| Objective:Diabetic peripheral neuropathy（DPN）is one of the maior and serious complications of diabetes mellitus.Common pathological changes in diabetic peripheral neuropathy include axonal degeneration,axonal loss,myelin sheath abnormalities and demyelination.Schwann cells are the primary cells of peripheral nerves and provide neurotropic support for maintaining normal axonal impulse conduction and promoting regeneration of damaged axons.Brain-derived neurotrophic factor（BDNF）and Neurotrophin 3 belong to a family of protein polypeptide nerve growth factors secreted by Schwann cells.They are essential for the survival,proliferation,migration and differentiation of neurons and neurogliocytes in the peripheral nervous systems.BDNF and Neurotrophin 3 deficiency in Schwann cells plays an important role in the pathogenesis of diabetic peripheral neuropathy,but the exact mechanism of downregulation of BDNF and Neurotrophin 3 expression in Schwann cells of diabetic peripheral neuropathy has not been fully elucidated.DNA methylation is an epigenetic modification that plays a key role in regulating gene transcription,chromatin remodeling,genome imprinting and genome integrity.DNA methylation usually occurs on the cytosines of Cp G dinucleotides in the 5’promoter of genes,and this process is primarily regulated by DNA methyltransferase（DNMT）and ten-eleven translocation（TET）family enzymes.At present,whether DNA methylation mediates BDNF expression in Schwann cells in diabetic peripheral neuropathy has not been explored.Mammalian target of rapamycin（mTOR）is a multifunctional pathway that regulates cell proliferation,metabolism,autophagy and apoptosis,and is a Serine/Threonine protein kinase.A variety of stimulis,including growth factors,glucose,amino acids and other nutrients,will affect the mTOR signaling pathway.The mTOR pathway is commonly controlled by distinct upstream pathways to enable cells to respond to different stimulis,including protein kinase B（PKB/Akt）and AMP activated protein kinase（AMPK）.However,there is no relevant report on whether there is a network interaction between the express ion of BDNF and Neurotrophin 3,DNA methylation,and mTOR signaling pathway and upstream regulatory molecules in Schwann cells in diabetic peripheral neuropathy.Therefore,in the present study,Male CD1 mice were used to induce experimental diabetes model,rat Schwann cell lines（RSC96）were cultured in high glucose（H）in vitro.First,the expression of BDNF and Neurotropin 3and the accompanied changes of myelin sheath structure in sciatic nerves tissue of diabetic mice model were detected.Second,the m RNA and protein expression of BDNF and Neurotropin 3 in RSC96 cells cultured in vitro with high glucose were detected.Third,the methylation status of BDNF promoter DNA and the expression of DNA methyltransferase1（DNMT1）were demonstrated.Meanwhile,DNMT inhibitor（5-Aza-2’-deoxycytidine,5-Aza）and DNMT1 sh RNA were used to measure the change of accordingly target.Finally,the effect of mTOR signaling pathway and its upstream regulator Akt and AMPK pathways on the expression of DNMT1 and BDNF in RSC96 cells induced by high glucose were examined.Thus,the effect of DNA methylation and Akt/mTOR signaling pathway on BDNF expression in Schwann cells in diabetic peripheral neuropathy were systematically explored,providing new research ideas and exploring new intervention targets for the prevention and treatment of diabetic peripheral neuropathy.Methods:1. The expression of BDNF and Neurotrophin 3 and myelin sheath abnormalities in the sciatic nerves of STZ-induced diabetic miceThe male CD1 mice were divided randomly into 3 groups:normal mice and diabetic mice.Diabetic mice models were induced by an intraperitoneal injection of streptozotocin at a dose of 150 mg/kg body weight（sodium citrate solution,p H 4.4）and normal mice received the same amount of sodium citrate solution.Those animals with a blood glucose level higher than 16.7 mmol/L were defined as diabetic after 72 h of the injection.Blood was obtained from tail vein and blood glucose was monitored every week.Those who did not meet the standard were discarded.At the age of 16 weeks,the mice were sacrificed,sciatic nerve tissues were separated and fixed in 4%paraformaldehyde or 4%glutaraldehyde for immunohistochemical,myelin sheath luxol fast blue staining and electron microscopy examinations.One portion of sciatic nerve tissues were snap frozen in liquid nitrogen and kept at-80℃for the extraction of protein and m RNA.Immunohistochemistry and Western blot were used to detect the protein expression of BDNF and Neurotrophin 3 in sciatic nerve.The changes of myelin sheath structure of sciatic nerve in mice were observed by nerve luxol fast blue staining and electron microscopy.2.Expression of BDNF,Neurotrophin 3 protein and m RNA and detection of DNA methylation status of BDNF promoter in vitro-high glucose cultured RSC96 cellsThe rat Schwann cell line RSC96 were cultured in DMEM medium containing 10%fetal bovine serum and 1%penicillin-streptomycin in a 37℃,5%CO2 incubator.（1）To detect the effect of high glucose on the BDNF and Neurotrophin 3 expression in RSC96 cells:RSC96 cells were randomly divided into 3 groups:normal glucose group（5.5 mmol/L glucose,normal glucose,N）,high glucose group（25 mmol/L glucose,high glucose,H）and mannitol hypertonic control group（5.5 mmol/L glucose+19.5 mmol/L mannitol,M）.RSC96 cells were cultured in serum-free medium for 12 hours to synchronize the cells growth.The serum-deprived RSC96 cells were respectively stimulated for 1 day,2 days and 3 days.The protein expression of BDNF and Neurotrophin 3 were detected by immunofluorescence and Western blot.The level of BDNF m RNA was detected by Real-time PCR.（2）To investigate the effect of epigenetic modification on BDNF protein expression in high glucose-induced RSC96 cells:After synchronizing cells with serum-free medium for 12 hours,the RSC96 cells were treated with DNA methyltransferase inhibitor（10μmol/L 5-Aza）or histone deacetylase inhibitor（400 nmol/L TSA）.Then RSC96 cells were divided into 4 groups:normal glucose group（N）,high glucose group（H）,high glucose+10μmol/L 5-Aza group（H+5-Aza）and high glucose+400 nmol/L TSA group（H+TSA）.The expression of BDNF protein were detected by immunofluorescence and Western blot after 3 days.（3）To detect the DNA methylation status of BDNF promoter by methylation specific PCR（MSP）technology:The RSC96 cells were divided into normal glucose group（N）,high glucose group（H）and high glucose+10μmol/L 5-Aza（H+5-Aza）group after synchronizing the cells with serum-free medium.Genomic DNA was extracted from RSC96 cells following the instructions of the TIANamp Genomic DNA kit after 3 days.After sulfite modification,DNA purification and recovery.Next,primer pairs of BDNF promoters for both methylated and unmethylated sequences were designed at the website.Methylation-specific PCR kit was used to performe the MSP reactions.The ratio of methylated DNA to total methylated and unmethylated DNA was calculated to represent the degree of methylation.3.Effect of DNMT on BDNF protein expression in RSC96 cells induced by high glucose（1）To detect the expression of DNMT and TET m RNA in high glucose-stimulated RSC96 cells:The RSC96 cells were divided into normal glucose group（N）and high glucose group（H）.Total RNA was isolated from RSC96 cells treated with normal glucose or high glucose medium after 3 days.After purification,m RNA was fragmented into small pieces by metal hydrolysis.Frag mented RNA was used to synthesize first strand c DNA using reverse transcriptase and random primers,which was followed by second strand c DNA synthesis using DNA polymerase I.These c DNA fragments went through an end repair process and ligation of the adapters to create the final c DNA library.After quantification of c DNA,sequencing was performed on the Illumina Hi Seq 2500.（2）To detect the effect of high glucose on DNMT1expression of RSC96 cells:RSC96 cells were randomly divided into normal glucose group（N）,high glucose group（H）and mannitol hypertonic control group（M）.After synchronizing cells with serum-free medium for 12 hours,the RSC96 cells were collected after 1 day,2 days and 3 days stimulated by high glucose,and DNMT1 protein expression was detected by immunofluorescence and Western blot.（3）To investigate the effect of DNMT1 inhibition on expression of BDNF protein in RSC96 cells:After synchronizing cells with serum-free medium for 12 hours,the RSC96 cells were divided into normal glucose group（N）,normal glucose+10μmol/L5-Aza group（N+5-Aza）,high glucose group（H）,high glucose+10μmol/L5-Aza group（H+5-Aza）.After 3 days,Western blot was used to detect the expression of DNMT1 and BDNF proteins.（4）To investigate the effect of DNMT1 knockdown on expression of BDNF protein:RSC96 cells in 6-well plates grew for 24 hours in the absence of antibiotics and were transfected with negative control sh RNA（p Genesil-1）or specific DNMT1 sh RNA using Lipofectamine?3000.Then the RSC96 cells were randomly divided into untransfection group（untransfection）,negative control group（p Genesil-1）and p Genesil-1-DNMT1 transfected group（p Genesil-1-DNMT1）.After transfection 24 h,RSC96 cells received high glucose treatment for 48 h,Western blot was used to detect the expression of DNMT1 and BDNF protein.4.Effect of Akt/mTOR signal pathway on expression of DNMT1 and BDNF in vitro-high glucose cultured RSC96 cells（1）To detect the effect of high glucose on mTOR signaling pathway in RSC96 cells:RSC96 cells were randomly divided into normalglucosegroup（N）,high glucose group（H）and mannitol hypertonic control group（M）.After3 days,Western blot was used to detect the expressions of mTOR and phospho-mTOR（Ser 2448）.（2）To detect the effects of mTOR pathway inhibitor（Torin1）and activator（MHY1485）on DNMT1 and BDNF protein expression in high glucose-stimulated RSC96 cells:The RSC96 cells were randomly divided into normal glucose group（N）,normal glucose+500 nmol/L Torin 1 group（N+Torin1）;normal glucose group（N）,high glucose group（H）and high glucose+10μmol/L MHY1485 group（H+MHY1485）.After 3 days,Western blot and immunofluorescence were used to detect the expressions of S6K,phospho-S6K（Thr 389）,DNMT1 and BDNF protein.（3）To detect the effect of high glucose on Akt and AMPK signaling pathway in RSC96 cells:The RSC96 cells were randomly divided into normal glucose group（N）,high glucose group（H）and mannitol hypertonic control group（M）.After 3 days,Western blot was used to detect the expression of Akt,phospho-Akt（Ser 473）,phospho-Akt（Thr 308）,AMPK and AMPK proteins.（4）To detect the effects of Akt and AMPK pathway inhibitors（LY294002 and Dorsomorphin）on mTOR,phospho-mTOR（Ser 2448）,DNMT1 and BDNF protein expression in high glucose-stimulated RSC96 cells:The RSC96 cells were randomly divided into normal glucose group（N）,normal glucose+DMSO group（N+DMSO）,normal glucose+LY294002/Dorsomorphin group（N+LY294002/Dorsomorphin）,and high glucose group（H）,high glucose+DMSO（H+DMSO）,highglucose+LY294002/Dorsomorphin group（H+LY294002/Dorsomorphin）.After 3 days,Western blot was used to detect the expression of mTOR,phospho-mTOR（Ser 2448）,DNMT1 and BDNF.Results:1.The expression of BDNF and Neurotrophin 3 and myelin sheath abnormalities in the sciatic nerves of STZ-induced diabetic mice.（1）The results of the Western blot showed that the expression of Pro-BDNF,mature-BDNF and Neurrophin 3 protein levels were decreased by 74.4%,40.1%and 59.0%respectively in sciatic nerves of diabetic mice compared with normal mice（P<0.05）.（2）The results of the immunohistochemistry showed that positive staining（brownish-yellow）of BDNF and Neurotrophin 3total protein were observed mainly in cytoplasim and nucleus of cells and weak positive staining of BDNF and Neurotrophin 3（The protein expression levels decreased 72.8%、36.6%,respectively）in the sciatic nerves of diabetic mice compared to normal mice.（3）Luxol fast blue staining revealed the thinned myelin sheath of sciatic nerves in diabetic mice compared to normal mice.（4）The results of the electronic microscopy demonstrated a regular myelin sheath in the sciatic nerves of normal mice;however,in-folding of myelin sheaths was found in the sciatic nerves of diabetic mice.2. Expression of BDNF,Neurotrophin 3 protein and m RNA and detectionof DNA methylation status of BDNF promoter in vitro-high glucose cultured RSC96 cells.（1）The effect of high glucose on the BDNF and Neurotrophin 3expression in RSC96 cells:The results of the Western blot showed that Pro-BDNF expression was decreased by 46.5%and 48.3%in the cells treated with high glucose for 2 d and 3 d,respectively,compared to the corresponding mannitol group（P<0.05）.Similarly,mature-BDNF expression was reduced by32.1%and 23.7%with high glucose treatment for 2 d and 3 d,respectively,compared to the mannitol group（P<0.05）.However,high glucose treatment for 1 d did not cause evident differences in Pro-BDNF and mature-BDNF expression compared to normal high or mannitol treatment（P>0.05）.In contrast,no evident difference in Neurotrophin 3 expression was observed in cells from any group at the indicated time points.Furthermore,immunofluorescence showed similar results to Western blot analysis.Both BDNF and Neurotrophin 3 were located in the nucleus and cytoplasm of RSC96 cells,as indicated by green fluorescence.As observed previously,3 d-high glucose stimulation led to reduced expression of BDNF,but notNeurotrophin 3,compared to normal glucose and mannitol treatment groups in RSC96 cells.Real-time PCR results showed that BDNF m RNA was decreased by 61.4%in cells treated with high glucose for 3 d compared to those treated with normal glucose（P<0.05）.（2）The effect of epigenetic modification on BDNF protein expression in high glucose-induced RSC96 cells:The results of the Western blot results showed that 5-Aza treatment enhanced expression of Pro-BDNF and mature-BDNF by 1.23 and 1.26 times,respectively,in high glucose-treated RSC96 cells（P<0.05）.However,addition of TSA did not increase the expression of BDNF protein.Similar to the Western blot detection,immunofluorescence was also used to determine the e ect of 5-Aza on BDNF expression in high glucose-stimulated RSC96 cells.Compared to cells in the high glucose group,those in the high glucose plus 5-Aza group presented strong positive expression of BDNF.In contrast,TSA treatment did not result in distinct alterations of BDNF expression in high glucose-stimulated RSC96cells.（3）DNA methylation status of BDNF promoter:The results of the methylation specific PCR（MSP）showed that the degree of methylation（methylation/methylation+unmethylation）of exon I and exon II promoters was significantly increased in high glucose-induced RSC96 cells compared to normal glucose-treated cells（P<0.05）.Moreover,5-Aza treatment reduced the degree of methylation in exon I and exon II promoters.In contrast,there were no di erences in the degree of methylation in exon IV（complete unmethylation）and exon IX（almost complete methylation）promoters between normal glucose and high glucose groups（P>0.05）.3. Effect of DNMT on BDNF protein expression in RSC96 cells inducedby high glucose.（1）The expression of DNMT and TET m RNA in high glucose-stimulated RSC96 cells:The RNA sequencing results showed that DNMT1 m RNA and DNMT3a m RNA,particularly DNMT1 m RNA,were enhanced in response to high glucose treatment in RSC96 cells.In contrast,DNMT3b,TET1,TET2 and TET3 m RNA showed no marked di erences between normal glucose-treated cells and high glucose-treated cells.（2）The effect of high glucose on DNMT1 expression of RSC96 cells:Western blot results showed that the high glucose increased DNMT1 protein expression in RSC96 cells.Statistical analysis revealed that DNMT1 was enhanced by 2.13,2.22 and 2.44 times at 1 d,2 d and 3 d,respectively,after high glucose stimulation compared to the normal glucose group（P<0.05）.As previously observed,there was no di erence in DNMT1 expression between normal glucose and mannitol control groups.In addition,immunofluorescence detection showed that DNMT1 was primarily localized to the nucleus in RSC96 cells.High glucose treatment did not alter the subcellular localization of DNMT1;however,DNMT1 expression levels were significantly increased in RSC96 cells treated with high glucose compared to those treated with normal glucose medium.（3）The effect of 5-Aza inhibition on expression of BDNF protein in RSC96 cells:Western blot showed that 5-Aza e ectively suppressed DNMT1 expression in RSC96 cells cultured in normal or high glucose.In detail,DNMT1 was decreased by 55.9%in response to 5-Aza treatment in normal glucose-stimulated cells and by 59.3%in response to5-Aza treatment in high glucose-stimulated cells（P<0.05）.As before,5-Aza addition increased Pro-BDNF by 1.67 times（P<0.05）and mature-BDNF by1.35 times（not statistically significant）in high glucose-treated RSC96 cells.In contrast,this e ect of 5-Aza on Pro-BDNF and mature-BDNF was absent in normal glucose-treated RSC96 cells.（4）The effect of DNMT1 knockdown on expression of BDNF protein:Western blot showed that compared with the negative control group（p Genesil-1）,the expression of DNMT1 protein in RSC96 cells in p Genesil-1-DNMT1 group decreased by 70.5%（P<0.05）;The expression of Pro-BDNF and mature-BDNF protein were increased by 29.3%and 82.9%,respectively,compared with the negative control group（p Genesil-1）（P<0.05）.4. Effect of Akt/mTOR signal pathway on expression of DNMT1 andBDNF in vitro-high glucose cultured RSC96 cells.（1）The effect of high glucose on mTOR signaling pathway in RSC96 cells:Western blot results showed that phospho-mTOR（Ser 2448）were decreased by 45.3%（P<0.05）in RSC96 cells treated with high glucose for 3 days compared with normal glucose group and mannitol hyperosmotic control group.There was no difference in expression of phospho-mTOR（Ser 2448）between cells of normal glucose group and mannitol hyperosmotic control group（P>0.05）.（2）The effect of mTOR pathway inhibitor Torin1 and activator MHY1485 on DNMT1 and BDNF protein expression in high glucose cultured RSC96 cells:The results of Western blot showed that Torin 1 could significantly inhibit mTOR pathway activation in normal glucose RSC96 cells,and the expression of phophopho-S6K（Thr 389）was significantly reduced（P<0.05）,a known mTOR pathway down-stream target.At the same time,DNMT1 expression was increased by 1.04 times（P<0.05）,and Pro-BDNF and mature-BDNF expression were decreased by 37.8%（P<0.05）and 29.3%（P>0.05）,respectively,in RSC96 cells treated with Torin 1 compared to those in the normal control group.The results of immunofluorescence showed that Torin 1group increased the expression of DNMT1,showing strong green fluorescence,in RSC96 nucleus compared with normal glucose control group.MHY1485can promote mTOR pathway activation in high glucose RSC96 cells,and the expression of phospho-S6K（Thr 389）is significantly increased（P<0.05）,while the expression of DNMT1 protein is decreased by 49.9%（P<0.05）,and the expression of Pro-BDNF and mature-BDNF are significantly increased（P<0.05）in high glucose plus MHY1485 treated cells.（3）The effect of high glucose on Akt and AMPK signaling pathway in RSC96 cells:Western blot results showed that compared with normal glucose group and mannitol hyperosmotic control group,the expression of phospho-Akt（Ser 473）and phosphor-Akt（Thr 308）proteins were decreased by 74.1%and 80.6%,respectively,and the expression of phosphor-AMPK（Thr 172）protein was increased by 46.5%（P<0.05）in high glucose group.There was no significant difference in the expression of total Akt and AMPK proteins among the three groups.（4）The effects of Akt and AMPK pathway inhibitors（LY294002 and Dorsomorphin）on mTOR,phospho-mTOR（Ser 2448）,DNMT1 and BDNF protein expression in high glucose-stimulated RSC96 cells:The results of Western blot showed that LY294002 were significantly reduced the expression of phospho-mTOR（Ser 2448）in normal and high glucose treated RSC96 cells（P<0.05）,and the expression of total mTOR has no obvious change.It was further found that LY294002 was increased the expression of DNMT1 and decreased the expression of BDNF in high glucose group（P<0.05）,but LY294002 did not affect the expression of DNMT1 and BDNF in normal glucose group.These data suggest that Akt pathway affects the mTOR pathway and the expression of DNMT1 and BDNF in high glucose treated RSC96 cells.Dorsomorphin was significantly increased phosphorylation of normal and high glucose treated mTOR.Western blot results showed that the expression of phospho-mTOR（Ser 2448）was significantly increased（P<0.05）,while the expression of total mTOR was not significantly changed.It was further found that Dorsomorphin was decreased the expression of DNMT1 in high glucose treated group（P<0.05）,while the expression of DNMT1 in normal glucose group did not change significantly.Compared with the control group,the expression levels of Pro-BDNF and mature-BDNF in RSC96 cells of high glucose group or normal glucose group did not change significantly.Conclusions:1.High glucose can decreases BDNF expression in Schwann cells in diabetes mellitus by increasing promoter DNA methylation of BDNF exons I and II.2.DNMT1 mediates high glucose-induced downregulation of BDNF expression in Schwann cells.3.The Akt/mTOR pathway participates in high glucose-induced increase of DNMT1 and decrease of BDNF in Schwann cells in DPN. |
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