The Role And Mechanism Of AMPK Regulating SIRT1 In The Occurrence And Development Of Acute Pancreatitis

Objective Acute pancreatitis(AP)is a common inflammatory disorder,characterized by high morbidity and mortality.AMPK-SIRT1 pathway is involved in a variety of diseases,but its role in AP remains elusive.This study was aimed to investigate the role of AMPK-SIRT1 pathway in AP.Methods A total of 20 healthy adult male SD rats with a body weight of 300 ～ 350 g were randomly divided into two groups: normal control(NC)group(n = 10)and acute pancreatitis(AP)group(n = 10).AP group was intraperitoneally injected with20% L-arginine(1ml/100g).The NC group was injected with the same amount of normal saline(1ml/100g).The rats in each group were anesthetized with 10% chloral hydrate(1 m L /350g)24 hours after the modeling was completed.Cardiac blood was collected and pancreas was dissected.AR42 J cells were cultured to the log-phase,and inoculated into the six-well plate.The cells were pretreated with AMPK inhibitor(Compoundc,10 u M)and AMPK activator(Metformin,10 m M),respectively.After 24 hours,AR42J cells were stimulated with Caerulein(100n M)to establish an in vitro model of acute pancreatitis.The levels of AMY,TNF-α and IL-1β in serum and cell supernatant of rats were determined by ELISA.The m RNA expression levels of AMPK and SIRT1 in rat pancreatic tissue and AR42 J cells were detected by q RT-PCR.Western blot analyses were used to detect the protein expression levels,respectively,of AMPK,p-AMPK,SIRT1,Beclin1,LC3Ⅱ/Ⅰ,p62,TNF-α and IL-6 in rat pancreatic tissue and AR42 J cells.The number of autophagosomes was observed by transmission electron microscopy.Results(1)In the AP model of rats,pathological results showed that compar ed with the NC group,the pancreas of the AP group was normal in the NC g roup,and the pancreatic parenchyma of the AP group had a large number of n ecrosis,interstitial congestion and edema,and a large number of inflammatory cell infiltration.It indicated that the animal model was successfully constructed.(2)The results of ELISA showed that the levels of AMY in serum and cell w ere increased,and the levels of TNF-α and IL-1β were increased significantly.T his indicated that the model of acute pancreatitis was successfully constructed i n vivo and in vitro.(3)In animal models,compared with NC group,the AP group AMPK,SIRT1 expression level decreased,Beclin1,LC3 Ⅱ / Ⅰ,p62 expr ession levels were increased significantly(P<0.05).(4)In vitro AP model,thr ough the activation of AMPK,compared with the Control group,AMPK and S IRT1 levels were increased significantly,Beclin1,LC3 Ⅱ / Ⅰ,p62,TNF-α and IL-6 expression levels were decreased significantly(P<0.05).The inhibition of AMPK by Compound C obtained opposite results.(5)The number of autopha gosomes in pancreatic AR42 J cells after 24 hours of stimulation with Caerulein was significantly higher than that of control group(P<0.05).The inhibition o f AMPK by Compound C obtained opposite results.Conclusion During acute pancreatitis occurrence,p-AMPK and SIRT1 were down-regulated,leading to the accumulation of p62,the increase of autophagic vacuoles,an d the damage of autophagy,the occurrence of inflammation.It hinted that activation o f AMPK restored impaired autophagy and inhibited inflammation reaction by up-regul ating SIRT1.The results of this study might provide important theoretical basis for ex plaining the pathogenesis of AP and investigating therapeutic target to treat and preve nt AP.