| Objective: Schwann cells secreted brain-derived neurotrophic factor(BDNF)deficiency is one of the pathogenesis of diabetic peripheral neuropathy.The mammalian target of rapamycin(mTOR)regulates a variety of cellular functions such as proliferation,autophagy,migration and apoptosis.However,whether the mTOR pathway is involved in the reduction of BDNF expression in Schwann cells has not been reported yet.DNA methyltransferase 3a(DNMT 3a)is an important epigenetic regulator whose expression is regulated by the mTOR pathway.However,it is unclear whether mTOR/DNMT 3a is involved in the regulation of BDNF reduction in diabetic peripheral neuropathy.Therefore,in this research,a in vitro cultured rat Schwann cell RSC 96 was used to investigate the effect of mTOR/DNMT 3a on the expression of BDNF in diabetic peripheral neuropathy.Methods:1.Western blot,IF and qPCR were used to detect the effects of high glucose on the expression of mTOR,DNMT 3a and BDNF in RSC 96 cellsRSC 96 cells were randomly divided into three groups: normal glucose group(N),mannitol group(M)and high glucose group(H),which were cultured for 1 day(1 d),2 day(2 d)and 3 day(3 d),respectively.Western blot was used to detect the expression of phospho-mTOR(Ser 2448),mTOR,DNMT 3a and BDNF in RSC 96 cells;immunofluorescence(IF)was used to investigate the expression of DNMT 3a and BDNF in RSC 96 cells;qPCR was used to detect the mRNA expression of BDNF.2.Western blot and qPCR were used to detect the effects of inhibition of DNMT 3a on the expression of BDNF in RSC 96 cells cultured with high glucoseIn the high glucose environment,DNMTs inhibitor 5-azacittosine(5-Aza)was used to stimulate RSC 96 cells or shRNA plasmid targeting DNMT 3a gene was transfected into RSC 96 cells.Western blot was used to detect DNMT 3a and BDNF expression;qPCR was used to detect BDNF mRNA.3.Western blot,IF and qPCR were used to detect the effects of inhibition of mTOR on the expression of DNMT 3a and BDNF in normal glucose cultured RSC 96 cellsRSC 96 cells were treated with mTOR pathway inhibitor Torin 1 in normal glucose conditions for 0 h,2 h,4 h,6 h,12 h and 24 h,respectively.Western blot was used to detect phospho-mTOR(Ser 2448),mTOR,phosphoS6 K 1(Thr 389),S6 K 1,phospho-4E-BP 1(Thr 37/46),4E-BP 1,DNMT 3a and BDNF protein expression;IF was used to detect DNMT 3a and BDNF expression;qPCR was used to detect BDNF mRNA expression.4.Western blot,IF and qPCR were used to detect the effects of activation of mTOR on the expression of DNMT 3a and BDNF in high glucose-induced RSC 96 cellsThe cells were treated with mTOR pathway activator MHY 1485 in high glucose-induced RSC 96 cells for 0 h,2 h,4 h,6 h,12 h and 24 h,respectively.Western blot was used to detect phospho-mTOR(Ser 2448),mTOR,phospho-S6 K 1(Thr 389),S6 K 1,phospho-4E-BP 1(Thr 37/46),4E-BP 1,DNMT 3a and BDNF protein expression;IF was used to detect DNMT 3a and BDNF expression;and qPCR was used to detect BDNF mRNA expression.Results:1.Effect of high glucose on the expression of mTOR,DNMT 3a and BDNF in RSC 96 cellsWestern blot analysis showed that the phospho-mTOR(Ser 2448)protein in the high glucose group was significantly lower than that in the mannitol group on 2nd and 3rd,which decreased by 40.62%(P<0.05)and 56.56%(P<0.01),respectively.There was no significant difference in the total mTOR expression between the two groups.High glucose significantly upregulated DNMT 3a expression.Western blot results showed that the DMNT3 a protein in the high glucose group increased by30.29 % and39.33 % on 2nd and 3rd(P<0.01),respectively,compared with the mannitol control group.IF results showed that DNMT 3a was localized in the nucleus of RSC 96 cells,and the fluorescence intensity of DNMT 3a was significantly enhanced in the high glucose group compared with the mannitol group.Conversely,high glucose down-regulated the expression of BDNF in RSC 96 cells.Western blot results showed that compared with the mannitol control group,the expression of Pro-BDNF protein in the high glucose group decreased significantly on the 2nd and the 3rd,respectively,which were decreased by 12.83% and 24.89 %(P<0.05),the expression of mature BDNF protein in the high glucose group decreased significantly on the 2nd and the 3rd,respectively,which were decreased by25.9% and 40.5 %(P<0.05).The qPCR results showed that the ratio of BDNF mRNA in the high glucose group was reduced by23.25 % on the second day(P<0.01),compared with mannitol group.IF results showed that BDNF was mainly located in the cytoplasm and nucleus of RSC 96 cells,and the fluorescence intensity of BDNF in the high glucose group was significantly lower than that in the mannitol control group.2.Inhibition of DNMT 3a on the expression of BDNF in RSC 96 cellsHigh glucose cultured RSC 96 cells were treated with 5-Aza for 2 days.Western blot results showed a 21.57% decrease in DNMT 3a and a 22.94% increase in Pro-BDNF and a 53.31% increase in mature BDNF in the 5-Aza treatment group compared with the DMSO group(P<0.05),the difference was statistically significant.qPCR results showed a 64.22% increase in BDNF mRNA expression compared to the DMSO group.Similarly,shRNA targeted to knockdown DNMT 3a gene were transfected into high glucose cultured RSC 96 cells.Western blot results showed that DNMT 3a was decreased by 37.44%,Pro-BDNF expression was increased by 30.42 % and mature BDNF expression was increased by 91.05% compared with the empty plasmid control group.The results of qPCR showed that the expression of BDNF mRNA in the DNMT 3a shRNA group was increased by 33.08% compared with the empty plasmid control group.3.Effect of inhibition of mTOR on the expression of DNMT 3a and BDNF in normal glucose cultured RSC 96 cellsTorin 1 was given to stimulate normal glucose cultured RSC 96 cells for 0 h,2 h,4 h,6 h,12 h,and 24 h,respectively.Compared with normal glucose 0 h,phospho-mTOR(Ser 2448),phospho-S6 K 1(Thr 389),phospho-4E-BP 1(Thr 37/46)decreased significantly at 2 h,DNMT 3a began to increase at 2 h,and BDNF began to decrease at 6 h.The Western blot results of Torin 1 stimulation for 24 h showed that DNMT 3a was 2.01 times higher,Pro-BDNF was 2.43 times lower than 0 h and the mature BDNF was 2.97 times lower than 0 h(P<0.01).The IF results indicated that the DNMT 3a fluorescence intensity was increased and the BDNF fluorescence intensity was weakened in the Torino1 treatment group compared with the DMSO control group.The qPCR results indicated that the expression of BDNF mRNA in the Torin 1 group was decreased by 72.02% compared with the DMSO group.4.Effect of activation of mTOR on the expression of DNMT 3a and BDNF in high glucose-induced RSC 96 cellsThe high-glucose cultured RSC 96 cells were stimulated with mTOR pathway activator MHY 1485 for 0 h,2 h,4 h,6 h,12 h,and 24 h,respectively.Compared with high glucose 0 h,phospho-mTOR(Ser 2448),phospho-S6 K 1(Thr 389),phospho-4E-BP 1(Thr 37/46)increased significantly at 4 h,DNMT 3a began to decrease at 6 h,and BDNF began to increase at 6 h.Western blot results showed that DNMT 3a was reduced by 2.12 times,Pro-BDNF was increased by2.31 times and mature BDNF was increased by 2.15 times after MHY 1485 stimulation for 24 hours compared with high glucose 0 h.The IF results indicated that the fluorescence intensity of DNMT 3a was decreased and the BDNF fluorescence intensity was increased in the MHY 1485 treated group compared with the DMSO control group.qPCR results showed a 121 % increase in BDNF mRNA in the MHY 1485 group compared to the DMSO group.Conclusions:1.The expression of phospho-mTOR(Ser 2448)was decreased,DNMT 3a expression was increased and BDNF expression was decreased in high glucose-induced RSC 96 cells.2.Down-regulation of DNMT 3a can increase the expression of BDNF in RSC 96 cells.3.In normal glucose cultured RSC 96 cells,the expression of DNMT 3a was increased and BDNF was decreased by administration of the mTOR pathway inhibitor Torin 1.4.High glucose-induced RSC 96 cells were given mTOR pathway activator MHY 1485,DNMT 3a expression was decreased,and BDNF expression was increased. |
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