The Role And Potential Mechanism Of Impaired Autophagy In The Pathogenesis Of Acute Pancreatitis Mediated By Hydrogen Sulphide

Background and purposeAcute pancreatitis?AP?is a common surgical abdomen.Despite the rapidly developed medical advances in such era,AP continues to be a clinical challenge due to there is no specific medicine or protocol to cure it.Although the exact mechanisms involved in the pathogenesis of AP have not been well elucidated,accumulation of cytoplasmic vacuoles and pre-mature activation of trypsinogen within acini were long noticed as the two early-phase features of AP.Recently,in addition to that the accumulated vacuoles were defined as autophagic in origin,the pre-mature activation of trypsinogen was shown to be a consequence of the impaired autophagy.Hydrogen sulphide?H₂S?is the third most common endogenously produced gaseous signalling molecule.Generally,H₂S is believed to increasingly synthesized and to play a pro-inflammatory role during AP,but the mechanisms involved remain obscure and controversial.Previously,we have shown that inhibition of H₂S synthesis effectively alleviated the necrotic injuries in AP rats by significantly promoting acini apoptosis.To further investigate the relevance of H₂S in AP,the effects of H₂S on impaired autophagy and the possible mechanisms that account for them were evaluated in the present study.MethodsIn vivo,one hundred and twenty male Wistar rats were randomized into four groups?n=30?:AP group,AP was induced by a retrograde infusion of 3.5%sodium taurocholate?Na-TC,0.15 m L/100 g?into the pancreaticobiliary duct.Sham group,underwent a midline laparotomy and separation of pancreaticobiliary duct without AP induction.Na HS group,administered an intra-peritoneal injection of a Na HS solution?1 m L;28?mol/kg weight?1 h after AP induction.DL-propargylglycine?PAG?group,administered an intra-peritoneal injection of a PAG solution?1 m L;80 mg/kg weight?1 h after AP induction.The surviving rats of each group were randomly sacrificed at 3h,6 h and 12 h since AP induction,respectively.The blood and pancreatic tissue samples were processed and collected until being assayed.In vitro,AR42J cells or m GFP-RFP-microtubule-associated protein light chain 3?LC3?-tagged AR42J cells were allocated into four groups similar to that in vivo:AP group,AP was stimulated by an incubation of 500?mol/L of Na-TC.Control group,incubated with PBS solution equivalent to that volume of Na-TC administered in AP group.Na HS group,administered a treatment with Na HS?100?mol/L?for 30 min before AP stimulation.PAG group,administered a treatment with PAG?3 mmol/L?for 60 min before AP stimulation.Alternatively,the cells were incubated with 10?mol/L of CQ for 2 h or20?mol/L of CC for 1 h before the experiments to manually alter the activity of autophagy.The cells were harvested for immunoblot at 1 h,3 h and 6 h or assayed for trypsinogen activation at 40 min since AP stimulation,respectively.ResultsThe pancreatic histopathological score,levels of inflammatory cytokines,extents of autophagic vacuelos accumulation,LC3 turnover and serum levels of amylase and lipase were all significantly increased at 6 h since AP induction.These above-mentioned increases could be potentialed by treatment with Na HS and resotred by treatment with PAG.After AP stimulation for 40 min,the trypsinogen activation was significantly increased.Treatment with Na HS significantly enhanced the trypsinogen activation caused by AP induction alone,whereas treatment with PAG significantly reversed the trypsinogen activation caused by AP induction alone.Both immunocolocalization between lysosome-associated membrane protein-2?LAMP-2?and LC3 assay in vivo and autophagy flux assay mediated by m GFP-RFP-LC3 in vitro indicated that the autophagosome-lysosome fusion was not hampered in AP.With the introduction of CQ,the inceased upstream activity of autophagy during AP was indentified,and its extent was positively correlated with the incubated level of H₂S.In vitro,the phosphorylation of AMPK,AKT and m TOR was examed by Western blot:the phosphorylation of AMPK was up-regulated while the phosphorylation of m TOR was down-regulated after AP stimulation,and these effects were positively correlated with the incubated level of H₂S.At last,with the introduction of CC,not only the increased phosphorylation of AMPK,but also the decreased phosphorylations of P70S6K and ULK1 mediated by the administration of Na HS after AP induction were all significantly suppressed.ConclusionH₂S exacerbates AP by over-activating autophagy via AMPK/m TOR pathway.An active and promt suppression of H₂S might be a promising therapeutic approach against AP-related injuries in the future.