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. 2022;13(2):483-500.
doi: 10.1016/j.jcmgh.2021.09.008. Epub 2021 Sep 23.

Trypsin-Mediated Sensitization to Ferroptosis Increases the Severity of Pancreatitis in Mice

Ke Liu  1 , Jiao Liu  2 , Borong Zou  2 , Changfeng Li  3 , Herbert J Zeh  4 , Rui Kang  4 , Guido Kroemer  5 , Jun Huang  6 , Daolin Tang  7
Affiliations

Trypsin-Mediated Sensitization to Ferroptosis Increases the Severity of Pancreatitis in Mice

Ke Liu et al. Cell Mol Gastroenterol Hepatol. 2022.

Abstract

Background & aims: Pancreatitis is characterized by acinar cell death and persistent inflammation. Ferroptosis is a type of lipid peroxidation-dependent necrosis, which is negatively regulated by glutathione peroxidase 4. We studied how trypsin, a serine protease secreted by pancreatic acinar cells, affects the contribution of ferroptosis to triggering pancreatitis.

Methods: In vitro, the mouse pancreatic acinar cell line 266-6 and mouse primary pancreatic acinar cells were used to investigate the effect of exogenous trypsin on ferroptosis sensitivity. Short hairpin RNAs were designed to silence gene expression, whereas a library of 1080 approved drugs was used to identify new ferroptosis inhibitors in 266-6 cells. In vivo, a Cre/LoxP system was used to generate mice with a pancreas-specific knockout of Gpx4 (Pdx1-Cre;Gpx4flox/flox mice). Acute or chronic pancreatitis was induced in these mice (Gpx4flox/flox mice served as controls) by cerulein injections or a Lieber-DeCarli alcoholic liquid diet. Pancreatic tissues, acinar cells, and serum were collected and analyzed by histology, immunoblot, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, or immunohistochemical analyses.

Results: Supraphysiological doses of trypsin (500 or 1000 ng/mL) alone did not trigger significant cell death in 266-6 cells and mouse primary pancreatic acinar cells, but did increase the sensitivity of these cells to ferroptosis upon treatment with cerulein, L-arginine, alcohol, erastin, or RSL3. Proteasome 26S subunit, non-adenosine triphosphatase 4-dependent lipid peroxidation caused ferroptosis in pancreatic acinar cells by promoting the proteasomal degradation of glutathione peroxidase 4. The drug screening campaign identified the antipsychotic drug olanzapine as an antioxidant inhibiting ferroptosis in pancreatic acinar cells. Mice lacking pancreatic Gpx4 developed more severe pancreatitis after cerulein infection or ethanol feeding than control mice. Conversely, olanzapine administration protected against pancreatic ferroptotic damage and experimental pancreatitis in Gpx4-deficient mice.

Conclusions: Trypsin-mediated sensitization to ferroptotic damage increases the severity of pancreatitis in mice, and this process can be reversed by olanzapine.

Keywords: Acinar Cells; Animal Model; Cell Death; Digestive Enzyme; Sterile Inflammation.

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Figures

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Graphical abstract
Figure 1
Figure 1
Trypsin increases the sensitivity of pancreatic acinar cells to ferroptosis. (A) Analysis of cell death in indicated acinar cells after treatment with trypsin (500–4000 ng/mL) for 24 hours. Data are presented as means ± SD; n = 3 biologically independent samples; 2-way analysis of variance with the Tukey multiple comparisons test on all pairwise combinations. (B and C) Analysis of cell death and MDA in indicated acinar cells after treatment with cerulein (100 nmol/L), L-arginine (5 mg/mL), alcohol (50 mmol/L), erastin (2 μmol/L), and RSL3 (500 nmol/L) in the absence or presence of trypsin (500 ng/mL), STI (500 ng/mL), ferrostatin-1 (1 μmol/L), liproxstatin-1 (500 nmol/L), 2-mercaptoethanol (20 μmol/L), Z-VAD-FMK (10 μmol/L), or necrosulfonamide (1 μmol/L) for 24 hours. Data are shown in a heat map as the mean of 3 biologically independent samples. (D) Analysis of cell death in indicated acinar cells after treatment with staurosporine (STS, 500 nmol/L) or CCT137690 (1 μmol/L) in the absence or presence of Z-VAD-FMK (10 μmol/L) or necrosulfonamide (NSA, 1 μmol/L) for 24 hours. Data are presented as means ± SD; n = 3 biologically independent samples; 2-way analysis of variance with the Tukey multiple comparisons test on all pairwise combinations. Ctrl, control.
Figure 2
Figure 2
PSMD4-dependent lipid peroxidation promotes ferroptosis in acinar cells. (A) Analysis of mRNA expression of 43 proteasome subunits in 266-6 cells after treatment with cerulein (100 nmol/L) in the presence of trypsin (500 ng/mL) for 24 hours. Data are shown in a scatter map as the mean of 3 biologically independent samples. Psmd4 ranked as the most up-regulated gene as shown in red. (B) Analysis of mRNA expression of Psmd4 in 266-6 cells and mPACs after treatment with cerulein (100 nmol/L) in the absence or presence of trypsin (500 ng/mL) for 24 hours. Data are presented as means ± SD; n = 3 biologically independent samples; 2-way analysis of variance (ANOVA) with the Tukey multiple comparisons test on all pairwise combinations. (C) Analysis of protein expression of PSMD4 in indicated 266-6 cells. Semiquantitative data are presented as means ± SD; n = 3 biologically independent samples; 1-way ANOVA test on all pairwise combinations. (D–I) Analysis of (D) cell death, (E) MDA, (F) 8-hydroxy-2-deoxyguanosine (8-OHG), (G) C20:4, (H) C22:4, or (I) lipid peroxidation in indicated 266-6 cells after treatment with cerulein (100 nmol/L), L-arginine (5 mg/mL), alcohol (50 mmol/L), erastin (2 μmol/L), and RSL3 (500 nmol/L) in the presence of trypsin (500 ng/mL) for 24 hours. Data are presented as mean ± SD; n = 3 biologically independent samples; 2-way ANOVA test on all pairwise combinations (∗P < .0001 vs control shRNA group or Psmd4 cDNA group). (J) Analysis of cell death in indicated 266-6 cells after treatment with staurosporine (500 nmol/L), CCT137690 (1 μmol/L), erastin (2 μmol/L), or RSL3 (500 nmol/L) in the absence of trypsin for 24 hours. Data are presented as means ± SD; n = 3 biologically independent samples; 2-way ANOVA with the Tukey multiple comparisons test on all pairwise combinations. Ctrl, control.
Figure 3
Figure 3
PSMD4-dependent GPX4 degradation promotes ferroptosis in acinar cells. (A and B) Analysis of (A) protein or (B) mRNA expression of GPX4 in control or Psmd4-knockdown 266-6 cells after treatment with cerulein (100 nmol/L) in the absence or presence of trypsin (500 ng/mL) for 24 hours. Data for mRNA are presented as means ± SD; n = 3 biologically independent samples. Two-way analysis of variance (ANOVA) test on all pairwise combinations. (C–G) Analysis of (C) protein expression, (D) intracellular Fe2+, (E) intracellular GSH, (F) intracellular CoQ10, and (G) Egln2 mRNA in control or Psmd4-knockdown 266-6 cells after treatment with cerulein (100 nmol/L) in the presence of trypsin (500 ng/mL) for 24 hours. Data are presented as means ± SD; n = 3 biologically independent samples. One-way ANOVA test on all pairwise combinations. (H) Immunoprecipitation (IP) analysis of the interaction between PSMD4 and GPX4 in 266-6 cells after treatment with cerulein (100 nmol/L) in the presence of trypsin (500 ng/mL) for 6 hours. Semiquantitative data are presented as means ± SD; n = 3 biologically independent samples; t test. (I) Analysis of protein expression in indicated 266-6 cells after treatment with cerulein (100 nmol/L) in the presence of trypsin (500 ng/mL) for 24 hours. Semiquantitative data are presented as means ± SD; n = 3 biologically independent samples; 1-way ANOVA test on all pairwise combinations. (J–M) Analysis of (J) cell death, (K) lipid peroxidation, (L) C20:4, or (M) C22:4 in indicated 266-6 cells after treatment with cerulein (100 nmol/L), L-arginine (5 mg/mL), alcohol (50 mmol/L), erastin (2 μmol/L), and RSL3 (500 nmol/L) in the presence of trypsin (500 ng/mL) for 24 hours. Data are presented as means ± SD; n = 3 biologically independent samples; 2-way ANOVA test on all pairwise combinations. ∗P < .0001 vs control sh or Psmd4/Gpx4 sh group. ACTB, actin beta; Ctrl, control; IB, immunoblotting; sh, shRNA.
Figure 4
Figure 4
Olanzapine is a new ferroptosis inhibitor in acinar cells. (A) Chemical structures of 5 potential ferroptosis inhibitors. (B) Analysis of cell death in 266-6 cells after treatment with cerulein (100 nmol/L), L-arginine (5 mg/mL), alcohol (50 mmol/L), erastin (2 μmol/L), and RSL3 (500 nmol/L) in the absence or presence of trypsin (500 ng/mL), olanzapine (10 μmol/L), idebenone (10 μmol/L), telmisartan (10 μmol/L), nisoldipine (10 μmol/L), and azelnidipine (10 μmol/L) for 24 hours. Data are shown in a heat map as the mean of 3 biologically independent samples. (C) Analysis of cell death in 266-6 cells after treatment with cerulein (100 nmol/L)/trypsin (500 ng/mL) in the absence or presence of olanzapine (10 μmol/L), risperidone (10 μmol/L), SB204741 (10 μmol/L), and SB242084 (10 μmol/L) for 24 hours. Data are presented as means ± SD; n = 3 biologically independent samples; 2-way analysis of variance (ANOVA) with the Tukey multiple comparisons test on all pairwise combinations. (D) Analysis of mRNA expression in indicated gene knockdown 266-6 cells. Data are presented as means ± SD; n = 3 biologically independent samples. (E) Analysis of cell death in indicated gene knockdown 266-6 cells after treatment with cerulein (100 nmol/L)/trypsin (500 ng/mL) for 24 hours. Data are presented as means ± SD; n = 3 biologically independent samples; 2-way ANOVA with the Tukey multiple comparisons test on all pairwise combinations. (F) Scavenging DPPH free radical activity of indicated drugs. Data are presented as means ± SD; n = 3 biologically independent samples; 1-way ANOVA test on all pairwise combinations. (G–I) Analysis of (G) lipid peroxidation, (H) C20:4, and (I) C22:4 in 266-6 cells after treatment with cerulein (100 nmol/L)/trypsin (500 ng/mL) in the absence or presence of ferrostatin-1 (1 μmol/L), olanzapine (10 μmol/L), risperidone (10 μmol/L), SB204741 (10 μmol/L), and SB242084 (10 μmol/L) for 24 hours. Data are presented as means ± SD; n = 3 biologically independent samples; 1-way ANOVA test on all pairwise combinations. (J) Analysis of GPX4 protein expression in 266-6 cells after treatment with cerulein (100 nmol/L)/trypsin (500 ng/mL) in the absence or presence of olanzapine (10 μmol/L) for 24 hours. Semiquantitative data are presented as means ± SD; n = 3 biologically independent samples; 1-way ANOVA test on all pairwise combinations. ACTB, actin beta; Ctrl, control; sh, shRNA.
Figure 5
Figure 5
Ferroptotic damage increases the severity of acute pancreatitis in mice. (A) Representative images of pancreatic histology in cerulein-induced pancreatitis in Gpx4 WT and KO mice with or without olanzapine (5 mg/kg) treatment. Histologic scores. for acinar cell death, leukocyte infiltration, and edema at 12 hours after the last cerulein treatment were evaluated. Data are presented as means ± SD; n = 5 mice/group; 1-way analysis of variance (ANOVA) test on all pairwise combinations. Bar=100 µm (B–L) In parallel, (B) pancreatic Hspa5 mRNA, (C) serum amylase, (D) pancreatic trypsin activity, (E) pancreatic MPO activity, (F) serum LDH, (G) serum HMGB1, (H) serum trypsin activity, (I) pancreatic Ptgs2 mRNA, (J) pancreatic Acsl4 mRNA, (K) pancreatic cleaved caspase-3 (C-CASP3), (L) pancreatic p-MLKL, and (M) pancreatic MDA at 12 hours after the last cerulein treatment were assayed. Data are presented as means ± SD; n = 5 mice/group; 1-way ANOVA test on all pairwise combinations.
Figure 6
Figure 6
Ferroptotic damage increases the severity of chronic pancreatitis in mice. (A) Representative images of pancreatic histology in Lieber–DeCarli alcoholic liquid diet–induced pancreatitis in Gpx4 WT and KO mice with or without olanzapine (5 mg/kg) treatment. Histologic scores for acinar cell death, leukocyte infiltration, edema, and fibrosis at 4 weeks after Lieber–DeCarli alcoholic liquid diet were evaluated. Data are presented as means ± SD; n = 20 mice/group; 1-way ANOVA test on all pairwise combinations. Bar=100 µm (B–O) In parallel, (B) pancreatic Hspa5 mRNA, (C) serum amylase, (D) pancreatic trypsin activity, (E) pancreatic MPO activity, (F) serum LDH, (G) serum HMGB1, (H) pancreatic Ptgs2 mRNA, (I) pancreatic Acsl4 mRNA, (J) serum trypsin activity, (K) serum tumor necrosis factor (TNF), (L) serum IL6, (M) serum IL1B, (N) pancreatic cleaved caspase-3 (C-CASP3), and (O) pancreatic p-MLKL at 4 weeks after Lieber–DeCarli alcoholic liquid diet were assayed. Data are presented as means ± SD; n = 5–8 mice/group; 1-way ANOVA test on all pairwise combinations.
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References

    1. Beck I.T., Kahn D.S., Solymar J., McKenna R.D., Zylberszac B. The role of pancreatic enzymes in the pathogenesis of acute pancreatitis. III. Comparison of the pathologic and biochemical changes in the canine pancreas to intraductal injection with bile and with trypsin. Gastroenterology. 1964;46:531–542. - PubMed
    1. Hegyi E., Sahin-Toth M. Genetic risk in chronic pancreatitis: the trypsin-dependent pathway. Dig Dis Sci. 2017;62:1692–1701. - PMC - PubMed
    1. Ji B., Logsdon C.D. Digesting new information about the role of trypsin in pancreatitis. Gastroenterology. 2011;141:1972–1975. - PMC - PubMed
    1. Neoptolemos J.P., Kemppainen E.A., Mayer J.M., Fitzpatrick J.M., Raraty M.G., Slavin J., Beger H.G., Hietaranta A.J., Puolakkainen P.A. Early prediction of severity in acute pancreatitis by urinary trypsinogen activation peptide: a multicentre study. Lancet. 2000;355:1955–1960. - PubMed
    1. Gudgeon A.M., Heath D.I., Hurley P., Jehanli A., Patel G., Wilson C., Shenkin A., Austen B.M., Imrie C.W., Hermon-Taylor J. Trypsinogen activation peptides assay in the early prediction of severity of acute pancreatitis. Lancet. 1990;335:4–8. - PubMed

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