| Part One TXNIP was expressed in the sciatic nerve of diabetic mice and Schwann cell treated with high glucoseObjective: The experiment takes STZ-induced type I diabetes mice model and high-glucose cultured Schwann cell as the research objects to detect the expression of TXNIP protein and mRNA in diabetic Schwann cells.Methods:1.TXNIP is expressed in the sciatic nerve of type I diabetic miceMale C57BL/6J mice were randomly divided into normal mice and diabetic mice;C57BL/6J mice,at 6–8 weeks of age were made to be type I diabetic mice models by the intraperitoneal injection of streptozotocin(STZ).Those mice with the random blood glucose more than 16.7 mmol/L were considered as diabetic mice.The sciatic nerve was isolated and extracted at 16 weeks.The part of sciatic nerve was fixed in 4% paraformaldehyde fixative solution and glutaraldehyde solution;the remaining sciatic nerve tissue was stored in a refrigerator at-80 for Western blot and Real℃-time PCR and other technology detection.Western blot and statistical analysis of TXNIP expression in the sciatic nerves of normal mice and diabetic mice;Immunohistochemistry of TXNIP expression in the sciatic nerves of normal mice and diabetic mice;Real-time PCR of TXNIP mRNA expression in the sciatic nerves of normal mice and diabetic mice;Double staining immunofluorescence of TXNIP and MBP in the sciatic nerves of diabetic mice;Electron microscope observation of myelin sheath structure in the sciatic nerves of normal mice and diabetic mice;Electrophysiological detection of action amplitude and conduction velocity of the sciatic nerves of normal mice and diabetic mice.2.TXNIP is expressed in Schwann cells stimulated by high glucose in vitroRat Schwann cells(RSC96,Peking Union Medical College Cell Resource Center of Chinese Academy of Medical Sciences)and human Schwann cells(HSC,Beina Chuanglian Biotechnology,Beijing,China)were cultured in DMEM supplemented with 10% fetal bovine serum(FBS),1% penicillin and 1% streptomycin with 5% CO2 and 37°C.Primary rat Schwann cells(PRSC)were isolated from the sciatic nerves of neonatal rats and maintained under DMEM medium containing 10% FBS,20 ng/m L BFGF and 2 μmol/L forskolin.To explore the effect of high glucose,RSC96 cells,HSC cells and PRSC cells were respectively divided into three groups: normal glucose group(5.5 mmol/L glucose),high glucose group(25 mmol/L glucose)and mannitol group(25 mmol/L mannitol);The protein expression of TXNIP was dectected after Schwann cells treated with high glucose for 24,48 and 72 h by Western blot;The protein expression of TXNIP was dectected after Schwann cells treated with high glucose for 24 h by immunofluorescence;We investigated the expression of TXNIP in the sciatic nerves of diabetic mice and normal mice by real-time PCR.To explore the effect of high glucose,RSC96 cells were respectively divided into two groups: normal glucose group(5.5 mmol/L glucose)and high glucose group(25 mmol/L glucose),the cells were collected after Schwann cells treated with high glucose for 48 h and then the expression of related indicators was analyzed by RNA-seq.Results:1.The expression of TXNIP in the sciatic nerve of type I diabetic mice Western blot results showed that compared with normal mice,the expression of TXNIP protein in sciatic nerve of diabetic mice was significantly increased(P<0.05);The results of IHC staining showed that compared with the normal group,the expression of IOD in the TXNIP positive area of the sciatic nerve of diabetic mice increased by 67.11%(P<0.05);Real-time PCR results showed that compared with normal mice,the expression of TXNIP mRNA in the sciatic nerve of diabetic mice was increased by 86.88%(P<0.05);The results of immunofluorescence double staining confirmed that Schwann cell biomarkers MBP and TXNIP were co-localized.2.The changes of nerve conduction function and myelin sheath structure of sciatic nerve in type I diabetic miceThe results of electron microscope observation showed that compared with the myelin sheath of normal mice,the structure of the myelin sheath of the sciatic nerve of diabetic mice was abnormal and the myelin sheath was folded or broken inward;Electron microscopy observation found that compared with normal mice,action amplitude and conduction velocity in sciatic nerve of diabetic mice was decreased by 37.76% and 27.36% respectively(P<0.05).3.TXNIP expression in Schwann cells stimulated by high glucose in vitroThe results of RNA-seq showed the top 20 significantly differential expression mRNA between RSC96 cells treated with normal glucose and those treated with high glucose for 48 h.Among those,TXNIP mRNA was markedly increased by 339.87 times;Then,western blot detection found that TXNIP protein was respectively significantly increased by 13.47 times and 13.20 times with high glucose treatment for 24 h and 48 h in RSC96 cells compared to normal glucose treatment(P<0.05).Similarly,immunofluorescence observation found that TXNIP was located in the nuclear and cytoplasm of RSC96 cells and high glucose stimulation increased TXNIP expression in the cytoplasm.The results showed that TXNIP protein was also enhanced in HSC cells by high glucose treatment for 24 h,48 h and 72 h.In line,immunofluorescence detection also presented the increased TXNIP expression in the cytoplasm of HSC cells;The results were seen that PRSC cells from newly born rats grew gradually with the time.Immunofluorescence of S-100 known as marker of Schwann cells confirmed the successful culture of PRSC cells.In addition,TXNIP protein expression was increased by 4.40 times in 24 h high glucose-treated PRSC cells versus normal glucose-treated cells(P<0.05).Summary:TXNIP expression was increased in the sciatic nerves of diabetic mice and Schwann cell lines treated with high glucosePart Two TXNIP upregulation mediated high glucose-induced cell autophagy and apoptosis in RSC96 cellsObjective: The STZ-induced knockout of TXNIP diabetes mice and RSC96 cells cultured with high glucose were used to detect the relationship between TXNIP and apoptosis and autophagy-related proteins in diabetic Schwann cells.Methods:1.The effect of TXNIP on sciatic nerve conduction function,autophagy and apoptosis in diabetic miceThe mice were randomly divided into normal mice(TXNIP +/+),diabetic mice(TXNIP +/+)and diabetic mice(TXNIP-/-).The method of diabetic modeling was the same as the first part.The mice sciatic nerve material and tissue processing were the same as the first part.The expression of LC3,cleaved caspase 3 and BAX protein in sciatic nerve were detected by immunohistochemistry.The morphological changes of mice sciatic nerve myelin were observed by electron microscopy.The nerve conduction function of sciatic nerve was detected by electrophysiology.2.The effect of TXNIP on apoptosis and autophagy in RSC96 cellTo explore the effect of high glucose on apoptosis and autophagy,RSC96 cells were respectively divided into three groups: normal glucose group(5.5 mmol/L glucose)and high glucose group(25 mmol/L glucose).Western blot detection of LC3-II/LC3-I and cleaved caspase3/caspase3 ratio expression of cells.To detect the effect of overexpression of TXNIP on the apoptosis and autophagy in RSC96 cell,RSC96 cells were randomly divided into NC group and TXNIP OE group.After 48 h of plasmid transfection,the expression of BAX,LC3-II/LC3-I and cleaved caspase 3 protein were detected by Western blot and immunofluorescence.To detect the effect of TXNIP knockdown on the apoptosis and autophagy in high glucose stimulated Schwann cells RSC96 cells were randomly divided into control sh RNA plasmid group and TXNIP sh RNA plasmid group.After 48 h of plasmid transfection,the expression of LC3-II/LC3-I and cleaved caspase3/caspase3 ratio were detected by Western blot,the expression of LC3,BAX and cleaved caspase3 were detected by immunofluorescence.The expression of autophagy and apoptosis-related proteins were dectected by immunofluorescence.Results:1.Knockout of TXNIP in vivo ameliorated cell autophagy and improved neuron transduction function in the sciatic nerve of diabetic miceWe further elucidated the in vivo effect of TXNIP on diabetic peripheral neuropathy using TXNIP(-/-)mice receiving STZ for diabetic model construction.Compared with diabetic mice(TXNIP +/+),action amplitude was increased by 1.33 times in diabetic mice(TXNIP-/-)(P<0.05).Also,conduction velocity of sciatic nerves was enhanced by 1.78 times in diabetic mice(TXNIP-/-)versus diabetic mice(TXNIP +/+)(P<0.01).However,TXNIP knockout prevented the decrease of autophagosome caused by diabetes mellitus.In line,abnormality of myelin sheath of the sciatic nerves was also improved in diabetic mice(TXNIP-/-)compared with diabetic mice(TXNIP +/+).Again,we further detected the expression of autophagy and apoptosis-related proteins in the sciatic nerves of diabetic mice(TXNIP-/-)and diabetic mice(TXNIP +/+).The results of immunohistochemistry showed that the positive LC3 staining in the sciatic nerves of diabetic mice(TXNIP-/-)was more than those of diabetic mice(TXNIP +/+).Similarly,cleaved caspase 3 and Bax expression was reduced in the sciatic nerves of diabetic mice(TXNIP-/-)versus diabetic mice(TXNIP +/+).Statistical analysis revealed that IOD of positive cleaved Caspase 3 staining and Bax staining were respectively decreased by 99.74% and 98.23%(P<0.05).2.TXNIP upregulation mediated cell autophagy and apoptosis in RSC96 cellsIn addition,we detected the function of TXNIP in high glucose-cultured RSC96 cells.The results were shown that upregulation of TXNIP in normal glucose-cultured RSC96 cells decreased LC3-II/LC3-I ratio,as well as increased pro-apoptotic factor Bax expression.Statistical analysis revealed that LC3-II/LC3-I ratio was reduced by 74.81% in TXNIP OE(overexpression)group versus NC group(P<0.05)and Bax was increased by 1.22 times by TXNIP overexpression(P<0.05).The results of immunofluorescence also found that overexpression of TXNIP in RSC96 cells enhanced cleaved caspase 3 expression indicated in red granules.3.Knockdown of TXNIP mediated apoptosis and autophagy in high glucose cultured RSC96 cellsSubsequently,in high glucose-cultured RSC96 cells,knockdown of TXNIP effectively prevented high glucose-induced LC3-II/LC3-I ratio downregulation and cleaved caspase 3/total caspase 3 ratio upregulation.Statistical analysis revealed that LC3-II/LC3-I ratio was increased by 4.12 times in TXNIP sh RNA plasmid-transfected RSC96 cells versus control sh RNA plasmid group(P<0.05).Then cleaved caspase 3/total caspase 3 ratio was reduced by 25.94% in TXNIP sh RNA plasmid-transfected RSC96 cells versus control sh RNA plasmid group(P<0.05).In addition,LC3 expression was detected in RSC96 cells by immunofluorescence and it could be seen that compared with control plasmid group the clustered LC3 significantly increased in those cells transfected with TXNIP sh RNA plasmid.As well,we explored the cleaved caspase 3 and Bax expression by the method of immunofluorescence,and the results were shown that both cleaved caspase 3 and Bax were decreased by the downregulation of TXNIP in RSC96 cells.Summary:Knockout of TXNIP in vivo ameliorated cell autophagy and improved neuron transduction function in the sciatic nerve of diabetic mice and TXNIP upregulation mediated high glucose-induced cell autophagy and apoptosis in RSC96 cellsPart Three DNMT mediated TXNIP expression,apoptosis and autophagy of Schwann cells in diabetesObjective: Diabetes mice and RSC96 cells cultured with high glucose were used to detect the effect of DNMT on the expression of TXNIP,apoptosis and autophagy related proteins in diabetic Schwann cells.Methods:1.The effect of 5-Aza on the nerve conduction function,TXNIP expression and apoptosis and autophagy of the sciatic nerve in diabetic miceC57BL/6J mice were respectively divided into two groups: diabetic group and diabetic mice+5-Aza group.Diabetes model building method was the same as the first part.To explore the in vivo effect of 5-Aza,diabetic mice were treated with 5-Aza at a 600 μg/kg weight by the intraperitoneal injection every other day.The nerve conduction function of the sciatic nerve was detected by electrophysiological;the material and tissue processing of the sciatic nerve were the same as the first part.The expression of LC3,BAX,cleaved caspase 3 and TXNIP protein in sciatic nerve was detected by immunohistochemical.2.The effect of 5-Aza on the expression of TXNIP,apoptosis and autophagy in RSC96 cells stimulated by high glucoseRSC96 cells were respectively divided into three groups: normal glucose group,DMSO-treated group and 5-Aza-treated group.Western blot and Immunofluorescence were used to detect the expression of LC3-II/LC3-I,BAX and TXNIP with high glucose stimulation for 24 h in RSC96 cells.Real-time PCR was used to detect the expression of TXNIP mRNA with high glucose stimulation for 24 h in RSC96 cells.3.The effect of high glucose on the expression of DNMT1 and DNMT3 a in RSC96 cellsRSC96 cells were respectively divided into two groups: normal glucose group(5.5 mmol/L,normal glucose,N)and high glucose group(25 mmol/L,high glucose,H).After 48 h of high glucose stimulation,the changes of DNMT mRNA were detected by RNA-sequence;DNMT1 and DNMT3a protein expressions in RSC96 cells cultured with high glucose were detected by Immunofluorescence.4.The effect of DNMT1 and DNMT3 a on the expression of TXNIP in RSC96 cells cultured with high glucoseTo investigate the effect of DNMT1 and DNMT3 a,RSC96 cells were randomly divided into pc DNA3.1 group,pc DNA3.1-DNMT1 group,pc DNA3.1-DNMT3 a group,p Genesil-1 group,p Genesil-1-DNMT1 group and p Genesil-1-DNMT3 a group.Western blot and Immunofluorescence detected TXNIP protein expression;Real-time PCR detected TXNIP mRNA expression.5.The effect of DNA methyltransferase inhibitor on the stability of TXNIP protein in RSC96 cells cultured with high glucoseTo explore the effect of 5-Aza,a 10 μmol/L 5-Aza was used for RSC96 cells.To investigate the effect of DNMT1 and DNMT3 a,RSC96 cells were randomly divided into pc DNA3.1 group,pc DNA3.1-DNMT1 group,pc DNA3.1-DNMT3 a group,p Genesil-1 group,p Genesil-1-DNMT1 group and p Genesil-1-DNMT3 a group.To elucidate TXNIP protein degradation,a 10 μmol/L MG132 and a 10 μmol/L chloroquine were used to treat RSC96 cells for 1 h.After transfected with plasmids for 48 h,TXNIP protein was detected by Western blot.Results:1.5-Aza inhibited TXNIP expression in the sciatic nerves of diabetic miceFurthermore,we explored the in vivo effect of 5-Aza on the sciatic nerves and found that the administration of 5-Aza decreased TXNIP expression in the sciatic nerves of diabetic mice,accompanied with the improved conduction velocity and action amplitude of the sciatic nerves.Immunohistochemistry results also revealed that LC3 expression was enhanced and cleaved caspase 3 expression was decreased in the sciatic nerves of diabetic mice treated with 5-Aza.Collectively,these data suggested that DNA methylation modification inhibitor reduced TXNIP expression in diabetic condition-cultured Schwann cells by affecting TXNIP protein not TXNIP mRNA.2.5-Aza inhibited TXNIP expression in high glucose-treated RSC96 cells In order to elucidate whether DNA methylation modification was involved in TXNIP expression in Schwann cells of diabetic peripheral neuropathy,we first explored the effect of 5-Aza known as DNA methyltransferase inhibitor on TXNIP expression in high glucose-cultured RSC96 cells.Then it could be found that 5-Aza treatment markedly suppressed TXNIP protein expression in high glucose-cultured RSC96 cells.Compared with solvent control group,TXNIP expression was decreased by 77.33% in 5-Aza treatment group(P<0.05).In line with the results of Western blot,immunofluorescence also revealed the inhibitory effect of 5-Aza on TXNIP protein in RSC96 cells treated with high glucose.Unexpectedly,there was no significant alteration in TXNIP mRNA expression between DMSO-treated group and 5-Aza-treated group in high glucose-cultured RSC96 cells.Additionally,5-Aza treatment also reversed the effect of high glucose on LC3 and Bax.LC3-II/LC3-I ratio was increased by 2.16 times and Bax was decreased by 32.45% in 5-Aza-treated group versus DMSO-treated group(P<0.05)3.DNMT1 and DNMT3 a expression was enhanced in RSC96 cells cultured with high glucoseConsidering that DNA methyltransferase is the writer of DNA methylation and the targets of 5-Aza,we further investigated which DNA methyltransferase was involved in 5-Aza-inhibited TXNIP expression in high glucose-cultured RSC96 cells.As illustrated that RNA sequencing results showed that DNMT1 mRNA and DNMT3 a mRNA were enhanced in RSC96 cells treated with high glucose versus those treated with normal glucose,not DNMT3 b.In line,Western blot detection also proved that DNMT1 protein was respectively increased by 49.27% and 54.17% with high glucose stimulation for 24 h and 48 h in RSC96 cells.Again,DNMT3 a was also respectively enhanced by 26.31% and 42% with high glucose treatment for 24h and 48 h compared to the normal glucose group.In addition,immunofluorescence assay also verified the results of Western blot,48 h high glucose treatment evidently enhanced DNMT1 and DNMT3 a expression,located in the nuclear of RSC96 cells.4.DNMT1 and DNMT3 a mediated 5-Aza-inhibited TXNIP expression in high glucose-treated RSC96 cellsTo further demonstrate the direct relationship between DNMT1,DNMT3 a and TXNIP in high glucose-cultured RSC96 cells,we transfected RSC96 cells with DNMT1 and DNMT3 a expression plasmid(pc DNA3.1-DNMT1 and pc DNA3.1-DNMT3a)and detected TXNIP expression at 48 h after transfection.The results showed that TXNIP protein expression was increased in response to transfection with pc DNA3.1-DNMT1 and pc DNA3.1-DNMT3 a compared to pc DNA3.1 in high glucose-cultured RSC96 cells(P<0.05).In turn,high glucose-cultured RSC96 cells transfected with DNMT1 and DNMT3 a sh RNA plasmid(p Genesil-1-DNMT1 and p Genesil-1-DNMT3a)presented the downregulation of TXNIP protein.Statistical analysis revealed that TXNIP protein was respectively decreased by 29.3% and 58.86% in high glucose-cultured RSC96 cells transfected with p Genesil-1-DNMT1 and p Genesil-1-DNMT3 a in comparison with those transfected with p Genesil-1(P<0.05).Confocal microscope observation also confirmed that RSC96 cells successfully transfected with p Genesil-1-DNMT1 and p Genesil-1-DNMT3 a showed the less expression of TXNIP.Unexpectedly,knockdown of DNMT1 or DNMT3 a in high glucose-cultured RSC96 cells increased TXNIP expression at the level of mRNA.In detail,TXNIP mRNA was increased by 1.8 times in p Genesil-1-DNMT1-transfected cells and 2.16 times in p Genesil-1-DNMT3a-transfected cells versus p Genesil-1-transfected cells.These findings suggested that DNMT1 and DNMT3 a knockdown decreased TXNIP protein independent of transcription in high glucose-cultured Schwann cells.5.DNMTs was involved in the regulation of TXNIP expression in Schwann cells of diabetes mellitus by the ubiquitin-proteasome pathwayBased on the above findings,we speculated that DNMT1 and DNMT3 a might regulate TXNIP protein degradation in high glucose-stimulated RSC96 cells.Firstly,MG132(proteasome inhibitor)and chloroquine(autophagy inhibitor)were used to treat RSC96 cells-stimulated by high glucose,and the results showed that TXNIP protein suppressed by 5-Aza was effectively reversed by MG132 treatment not chloroquine.In addition,DNMT3 a knockdown-induced TXNIP expression downregulation was also prevented with MG132 treatment.Collectively,the above experiments suggested that DNMTs was involved in the regulation of TXNIP expression in Schwann cells of diabetes mellitus by the ubiquitin-proteasome pathway.Summary:DNA methylation modification inhibitor DNA methylation modification inhibitor reduced TXNIP expression in diabetic condition-cultured Schwann cells by affecting TXNIP protein not TXNIP mRNAPart Four DNA methyltransferase regulates TXNIP protein degradation through ITCH and NEDL3Objective: The experiment takes the rat Schwann cell line cultured with high glucose to detect the expression of ITCH and NEDL3 in diabetic Schwann cells,as well as the relationship with DNMT1,DNMT3 a and TXNIP;it preliminarily explores the mechanism of TXNIP protein stability by the high expression of DNMT1 and DNMT3 a in Schwann cells.Methods:1.ITCH and NEDL3 protein and mRNA expression in RSC96 cells cultured with high glucoseRSC96 cells were randomly divided into normal glucose group(5.5 mmol/L glucose,normal glucose,N)and high glucose group(25 mmol/L glucose,high glucose,H).After 48 hours of cell culture treatment,the expression of ITCH and NEDL3 mRNA was detected by RNA-sequence technology.RSC96 cells were randomly divided into normal glucose group(5.5 mmol/L,normal glucose,N)and high glucose group(25 mmol/L,high glucose,H).After high glucose stimulation for 12,24 and 48 h,the changes of ITCH and NEDL3 protein were detected by Western blot.2.TXNIP protein expression after down-regulation of ITCH and NEDL3 in RSC96 cellsRSC96 cells were randomly divided into p Genesil-1 group,p Genesil-1-ITCH group,and p Genesil-1-NEDL3 plasmid knockdown group.After 48 hours of plasmid transfection,TXNIP protein expression in RSC96 cells were detected by Western blot.3.Co-localization of ITCH and NEDL3 with TXNIP in RSC96 cellsRSC96 cells were respectively transfected with PEGFP-N1-TXNIP+ pm Cherry-ITCH plasmid and PEGFP-N1-TXNIP+pm Cherry-N1-NEDL3 plasmid.After 48 hours of plasmid transfection,the expression of ITCH,NEDL3 and TXNIP in RSC96 cells was observed by the confocal microscope.4.The effect of TXNIP protein expression with NEDL3 upregulation in RSC96 cells cultured with high glucoseRSC96 cells were randomly divided into PCDNA3.1 group,PCDNA3.1-NEDL3 group,PCDNA3.1-NEDL3+HA-Ub group and PCDNA3.1-NEDL3+HA-Ub+MG132 group.After 48 hours of plasmid transfection,TXNIP expression in RSC96 cells were detected by Western blot.5.Detection of ITCH and NEDL3 mRNA expression after down-regulation of DNMT1 and DNMT3 a in RSC96 cells stimulated by high glucoseThe RSC96 cells were randomly divided into p Genesil-1 group,p Genesil-1-DNMT1 group and p Genesil-1-DNMT3 a group;After 48 hours of plasmid transfection,the expression of ITCH and NEDL3 mRNA was detected by Real-time PCR.Results:1.ITCH and NEDL3 mRNA and protein expression in high glucose stimulated RSC96 cellsAfter RSC96 cells were cultured in normal and high glucose for 48 hours,the expression of ITCH family proteins was detected by RNA-seq.The results of the study found that the expression of ITCH and NEDL3 mRNA decreased in RSC96 cells stimulated by high glucose.The expression of ITCH and NEDL3 protein in RSC96 cells was detected by Western blot after high glucose stimulation for 12,24 and 48 h.The results showed that the expression of ITCH and NEDL3 protein in RSC96 cells was gradually down-regulated by high glucose over time(P<0.05).2.The effect of downregulation of ITCH and NEDL3 on the expression of TXNIP protein in RSC96 cells cultured with normal glucoseDownregulation ITCH and NEDL3 of RSC96 cells cultured with normal glucose significantly upregulated the expression of TXNIP protein.The results showed that TXNIP protein expression was increased in response to transfection with ITCH sh RNA and NEDL3 sh RNA compared to NC group in RSC96 cells(P<0.05).3.Co-expression of ITCH,NEDL3 and TXNIP in RSC96 cellsConfocal results showed that: pm Cherry-N1-ITCH and PEGFP-N1-TXNIP plasmids were co-transfected in RSC96 cells cultured with normal glucose;pm Cherry-N1-NEDL3 and PEGFP-N1-TXNIP plasmids were co-transfected in RSC96 cells cultured with normal glucose;the results showed that ITCH and TXNIP were co-expression in RSC96 cells;NEDL3 and TXNIP were co-expression in RSC96 cells.4.The effect of TXNIP protein upregulation NEDL3 in RSC96 cells stimulated by high glucoseWestern blot results showed that: compared with the KB group,the expression of TXNIP protein in the NEDL3 OE overexpression group was significantly increased.Compared with the NEDL3 OE overexpression group,the expression of TXNIP protein in the NEDL3 OE+HA-Ub plasmid transfection group was significantly decreased and the expression of TXNIP protein increased significantly after treatment with MG132(P<0.05).5.The expression of ITCH and NEDL3 mRNA after downregulating DNMT1 and DNMT3 a in RSC96 cells stimulated by high glucoseReal-time PCR results showed that knocking DNMT1 and DNMT3 a in RSC96 cells stimulated by high glucose can significantly increase the expression of ITCH and NEDL3 mRNA.Statistical analysis revealed that compared with the NC control group,the expression of ITCH mRNA in the Sh-DNMT1 plasmid group and the Sh-DNMT3 a plasmid group was respectively increased by 80.79% and 91.72%(P<0.05).Compared with the KB control group,the expression of NEDL3 mRNA in the Sh-DNMT1 plasmid group and Sh-DNMT3 a plasmid group was respectively increased by 62.90% and 62.33%(P<0.05).Summary:The upregulation of DNMT1 and DNMT3 a reduced TXNIP at the level of protein by regulating protein degradation.Downregulation of ITCH and NEDL3 of TXNIP might be involved in DNMTs-regulated TXNIP protein in Schwann cells.Conclusions:In this study,we first reported that DNMT1 and DNMT3 a expression was significantly increased in the sciatic nerves of diabetic mice and high glucose-treated Schwann cells,accompanied by inhibiting ITCH and NEDL3 gene transcription and improving TXNIP protein stability.Taken together,the upregulation of DNMT1 and DNMT3 a inhibited high glucose-induced ITCH and NEDL3 expression,leading to cell autophagy inhibition and apoptosis via TXNIP protein upregulation in Schwann cells of DPN. |
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