT signaling pathway prevented high-glucose exposure-caused SREBP-1 downregulation in RSC96 cellsCells in the cultured medium absent of high glucose showed a slight increase in p-Akt(Ser 473)and p-Akt(Thr 308)expression under the stimulation of insulin(compared with normal glucose group,P<0.05,P<0.05).In contrast,cells cultured with medium present of high glucose showed evident increase in the expression of p-Akt(Ser 473)and p-Akt(Thr 308)after the stimulation of insulin(compared with high glucose group,P<0.05,P<0.05).As for Akt,insulin has no obvious effect on Akt expression either in cells cultured with normal glucose medium or in those cultured with highglucose medium(P>0.05).In sequence,high glucose reduced SREBP-1 expression was reversed with the treatment of insulin in RSC96 cells(P<0.05).Conclusion:1.High-glucose stimulation inhibited the PI3K/Akt signal pathway and reduced the SREBP-1 expression both in Schwann cells of diabetic mice and in cultured RSC96 cells in vitro.2.Insulinprevented high-glucose-exposure-induced SREBP-1 downregulation in RSC96 cells through the activation of PI3K/AKT signaling pathway.3.PI3K/AKT signaling pathway may be involved in the SREBP-1 downregulation in diabetic peripheral neuropathy.