Fig 1: LRG1 regulates acinar cell function through AKT-mediated CCK1R expression. (A) qRT-PCR analysis of cholecystokinin A receptor (Cck1r) mRNA levels and (B) Western blot (top) and densitometry analysis (bottom) of CCK1R protein levels in the pancreas of adult wild-type and Lrg1-/- mice. (C) Western blot (left) and densitometry analysis (right) of phosphorylated and total levels of ALK5, AKT, ERK, and SMAD2 in the pancreatic of wild-type and Lrg1-deficient mice. (D) Western blot (left) and densitometry analysis (right) of phosphorylated and total levels of ALK5 and AKT protein in primary acinar cells isolated from wild-type or Lrg1-/- mice in the presence or absence of rhLRG1. (E) qRT-PCR analysis Cck1r mRNA levels and (F) Western blot (left) and densitometry analysis (right) of CCK1R protein levels in primary acinar cells isolated from wild-type or Lrg1-/- mice in the presence or absence of rhLRG1. (G) Western blot (left) and densitometry analysis (right) of pALK5, ALK5, pAKT, and AKT levels in primary acinar cells isolated from Lrg1-/- mice subjected to the treatment with ALK5 (SB 431542) or AKT(MK2206) specific inhibitor. (H) qRT-PCR analysis of Cck1r mRNA levels and (I) Western blot (left) and densitometry analysis (right) of CCK1R protein levels in primary acinar cells isolated from Lrg1-/- mice subjected to the treatment with ALK5 or AKT inhibitor. All images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak's multiple comparisons test or unpaired, two-tailed Student's t-test of n ≥ 4 mice or independent experiments; *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Fig 2: Loss of LRG1 does not affect the structure and function of the normal mouse pancreas. (A) Immunofluorescence staining of LRG1 (red), CD31 or NG2 (green), and DAPI (blue) in the normal mouse pancreas. (B) Pancreas weight to body weight ratio of wild-type and Lrg1-/- mice. (C) H&E staining showing vasculature of wild-type and Lrg1-/- mouse pancreas. (D) Immunofluorescence staining of CD31 (red) and DAPI (blue) (left) and quantification of vessel density (right) in wild-type and Lrg1-/- mouse pancreas. (E) H&E staining showing exocrine component of wild-type and Lrg1-/- mouse pancreas. (F) Immunofluorescence staining of amylase (AMY, green) and DAPI (blue) in wild-type and Lrg1-/- pancreas. (G) Serum amylase activity in wild-type and Lrg1-/- mice. (H) Immunofluorescence staining of cytokeratin 19, KRT19 (red), and DAPI (blue) (left) and quantification of ductal density (right) in wild-type and Lrg1-/- pancreas. (I) H&E staining showing endocrine component of wild-type and Lrg1-/- mouse pancreas. (J) Immunofluorescence staining of glucagon (GCG, green) and DAPI (blue) in wild-type and Lrg1-/- pancreas. (K) Fasting glucagon levels in wild-type and Lrg1-/- mice. (L) Immunofluorescence staining of insulin (INS, magenta) and DAPI (blue) in wild-type and Lrg1-/- pancreas. (M) Fasting blood glucose levels in wild-type and Lrg1-/- mice following the intraperitoneal delivery of insulin. Intraperitoneal insulin tolerance (ITT) is indicated as (N) the percentage of basal glucose and (O) the area under the curve (AUC). All images are representative, scale bar: 50μm for H&E images and 20μm for immunofluorescence images. Data are presented as mean ± s.e.m. Significance was determined by unpaired, two-tailed Student's t-test of n ≥ 4 mice; n.s.: not significant.
Fig 3: LRG1 is highly induced in humans and mice during AP. (A) ELISA analysis of LRG1 levels in the serum of healthy controls (n = 10) and AP patients (n = 18). (B) Correlation analysis with regression line (95% confidence intervals) of serum LRG1 and CRP in AP patients. (C) Schematic diagram of caerulein-induced AP in mice. (D) ELISA analysis of serum LRG1 levels in mice subjected to caerulein-induced AP. (E) qRT-PCR analysis of pancreatic Lrg1 levels at various time points during AP progression. (F) Western blot (top) and densitometry analysis (bottom) of pancreatic LRG1 levels at various time points during AP progression. (G) Immunofluorescent staining of LRG1 (red), CD31 or AMY or MPO (green), and DAPI (blue) in mouse pancreas 24 hours following AP. Scale bar (CD31 and MPO): 10μm, Scale bar (AMY): 40μm. (H) Immunofluorescent staining of LRG1 (red), CD31 or AMY or MPO (green), and DAPI (blue) in control or human AP pancreas. Scale bar: 10μm. (I) qRT-PCR analysis of Lrg1 in the pancreas of saline or caerulein-treated wild-type recipient mice transplanted with wild-type or Lrg1-/- BMCs and Lrg1-/- recipient mice transplanted with wild-type BMCs 24 hours post AP induction. (J) qRT-PCR or (K) Western blot (top) and densitometry analysis (bottom) of Lrg1 in isolated acinar cells 24 hours following the first caerulein injection. All images are representative. Data are presented as mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak's multiple comparison test or unpaired, two-tailed Student's t-test of n ≥ 3 mice or independent experiments unless stated otherwise; *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Fig 4: Non-myeloid cell-derived LRG1 protects against AP-induced damage. (A) Schematic diagram of bone marrow transplantation with wild-type mice reconstituted with wild-type BMCs (Wild-type → Wild-type), Lrg1-/- mice reconstituted with wild-type BMCs (Wild-type → Lrg1-/-), wild-type mice reconstituted with Lrg1-/- BMCs (Lrg1-/- → Wild-type) and Lrg1-/- mice reconstituted with Lrg1-/- BMCs (Lrg1-/- → Lrg1-/-). (B) H&E staining and (C) histopathological scoring of overall pancreatic damage. Scale bar: 100μm, scale bar of boxed region: 50μm. (D) Immunofluorescent staining against MPO (red) and DAPI (blue) (left) and quantification (right) of infiltrated MPO+ inflammatory cells (arrow) in the pancreas. Scale bar: 25μm. qRT-PCR analysis of mRNA levels of (E) Cxcl1, (F) Ccl2, (G) Tnfa, (H) Il6 and (I) Amy2 in the pancreas. (J) Western blot (top) and densitometry analysis (bottom) for cleaved caspase 3 levels in primary wild-type or Lrg1-/- acinar cells subjected to saline or caerulein treatment. qRT-PCR analysis of mRNA levels of (K) Tnfa, (L) Il6, and (M) Cxcl1 in primary acinar cells isolated from wild-type or Lrg1-/- mice subjected to saline or caerulein treatment. All analyses were performed 24 hours after the induction of AP. Images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak's multiple comparisons test of n ≥ 4 mice; *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Fig 5: LRG1 is regulated by IL-6 in pancreatic acinar cells during AP. (A) ELISA analysis of IL-6 levels in the serum of healthy controls (n = 10) and AP patients (n = 18). (B) Correlation analysis with regression line (95% confidence intervals) of serum LRG1 and IL-6 in AP patients. (C) ELISA analysis of serum IL-6 levels in mice subjected to caerulein-induced AP. (D) qRT-PCR analysis of Il6 in mouse pancreas at various time points during AP progression. (E) qRT-PCR analysis of Il6 in isolated acinar cells 24 hours following the first caerulein injection. (F) Western blot (left) and densitometry analysis (right) of phosphorylated and total STAT3, and LRG1 in wild-type acinar cells subjected to DMSO or STATTIC treatment with or without addition of IL-6. (G) Schematic indicating organization of the mouse LRG1 promoter containing two putative STAT3 transcription factor binding sites. (H) DNA agarose gel (left) and quantitative analysis (right) of chromatin immunoprecipitation assay for STAT3 and LRG1 promoter association in the presence or absence of IL-6 in primary acinar cells. All images are representative. Data are presented as mean ± s.e.m. Significance was determined by one-way ANOVA followed by Holm-Sidak's multiple comparison test or unpaired, two-tailed Student's t-test of n ≥ 3 mice or independent experiments unless stated otherwise; *: p < 0.05, **: p < 0.01.
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