Promoting Repairing Effect Of Taurine On Axons Of Diabetic Peripheral Neuropathy Rats And Mechanism Research

Objective: To investigate the promoting repairing effect and mechanism of taurine on damaged axons of type 2 diabetic peripheral neuropathy.Materials and Methods: Sixty adult SPF male Sprague-Dawley rats were used with a body weight of 140-180 g.Randomly grouped into two groups which were normal Control group(Con)and Diabetic peripheral neuropathy(DPN)group.12 rats in the Con group with fed normal diet;48 rats in the diabetic model group with fed high fat and high sugar diet(25% sucrose + 15% lard + 1.5% cholesterol + 1% sodium cholate + 57.5% normal rat diet).After 4 weeks of high-fat and high-sugar feeding,the rats in the DPN model group were intraperitoneally injected with streptozotocin(STZ)at a dose of 25 mg/kg,and the normal Control group was injected with the same amount of citrate buffer.After 72 hours,the random blood glucose of rats was measured in the tail vein.If the random blood glucose was higher than 16.7 mmol/l and significant changes in neuroelectrophysiology,the DPN model was established.If DPN model was not established,the STZ was injected in small doses once again.After successful made DPN model,they were randomly divided into four groups of 12 rats each group.In the normal Control group,0%(DPN group),0.5%(0.5% T group),1%(1% T group),2%(2% T group)of taurine were added into the drinking water of the DPN model group with eight weeks.After 8 weeks,3 rats in each group were sacrificed by 4%paraformaldehyde perfusion,and the sciatic nerve tissue was extracted.One rat was selected in each group to extract the sciatic nerve tissue and quickly placed it into 2.5%glutaraldehyde with small pieces.The rest rats of each group were killed by decapitation,and the sciatic nerve tissue was quickly extracted at a low temperature and stored at-80 °C.Sciatic nerve tissue was observed by the techniques of transmission electron microscopy and immunofluorescence.The expression of GAP-43,NGF,TrkA/p-TrkA,Akt/p-Akt and mTOR/p-mTOR protein were detected by Western Blot.In vitro cell experiments were performed on Schwann cells(SCs)with 50 mM high glucose(HG),followed by treatment with 10 mM,20 mM and 40 mM taurine for 48 hours.The expression of NGF in SCs was measured.The supernatants of the different treatment groups were collected and the supernatant was defined as "SC-CM".Western blot analysis of GAP-43,NGF,TrkA/p-TrkA,Akt/p-Akt,mTOR/p-mTOR in rat Dorsal root ganglion(DRG)neurons cultured in SC-CM medium.For the study of cellular pathways,antiNGF,K252a(TrkA inhibitor),MK2206(Akt inhibitor),and Rapamycin(mTOR inhibitor)were added to DRG neurons.The growth of DRG neuron axons was observed by immunofluorescence staining.Statistical analysis was performed on the experimental results using Graphpad Prism 5 statistical analysis software.Results: 1.The effect of taurine on blood glucose,body weight and electrophysiology of DPN rats The blood glucose of the initial DPN rats treated with taurine was significantly higher than that of the Con group.The latency of MCV(motor nerve Conduction velocity)was significantly prolonged compared with the Con group.The MCV was lower than that in the Con group,and the body weight was significantly lower than that in the Con group,and the difference was statistically significant(p<0.05).After 8 weeks of taurine treatment,the blood glucose was lower than that in the DPN rats.The comparison of the group was lower,the motor nerve Conduction latency was lower compared with DPN group,the motor nerve Conduction velocity increased faster than the DPN group,and the body weight was increased,and the difference was statistically significant(p<0.05).2.Effects of taurine on axonal injury of sciatic nerve in diabetic rats Transmission electron microscopy showed that the sciatic nerve of DPN group was significantly damaged compared with the Con group,and the diameter of axon was significantly reduced(p<0.05).The degree of damage was reduced,and the diameter of the axons increased(p<0.05).The double immunofluorescence staining of rat sciatic nerve showed that the fluorescence intensityof SMI312 in DPN group was significantly lower than that in the Con group(p<0.05),and it was increased in each taurine treatment group gradually(p<0.05).The results of GAP-43 protein detection in each group of sciatic nerve were Consistent with the results of immunofluorescence.The immunofluorescence double staining of DRG neurons showed that the length of axon of DRG neurons in HG group was significantly lower than that in the Con group(p<0.05).The length of axons in the taurine treatment groups increased obviously(p<0.05).The GAP-43 protein detection results of each group of DRG neurons were Consistent with the immunofluorescence results.3.The effect of taurine on NGF and TrkA in serum and sciatic nerve of diabetic rats The results of ELISA on NGF Concentration in serum showed that the serum NGF Content in DPN group was significantly decreased(p<0.05),but increased in taurine groups(p<0.05).The result Concentration of NGF in the sciatic nerve is Consistent with that in serum.The NGF Concentration measurements in SCs and medium supernatants were Consistent with the in vivo measurements.The phosphorylation level of TrkA protein in the sciatic nerve was significantly lower in the DPN group than in the Con group(p<0.05),and increased in the taurine-treated groups(p<0.05).The expression of p-TrkA protein in dorsal root ganglion neurons have the same trend with the sciatic nerve detected by western blotting.However,after adding anti-NGF in the taurine treatment group,the protein expression level of p-TrkA was significantly decreased(p<0.05).4.The effect of taurine on Akt/mTOR signaling pathway in sciatic nerve of diabetic rats The expression of Akt protein in sciatic nerve showed that p-Akt expression was significantly lower in the DPN group than in the Con group(p<0.05),in each taurine treatment group.The expression of Akt protein in DRG neurons was Consistent with that in sciatic nerve.However,after intervented with K252 a,the expression level of p-Akt protein was significantly decreased(p<0.05).The protein expression of mTOR in each group of sciatic nerve and DRG neurons was the same as that expression of Akt.5.Taurine amplifies the growth of DRG neurons by regulating NGF expression Immunofluorescence results show tha medium can inhibit the growth of neurons after adding NGF and the axon length of DRG neurons issignificantly higher than that of high glucose cultured group(p<0.05).Similarly,after the intervention of NGF-Ab,the effect of taurine on promoting axonal growth of damaged DRG neurons was blocked,and the axon length and taurine group ratio were significantly reduced(p<0.05).The expression of GAP-43 protein in each group of DRG neurons was also detected,and the results were Consistent with the fluorescence results.6.Taurine regulates Akt/mTOR signaling pathway by NGF to promote axonal growth of DRG neurons Immunofluorescence results show that taurine with the intervention of K252 a,MK2206 and Rapamycin,respectively,promotes axonal growth.The effect of taurine promoting axonal growth was suppressed and the axon length was significantly shortened(p<0.05).The expression of GAP-43 protein in each group of DRG neurons was Consistent with the results of immunofluorescence.Conclusion: 1.Taurine can promote the axon growth of sciatic nerve in DPN rats;2.Taurine can up-regulate the expression of NGF protein in sciatic nerve of DPN rats;3.Taurine can promote the regeneration of damaged axons of diabetes by up-regulating the protein expression level of NGF in peripheral nerves and activating the Akt/mTOR signaling pathway.