Protective Effect Of Quercetin On The ER Stress-related Apoptosis Induced By Thapsigargin In Macrophages

Background and purposeEndoplasmic reticulum（ER） stress has been attracting considerable attention since ERstress triggers such disorders as inflammation and apoptosis. ER stress occurs in macropha-ge-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosisand subsequent plaque necrosis. Therefore, signaling pathways that alter ER stress-inducedapoptosis may affect advanced atherosclerosis. Here the purpose of the present study is toinvestigate the effects of quercetin（Que） on ER stress-related prolifera-tion and apoptosisinduced by thapsigargin（TG）, the expression of glucose-regulated protein78（GRP78） andC/EBP homologous protein（CHOP） in RAW264.7cells, to examine the influence of caveo-lin-1expression and the activation of PI3K on the regulation effects of Que and to attemptto widen the thinking of mechanism study of presentation of Que for AS.Materials and Methods1. Establishment of the macrophage-derived ER stress model: RAW264.7cells werecultivated with TG in concentration of0.3μmol/L,1μmol/L and3μmol/L respectively. Th-en the cell viability was measured at12h,24h,36h and48h. The less than50%survival ra-te regarded as the mark of ER stress-derived, the optimum condition can be chosen to esta-blish the mode of ER stress-related apoptosis induced by TG in RAW264.7.2. To investigate the effects and possible mechanisms of Que on ER stress-relatedapoptosis induced by TG, RAW264.7cells were pretreated with various concentrations ofQue for30min, and then treated with1μmol/L of TG for24h. Cell viability was determined by MTT; Used the method of Annexin V-FITC/PI to determined the apoptosis rate byflow cytometry assay and observed the cell apoptotic morphology by laser scanning confo-cal microscopy; The changes of intracellular Ca²⁺concentration ([Ca²⁺]i) were determined by flow cytometry assay in way of Fluo-3/AM; the protein levels of GRP78and CHOPwere determined by Western blotting; the effects of Que on GRP78and CHOP inductionby TG was determined by Western blotting after incubating cells with phosphatidylinositol3-kinase （PI3K） inihibitors（15nmol/L of LY294002）, so as to determine whether Que med-iates ER stress in the RAW264.7cells through PI3K signaling pathways.3. In order to make sure whether caveolin-1participate in adjusting the effects of Queon ER stress, RAW264.7cells were treated with various concentrations of TG for processi-ng time, western blotting detected the varieties of caveolin-1expression. RAW264.7cellswere pretreated with2.5μg/mL of filipin（III） for30min and treated with160μmol/L ofQue for24h; then using Annexin V-FITC/PI dyeing method to observe the change of cellapoptosis rate of RAW264.7cells induced by TG through flow cytometric and laser confo-cal microscope;CHOP protein was determined by western blotting, observe whethercaveolin-1take an active part in adjusting the cell apoptosis induced by TG.Results1. Establishment of macrophage-derived apoptosis cells: RAW264.7cells were cultu-red with1μmol/L TG for24h and the survival rate is41.07±3.35%. Accord to the sign ofless than50%survival rate, this time the cells have translated into the ER stress related cellapoptosis. Thus, choose1μmol/L TG to react on24h to establish the ER stress apoptoticcells model to conduct the follow-up experiment.2. Que can suppress ER stress related injury induced by TG in RAW264.7cells. Co-mpared with TG group, cell viability increased （P<0.05）, apoptotic rate and [Ca²⁺]i decre-ased（P<0.05） and changes of apoptotic morphology alleviated. The increasing of GRP78and CHOP induced by TG as an ER stress marker was suppressed by Que （P<0.05）. Thesuppressive effect of Que on GRP78and CHOP was reproduced by LY294002（P<0.05）,but they failed to exhibit additive suppression.3. Caveolin-1expression in RAW264.7cells had a marked increase at an early stageof TG treatment （P<0.05） and then decreased with prolonged or severe treatment. The inc-reasing of caveolin-1expression induced by TG in RAW264.7cells was abolished afterinhibition of caveolae by filipin（III）（P<0.05）. Conversely, no matter cell morphology usi-ng laser scanning confocal microscopy or apoptosis rate using flow cytometry assay, theeffect of TG on apoptosis of RAW264.7cells was further augmented after pretreatmentwith filipin（III）（P<0.05）. Western blotting showed that CHOP induced by TG was augm-ented by filipin（III） in RAW264.7cells （P<0.05）. Pretreatment with filipin（III） can suppr-ess the inhibitory effect of Que on aopotosis rate and CHOP expression. Conclusion1. Que can suppress the ER stress induced by TG in RAW264.7cells and theprotective effect may be related with its suppression on PI3K signal pathway.2. The expression of caveolin-1had a marked increase at an early stage of ER stressinduced by TG in RAW264.7cells and can promote the inhibitory effect of Que throughthe PI3K signaling pathways on ER stress.