Study On Taurine Improving Myelin Injury Of Sciatic Nerve In Type 2 Diabetic Rats

Objective: Diabetic peripheral neuropathy(DPN)is one of the most common chronic complications of diabetes.The myelin injury of sciatic nerve caused by apoptosis of Schwann cell is the main pathological feature of DPN.To observe the protective effect of taurine on myelin injury in diabetic peripheral neuropathy(DPN)rats and explore its mechanism.Materials and Methods: Sixty male Sprague Dawley(SD)rats were randomly assigned to Control group(common feed)and model group.After four weeks in model group feeding with high-fat and high-sugar diet,the rats were intraperitoneal injected streptozotocin(STZ)at 25 mg/kg,after 72 hours of STZ injection,the random blood glucose values of ?16.7 mmol/L and neurotransmission disorders were considered as the DPN model.Control group was injected with the same dose of citrate buffer.After successful modeling,it will be divided into DPN group and taurine protection group(0.5%,1% and 2% Tau group).Control group and DPN group will be fed with normal drinking water,and taurine protection group will add three doses of taurine to drinking water for eight weeks.After eight weeks,the rats was sacrificed.In our study,we selected the electron microscope experiment to observe myelin injury in the sciatic nerve and the apoptosis of Schwann cell.Then the apoptosis of schwann cells in rats was detected by TUNEL and immunofluorescence double staining;the concentration of NGF in the serum of rats was detected by ELISA;the protein expression changes of NGF,TrkA,p-TrkA,Akt,p-Akt,GSK3?,p-GSK3? in sciatic nerve of rat were detectedby western blot.At the same time,RSC96 cells were used as experimental cells in vitro,and the cells were divided into Control group,HG group,T1 group,T2 group and T3 group.To further examine the relationship between each pathway protein,we added corresponding inhibitors such as NGF neutralizing antibody(ab16161),Trk A inhibitor(K252a),Akt inhibitor(perifosine)and GSK3? inhibitor(TDZD-8).The apoptosis of RSC96 cells was detected by TUNEL and flow cytometry.The concentration of NGF in the supernatant of RSC96 cells was detected by ELISA.Each protein expression changes was detected by Western blot.The RSC96 cells activity of Caspase-3 was detected by activity kit.The experimental data was analyzed by using the Graph Pad Prism software 5.0.Results: 1.Effect of taurine on myelin injury of sciatic nerve in DPN rats Transmission electron microscopy(TEM)showed that the myelin structure of the DPN group was disordered and expanded toward both axon and stromal side,forming a myelin fold.In addition,as the myelin layer partially dissolved,the gap increased,vacuoles appeared,and slight demyelination.However,the taurine protection group improved the pathological changes of the sciatic nerve of DPN rats and maintained their structural integrity.Compared with Control group,it showed a significant decrease of MBP expression in sciatic nerve of DPN group.In contrast,MBP levels were significantly increased after taurine treatment(P <0.05),reversing the DPN mediated decrease.2.Effect of taurine on apoptosis of Schwann cells in sciatic nerve of DPN rats Apoptosis of Schwann cells was observed by TUNEL and S100 double staining.The number of TUNEL+/S100+ cells was significantly increased in DPN group compared to Control group(P <0.05).On the contrary,the increased amount of TUNEL+/S100+ cells in DPN group was significantly reduced after taurine treatment.Moreover,with the increase in the concentration of taurine supplements,the double positive cells were significantly reduced(P <0.05).The ultrastructure of SCs in the sciatic nerve was observed by TEM.It was found that the SCs in sciatic nerve of DPN group showed chromatin condensation and crescent accumulation around the nuclear membrane.However,after taurine treatment,the chromatin of SCs was uniformlystained without pyknosis,suggesting taurine attenuate the apoptosis of Schwann cells in sciatic nerve of DPN rats.3.Effect of taurine on apoptosis of RSC96 cells exposed to high glucose The result of TUNEL,Caspase-3 activity and flow cytometry showed that the apoptotic index of TUNEL+ cells and the activity of Caspase-3 in HG group were significantly higher than those in Control group(P <0.05),and early apoptotic rate was significantly increased(P <0.05).After taurine treatment,the number of TUNEL+ cells,Caspase-3 activity and early apoptotic rate were effectively reduced(P <0.05).4.Effects of taurine on NGF and Trk A in sciatic nerve of DPN rats and RSC96 cells Western blot showed that the protein expression of NGF and p-Trk A in DPN group and HG group was significantly lower than those in Control group(P <0.05).However,after taurine treatment,the expression of NGF and p-Trk A was significantly higher than that of DPN group or HG group(P <0.05).Furthermore,the increased level of p-Trk A by taurine was decreased after the addition of ab16161(P <0.05).5.Taurine mediated NGF antagonizes apoptosis induced by high glucose in RSC96 cells TUNEL,Caspase-3 activity assays and flow cytometry showed that taurine significantly reduced the apoptotic rate and Caspase-3 activity in HG-exposed RSC96 cells(P <0.05).However,the addition of ab16161 blocked the anti-apoptotic effect of taurine,resulting in increased apoptosis rate and Caspase-3 activity in RSC96 cells induced by HG.6.Effect of taurine on Akt/GSK3? pathway in sciatic nerve of DPN rats and RSC96 cells Western blot showed that the expression of p-Akt and p-GSK3? in DPN group and HG group was significantly lower than that in Control group(P <0.05),and taurine could reverse the decrease of phosphorylation of Akt and GSK3?.In the presence of K252 a,taurine up-regulated the level of p-Akt in HG-exposed RSC96 cells.When perifosine was added to HG-exposed RSC96 cells,the ability of taurine to up-regulate p-GSK3? was blocked.7.Taurine activates Akt/GSK3? pathway to inhibit apoptosis of RSC96 cells exposed to HG TUNEL,Caspase-3 activity assays and flow cytometry showed increased apoptotic rate and Caspase-3 activity after K252 a,perifosine and TDZD-8 were respectively added to T3 group(P <0.05).LDH experiments showed that after the addition of inhibitors,LDH activity was increased(P <0.05)and positivelycorrelated with changes in apoptotic index.Conclusion: Taurine inhibits apoptosis of Schwann cells in DPN rats and improves sciatic myelin sheath injury via up-regulate NGF and Trk A receptors,activate Akt/GSK3? signaling pathway.