| Objective: Diabetic peripheral neuropathy(DPN)is a chronic complication of diabetes.Schwann cells are the main supporting cells of peripheral nerves.Schwann cells autophagy were inhibited in diabetic peripheral neuropathy,and the exact mechanism has not been fully elucidated.Histone deacetylase(HDAC)is an enzyme that catalyzes the removal of acetyl functional groups from lysine residues of histones and non-histones,and Histone deacetylase can regulate cell autophagy.To investigate whether HDAC participates in autophagy inhibition of diabetic Schwann cells(RSC96).In this study,diabetes mice and Schwann cells of rats cultured in vitro were used to investigate the effect of HDAC on Schwann cell autophagy.Methods:1.Immunohistochemistry,Luxol Fast Blue and electron microscopy were used to detect the Structure of sciatic nerve and the expression of autophagy-related proteins in diabetic miceMale CD1 mice were randomly divided into a normal control group(Control)and a diabetes mellitus group(DM).diabetic model mouse was constructed by a single intraperitoneal injection of streptozotocin(STZ).Mice were sacrificed after feed 8 weeks,and the sciatic nerve was retained.Immunohistochemistry was used to detect the expression of LC3 and P62 in the sciatic nerve.Luxol Fast Blue and Electron microscopy were used to detect changes in the sciatic nerve structure.2.Western blot and immunofluorescence were used to detect the effects of high glucose on the expression of LC3-Ⅱ,LC3-and P62 in RSC96 cellsⅠRSC96 cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin in a 5% CO2 incubator at 37℃.RSC96 cells were stochastic divided into normal glucose group(N),mannitol group(M)and high glucose group(H).Cells were cultured for 24 h,48 h and 72 h,Western blot was used to detect the expression of LC3-Ⅰand LC3-Ⅱin RSC96 cells.Immunofluorescence was used to investigate the expression of P62 in RSC96 cells.3.Western blot,immunofluorescence and transcriptome sequence were used to detect the effects of HDAC on autophagy of RSC96 cells cultured in high glucose(1)The effect of the HDAC inhibitor trichostatin A on the expression of LC3-Ⅰ,LC3-Ⅱand P62 proteins in RSC96 cells.The cells were stochastic divided into: normal glucose group(N),normal glucose + DMSO group(N+DMSO),normal glucose + TSA(N+TSA),high glucose group(H),high glucose + DMSO group(H+DMSO),High glucose + TSA group(H+TSA).After termination of culture,Western blot was used to detect the expression of LC3-Ⅰ,LC3-Ⅱand P62,and immunofluorescence was used to detect the expression of P62.(2)Detect the effect of high glucose on the m RNA expression of HDAC family in RSC96 cells: RSC96 cells were randomly divided into normal glucose group(N)and high glucose group(H).After 72 hours,RNA was extracted from transcriptome sequence.Detection the effect of high glucose on the protein expression of HDAC family in RSC96 cells: The cells was stochastic divided into normal glucose group(N),mannitol group(M)and high glucose group(H),which were cultured for 24 h,48 h and 72 h,Western blot and Immunofluorescence were used to detect the expression of HDAC1,HDAC5 and HDAC8 in RSC96 cells.(3)The direct effect of down-regulating HDAC1 or HDAC5 on RSC96 cells the proteins of LC3-Ⅰand LC3-Ⅱwere detected.Lipofectamine3000 transfection reagent was used to transfect p Genesil-1-HDAC1,p Genesil-1-HDAC5 and p Genesil-1-NC(negative control)into RSC96 cells,respectively,and they were stimulated with high glucose for 48 h.Western blot was used to detect the expression of HDAC1,HDAC5,LC3-Ⅰand LC3-Ⅱ.4.Western blot,immunofluorescence and transcriptome sequence were used to detect the effects of HDAC1 on the expression of Atg3,Atg5 and Atg7 expression and autophagy in high glucose cultured RSC96 cells(1)Detect the effect of high glucose on the expression of Atg3,Atg5 and Atg7 in RSC96 cells.RSC96 cells were stochastic divided into normal glucose group(N),mannitol group(M)and high glucose group(H),After 72 h,RNA was extracted for transcriptome sequence.RSC96 cells were stochastic divided into normal glucose group(N),mannitol group(M)and high glucose group(H),After 72 h,RSC96 cells were collected,Western blot and Immunofluorescence were used to detect the expression of Atg3,Atg5 and Atg7 in RSC96 cells.(2)The direct effect of down-regulating HDAC1 on Atg3,Atg5 and Atg7 in RSC96 cells were detected.RSC96 cells were stochastic divided into p Genesil-1-NC group and p Genesil-1-HDAC1 group,and were stimulated with normal glucose and high glucose for 48 h after transfection.Western blot was used to detect expression of Atg3,Atg5 and Atg7 in RSC96 cells.(3)Autophagy was improved by knockdown HDAC1,the effect of down-regulating Atg3 protein on autophagy function of RSC96 cells cultured in high glucose was examined.RSC96 cells were stochastic divided into p Genesil-1-HDAC1 group and p Genesil-1-HDAC1+p Genesil-1-Atg3 group.RSC96 cells were cultured for 48 h in high glucose,Western blot was used to detect expression of LC3-Ⅰ,LC3-Ⅱ,cleaved caspase-3 and total caspase-3 in RSC96 cells.Results:1.The expression of LC3 and p62 proteins and the structure abnormal in the sciatic nerve of diabetic miceImmunohistochemical analysis indicate that the LC3 and P62 protein in the sciatic nerve of the diabetic mellitus group was lower than that in the normal control group,Luxol Fast Blue showed that myelinated nerve fibers atrophy in the diabetic mellitus group,Electron microscopy indicate that the myelin sheath structure of the diabetic mellitus group indicate stratification and inward folding.2.Effect of high glucose on the expression of LC3-Ⅰ,LC3-and P62 Ⅱin RSC96 cellsWestern blot analysis indicate that the ratio of LC3-Ⅱ/ LC3-Ⅰ in the high glucose group was 1.73 times higher than that in the normal group at 24 hours,and the ratio of LC3-Ⅱ/ LC3-Ⅰ in the high glucose group was 54.65% and 80.58% lower than that in the normal group of 48 hours and 72 hours(P<0.05),and the expression of P62 in the high glucose group was lower than that in the normal glucose group on 24 h,48 h and 72 h,which decreased by 32.67%,58.09% and 48.08%(P<0.05).The results of immunofluorescence indicated that P62 was mainly localized in the cytoplasm of RSC96 cells,and the fluorescence intensity of P62 in the high glucose group was significantly lower than that in the normal glucose group.3.Effect of HDAC on autophagy of RSC96 cells cultured in high glucose(1)Effect of HDAC inhibitor TSA on the expression of LC3-Ⅰ,LC3-Ⅱand P62 proteins in RSC96 cells.RSC96 cells cultured in normal glucose were treated with HDAC inhibitor TSA,Western blot analysis indicate that the ratio of LC3-Ⅱ/ LC3-Ⅰin N+TSA group was 1.74 times higher than that in N+DMSO group(P<0.05),and there was no significant difference in p62 protein expression of the two groups.RSC96 cells cultured in high glucose were treated with HDAC inhibitor TSA,Western blot analysis indicate that the ratio of LC3-Ⅱ/ LC3-in H+TSA group was 1.60 times higher than that in ⅠH+DMSO group(P<0.05),and there was no significant difference in p62 protein expression of the two groups.The results of immunofluorescence indicate that there was no significant difference in the fluorescence intensity of P62 between the TSA group and the DMSO group in RSC96 cells cultured in high glucose.(2)Effect of high glucose treatment of m RNA and protein expression of HDAC family members in RSC96 cells.Transcriptome sequence results indicate that HDAC1,HDAC5 and HDAC8 m RNA in the high glucose group were 3.47 times,2.94 times and 2.93 times higher than that in normal glucose group.Western blot analysis indicated that Compared with the normal glucose group,the expression of HDAC1 protein increased by 2.15 times,1.79 times,and 3.48 times at 24 h,48 h and 72 h(P<0.05),respectively.Compared with the normal glucose group,the expression of HDAC5 protein increased by 1.58 times,1.74 times,and 3.00 times at 24 h,48 h and 72 h(P<0.05),respectively.and there was no significant difference in HDAC8 protein expression.The results of immunofluorescence indicate that HDAC1 was mainly localized in the nucleus of RSC96 cells,and the HDAC1 fluorescence intensity in the high glucose group was higher than that in the normal glucose group.HDAC5 is mainly localized in the nucleus and cytoplasm of RSC96 cells.The fluorescence intensity of HDAC5 in the high glucose group is higher than that in the normal glucose group.(3)Down-regulating of HDAC1 or HDAC5 on the expression of LC3-Ⅰand LC3-in RSC96 cells.sh RNAs targeted to knockout HDAC1 gene was Ⅱtransfected into high glucose cultured RSC96 cells.Western blot analysis indicated that the ratio of LC3-II/ LC3-Ⅰin the p Genesil-1-HDAC1 group was higher than that in the p Genesil-1-NC group,which increased by 2.64 times(P<0.05).sh RNAs targeted to knockout HDAC5 gene was transfected into high glucose cultured RSC96 cells.Compared with the control group,the ratio of LC3-Ⅱ/ LC3-indicateⅠ d no significant difference.4.Effect of HDAC1 on the expression of Atg3,Atg5 and Atg7 expression and autophagy in high glucose cultured RSC96 cells(1)Effect of high glucose on the expression of Atg3,Atg5 and Atg7 in RSC96 cells.Transcriptome sequence indicate showed that Atg3 and Atg5 in the high glucose group of RSC96 cells were reduced by 30.6% and 67.4%,respectively,and Atg7 increased by 1.33 times compared with the normal glucose group.Western blot analysis indicated that compared with the normal glucose group,the expression of Atg3 protein was reduced by 26.70%(P<0.05),the expression of Atg7 protein was increased by 1.72 times(P<0.05),and the expression of Atg5 protein was not significantly different in RSC96 cells.The results of immunofluorescence indicate that Atg3,Atg5 and Atg7 was mainly localized in the cytoplasm of RSC96 cells,and the Atg3 fluorescence intensity in the high glucose group was lower than that in the normal glucose group.The Atg7 fluorescence intensity in the high glucose group was higher than that in the normal glucose group.and there was no significant difference in Atg5 fluorescence intensity.(2)Down-regulation of HDAC1 directly affects Atg3,Atg5 and Atg7 in RSC96 cells.Western blot results indicate that in normal glucose cultured RSC96 cells,the expression of Atg3 protein in the p Genesil-1-HDAC1 group was 1.33 times higher than that of the control group(P<0.05),but there was no significant difference in the expression of Atg5 and Atg7.RSC96 cells cultured in high glucose,the expression of Atg3 protein in the HDAC1 knockdown group was 1.31 times higher than that of the control group(P<0.05),and there was no significant difference in the expression of Atg5 and Atg7.(3)Under the premise of knocking down HDAC1 to improve autophagy,the effect of down-regulating Atg3 on the autophagy function of RSC96 cells cultured in high glucose was examined.Western blot results indicate that compared with the p Genesil-1-HDAC1 transfection group alone,the expression of Atg3 was reduced by 43.94%(P<0.05),and the ratio of LC3-Ⅱ/ LC3-Ⅰwas reduced by 22.93%(P<0.05).The cleaved caspase-3/total caspase-3 ratio increased by 1.91 times(P<0.05).Conclusions:1.In sciatic nerve of diabetic mice,the expression of autophagy-related proteins was decreased.2.The ratio of LC3-Ⅱ/ LC3-and the expression of P62 were reduced Ⅰby high glucose,and high glucose caused a decrease in autophagy.3.The expression of HDAC1 was increased in RSC96 cells culture in high glucose,and the ratio of LC3-/ LC3Ⅱ-Ⅰwas decreased.4.High glucose-induced reduction of Atg3 expression in RSC96 cells,mediates the effects of HDAC1 on LC3-transformation and autophagy... |
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