Calcium/P53/Ninjurin 1 Signaling Mediates Plasma Membrane Rupture of Acinar Cells in Severe Acute Pancreatitis

Abstract

Ninjurin 1 (NINJ1) is a double-transmembrane cell-surface protein that might mediate plasma membrane rupture (PMR) and the diffusion of inflammatory factors. PMR is a characteristic of acinar cell injury in severe acute pancreatitis (SAP). However, the involvement of NINJ1 in mediating the PMR of acinar cells in SAP is currently unclear. Our study has shown that NINJ1 is expressed in acinar cells, and the expression is significantly upregulated in sodium-taurocholate-induced SAP. The knockout of NINJ1 delays PMR in acinar cells and alleviates SAP. Moreover, we observed that NINJ1 expression is mediated by Ca²⁺ concentration in acinar cells. Importantly, we found that Ca²⁺ overload drives mitochondrial stress to upregulate the P53/NINJ1 pathway, inducing PMR in acinar cells, and amlodipine, a Ca²⁺ channel inhibitor, can reduce the occurrence of PMR by decreasing the concentration of Ca²⁺. Our results demonstrate the mechanism by which NINJ1 induces PMR in SAP acinar cells and provide a potential new target for treatment of SAP.

Keywords: nerve-injury-induced protein 1; plasma membrane rupture; severe acute pancreatitis.

Figure 1

NINJ1 accumulates on STC-SAP mouse acinar cells and affects PMR. (A) Primary mouse acinar cell treated with 5 mM STC for 50 min, then stained with IF. IF: NINJ1 observed; scale bar = 100 μm. (B) The quantitative plot of NINJ1 fluorescence levels (n = 5). (C) NINJ1_26-37 was intraperitoneally injected before modeling (3 and 6 mg/kg), once a day, three times in total; the SAP model was induced by retrograde injection of 3.5% STC into the pancreaticobiliary duct. Samples were taken 24 h later. Yellow square = enlarged area. White arrow = oligomerization. Scale bar = 100 µm. (D) The quantitative plot of NINJ1 fluorescence levels. (n = 3). (E) Mouse pancreatic acinar cells 266-6 were photographed using HCS real-time imaging. Scale bar = 50 µm. (F) An HCS data analysis system was used to analyze the changes in the cell perimeter at different time points. SAP: severe acute pancreatitis, STC: sodium taurocholate, PMR: plasma membrane rupture, IF: immunofluorescence, HCS: high-content screening. All data are presented as mean ± SEM; * p < 0.05, ** p < 0.01 vs. the control group. ^# p < 0.05 vs. the STC group.

Figure 2

Protective effect of NINJ1^−/− on acinar cells. (A) NINJ1 DNA expression in acinar cells from NINJ1^−/− mice. (B) Western blot detected NINJ1 protein expression in the NINJ1^−/− mice acinar cells. GAPDH was used as the loading control (n = 3). (C) Representative fluorescence plot of PI/Hoechst 33,342 staining in primary mouse acinar cells. Scale bar = 200 µm. (D) PI/Hoechst 33,342 staining necrosis bar chart of primary mouse acinar cells (n = 3). Data are presented as mean ± SEM, ** p < 0.01 indicates WT-CON vs. WT-STC, ^# p < 0.05 indicates WT-STC vs. KO-STC; ^{^} p < 0.05 indicates WT-STC vs. NINJ1^−/−-STC. n.s = no significance. (E) HCS real-time imaging of mouse pancreatic acinar cell and representative pictures were selected from the periods of 300, 600, and 900 min in WT-CON group, WT-STC group, NINJ1^−/−-CON group, NINJ1^−/−-STC group (n = 3). STC: sodium taurocholate. HCS: high-content screening. Scale bar = 50 µm.

Figure 3

NINJ1 expression is mediated by Ca²⁺ concentration. (A) Primary mouse acinar cells were incubated with different metal ions for 50 min, and the proteins were extracted for Western blot experiments to detect the effects of Ca²⁺, K⁺, Mg²⁺, and Na⁺ on NINJ1. (B) The quantitative plot of NINJ1 protein levels (n = 3). (C) Ca²⁺ (10 mM) was added with mouse primary acinar cells and incubated with BAPTA-AM for 50 min, and the protein was extracted for Western blot analysis to detect the effect of BAPTA-AM on NINJ1. (D) The quantitative map of NINJ1 protein level (n = 3). (E) Primary mouse acinar cells were incubated with AML (5 and 15 μM) and incubated with STC (5 mM) for 50 min. Representative fluorescence plot of PI/Hoechst 33342 staining in primary mouse acinar cells. Scale bar = 200 µm. (F) Column of PI/Hoechst 33342 staining necrosis in primary mouse acinar cells (n = 5). (G) Primary mouse acinar cells were incubated with AML (15 μM) combined with STC (5 mM) for 50 min, and the proteins were extracted for Western blot experiments. (H) The quantitative lot of NINJ1 protein levels (n = 3). STC: sodium taurocholate; AML: amlodipine. All data are presented as mean ± SEM, * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. the control group. ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001, ^{^^} p < 0.01 vs. the STC group.

Figure 4

Effect of AML on pancreatic tissue in STC-SAP mice. (A) AML (6 mg/kg) was intraperitoneal injected once a day three times before modeling; SAP was induced by retrograde injection of 3.5% STC into the pancreaticobiliary duct. Samples were taken 24 h later. Scale bar = 50 µm. (B) The histopathological score of mouse pancreas (n = 5). (C) Serum levels of lactate dehydrogenase, lipase, and amylase were measured (n = 3). (D) Immunofluorescence plot of NINJ1 on pancreatic tissue, Yellow square = enlarged area. White arrow = oligomerization. Scale bar = 100 µm. (E) The quantitative map of NINJ1 fluorescence levels (n = 5). STC: sodium taurocholate. SAP: severe acute pancreatitis. AML: amlodipine. All data are presented as mean ± SEM, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the control group, ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001 vs. the STC group.

Figure 5

AML via inhibition of intracellular Ca²⁺ to inhibit ROS-P53. (A) Mouse primary acinar cells incubated with 15 μM AML for 30 min; Fluo-4 AM was added for another 20 min; STC (5 mM) was added before microscopy, and then observed under confocal microscope. A representative Fluo-4 AM staining. Scale bar = 100 μm. (B) The quantitative plot of Fluo-4AM fluorescence levels (n = 3). (C) Primary mouse acinar cells were incubated with 15 μM AML for 30 min, followed by the addition of TMRM for another 20 min, and then observed under confocal microscope. The representative TMRM staining. Scale = 100 μm. (D) A quantitative map of TMRM fluorescence levels (n = 3). (E) Primary mouse acinar cells were incubated with 15 μM AML for 30 min; then, DCFH-DA was added and co-incubated for another 20 min before observation under confocal microscope. The representative DCFH-DA staining. Scale = 100 μm. (F) The quantitative plot of DCFH-DA fluorescence levels (n = 3). (G) The primary mouse acinar cells were incubated with AML and STC for 50 min, and the protein was extracted for Western blot analysis to detect the effect of AML on P53. (H) The quantitative plot of P53 protein levels (n = 4). AML: amlodipine, STC: sodium taurocholate. All data are presented as mean ± SEM, ** p < 0.01 vs. the control group; ^# p < 0.05 vs. the STC group.

Figure 6

NINJ1 is regulated by P53 and affects PMR. (A) Primary mouse acinar cells were incubated with STC, RITA, and PFT-α for 50 min. The effects of STC on P53 and NINJ1 were detected by Western blot. (B) The quantitative plot of P53 (n = 4) and NINJ1 (n = 5) protein levels. (C) 266-6 mouse pancreatic acinar cells were captured by HCS real-time imaging. Scale bar = 50 µm (D) An HCS data analysis system was used to analyze the changes in cell perimeter at different time points. STC: sodium taurocholate; RITA: P53 agonist; PFT-α: P53 inhibitor. HCS: high-content screening. PMR: plasma membrane rupture. All data are presented as mean ± SEM, ** p < 0.01, *** p < 0.001 vs. the control group, ^# p < 0.05, ^## p < 0.05, ^{^} p < 0.05, ^{^^} p < 0.05 vs. the STC group.