Studies On Molecular Mechanisms Of Resveratrol’s Inhibition Of HsBAFF-stimulated B-cell Proliferation And Survival By Regulating Akt/mTOR-autophagy Pathway

The present study,using cellular and molecular biology techniques and methods including trypan blue exclusion,cell counting,MTS assay,flow cytometry,RNA interference and Western blotting,etc.,and employing Raji cells and mouse primary spleen B lymphocytes as experimental objects,investigated resveratrol’s inhibition of hsBAFF-stimulated B-cell proliferation and survival,resveratrol’s inhibition of hsBAFF-stimulated B-cell proliferation and survival via regulating mTOR-autophagy pathway,association of resveratrol blockage of hsBAFF-induced Akt activation with autophagy inhibition-dependent proliferation and survival in B cells.The detailed results were summarized as follows:1 Resveratrol inhibits hsBAFF-stimulated B-cell proliferation and survivalRaji cells and primary B lymphocytes were pretreated with different concentrations of resveratrol(2.5-10 μM)for 1h,and then stimulated with/without 2.5μg/ml hsBAFF for 48 h.After that,cell counting,MTS assay,trypan blue exclusion and flow cytometry was conducted to evaluate manifestation of cell proliferation and survival.The results showed that resveratrol inhibited hsBAFF-stimulated B-cell proliferation and viability in a dose-dependent manner.Pretreatment with resveratrol attenuated hsBAFF-induced live B-cell numbers dose-dependentlly.Our findings indicate that resveratrol may inhibit hsBAFF-stimulated B-cell proliferation and survival.2 Resveratrol inhibits hsBAFF-stimulated B-cell proliferation and survival via regulating mTOR-autophagy pathwayRaji cells and primary B lymphocytes,Raji cells infected with shRNA mTOR,shRNA LC3-Ⅰ/Ⅱ and shRNA GFP(as control),or Raji cells infected with Ad-GFP-LC3,were pretreated with/without resveratrol(2.5-10 μM)for 1 h,and then stimulated with/without 2.5 μg/ml hsBAFF for 12 h or 48 h;or pretreated with 100 ng/ml rapamycin for 2 h and then co-pretreatment with 10 μM resveratrol for 1 h,followed by stimulating with/without 2.5 μg/ml hsBAFF for 12 h or 48 h.After that,the cell proliferation and viability were evaluated by cell counting and MTS assay;the autophagosome performance was assessed by GFP-LC3 imaging or MDC staining;the changes of protein expression for the related pathway were determined by Western blotting.The results showed that resveratrol inhibited hsBAFF-activated mTOR pathway involved in autophagy changes in B cells;Rapamycin potentiated resveratrol’s regulation of mTOR-autophagy pathway,thereby suppressing hsBAFF-stimulated B-cells proliferation and viability;Downregulaiton of mTOR enhanced resveratrol’s inhibition of hsBAFF-stimulated B-cell proliferation and survival.The findings suggest that resveratrol blocks hsBAFF-stimulated proliferation and survival by regulating mTOR-autophagy pathway in B cells.3 Resveratrol blocks hsBAFF-induced Akt activation associated with autophagy inhibition-dependent proliferation and survival in B cellsRaji cells and primary B lymphocytes were pretreated with/without resveratrol(2.5-10 μM)for 1 h and then stimulated with/without 2.5 μg/ml hsBAFF for 12 h,or co-pretreatment of Akt inhibitor X(20 μM)with resveratrol(10 μM)for 1 h and then stimulated with 2.5 μg/ml hsBAFF for 12 h or 48 h.After that,the cell proliferation and viability were evaluated by cell counting and MTS assay;the autophagosome performance was assessed by GFP-LC3 imaging;the changes of protein expression for the related pathway were determined by Western blotting.The results showed that resveratrol inhibited hsBAFF-stimulated B-cell Akt activation in a dose-dependent manner;Inhibition of Akt with Akt inhibitor X strengthened resveratrol’s blockage of hsBAFF-induced autophage inhibition-dependent proliferation and viability in B cells.The findings imply that resveratrol may block hsBAFF-induced Akt activation associated with autophagy inhibition-dependent proliferation and survival in B cells.