Hesperetin induces glyoxalase 1 enhancement in SH-SY5Y cells cultured with high glucose via Nrf2/ARE activation

OBJECTIVE To investigate the neuroprotective effects of hesperetin on central neurons under chronic high glucose,and the relationship to glyoxalase 1(Glo-1),a cytoprotective enzyme.METHODS The human neuroblas⁃toma SH-SY5Y cells were divided into 5 groups:normal glucose,high glucose(HG),HG plus low,middle,or high concentra⁃tion of hesperetin(1,5,25μmol·L^-1).After treatment for 72 h,neuron damages,Glo-1 expressions and functions,as well as Nrf2/ARE pathway and its regulating mechanisms were examined.RESULTS Hesperetin increased cell viability and decreased lactate dehydrogenase release,which was accompanied by the elevated activity,protein,and mRNA levels of Glo-1 as well as the enhanced Glo-1 functions in SH-SY5Y cells cultured with HG.Moreover,hesperetin activated Nrf2/ARE pathway as evidenced by the raised Nrf2 and p-Nrf2 levels in nucleus and up-regulation of γ-glutamycysteine synthase(γ-GCS),a well-known target gene of Nrf2/ARE pathway.Nevertheless,pretreatment with a PKC inhibitor(Go 6983)or an Akt inhibitor(MK-22062HCl,reflecting GSK-3β activation)abolished the effect of hesperetin on protein expressions of Glo-1 and γ-GCS.CONCLUSION Hesperetin exerted the neuroprotection by promoting Glo-1 function in central neurons in long-term HG condition,which was mediated by activation of Nrf2/ARE pathway;moreover,the increased Nrf2 phosphorylation and nuclear translocation mediated by PKC activation and/or GSK-3β inhibition were involved in the activation of Nrf2/ARE pathway by hesperetin.

Neuroprotection of quercetin on central neurons against chronic high glucose through enhancement of Nrf2/Glo-1 mediated by phosphorylation regulation

OBJECTIVE To investigate the neuroprotective effects of quercetin on central neurons against chronic high glucose in central neurons,in relation to Nrf2/ARE/Glo-1 activation.METHODS SH-SY5Y cells were cultured with high glucose(HG,70 mmol·L^(-1)),4-fold of the normal glucose(17.5 mmol·L^(-1)).Quercetin was set three concentrations(5,10,20μmol·L^(-1)),with Nrf2 activator sulforaphane(SFN)as a positive group(2.5μmol·L^(-1)).After 72 h,cells were collected for glyoxalase 1(Glo-1)activity and GSH level were by spectrophotometry;advanced glycation end-products(AGEs)as well as nuclear Nrf2 and p-Nrf2 levels by immunofluorescence;Glo-1,γ-glutamycysteine synthase(γ-GCS),Nrf2 and p-Nrf2 protein levels by Western blotting,and Glo-1 andγ-GCS m RNA levels by real-time qP CR.RESULTS Quercetin increased the cell viability of SH-SY5Y cells,and upregulated the levels of Glo-1 activity,protein,and m RNA in SH-SY5Y cells cultured with HG,accompanied by the elevated levels of glutathione,a cofactor of Glo-1 activity,and the reduced levels of AGEs.Meanwhile,quercetin could increase p-Nrf2 and Nrf2 levels in nucleus as well as p-Nrf2 levels in cytosol of SH-SY5Y cells exposed to chronic HG,accompanied by the elevated protein expression and m RNA levels ofγ-GCS,a known target gene of Nrf2/ARE signaling.Moreover,a PKC activator or a p38MAPK inhibitor pretreatment could significantly increase the protein expression ofγ-GCS in HG condition,but an alkylating agent for sulfydryl of cysteine in Keap 1,a negative regulator of Nrf2,pretreatment only showed an increased tendency ofγ-GCS protein,compared with without pretreatment;however,after pretreatment with those tool drugs,co-treatment with quercetin and HG had similar results to those of single tool drug pretreatment followed by HG exposure.CONCLUSION Firstly,quercetin can enhance Glo-1 function in central neurons,which is mediated by activation of Nrf2/ARE pathway,then exerts the neuroprotection against HG induced damage;moreover,PKC and p38 MAPK pathways may be involved in Nrf2 inactivation in chronic HG condition.