The Role And Mechanism Of Wip1 In The Progression Of Acute Pancreatitis Models Through STING-TBK1 Pathway

Objective: This study was aimed to explore the molecular mechanism of Wip1 involving in acute pancreatitis through cerulein-induced models in vivo and in vitro.Methods: Twenty 8-week-old SD rats were randomly divided into two groups.Rats in the acute pancreatitis group were injected intraperitoneally with 50 μg/kg of caerulein,seven consecutive times with an interval of 1 hour each time,and then serum and pancreatic tissue were extracted 6 hours after the last injection.Rats in the blank control group were intraperitoneally injected with an equal amount of sterilized water,the method was the same as that in the AP group.Then the levels of lipase,amylase,IFNβ and TNFα in the serum of the rats were analyzed by ELISA.Meanwhile,the levels of Wip1,STING,TBK1 and IRF3 proteins were analyzed by Western blot.AR42 J cells were stimulated by caerulein 100 nmol/L to construct an AP model in vitro.After treatment with 100 nmol/L caerulein at different time points(0 h,1 h,3 h,6 h,12 h,24h),the levels of amylase,IFNβ and TNFα in culture medium were analyzed by EILSA,and the levels of Wip1,STING,TBK1 and IRF3 proteins were analyzed by Western blot.Lentiviral vector expressing sh RNA against Wip1 was applied to knockdown Wip1 expression.After treatment with 100 nmol/L caerulein in lentiviral-transfection AR42 J cells at different time points(0 h,1 h,3 h,6 h,12 h,24h),the levels of amylase,IFNβ and TNFα in culture medium were analyzed by EILSA,and the levels of Wip1,STING,TBK1 and IRF3 proteins were analyzed by Western blot.Results:1.The AP model was established by caerulein stimulation: in the AP rat model,the levels of amylase and lipase in serum were increased after the caerulein stimulation and significantly higher than those in the normal group(p <0.05);in the AP cell model,after treatment of caerulein,the levels of amylase in the supernatant of AR42 J cells were increased in a time-dependent manner and reached a peak around 12 hours(p <0.05).2.The levels of IFNβ and TNFα in serum were upregulated by caerulein stimulation(P <0.05);the levels of IFNβ and TNFα in the supernatant of AR42 J cells were upregulated by caerulein stimulation in a time-dependent manner and reached a peak around 12 hours(P <0.05).3.The levels of Wip1,STING,TBK1 and IRF3 of pancreas tissues were upregulated by caerulein stimulation(P <0.05);the levels of Wip1,STING,TBK1 and IRF3 of AR42 J cells were upregulated by caerulein stimulation in a time-dependent manner and reached a peak around 12hours(P <0.05).4.The levels of amylase,IFNβ and TNFα upregulated by caerulein stimulation were declined by knockdown Wip1 in AR42 J cells.Also the levels of STING,TBK1 and IRF3 upregulated by caerulein stimulation were declined by knockdown Wip1(P <0.05).Conclusion: In the animal model and cell model of acute pancreatitis induced by caerulein,Wip1 and STING-TBK1 signaling pathways are activated.The molecular regulation mechanism may be that acinar cell necrosis causes Wip1 to be up-regulated and causes DNA damage to activate the STING-TBK1 signaling pathway,promote the secretion of pro-inflammatory cytokines including IFN β and TNF α,and thus promote the development of AP.