Neutrophil Gelatinase-Associated Lipocalin Protects Acinar Cells From Cerulein-Induced Damage During Acute Pancreatitis

Abstract

Objectives: Elevated neutrophil gelatinase-associated lipocalin (NGAL) is a promising marker for severe acute pancreatitis (SAP) and multiple organ failure, suggesting systemic and local contributions during pancreatitis. We investigated the role of NGAL locally on acinar cell biology.

Methods: Western blot, reverse transcriptase-polymerase chain reaction, and immunohistochemistry analysis were performed to analyze the levels of NGAL receptors, apoptotic and regeneration markers, and 4-hydroxynonenal (4HNE) levels, 3-[4,5-Dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide assay, and annexin V/propidium iodide staining were used to evaluate cell viability, and effect on endothelial cells was accessed by endothelial permeability assay.

Results: Cerulein treatment at 20 μM for 12 hours significantly reduced acinar cell viability by 40%, which was rescued by NGAL at 800 and 1600 ng/mL concentrations, observed during mild and SAP, respectively. Mechanistically, NGAL significantly reduced the levels of reactive oxygen species and 4HNE adduct formation in a 24p3R-dependent manner and upregulated the expression of acinar cell regeneration markers, like CDK-2, CDK-4, and C-myc. However, SAP levels of NGAL significantly increased endothelial permeability and downregulated the levels of ZO-1, and cerulein treatment in NGAL knockout mice showed increased levels of 4HNE adducts.

Conclusions: Neutrophil gelatinase-associated lipocalin rescues intracellular reactive oxygen species during pancreatitis and promotes survival and regeneration of acinar cells.

Conflict of interest statement

S.K.B. is a cofounder of Sanguine Diagnostics and Therapeutics Inc. The other authors declare no conflict of interest.

Figures

FIGURE 1.

Pancreatic acinar cells express high levels of the NGAL receptor 24p3R. A, Reverse transcriptase PCR analysis performed using RNA isolated from a murine acinar cell line (266.6) and pancreas demonstrated the higher expression of NGAL receptor 24p3R compared with LRP2. The white space separates the expression in a cell line (266.6) and mouse pancreas. B, Immunohistochemistry analysis showed the strong expression of 24p3R in murine pancreatic acinar cells. Scale bar, 100 μm. C, Quantification of intensity and total stained area from the randomly selected areas for 24p3R in the murine pancreas (n = 6). D, Effect on 24p3R expression upon treatment with the previously identified MAP (800 ng/mL) and SAP (1600 ng/mL) rmNGAL concentrations using immunofluorescence analysis. Scale bar, 20 μm.

FIGURE 2.

Neutrophil gelatinase–associated lipocalin rescues cerulein-induced cell death in mouse acinar cells. A, MTT assay for evaluating cerulein dose response in pancreatic acinar cell viability during a 12-hour treatment. B, Viability of acinar cells in the presence of 20 μM cerulein along with 800 and 1600 ng/mL levels of rmNGAL for 12 hours as measured by MTT assay. C, D, Effect of rmNGAL in preventing cerulein-induced acinar cell death as demonstrated by annexin V/PI live dead staining. E, The rmNGAL blocks cerulein-induced acinar cell death by modulating PARP expression as demonstrated by Western blot analysis. *P < 0 0.05, **P < 0.01, ***P < 0.001, ns, not significant.

FIGURE 3.

Neutrophil gelatinase–associated lipocalin prevents acinar cell death by scavenging cerulein-induced ROS. A, Pancreatic acinar cells were treated with cerulein alone, or in combination with 800 or 1600 ng/mL of rmNGAL for 12 hours and stained with DCFDA for the detection of intracellular ROS. Highly fluorescent 2′,7′-dichlorofluorescein (DCF) production from DCFDA after ROS-mediated oxidation was measured by fluorescence microscopy. Scale bar, 10 μm. B, Quantification of fluorescence intensity from different treatment groups from (A). C, Effect of cerulein alone or in combination with NGAL treatment on ROS induced 4HNE adducts, as assessed by Western blot analysis. D, MTT assay demonstrates the effect of rmNGAL in rescuing acinar cells from death by scavenging cerulein-induced intracellular ROS along with 0.5 mM NAC, a known ROS scavenger. E, Effect of 24p3R silencing on the formation of 4HNE adducts due to increased ROS levels after treatment with cerulein. *P < 0.05, **P < 0.01. Scr, scrambled; ShRNA, short hairpin RNA.

FIGURE 4.

Neutrophil gelatinase–associated lipocalin treatment promotes mouse acinar cell regeneration and alters endothelial permeability. A, Effect of rmNGAL on acinar cell growth as analyzed by MTT assay following treatment with MAP or SAP concentration for rmNGAL for 12 hours. B, Expression profile of acinar cell regeneration markers, CDK2, CDK4, and C-myc after treatment with rmNGAL at different time points. C, Expression of Ki-67 in acinar cells treated with MAP (800 ng/mL) and SAP (1600 ng/mL) levels of rmNGAL. D, Quantification of the percentage of acinar cells with Ki-67 nuclear staining after 12 and 24 hours demonstrated that rmNGAL treatment promotes proliferation of acinar cells. **P < 0.01, ***P < 0.001. Scale bar, 50 μm. E and F, Murine and human endothelial cells, mBMEC and hMEC-1, respectively, were used to evaluate the role of NGAL on endothelial permeability at indicated time points. High glucose levels (20 mmol/L) have been shown to increase endothelial permeability and, therefore, used as positive control. *P < 0.05, **P < 0.01. G and H, Effect of rhNGAL on the expression of tight junction proteins, ZO-1, and claudin 5 as assessed by Western blot (G) and IF experiments (H). Scale bar, 20 μm. UT, untreated.

FIGURE 5.

Neutrophil gelatinase–associated lipocalin KO increases ROS levels and 24p3R expression in the AP model. A, The method followed to induce AP in WT and NGAL KO mice by a series of intraperitoneal cerulein injections. B and D, Immunohistochemistry for the extent of 4HNE adduct formation (B) and 24p3R expression (D) on pancreatic tissues from WT and NGAL KO mice either treated with cerulein or vehicle control. C and E, Average H-score (n = 6) for the intensity and expression of 4HNE (C) and 24p3R (E), respectively, in cerulein- and saline-treated WT and NGAL KO mice. F, Schematic representation of NGAL-mediated rescue and regeneration of acinar cells by reducing the intracellular ROS locally at the site of inflammation. However, persistently high levels of NGAL in serum increase endothelial permeability systemically, resulting in drops in blood pressures and subsequent induction of MOF. *P < 0.05, **P < 0.01. i.p., intraperitoneal; ns, not significant.