| Objectives:1.To observe changes in sciatic nerve MNCV,serum neuritin,MDA and SODlevels in diabetic rats.2.To observe the effects of different glucose concentrations on neuritin expression of Schwann cells.3.To determine whether exogenous neuritin protects diabetic Schwann cells from undergoing apoptosis under high glucose in vitro.4.To observe the antioxidative effect of lipoic acid in Schwann cells.Methods:1.10 male rats were randomly divided into the normalgroup(n=5)and the diabetic group(n=5).The diabetic rats were induced by intraperitoneal injection of STZ.2.The sciatic nerve was stimulated proximally at the sciatic notch and distally at the ankle via bipolar electrodes with supramaximal stimuli(8 V)at 20 Hz.The latencies of the compound muscle action potentials were recorded via bipolar electrodes from the first interosseous muscle of the hindpaw and measured from the stimulus artifact to the onset of the negative M-wave deflection.MNCV was calculated by subtracting the distallatency from the proximallatency,and the result was divided into the distance between the stimulating and recording electrode.3.The content of neuritin was measured by ELISA kit,MDA was detected by TBA method,and SOD activity was assessed by dehydrogenase method.4.Under aseptic conditions,sciatic nerveswere obtained from SD rats.The epineurium was stripped off using fine-pointed forceps,and the sciatic nerve was cut into 2-3mm fragments.Then nerve fragments were digested in0.25%trypsin and 0.05%collagenase ? solution in the incubator for 90min.The cell pellet was washed using DMEM medium with 10%fetal bovine serum and centrifuged at 1200rpm for 6 min.The supernatant was discarded and the cell pellet was resuspendedwith Schwann cell-culture medium.5.Schwann cells were divided into 3 groups:control group(glucose concentration 5.56mmol/1,Schwann cells from normal rats),experimental group(glucose concentration 25mmol/l,Schwann cells from diabetic rats),hypertonic mannitol group(5.56mmol/1 glucose +20mmol/1 mannitol,Schwann cells from normal rats).Schwann cells neuritin was measured by qPCR and Western blot.6.Schwann cells were treated with neuritin(0,1 0,100ng/ml)for 48h and cell apoptosis was measured by Annexin V-FITC/PI.7.Schwann cells were treated with lipoic acid(0,0.5,1.0,1.5mmol/1)for 36h and cel1MDA was detected by TBA method.Results:1.Compared with normal rats,sciatic nerve MNCV,serum neuritin and SODlevels were decreased,MDA levels were increased in diabetic rats,respectively(P<0.05).2.QPCR results showed that Schwann cells neuritin mRNA expression decreased under high glucose(P<0.05).Western blot results also showed that neuritin protein expression decreased under high glucose(P<0.05).3.Schwann cells apoptosis increased signigficantly in high glucose media compared to normal glucose(P<0.05).Exogenous neuritin treatment could inhibit the apoptosis of Schwann cells exposed to high glucose in a dose-dependent manner.4.Schwann cellsMDA level decreased after lipoic acid treatment.Conclusion:1.Abnormal peripheral nerve function,decreased serum neuritinand oxidative stressoccurred in rats with diabetes.2.Neuritin expression was down-regulatedin Schwann cellsdissociated from sciatic nerves of the diabetic rats.Apoptosis of Schwann cells with down-regulated neuritin increased.3.The exogenous neuritin treatment could inhibit the apoptosis of Schwann cells in a dose dependent manner.4.Lipoic acid mitigated oxidative stress of Schwann cells.5.Lipoic acid treatment to improve survival of Schwann cells and thus diabetic neuropathy via countering hyperglycemia-oxidative stress induced neuritin reduction needs to be investigated. |
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