Fig 1.
Serum amylase and lipase activity.
UCN2 reduced serum (A) amylase activity by 43% and (B) lipase activity by 48% compared with caerulein treatment. Data are shown as mean ± SD (n = 3), one-way ANOVA and Tukey’s multiple comparisons. CR: caerulein. Amylase: ***: p<0.0001 vs. saline; #: p = 0.0038 saline vs. UCN2 + CR; *: p = 0.0145 Stressin1 +CR vs. UCN2 + CR. For Lipase: ***: p = 0.0001 vs. saline; #: p = 0.0107 saline vs. UCN2 + CR; *: p = 0.019 Stressin1 +CR vs. UCN2 + CR.
Fig 2.
Histopathology and effect of UCN2 on cell necrosis.
(A) Representative micrographs showing H&E staining of pancreatic tissue sections. Necrotic cells are shown within yellow circles and arrows point to inflammatory cell infiltrates. Scale bar: 20μm. (B) Necrosis measurement: Necrosis was assessed on H&E stained pancreatic tissue sections using the following criteria- swollen cytoplasm, loss of plasma membrane integrity, and organelle leakage into interstitium. Data are shown as mean ± SEM (n = 3) for each condition. One-way ANOVA and Tukey’s multiple comparisons. CR: caerulein. **: p = 0.0059 Saline vs. CR; #: p = 0.0030 Saline vs. Stressin1 +CR.
Fig 3.
UCN2 reduced neutrophil infiltration.
Number of inflammatory cell infiltration (neutrophils) was counted on H&E stained pancreatic tissue sections and expressed as percentage of total acinar cells. Data are shown as mean ± SEM (n = 3) for each condition. One-way ANOVA and Tukey’s multiple comparisons. CR: caerulein. *: p = 0.0112 Saline vs. CR; **: p = 0.0012 Saline vs. Stressin1 +CR.
Fig 4.
Loss of UCN2 expression in caerulein-induced pancreatitis.
UCN2 levels decreased in pancreatic lysates from UCN2 + caerulein-treated rats compared with saline-treated rats (p = 0.042), 1-way ANOVA, whereas UCN2 levels did not change in primary acinar cells.
Fig 5.
Effect of UCN2 on NF-κB and trypsin activity and IκB degradation.
Rats were pre-treated with saline or UCN2 for 30 min followed by treatment with caerulein for 1 hour. (A) NF-κB DNA binding activities were measured in the pancreatic nuclear extracts using ELISA. UCN2 treatment decreased NF-κB by nearly 42% in the nuclear extracts. (B) Western blot analysis showed decreased IκB-α and NF-κB levels in pancreatic tissue cytosolic extract after caerulein treatment compared with saline controls, suggesting either increased degradation IκB-α or increased translocation of NF-κB from the cytosol to the nucleus. UCN2 treatment prevented caerulein-induced changes in IκB-α degradation and NF-κB nuclear translocation. GAPDH, a housekeeping protein was used as a loading control (n = 3/condition). (C) Trypsin activity was increased after caerulein treatment and UCN2 treatment had a modest (15%) effect on trypsin activity. Values are means ± SEM (n = 5). One-way ANOVA and Tukey’s multiple comparisons. CR: caerulein.
Fig 6.
UCN2 evokes Ca2+ transients in primary acinar cells.
Primary acinar cells were loaded with Fura2AM and stimulated with secretagogues as shown. (A) Peak [Ca2+]i increased in response to stimulations with 0.1nM caerulein (CR), 10nM, and 100nM UCN2. Ca2+ peaks were normalized to ionomycin responses. Stimulation with UCN2 showed dose-dependent increases in Ca2+ responses. Lower panel shows a cluster of Fura2AM-loaded acini. (B) Primary acinar cells were first stimulated with 10nM or 100nM of UCN2 followed by stimulation with 0.1nM or 1.0nM CR as shown. 100nM of UCN2 completely abrogated CR to induce Ca2+ peak responses at 0.1nM and reduced Ca2+ peak responses by 50% at 1.0nM, whereas 10nM UCN2 was not effective at modulating CR-induced Ca2+ responses. (C) Representative traces of Ca2+ peaks generated after stimulation with CR, UCN2, and ionomycin. N = 6-8/condition. One-way ANOVA and Tukey’s multiple comparisons.
Fig 7.
UCN2 prevents F-actin redistribution and over secretion in the face of caerulein.
Pancreatic primary acinar cells were stained with Alexa Fluro 555 phalloidin to visualize F-actin bundles. (A) in unstimulated acini, F-actin was primarily seen at the apical poles. (B) stimulation with 1.0nM caerulein or (C) 100nM UCN2 for 30 minutes resulted in robust redistribution of F-actin from the apical to the basolateral pole, along with presence of secretory granules near the basolateral surface (arrows). (D) Pre-treatment with 100nM UCN2 followed by 1.0nM caerulein for 30 min restored apical F-actin expression.