Study Of The Role Of Polydatin On Methylglyoxal Induced Schwann Cell Damage

BackgroundRepair of refractory wounds is one of the most important problems in plastic surgery.Diabetic peripheral neuropathy?DPN?and its nutrient vessels dysfunction are common reasons for refractory wounds.Hyperglycemia-induced formation of advanced glycation end product?AGE?plays a vital role in the pathogenesis of DPN.Methylglyoxal?MGO?is the potent precursor of AGE,which is significantly increased under diabetic condition and is closely related to the development of DPN by injuring the cellular constitutent of the peripheral nerve system.Polydatin?PD?is a major active component isolated from Polygonum cuspidatum Sieb.et Zucc and has extensive pharmacological activities,especially strong effects on inhibiting oxidation and inflammation.PD has been shown to protect from injury of ischemia reperfusion,shock,heart failure and renal and cardiovascular dysfunction of experimental diabetic animals;however,it remains unclear whether PD has protective effect on DPN and what is the possible mechanism behind this,which deserves further investigation.ObjectiveIn the present study,rat Schwann cell line-RSC96 cells were exposed to MGO to simulate DPN in vivo.Before the exposure to MGO,the cells were pretreated with PD,and PD-mediated neuroprotection and relevant mechanism was investigated.The experiment is carried out from the following aspects:1.To explore the damage effects of MGO on RSC96 cells2.To clarify the protective effects of PD on RSC96 cells injury induced by MGO within a short time?6 h?3.To illuminate the protective effects of PD on RSC96 cells injury treated with long time MGO?24 h?4.To study the related mechanism of PD on cell protectionMethods1.RSC96 cells were maintained in DMEM with high glucose and 10%fetal bovine serum for 48 h and incubated for 12 h in serum-free medium,then the cells were treated with different concentrations of MGO?1000 ?M,1200 ?M,1400 ?M,1600?M,1800 ?M?for 6 h and 24 h.Then,CCK-8 was used to measure the cell viability and find the value of half inhibitory concentration?IC50?of MGO.2.RSC 96 cells were maintained in DMEM with high glucose and 10%fetal bovine serum for 48 h,then the cells were pretreated with different levels of PD?50 ?M,100 ?M,150 ?M?diluted with serum-free medium for 12 h.Subsequently,MGO in the concentration of IC50 was added into the medium for 6 h and 24 h.PD mediated neuroprotection was explored through CCK-8 assay,lactate dehydrogenase?LDH?release measurement,mitochondrial membrance potential measurement,ATP measurement,cell apoptosis assessment.3.The protein expression of glycation end product receptor?RAGE?,glyoxalase 1?GLO1?,Nrf2/Keapl were detected by Western blot assay to analyze the possible mechanism of PD.Results1.The assessment of RSC96 cells injury induced by MGO The viability of RSC96 cells treated with MGO was significantly decreased in a dose-dependent manner.The IC50 value of MGO was around 1650 ?M in 6 h and 24 h.There was also a significant abnormality in morphology of the cells after MGO treatment.2.PD protects RSC96 cells against MGO-induced cytotoxicity The cells pretreated with PD for 12 h led to a significant increase in cell viablility through CCK-8 assay.Besides,LDH levels increased obviously after exposure to MGO alone;Pretreatment with PD at a concentration of 50-200 ?M markedly attenuated the MGO-mediated increase in LDH release.These results demonstrated that preconditioned with PD produces a neuroprotective effect in the context of MGO-induced cytotoxicity.3.Treatment with PD inhibits MGO-induced oxidative toxicity Production of reactive oxygen species?ROS?was much higher in cells treated with MGO alone than in control cells;Likewise,pretreated with PD significantly reduced ROS production following MGO exposure.4.Treatment with PD reverses MGO-induced mitochondrial membrance potential dissipation MGO could induce mitochondrial dysfunction of RSC96 cells,characterized by the decrease in mitochondrial membrance potential and ATP levels.Pretreatment with PD restored the changes in mitochondrial membrance potential and promoted the synthesis of ATP.5.Treatment with PD attenuates MGO-induced RSC96 cell apoptosis Compared with the normal control group,the numbers of apoptotic cells clearly increased after MGO treatment for 6 h and 24 h byMuse analysis,whereas the levels of apoptotic cells were attenuated after pretreatment with PD.6.Effects of AGE-RAGE pathway and Nrf2/GLO1 pathway in PD protection Through Western blot,we found that under MGO treatment condition,PD could reduce RAGE expression to repress the AGE-RAGE interaction.In addition,PD also increase Nrf2 expression,decrease Keap1 expression as well asreversed GLO1?the target gene of Nrf2?downregulation induced by MGO to exert neuroprotection effects.Conclusions1.The damage effects of MGO on RSC96 cells were in a concentration-dependent manner and the IC50 value was 1650 ?M.2.The damage of MGO on RSC96 cells were characterized by oxidative toxicity and mitochondrial dysfunction,which were consistent with the Schwann cells injury of DPN₃.PD could reverse MGO-induced oxidative toxicity and mitochondrial dysfunction,which lead to a significant increase in cell viablility and decrease in apoptosis.4.The mechanism of PD protection may be involved in the inhibition of AGE-RAGE pathway and the activation of Nrf2/GLO1 pathway.