The Impact Of Endoplasmic Reticulum Stress On The Zymophagy Of Caerulein-induced Experimental Acute Pancreatitis

BackgroundAcute pancreatitis (AP) is a common disease of digestive system.Although generally self-limiting, it is a potentially fatal disease, becauseabout15%~20%cases will develop severe acute pancreatitis (SAP). Theprognosis of SAP is dismal, with a mortality rate as high as10%~30%.The pathogenesis of AP, although researched and explored by manyscholars, was still elusive. It is now widely recognized that the pancreaticacinar cells (PACs) play a vital role in the genesis of AP. Variouspathogenic factors can stimulate the PACs, leading to a cascade ofpathophysiological changes, including the injury of organelles (such asmitochondria, lysosomes, endoplasmic reticulum, etc.), and disturbanceof homeostasis with calcium overload. The excessive calcium induces theactivation of nuclear factor κB (NF-κB) and trypsinogen, leading toacinar cell damage and death, causing self-digestion and inflammation ofpancreatic tissue, end with organ failure and systemic inflammatoryreaction.Autophagy is a kind of programmed cell death, characterized with a largeamount of vacuole structure with cytoplasm and organelles wrappedinside, which are further degraded by lysosome. Autophagy can removemisfolded proteins and long-life proteins as well as excess, damagedorganelles and invading microbes. At the same time, autophagy is also a protective mechanism of cell to adapt to the environment since it candecompose and recycle cellular components to provide nutrients andmaterials for biosynthesis. Therefore, autophagy plays an important rolein the process of normal cell growth and development as well as thegenesis and development of many kinds of diseases. In recent years, theeffect autophagy has on the genesis of AP has become a hot topic in thefield of the pathogenesis of AP, although many studies have beenconducted to discuss the role played by autophagy in the process ofgenesis and development of AP, its specific effect and molecularmechanism is still a big controversy in AP. Zymophagy is a newlydiscovered protection mechanism of pancreas, which is discovered byforeign scholars in an experimental model of AP in2011for the first time.Zymophagy refers to the selective autophagy of zymogen granules inpancreatic acinar cells, with the effect of degradating abnormallyactivated trypsinogen. The study found the formation of zymophagyneeds vacuolar membrane protein1(VMP1). VMP1comes from theendoplasmic reticulum, and endoplasmic reticulum stress is involved inthe occurrence and development of AP. Whether the initiate ofzymophagy is associated with endoplasmic reticulum stress has not beenreported yet. Based on the above, this research choose caerulein-inducedpancreatic AR42J cells as study subject, observed the effects ofendoplasmic reticulum stress in zymophagy and its significance in the pathogenesis of experimental AP.ObjectiveTo explore the impact and significance of endoplasmic reticulum stresson zymophagy of caerulein-induced experimental AP model.Materials and methods1. Observe the changes of Zymophagy in AR42J cell caerulein-inducedexperimental AP model: culture rat pancreatic acinar cells AR42J, inducecell AP by caerulein, detect inflammatory cytokines IL-1, IL-6, TNF-α,detect trypsinogen activation (TAP) and amylase; detect the expression ofLC3, Beclin1mRNA and protein by the method of polymerase chainreaction (PCR) and Western blot; observe autophagosome andzymophagosome by transmission electron microscopy.2. Establish theendoplasmic reticulum stress model of AR42J cells, and observe cellularchanges and the change of PERK expression in cells: A tool reagentsthapsigargin is used to induce endoplasmic reticulum stress in AR42Jcells. The cells are collected at different time points after the treatment ofdrugs. Apoptosis is detected by flow cytometry; PERK expression in eachgroup is detected by real-time quantitative PCR; autophagosome andzymophagosome are observed by transmission electron microscopy.3Observe the effects of endoplasmic reticulum stress on autophagy andzymophagy in experimental AP: after the successful establishment of cellAP model, induce endoplasmic reticulum stress. Detect changes in inflammatory factors, activation of trypsinogen and cell injury. Detect theexpression of LC3, Beclin1mRNA and protein by the method ofpolymerase chain reaction (PCR) and Western blot. Observeautophagosome and zymophagosome by transmission electronmicroscopy.Results1. Autophagy was involved in the occurrence and development ofexperimental AP. Compared with the control group, inflammatorymarkers, amylase and autophagy markers were significantly increased ineach group of AP.2. Transmission electron microscopy foundzymophagosome in AR42J cells pancreatitis model.3. The endoplasmicreticulum stress model of AR42J cells were successfully established.PERK expression was significantly increased and reached a peak at12h.4. Intervention in experimental acute pancreatitis on endoplasmicreticulum stress conditions, cell injury and inflammation has no obviousincrease, but the quality of autophagosome and zymophagy increasedsignificantly.ConclusionAutophagy is involved in the occurrence of AP, with the expression ofautophagy-related proteins significantly increased, and autophagy playsan important role in early stage of this disease. By inducing endoplasmicreticulum stress in AP cell model by thapsigargin, found that the number of autophagosome and zymophagosome significant increased butinflammation was no severe increase compared with AP group.Thisindicated that endoplasmic reticulum stress induced autophagy disorder inthe process of AP. Zymophagy degrades the activated granules avoidingthe spreading of their contents into the cytoplasm,thus reducing theprogress of inflammation. Endoplasmic reticulum stress played a certainrole in the pathogenesis of pancreatitis through relative pathways.