Studies On Molecular Mechanisms Of Metformin’s Blockage Of HsBAFF-inhibited Autophagy Promoting B-cell Activation

The present study,using cellular and molecular biology techniques and methods including cell culture,RNA interference,GFP-LC3 fluorescent labeling,MDC staining,cell counting,trypan blue exclusion,MTS assay for cell viability,and Western blotting,etc.,and employing Raji cells and primary murine B lymphocytes as experimental objects,by establishing cellular models of human soluble B-cell activating factor(hsBAFF)induction,explored the molecular mechanism of metformin’s blockage of hsBAFF-inhibited autophagy promting B-cell activation via AMPK/mTOR pathway-ULK1/Beclin 1 signaling.The detailed results were summarized as follows:1 Metformin resists hsBAFF-inhibited autophagy promoting B-cell activationRaji cells and primary murine B lymphocytes were pretreated with or without different concentrations of metformin(0.5-2 m M)for 2 h and then stimulated with or without hsBAFF(2.5 μg/ml)for 12 h or 48 h.Manifestation of cell autophagosomes was evaluated by MDC staining and GFP-LC3 fluorescent labeling.Expression of related proteins was analyzed by Western blotting.Cell proliferation and viability were evaluated by cell counting,MTS assay and trypan blue exclusion.The results showed that metformin reversed hsBAFF-induced decrease in the number of autophagosomes in B cells in a concentration-dependent manner,suppressed hsBAFF-elevated expression of p-ULK1(Ser757)and Bcl-2,and resisted hsBAFF-inhibited expression of p-Beclin 1(Ser14),p-Beclin 1(Thr119)and LC3-II in the cells;Meanwhile,metformin also inhibited hsBAFF-stimulated B cell proliferation and viability concentration-dependently.These data suggest that metformin resists hsBAFF-inhibited autophagy promoting B-cell activation.2 Metformin blocks hsBAFF-inhibited autophagy promoting B-cell activation by regulating ULK1/Beclin 1Raji cells infected with lentiviral sh RNA to ULK1,to Beclin 1 mutants Ser14D(S14D)or Thr119E(T119E)were pretreated with or without metformin(1.5 m M)for2 h,then stimulated with or without hsBAFF(2.5 μg/ml)for 12 h or 48 h.Expression of related proteins was analyzed by Western blotting.Manifestation of cell autophagosomes was evaluated by GFP-LC3 fluorescent labeling.Cell proliferation and viability were evaluated by cell counting and MTS assay.The results revealed that knockdown of ULK1 resulted in a significant decrease in the expression of thebasal and metformin-induced p-Beclin 1(Ser14)and LC3-Ⅱ,as well as the number of autophagosomes,whereas there existed an opposite change in Bcl-2 expression,cell proliferation and viability,thereby potentiating hsBAFF-inhibited autophagy promoting B-cell activation.Overexpression of point mutants Beclin 1(T119E)or Beclin 1(S14D)significantly elevated the basal and metformin-induced LC3-Ⅱexpression and the number of autophagosomes,meanwhile Bcl-2 expression,cell proliferation and viability were declined,thereby resisting hsBAFF-inhibited autophagy promoting B-cell activation.These results imply that metformin blocks hsBAFF-inhibited autophagy promoting B-cell activation by regulating ULK1/Beclin 1.3 Metformin blocks hsBAFF-inhibited autophagy promoting B-cell activation by regulating AMPK/mTOR pathway via ULK1/Beclin 1 signalingRaji cells and primary murine B lymphocytes,or Raji cells infected with Ad-AMPKɑ-ca,Ad-dn-AMPKɑ or Ad-GFP,respectively,were pretreated with or without metformin(1.5 m M)for 2 h and then stimulated with or without hsBAFF(2.5μg/ml)for 12 h or 48 h,or pretreated with or without rapamycin(100 ng/ml),Compound C(20 μM)or AICAR(2 m M)for 2 h and then metformin(1.5 m M)for 2h,followed by stimulating with or without hsBAFF(2.5 μg/ml)for 12 h or 48 h.Expression of related proteins was analyzed by Western blotting,and cell proliferation and viability were evaluated by cell counting and MTS assay.The results exhibited that metformin repressed hsBAFF-induced AMPK activity reduction in B cells.Rapamycin strengthened metformin-inhibited mTOR pathway,and thus blocked hsBAFF-inhibited autophagy promoting B-cell proliferation and viability.Activation of AMPK with AICAR or overexpression of constitutively active AMPKα(AMPKα-ca)enhanced metformin’s blockage of hsBAFF-inhibited autophagy promoting B-cell proliferation and viability,yet inhibition of AMPK with compound C or expression of dominant negative AMPKα(dn-AMPKα)resisted metformin’s blockage of hsBAFF-inhibited autophagy promoting B-cell proliferation and viability.These findings underscore that metformin blocks hsBAFF-inhibited autophagy promoting B-cell activation by regulating AMPK/mTOR pathway via ULK1/Beclin 1 signaling.