| Objective To investigate the mechanisms by which quercetin protects pancreatic β cells from palmitate-induced apoptosis,and explore whether autophagy-lysosomal pathway is involved in this process.Methods The rat insulinoma β cell line INS-1 was stimulated with 0.5 m M palmitate(PA)for 24 hours to establish a pancreatic β cell apoptosis model in vitro.At the same time,different concentrations of quercetin(10 μM,25 μM,50 μM)were used for Intervention.After that,the CCK8 assay was used to detect cell viability,TUNEL staining was carried out to detect cell apoptosis,and western blot was performed to detect the protein expression levels of LC3 and P62.Based on the above experiment,we evaluated the effect of quercetin on the apoptosis and autophagy of pancreatic β cells;Pancreatic islets were isolated from Wistar rats and stimulated with 0.5 m M PA for 24 hours.At the same time,50 μM quercetin was used for intervention.Western blot was used to detect the protein expression levels of cleaved caspase 3,LC3 and P62,and to evaluate the effect of quercetin on PA-induced pancreatic β apoptosis and autophagy dysfunction.Atg5 small interfering RNA was used for INS-1 cell treatment to block autophagy or autophagic flux,and TUNEL staining was performed to detect cell apoptosis.Based on the above experiment,we investigated whether quercetin protects lipotoxic-induced pancreaticβ apoptosis by affecting autophagy or autophagic flux.To further explore this,we used two more robust methods to assess autophagic flux: m Cherry-GFP-LC3 analysis(LC3 dual fluorescent adenovirus m Cherry-GFP-LC3 B was transfected into INS-1 cells,and the number of yellow autophagosomes and red autophagolysosome spots were counted under confocal laser)and net LC3 II net flux analysis(Western blot determination of the difference in LC3 II expression before and after chloroquine treatment).Then we evaluated the effect of PA treatment and quercetin intervention on the autophagic flux of pancreatic β cells.Further study of the mechanism,using TFEB immunofluorescence localization,western blot detection of cytoplasmic and nuclear TFEB expression,RT-PCR detection of the TFEB target gene(BECN1,MCOLN1 and UVRAG),we investigated the effects of PA and quercetin on the pathway of lysosomal biogenesis;We also detected the effect of PA and quercetin on lysosomal leakage,function,autophagosome and lysosome fusion process by Lysotracker staining(lysosomal fluorescent probe),co-localization of lysosome(Lysotracker)and autophagosome(LC3),and co-localization of lysosome(Lamp2)and lysosomal enzyme cathepsin D under confocal laser,measurement of lysosomal enzyme cathepsin B enzyme activity and the number of the lysosomal enzyme cathepsin B/D punctate staining,to clarify whether quercetin affect autophagy flux by affecting lysosome biogenesis or lysosomal function.Autophagy was blocked by 3MA,LC3 expression was detected by Western blot and Lysotracker staining was used to detect lysosomal function to determine whether PA and quercetin affect lysosomal function through autophagy.Determine the number of reactive oxygen species in different treatment groups by DCFHDA staining,and use NAC to block the production of reactive oxygen species to verify whether PA can cause lysosome dysfunction and blockade of autophagic flux by generating reactive oxygen species,and whether this process is involved in the protective effect of quercetin on lipotoxicity-induced pancreatic β cells apoptosis.Result We found that quercetin treatment partially reduced palmitate-induced β cell apoptosis,but this protective effect was abolished by pharmacologic inhibition of autophagy,as well as by silencing of key autophagy gene.m Cherry-GFP-LC3 and net LC3 II flux analysis showed that PA treatment can block the autophagic flux of pancreatic β cells,while quercetin can improve it.Further mechanism studies found that PA can promote TFEB nuclear translocation and its mediated lysosome biogenesis,but quercetin has no further effect on PA-lysosomal biogenesis.Lysotracker staining found that quercetin can improve PA-induced lysosomal dysfunction,increase lysosomal enzyme activity and fusion of autophagosomes and lysosomes,thereby improving autophagic flux,and studies also found that long-term blockade to autophagy flux under PA treatment can lead to increased lysosomal membrane permeability(LMP)and release of lysosomal enzyme Cathepsin D,which in turn activates pancreatic β-cell apoptosis,while quercetin treatment can partially alleviate cell apoptosis mediated by lysosomal LMP.Finally,the study found that PA caused lysosomal dysfunction and autophagy flux blockade depends on the increase in peroxide production,and quercetin can reduce lysosomal dysfunction by reducing oxidative stress and improving autophagy flux,and subsequent lysosomal LMP,thereby alleviating PA-inducedβ cell apoptosis.Conclusion Our data show that impaired autophagic flux is a major factor contributing to βcell apoptosis in T2 D.Lipotoxicity causes blockage of autophagic flux by oxidative stressinduced lysosome dysfunction.This leads to accumulation of defective lysosomes,resulting in LMP and release of cathepsins into cytosol with subsequent induction of β cell apoptosis.Quercetin could improve lysosomal function,and further improve autophagic flux and LMP,which directly contributes to its anti-apoptotic effects.These findings provide new evidence regarding anti-apoptosis mechanism of quercetin in the treatment of T2 D,and also highlight autophagic flux and lysosomal regulation for the prevention and/or treatment of T2 D.Figure7;Table4;Reference 133... |
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