Pyrroloquinoline Quinone Protected Autophagy-dependent Apoptosis Induced By Mono(2-Ethylhexyl)Phthalate In INS-1 Cells

Objective: Di-2-ethylhexyl phthalate(DEHP)is an emerging endocrine disrupting compound and widely used as a plasticizer in daily necessities,such as personal care products,food packaging materials,Medical equipment,children's toys and building decoration materials.Mono-(2-ethylhexyl)phthalate(MEHP),as the main active product of DEHP in the metabolic process of organisms,has a damaging effect on human cells.Studies have shown that DEHP damages islet ? cells,which in turn causes autophagydependent apoptosis and induces insulin resistance(IR),which ultimately leads to type 2 diabetes.However,the mechanism of MEHP damage to islet ? cells has not been fully studied.Pyrroloquinoline quinone(PQQ)has the effects of preventing oxidative stress,reducing free radical levels and lipid peroxidation(LPO),but whether it can inhibit MEHP-induced islet ?-cell apoptosis has not been confirmed.Therefore,in this study,rat islet ?(INS-1)cells were used as experimental objects,in order to prove whether MEHP can cause autophagy-dependent apoptosis in INS-1 cells and explore how PQQ can protect in this process effect.Methods: After treatment of INS-1 cells with different concentrations(0,6.25,12.5,25 ?M)of MEHP and 5 m M PQQ + 25 ?M MEHP,western blot was used to detect changes in the expression of LC3,p62,cathepsin D,cytochrome c,caspase 9,3 and other related proteins;DCFH-DA fluorescent probes were used to detect changes in intracellular ROS content;Detection kits were used to measure intracellular GSH,MDA levels and SOD activities to reflect changes in oxidative stress;The changes of lysosomal membrane stability,mitochondrial transmembrane potential and apoptosis of INS-1 cells were observed after Ao staining,JC-1 staining and Tunel staining with Olympus BX-63 Fluorescence microscope;After 10 m M NAC pretreatment for 1 h,the relationship between ROS production and autophagy,Lysosomal membrane permeability(LMP),Mitochondrial outer membrane permeabilization(MOMP)and apoptosis was examined in MEHP treated cells;After pretreatment with 1 m M 3MA for 2 h,the relationship between autophagy and LMP,MOMP,and apoptosis was detected in MEHP-treated cells;After pretreatment with 100 ?M pepstatin A for 4 h,the effects of cathepsin D on MEHPinduced MOMP and apoptosis were examined.Results: With the increase of MEHP exposure concentration,ROS production increased and the degree of oxidative stress deepened;The expressions of LC3,p62,cathepsin D,cytochrome c,caspase 9,3 and other related proteins were up-regulated;LMP,MOMP,and apoptosis all increased significantly.And after PQQ action,above index all have apparent improvement.After the addition of NAC,the expression of autophagy marker proteins LC3 and p62 was down-regulated,the release of LMP and cathepsin D was decreased,the release of MOMP and cytochrome c was decreased,the expression of apoptosis marker proteins caspase 9 and 3 was down-regulated,and TUNEL showed that apoptosis was also alleviated.In the same way,after 3MA and pepstatin A pretreatment,the relevant detection indicators were also significantly improved.Conclusion: MEHP can induce autophagy-dependent apoptosis of INS-1 cells by oxidative stress-lysosomal mitochondrial axis,while PQQ can reduce oxidative stress and inhibit cell apoptosis by lysosomal mitochondrial axis.