The Mechanism Of ERK Pathway In High Glucose-induced Myelination Impairment And The Protective Effects Of Quercetin, Hirudin, Cinnamaldehyde And The Combinations Of Them

【Objective】The aim of the present study is to investigate the mechanism of ERK pathway in high glucose-induced myelination impairment of peripheral nervous system and the protective effects of quercetin, hirudin, cinnamaldehyde, and the combinations of them at the cellular and molecular level.【Methods】1. Explants-culture method, general hemi-explants-culture method and improved enzyme digestion combined with explants-culture method were adopted to culture Schwann cells(SCs) respectively. The purity of SCs was identified by S-100 immunofluorescence staining. The differences of the three methods in the total number and purity of cells obtained were compared.2. SCs were exposed to different concentrations of high glucose (50 mM,75 mM, 100 mM,125 mM, and 150 mM) for 24h,48h,72h and 96h, respectively. Cell viability and nuclear morphology were evaluated by Counting Kit-8 (CCK-8) assay and Hoechst assays, respectively. The expressions of P0, p-ERK and ERK protein and P0, Krox-20, MAG mRNA were detected by Western blot and quantitative real-time PCR analysis.3. SCs were exposed to quercetin(Q, different concentrations), hirudin(H), cinnamaldehyde(C), combination of quercetin and hirudin (QH), combination of quercetin and cinnamaldehyde (QC), combination of hirudin and cinnamaldehyde (HC), combination of quercetin, hirudin and cinnamaldehyde (QHC) and U0126 in the presence of 50 mM high glucose for 72h, respectively. Cell viability and differentiation were evaluated by CCK-8 assay and periaxin immunofluorescence staining, respectively. The expressions of P0, p-ERK, ERK, p-c-Jun, c-Jun, NICD protein and P0, Krox-20, MAG, Notchl, Jaggedl mRNA were detected by Western blot and real-time quantitative PCR analysis. The secretion of CNTF was determined by ELISA.4. Dorsal root ganglion neurons(DRGn) were dissociated by mixed enzyme solutions step by step and cell purity was identified by MAP-2 immunofluorescence staining. The purified SCs were added into the DRGn to build the co-cultures system. The formation of the myelin was detected by MBP immunofluorescence staining. DRGn/SCs cocultures were exposed to 50 mM high glucose for 3d,5d and 7d, respectively. The expressions of MBP, MAG, p-ERK and ERK protein and MBP, MAG mRNA were detected by Western blot and real-time quantitative PCR analysis.5. DRGn/SCs cocultures were exposed to Q, H, C, QH, QC, HC, QHC and U0126 in the presence of 50 mM high glucose for 7d, respectively. The number and length of the myelin segments were evaluated by MBP immunofluorescence staining. The expression of and location p-ERK were detected by MAG and p-ERK immunofluorescence double staining. The expressions of MBP, MAG, p-ERK and ERK protein and MBP, MAG mRNA were detected by Western blot and real-time quantitative PCR analysis.【Results】1. The total number of cells and SCs purity of improved method group were significantly higher than hemi-explants-culture method group and explants-culture method group, respectively(P<0.01).2. CCK-8 assay and Hoechst staining showed that high glucose inhibited SCs proliferation and increased apoptosis ratio in concentration dependent manner at 72h. Western blot and real-time quantitative PCR analysis revealed that compared with control(CON) group P0 protein expression and P0, Krox-20, MAG mRNA expressions were significantly reduced while phosphorylation level of ERK was increased obviously in SCs exposed to 50 mM high glucose for 72h(P<0.01).3. The effects of quercetin on SCs exposed to 50 mM high glucose①ompared with CON group, the cell proliferation ratio, periaxin positive cell percentage and CNTF secretion level were decreased significantly in high glucose (HG) group(P<0.01). The cell proliferation ratio, periaxin positive cell percentage and CNTF secretion level in Q groups and U0126 group were increased markedly than HG group(P<0.01) and the effects of quercetin were dose dependent (P<0.01).② Compared with CON group P0 protein expression and P0, Krox-20, MAG mRNA expressions were reduced significantly in HG group(P<0.01) while those myelin protein and gene expressions were increased obviously in Q groups and U0126 group than HG group(P<0.01). The effects were positively correlated with quercetin concentration (P<0.01).③ The phosphorylation level of ERK and c-Jun, NICD protein expression, Notch1 and Jagged1 mRNA were increased significantly in HG group compared to CON group(P<0.01) while those protein and gene expressions were reduced remarkably in Q groups and U0126 group compared to HG group(P<0.01). The effects were negatively correlated with quercetin concentration (P<0.01).4. The effects of quercetin, hirudin and cinnamaldehyde combinations on SCs exposed to 50 mM high glucose① The cell proliferation ratio, periaxin positive cell percentage and CNTF secretion level in Q, H, C, QH, QC, HC, QHC and U0126 group were increased markedly compared with HG group(P<0.01) and the above parameters of QHC group were increased significantly than the other treatment groups (P<0.01). The periaxin positive cell percentage and CNTF secretion level of combination of two monomers were increased obviously than the single monomer (P<0.01or P<0.05) and Q group were higher than H and C group (P<0.01).② P0 protein expression and P0, Krox-20, MAG mRNA expressions in Q, H, C, QH, QC, HC, QHC and U0126 group were increased significantly compared with HG group(P<0.01) and those myelin protein and gene expressions were increased obviously in QHC group than the other treatment groups (P<0.01). The above protein and gene expressions of combination of two monomers were increased obviously than the single monomer (P<0.01or P<0.05) and Q group were higher than H and C group (P<0.01).③ The phosphorylation level of ERK and c-Jun, NICD protein expression, Notchl and Jaggedl mRNA were decreased significantly in Q, H, C, QH, QC, HC, QHC and U0126 group compared with HG group(P<0.01) and those protein and gene expressions in QHC group were lowest among treatment groups (P<0.01). The above protein and gene expressions of combination of two monomers were decreased obviously than the single monomer (P<0.01or P<0.05) and Q group were lower than H and C group (P<0.01 or P<0.05).5. MAP-2 immunofluorescence staining showed that the purity of DRGn was 97.39±0.09%. Typical myelin fragments were visible by MBP immunofluorescence staining in DRGn/SCs cocultures.6. Compared with CON group, the MBP and MAG protein and mRNA expressions were decresaed significantly in DRGn/SCs cocultures exposed to 50 mM high glucose for 7d (P<0.01) while the phosphorylation level of ERK was increased obviously (P<0.01).7. The effects of quercetin on DRGn/SCs cocultures exposed to 50 mM high glucose①Compared with CON group, the number and length of the myelin segments in HG group were significantly reduced (P<0.01) while compared with HG group, the number and length of the myelin segments in Q groups and U0126 group were significantly increased (P<0.01). The effects of quercetin were dose dependent (P<0.01).② Compared with CON group, the MBP and MAG protein and mRNA expressions in HG group were significantly reduced (P<0.01) while compared with HG group, those myelin protein and gene expressions in Q groups and U0126 group were significantly increased (P<0.01). The effects of quercetin were dose dependent (P<0.01).③ Compared with CON group, the phosphorylation level of ERK and p-ERK expression in HG group were significantly increased (P<0.01) while compared with HG group, the phosphorylation level of ERK and p-ERK expression in Q groups and U0126 group were significantly decreased (P<0.01). The effects of quercetin were dose dependent (P<0.01).8. The effects of quercetin, hirudin and cinnamaldehyde combinations on DRGn/SCs cocultures exposed to 50 mM high glucose①Compared with HG group, the number and length of the myelin segments in Q, H, C, QH, QC, HC, QHC and U0126 group were significantly increased (P<0.01) and QHC group were higher than the other treatment groups(P<0.01). The number and length of the myelin segments in combination of two monomers were higher than the single monomer (P<0.01or P<0.05) and Q group were lower than H and C group (P<0.05).②Compared with HG group, the MBP and MAG protein and mRNA expressions in Q, H, C, QH, QC, HC, QHC and U0126 group were significantly increased (P<0.01) and QHC group were higher than the other treatment groups(P<0.01). Those myelin protein and gene expressions in combination of two monomers were higher than the single monomer (P<0.01or P<0.05) and Q group were higher than H and C group (P<0.05).③ompared with HG group, the phosphorylation level of ERK and p-ERK expression in Q, H, C, QH, QC, HC, QHC and U0126 group were significantly decreased (P<0.01) and QHC group were lower than the other treatment groups(P<0.01). The phosphorylation level of ERK and p-ERK expression in combination of two monomers were lower than the single monomer (P<0.01or P<0.05) P-ERK expression in QH group was lower than Q and H group (P<0.01 or P<0.05). The phosphorylation level of ERK in HC group was lower than H and C group (P<0.01). The phosphorylation level of ERK and p-ERK expression in Q group were lower than H and C group (P<0.01).【Conclusion】1. High glucose can inhibit the SCs proliferation and differentiation, reduce the CNTF secretion, decrease the myelin protein and genes expressions including P0, MAG and Krox-20 through inducing the activation of ERK pathway and its downstream targets c-Jun and Notch pathway, thus leading to the SCs myelination impairment.2. Quercetin, hirudin, cinnamaldehyde and the combinations of them can promote the SCs proliferation and differentiation, increase the CNTF secretion, upregulate the myelin protein and genes expressions including P0, MAG and Krox-20 through inhibiting activation of ERK pathway and its downstream targets c-Jun and Notch pathway, thus attenuating the SCs myelination impairment induced by high glucose.3. High glucose can downregulate the myelin protein and genes expressions including MBP and MAG, reduce the number and length of the myelin segments in DRGn/SCs cocultures by activating the ERK pathway in SCs, thus leading to the myelination impairment of DRGn/SCs cocultures.4. Quercetin, hirudin, cinnamaldehyde and the combinations of them can upregulate the myelin protein and genes expressions including MBP and MAG, increase the number and length of the myelin segments in DRGn/SCs cocultures by activating the ERK pathway in SCs, thus alleviating the myelination impairment of DRGn/SCs cocultures induced by high glucose.【Innovation】The present study is designed to investigate the mechanism of ERK pathway in high glucose-induced myelination impairment of peripheral nervous system and the protective effects of quercetin, hirudin, cinnamaldehyde and the combinations of them at the cellular and molecular level, which hasn’t been reported home and abroad. The present research can provide the theory and experimental basis for Jinmaitong application in DPN prevention and treatment.