NADPH oxidase 1 mediates caerulein-induced pancreatic fibrosis in chronic pancreatitis

Abstract

Inflammatory disorders of the pancreas are divided into acute (AP) and chronic (CP) forms. Both states of pancreatitis are a result of pro-inflammatory mediators, including reactive oxygen species (ROS). One of the sources of ROS is NADPH oxidase (Nox). The rodent genome encodes Nox1-4, Duox1 and Duox2. Our purpose was to assess the extent to which Nox enzymes contribute to the pathogenesis of both AP and CP using Nox-deficient mice. Using RT-PCR, Nox1 was found in both isolated mouse pancreatic acini and pancreatic stellate cells (PaSCs). Subsequently, mice with genetically deleted Nox1 were further studied and showed that the histo-morphologic characteristics of caerulein-induced CP, but not caerulein-induced AP, was ameliorated in Nox1 KO mice. We also found that the lack of Nox1 impaired caerulein-induced ROS generation in PaSCs. Using Western blotting, we found that AKT mediates the fibrotic effect of Nox1 in a mouse model of CP. We also found a decrease in phospho-ERK and p38MAPK levels in Nox1 KO mice with CP, but not with AP. Both CP-induced TGF-β up-regulation and NF-ĸB activation were impaired in pancreas from Nox1 KO mice. Western blotting indicated increases in proteins involved in fibrosis and acinar-to-ductal metaplasia in WT mice with CP. No change in those proteins were observed in Nox1 KO mice. The lack of Nox1 lowered mRNA levels of CP-induced matrix metalloproteinase MMP-9 and E-cadherin repressor Twist in PaSCs. CONCLUSION: Nox1-derived ROS in PaSCs mediate the fibrotic process of CP by activating the downstream redox-sensitive signaling pathways AKT and NF-ĸB, up-regulating MMP-9 and Twist, and producing α-smooth muscle actin and collagen I and III.

Keywords: Chronic pancreatitis; MMP-9; NADPH oxidase 1; NF-ĸB; Twist.

Fig. 1A.. The absence of Nox1 impairs the effect of CP on pancreas weight. Left panel:

Representative pictures demonstrating pancreas atrophy in WT mice with CP, but not in Nox1 KO mice with CP. Right panel: Comparison of PW/BW ratio. Mean ± SE. n: WT: 10; WT w/CP: 7; Nox1 KO: 9; Nox1 KO w/CP: 6. *:p < 0.05 vs control.

Fig. 1B.. The absence of Nox1 impairs the histological feature characteristics of CP.

Representative hematoxylin and eosin (H&E)-stained sections (total magnification: 400x) show moderate acinar loss, fibrosis, and inflammatory infiltration (arrows) in pancreas from caerulein-treated WT mice, while normal cytoarchitecture of the pancreas and lack of fibrosis in pancreas from caerulein-treated Nox1 KO mice. n: 3.

Fig. 2A.. The lack of Nox1 reduces fibrosis in CP.

Pancreatic tissue were fixed with 10% formalin and embedded in paraffin. Following incubation with antibody against α-SMA, positive cells were visualized using 3, 3-diaminobenzi-dine tetrahydrochloride (DAB) as a chromogen (color: brown). (Arrows: α-SMA). Total magnification: 200X. n: 3.

Fig. 2B.. The lack of Nox1 reduces the amount of collagen I and III fibers in CP.

Pancreatic tissue were fixed with 10% formalin and embedded in paraffin. Cryostat sections of the tissue fixed were put onto glass slides, which were incubated with Picro-Sirius Red solution and proceeded according to the instruction provided by the manufacturer (Abcam, Cambridge, MA). Total magnification: 400X. (color: red). n: 3.

Fig. 2C.. The lack of Nox1 reduces CP-increased collagen 1A levels in whole pancreatic tissues.

Whole pancreatic lysates were obtained from WT and Nox1 KO mice with or without CP. Representative immunoblots for collagen 1A1 (220 kDa) shows increased levels of collagen 1A1 in whole pancreatic lysates from WT mice with CP, but not from Nox1 KO mice with CP. A representative immunoblot for tubulin (52 kDa) indicated that the same amount of proteins was loaded in each well. (n: 4).

Fig. 3A.. The lack of Nox1 impairs nuclear translocation of NF-ĸB in CP. Left:

NF-ĸB was visualized using DAB as a chromogen (color: brown). Arrows: NF-ĸB positive nuclei. Total magnification: 400X. Right: Quantitative analysis of nuclear translocation of NF-ĸB is displayed as Box and whiskers plot. Values are means ± SE (n: 4 experiments). ***p < 0.001 vs. heathy WT mice.

Fig. 3B.. The lack of Nox1 impairs CP-induced NF-ĸB activation in whole pancreatic tissues.

Whole pancreatic lysates were obtained from WT and Nox1 KO mice with or without CP. Representative immunoblots for phospho–NF–ĸB (65 kDa) shows an activation of p65 NF-ĸB in whole pancreatic lysates from WT mice with CP, but not from Nox1 KO mice with CP. A representative immunoblot for total p65 NF-ĸB (65 kDa) indicated that the same amount of proteins was loaded in each well. (n: 4).

Fig. 4A.. The absence of Nox1 abolishes caerulein-induced TGF-β up-regulation.

Total RNA was isolated from mouse pancreas using Trizol and RNeasy® Mini kit. Data are expressed as fold change in gene expression (mean ± SE). 18S rRNA was used as a reference. n: 5 mice. *:p < 0.05 vs control.

Fig. 4B.. Left panel: Nox1 mediates caerulein-induced ADM.

Representative immunoblots for vimentin (57 kDa), α-SMA (42 kDa), amylase (45 kDa), keratin 19(44.5 kDa), and TGF-β (12 kDa, 45 kDa) are shown. Equivalent loading was confirmed using α-tubulin (52 kDa). n: 3. Right panels: Nox1 induces phosphorylation of AKT in a mouse model of CP, but not in a mouse model of AP. Representative immunoblots for p-AKT (60 kDa), total AKT (60 kDa), phospho-S6RP (32 kDa), p38MAPK (40 kDa), p-JNK (46, 54 kDa), total JNK (46, 54 kDa), p-ERK (44, 42 kDa), and total ERK (44, 42 kDa) are shown. Equivalent loading was confirmed using α-tubulin (52 kDa). n: 3.

Fig. 4C.. Nox1 induces phosphorylation of AKT in a mouse model of CP.

p-AKT was visualized using DAB as a chromogen (color: brown). The staining displayed a cytoplasmic localization of p-AKT in pancreatic acini and islets of Langerhans from WT mice with CP, but not from Nox1 KO mice with CP. Total magnification: 400X.

Fig. 5.. Nox1 mediates caerulein-induced ROS generation in PaSCs. Left panel:

H₂O₂ levels were tested using fluorescence by H₂DCFDA. n: 6 experiments. Right panel: Intracellular levels of O⁻₂ were determined using fluorescence by DHE. n: 3 experiments. (mean ± SE). *: p < 0.05 vs. control PasCS from WT mice.

Fig. 6.

A schematic model proposed for the role of Nox1 in the fibrotic process of CP.