Combination of Quercetin, Hirudin and Cinnamaldehyde Promotes Schwann Cell Differentiation and Myelination against High Glucose by Inhibiting ERK Signaling Pathway

Objective:To investigate the therapeutic and synergistic effects of QHC(combination of quercetin(Q),hirudin(H)and cinnamaldehyd(C))on Schwann cell differentiation and myelination against high glucose(HG)induced injury.Methods:Primary-culture Schwann cells exposed to HG(50 mmol/L)for 72 h and Schwann cell–dorsal root ganglion(DRG)neuron cocultures exposed to HG(50 mmol/L)for 7 days were employed as in vitro model of diabetic neuropathy.The cells were randomly divided into 10 groups:control(CON,25 mmol/L glucose),HG(50 mmol/L glucose),HG plus 10μmol/L quercetin(Q),HG plus 0.04 IU/mL hirudin(H),HG plus 100 nmol/L cinnamaldehyd(C),HG plus 10μmol/L quercetin and 0.04 IU/mL hirudin(QH),HG plus 10μmol/L quercetin and 50 nmol/L cinnamaldehyd(QC),HG plus 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd(HC),HG plus 10μmol/L quercetin,0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd(QHC)or 10μmol/L U0126.Cell differentiation was evaluated by periaxin immunofluorescence staining.The protein expression levels of myelin protein zero(P0),myelin basic protein(MBP),myelin-associated glycoprotein(MAG),extracellular signal-regulated kinase(ERK),p-ERK,p-c-Jun,c-Jun,notch intracellular domain(NICD)and the mRNA expression levels of P0,MBP,MAG,Krox-20,Notch1 and Jagged1 were detected by Western blotting and real-time quantitative PCR analysis.The secretion of ciliary neurotrophic factor(CNTF)was determined by enzyme-linked immunosorbent assay(ELISA).The number and length of the myelin segments were evaluated by MBP immunofluorescence staining.The expression and the location of p-ERK in cocultures were detected by MAG and p-ERK immunofluorescence double staining.Results:Co-treatment with Q,C,H and their combination promoted Schwann cell differentiation,increased CNTF secretion,up-regulated the protein and m RNA expressions of myelin,and increased the number and length of the myelin segments(P<0.01 or P<0.05).In particular,the combination therapy of Q,H and C was superior to the respective monotherapy(P<0.01).Combination therapy of QHC exhibited higher inhibitory activities for ERK signaling related molecules than each monomer or the combination of the two monomers(P<0.01).Conclusions:QHC combination yielded synergy in promoting Schwann cell differentiation and myelination and the protective effect may involve in the inhibition of ERK signaling pathway,providing scientific evidence for better understanding of combination of Q,H and C in clinical applications.