| Objectives:Hypertriglyceridemia is one of the causes of acute pancreatitis. Thepathogenesis of hypertriglyceridemic pancreatitis has not been eluci-dated. Firstly, this study was aimed to investigate the damage ofpancreatic acinar cells when the cells were incubated with palmitic acidin vitro and the mechanism of endoplastic recticulum stress in the damageof acinar cells. In vivo study, the rat model of hyperlipidemia was inducedby feeding with high fat diet for eight weeks. Acute pancreatitis modelwas induced by intraperitoneal injection of caerulein. The effect ofendoplasmic recticulum stress was clarified in pathogenesis of acutepancreatitis associated with hyperglyceridemia in rat.Methods:1. In vitro study:(1) Pancreatic acinar cells were isolated by collagenase digestion. Tostudy the exocrine function of the cells, the acinar cells were stimulatedwith CCK-8.(2) Pancreatic acinar cells which were isolated by collagenasedigestion were incubated with 0.05mmol/L, 0.1mmol/L palmitic acid for1,2,3 hours, respectively. Afterwards, the acinar cells were stimulatedwith CCK-8 for 0.5h. Then the exocrine function of pancreatic acinar cellswere studied when the cells were incubated with palmitic acid. (3) The apoptosis of pancreatic acinar cell was assessecd bytransmission electron microscopy when incubated with palmitic acid.(4) GRP78/Bip153, splicing of XBP-1, GADD153/CHOP and caspase-12 mRNAwere detected by semi-quantitative RT-PCR.(5) By loading pancreatic acinar cells with the fluorescentratio-metric calcium indicator Fluo-3-AM, intracecellular free calciumlevels of pancreatic acinar cells were determined on laser scanningconfocal microscope. These pancreatic acinar cells were incubated with0.05mmol/L, 0.1mmol/L pancreatic acinar cells for 1,2,3 hours respective-ly, then stimulated with 100pmol/L CCK-8.2. In vitro study:(1) Male SD rats were chronically fed with a high-fat diet for eightweeks. Blood samples were taken to measure serum triglyceride, choles-terol to establish the rat model of hyperlipidemia.(2) Twenty-four hyperlipidemia rats and 24 normal rats were dividedinto four groups: normal diet group(n=6), normal diet+acute pancreatitisgroup(n=18), high-fat diet group(n=6) and high-diet+acute pancreatitisgroup(n=18). Acute pancreatitis was induced in rats by twice i.p.injection of 40μg/kg caerulein every 2 hours. The rats were sacrificedat 9,12,24 hours, respectively. The serum amylase and pancreatichistological changes were studied to determine whether hyperglyceridemiaaggravated acute pancreatitis in rats.(3) The endoplasmic reticulum in pancreatic acinar cell was analyzedby transmission electron microscopy. The apoptosis of pancreatic acinarcell was analyzed by TdT-mediated dUTP-biotin nick end labeling (TUNEL).(4) GRP/Bip, XBP-1 splicing, GADD153/CHOP, caspase-12 mRNA were detect-ed by semi-quantitative RT-PCR. The protein expression of GRP78/Bip wasassessed by immunohistochemical staining. The protein expression ofGRP78/Bip and cleaved caspase-12 were detected by Western blot. Results:1. After isolated pancreatic acinar successfully, the cells wereincubated with 0.05mmol/L and 0.1mmol/L palmitic acid for 1,2,3hours, respectively. Given 100pmol/L CCK-8 stimulation, the amylasereleasing rates of cells were increased as incubating time andconcentration of palmitic acid increased. And releasing rates of cellswere significantly higher than those absent of palmitic acid in incubation(p＜0.05). After pancreatic acinar cells were incubated with 0.1mmol/Lpalmitic acid for 3 hours and stimulated with CCK-8 for 0.5 hourafterwards, the mRNA of GRP78/Bip, GADD153/CHOP, splicing of XBP-1 andcaspase-12 were detected by semi-quantitative RT-PCR. The apoptosis ofpancreatic acinar cells were also observed under transmission electronmicroscope.2. After isolated in vitro, the pancreatic acinar cells were incubatedwith 0.1mmol/L palmitic acid for 2 hours, intracellular calcium plateaulevels was maintained higher than those of the cells without palmitic acidincubation(p＜0.05). When the pancreatic acinar cells were incubated with0.1mmol/L palmitic acid for 3 hours, intracellular calcium plateaulevels and peaking value are higher than those of the cells withoutpalmitic acid incubation(p＜0.05).3. When fed for eight weeks, serum triglyceride and cholesterol inhigh-fat diet rats were significantly higher than normal dietgroup(p＜0.05). After given caerulein to induced acute pancreatitis, serumamylase level in hyperlipidemia rats are higher than the normal group. Andinjury scores of pancreatic histology of hyperlipidemia rats in acutepancreatitis are higher than those without hyperlipidemia(p＜0.05).4. When hyperlipidemia rat were induced acute pancreatitis, mRNAexpression of GRP78/Bip, GADD153/CHOP and caspase-12 were higher thannormal diet acute pancreatitis group(p＜0.05). The splicing of XBP-1 mRNAwas also seen. The cleaved caspase-12 and GRP78/Bip were also detected byWestern blot. GRP78/Bip was also expressed in pancreatic tissue of hyperlipidemia rats in acute pancreatitis by immunohistochemical stain-ing. Under transmission electron microscopy, the endoplasmic reticulumswere exceedingly dilated and distorted. Extent of apoptosis by TUNEL wasmore increased in pancreatic acinar cell of high-fat diet +acutepancreatits than those in normal diet+acute pancreatitis(p＜0.05).Conclusions:1. The exocrine function of pancreatic acinar cells with stimulationof CCK-8 was increased when cells were incubated with palmiticacid. Meanwhile, intracellular calcium levels maintained high levels,endoplasmic reticulum stress and apoptosis associated endoplasmicreticulum were detected. These results indicated hypertriglyceridemia mayincreased sensitivity of pancreatic acinar cell to CCK-8 andintracellular calcium concentration to induce endoplastic reticulumstress through cleavage product free fatty acids. The apoptosis associatedendoplasmic recticulum stress may aggravate damage of pancreatic acinarcells.2. Eight weeks fed with high-fat diet can establish rat model ofhyperlipidemia successfully. Levels of serum triglyceride and totalcholesterol were significantly increased. Hyperglyceridemia canaggravate acute pancreatitis when induced with caerulein. The apoptosisindexes were increased in acute pancreatitis associated hyperglycerid-emia. Meanwhile, endoplasmic reticulum stress and related apoptosis werealso found in pancreatic tissue.
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