The Role Of IGF-1,IGF-1R And PI3K/Akt Signaling Pathway In Diabetic Peripheral Neuropathy And The Protective Effect Of Jinmaitong Capsule

[Objective]The aim of this experiment is to investigate the role of IGF-1,IGF-1R and PI3K/Akt signaling pathway in diabetic peripheral neuropathy and the protective effect of Jinmaitong capsule.[Methods]A single dose of streptozotocin(STZ)was used for the induction of diabetes in male Sprague-Dawley rats.These STZ-DM rats were randomly divided into 5 groups including Diabetes group(DM),JMT-L group(treated with JMT similar to the five-fold dose of adult recommended dosage),JMT-M group(treated with JMT similar to the ten-fold dose of adult recommended dosage),JMT-H group(treated with JMT similar to the twenty-fold dose of adult recommended dosage)and NTP group(treated with Neurotropin similar to the ten-fold dose of adult recommended dosage).10 normal SD rats with the matching size,age and gender were selected as a control group(CON).All rats were given intragastric administration for 16 weeks(the CON and DM groups were treated with distilled water)and then killed.Body weight and blood glucose were detected before and at the 4th,8th,12th,16th week after treatment.The pain threshold to mechanical stimulation with Von Frey filament were detected before death.Morphological changes in sciatic nerve were observed through optical microscopy and transmission electron microscopy.The expressions of IGF-1 in the serum were detected by ELISA.The expressions of IGF-1 in the sciatic nerve were detected by immunohistochemistry.The target genes of IGF-1,IGF-1R,P0 and PMP22 were validated by quantitative real time polymerase chain reaction(qRT-PCR).The expressions of IGF-1,p-IGF-1R,P0 and PMP22 were detected by western blot analyses.The target genes of PI3K and Akt were validated by quantitative real time polymerase chain reaction(qRT-PCR).The expressions of p-PI3K and p-Akt were detected by western blot analyses.[Results](1)Blood glucose,body weight and pain threshold to mechanical stimulation:The blood glucose levels of STZ-DM rats were much higher than those of normal rats(P<0.01).In all the treated groups,there was no significant difference among them compared with each other or with DM model group at the 4th,8th,12th week after treatment(P>0.05).At the 16th week after treatment,the blood glucose levels of all the treated groups were lower than those of DM model group(P<0.01).The body weight of STZ-DM rats were much lower than those of normal rats before and after the intervention(P<0.01).There was no significant difference among all the STZ-DM rats at the 4th,8th,12th,16th week after treatment(P>0.05).Compared with normal group,the pain thresholds in both sides of DM model group decreased extremely(P<0.01).Compared with DM group,the threshold values of all three JMT treated groups and NTP treated group significantly increased(P<0.05 or P<0.01).(2)Pathological morphology:H&E staining was performed to observe the histopathology of the rat sciatic nerves.The morphology of the sciatic nerves in CON group was dense and regular.The sciatic nerves of the DM group was loose and irregular.Some nerve fibers in the sciatic nerves appeared demyelinated.Transmission electron microscopy was used to observe the ultrastructure of myelin.In the CON group rats,the sciatic nerves had uniform and dense myelination with structural integrity presenting concentric light and dark circles in lamellar structures.In the DM group rats,the myelin sheath of the myelinated nerve fibers was loose,disorganized,and exhibited vacuolar-like defects.Lamellar spaces were expanded and separated from each other.(3)IGF-1 in the serum detected by ELISA:The protein levels of IGF-1 in serum of DM model group were much lower than that of control group(P<0.01).Compared with DM group,the protein levels of IGF-1 in serum of JMT-L,JMT-M and NTP groups remarkably increased(P<0.05 or P<0.01).There was no significant difference between all the treated groups(P>0.05).(4)IGF-1 in the sciatic nerve detected by immunohistochemistry:The average optical density(AOD)of IGF-1 expressions in DM group were much lower than those in control group(P<0.01).And the AOD of IGF-1 in all the treated groups increased remarkably compared with DM model group(P<0.05 or P<0.01).(5)The expressions of IGF-1,IGF-1R,P0 and PMP22 in sciatic nerve of DM rats:The levels of IGF-1,IGF-1R,P0 and PMP22 mRNA expression in DM group were much lower than those of the CON group(P<0.05 or P<0.01).Compared with DM group,IGF-1,IGF-1R,P0 and PMP22 mRNA expressions in all treated groups remarkably increased(P<0.05 or P<0.01).But the mRNA expression of IGF-1R showed no significant difference between JMT-H group and DM group(P>0.05).There was no significant difference between the mRNA expression of PMP22 in JMT-L group and DM group(P>0.05).The protein levels of IGF-I,p-IGF-1R,P0 and PMP22 in DM group were much lower than those of control group(P<0.05).Compared with DM group,the protein levels of IGF-1,p-IGF-1R and PO in all treated groups remarkably increased(P<0.05 or P<0.01).The protein levels of PMP22 in JMT-L,JMT-M and NTP groups were much higher than those of control group(P<0.05 or P<0.01),but there was no significant difference between JMT-H group and DM group(P>0.05).(6)The expressions of PI3K and Akt in sciatic nerve of DM rats:The levels of p-PI3K,p-Akt mRNA expression in DM group were much higher than those of the CON group(P<0.01).Compared with DM group,p-PI3K,p-Akt mRNA expressions in all treated groups remarkably decreased(P<0.05 or P<0.01).The protein levels of p-PI3K,p-Akt in DM group were much higher than those of control group(P<0.01).Compared with DM group,the protein levels of p-PI3K,p-Akt in all treated groups remarkably decreased(P<0.05 or P<0.01).[Conclusion](1)The blood glucose levels of DM rats were more than 16.7mmol/L after injection of STZ.Compared with normal group,the pain thresholds of DM rats decreased.The structures of sciatic nerves in DM rats were destroyed.All these demonstrateed that the DPN models were established.(2)Expressions of IGF-1,IGF-1R,P0 and PMP22 were significantly decreased in sciatic nerves of DPN rats.This indicated that reduction of IGF-1 and IGF-1R is related to the diminished abilities to repair and regenerate in diabetic peripheral nerves.(3)In the sciatic nerves of DPN rats,the PI3K/Akt signaling pathway was activated.(4)JMT treatment significantly ameliorated hyperalgesia in DPN rats.Besides,it could also up-regulated the expressions of IGF-1,IGF-1R,P0 and PMP22,down-regulated the expressions of PI3K and Akt in sciatic nerves of DPN rats.JMT stimulated neuroprotection as well as neuroregeneration through up-regulating the expression of IGF-1,IGF-1R,and inhibiting the PI3K/Akt signaling pathway in sciatic nerves.[Innovation]The present study was designed to investigate the effects of Traditional Chinese Medicine Jinmaitong Capsule on the IGF-1,IGF-1R and PI3K/Akt signaling pathway levels of sciatic nerve in STZ-DM rats at the integral and molecular level,which hasn't been reported home and abroad.The present research can provide the theory and experimental basis for the application of Jinmaitong Capsule in DPN prevention and treatment.