Lipopolysaccharide enhances TGF-β1 signalling pathway and rat pancreatic fibrosis

Abstract

Pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor-beta1 (TGF-β1) is a key regulator of extracellular matrix production and PSC activation. Endotoxin lipopolysaccharide (LPS) has been recognized as a trigger factor in the pathogenesis of ACP. This study aimed to investigate the mechanisms by which LPS modulates TGF-β1 signalling and pancreatic fibrosis. Sprague-Dawley rats fed with a Lieber-DeCarli alcohol (ALC) liquid diet for 10 weeks with or without LPS challenge during the last 3 weeks. In vitro studies were performed using rat macrophages (Mφs) and PSCs (RP-2 cell line). The results showed that repeated LPS challenge resulted in significantly more collagen production and PSC activation compared to rats fed with ALC alone. LPS administration caused overexpression of pancreatic TLR4 or TGF-β1 which was paralleled by an increased number of TLR4-positive or TGF-β1-positive Mφs or PSCs in ALC-fed rats. In vitro, TLR4 or TGF-β1 production in Mφs or RP-2 cells was up-regulated by LPS. LPS alone or in combination with TGF-β1 significantly increased type I collagen and α-SMA production and Smad2 and 3 phosphorylation in serum-starved RP-2 cells. TGF-β pseudoreceptor BAMBI production was repressed by LPS, which was antagonized by Si-TLR4 RNA or by inhibitors of MyD88/NF-kB. Additionally, knockdown of Bambi with Si-Bambi RNA significantly increased TGF-β1 signalling in RP-2 cells. These findings indicate that LPS increases TGF-β1 production through paracrine and autocrine mechanisms and that LPS enhances TGF-β1 signalling in PSCs by repressing BAMBI via TLR4/MyD88/NF-kB activation.

Keywords: TGF-β1; alcohol; lipopolysaccharide; pancreatic fibrosis; pancreatic stellate cell.

Figure 1

LPS was associated with increased expression of collagen and activation of PSCs. Aniline blue staining showing collagen deposition in the pancreas (blue) of alcohol (ALC, 15 g/kg/d)‐fed rat and ALC rat plus LPS (3 mg/kg once a week for 3 weeks) repeated injections (ALC + LPS) (A); Immunostaining showing the activated PSCs in the pancreas of ALC rat and ALC + LPS rat (B); The area of collagen deposition (C) or the content of Col1A1 (D) or the number of activated PSCs (E) in the pancreas of ALC + LPS rats was significantly more than that of ALC rats. Student's t test, ***P < .001 vs ALC rats (n = 11/group)

Figure 2

Enhanced expression of TLR4 in the pancreas of ALC‐fed rats with LPS injections. (A, B) Double‐immunofluorescence labelling using antibodies for TLR4 and F4/80 or α‐SMA. Yellow, colocalization of two antibodies. Scale bar = 50 μm; (A) Yellow staining indicates TLR4‐positive Mφs; (B) Yellow staining indicates TLR4‐positive PSCs. The number of TLR4‐positive Mφs/PSCs was higher in the pancreas of ALC + LPS rats than that of ALC rats (C, D); (E) The expression of TLR4 protein was examined by Western blot analysis (top panels) and the results, normalized with GAPDH, are the mean ± SEM of triplicate determinations (bottom panels). Student's t test, *P < .05, ***P < .001 vs ALC rats (n = 11/group)

Figure 3

Decreased expression of BAMBI in the PSCs of ALC‐fed rats with LPS injections. Immunohistochemical staining showing strong positive staining for BAMB in several PSCs of ALC rat (A), but weak staining in more PSCs of ALC + LPS rat (red) (B). The number of BAMBI‐positive PSCs in the pancreas of ALC and ALC + LPS rats (C); Computer image analysis showed that the integrated optical density (IOD) of BAMBI‐positive PSCs in the pancreas of ALC and ALC + LPS rats (D); The mean optical density (OD) per BAMBI‐positive PSC was used for comparing the expression intensity of BAMBI in PSCs of ALC and ALC + LPS rats (E). Student's t test, ***P < .001 vs ALC rats (n = 11/group)

Figure 4

TGF‐β1 production was enhanced by LPS in the pancreas of ALC‐fed rats and in the sera of ACP patients. (A, B) Double‐immunofluorescence labelling using antibodies for TGF‐β1 and F4/80 or α‐SMA. Yellow, colocalization of two antibodies. Scale bar = 50 μm; (A) Yellow staining indicates TGF‐β1‐positive Mφs; (B) Yellow staining indicates TGF‐β1‐positive PSCs. The number of TGF‐β1‐positive Mφs/PSCs was higher in the pancreas of ALC + LPS rats than that of ALC rats (C, D); (E) The expression of TGF‐β1 protein was determined by ELISA. Student t test, **P < .01, ***P < .001 vs ALC rats. (F) There was a positive correlation between TGF‐β1 and Col1A1 in the pancreas of LPS‐treated ALC rats. Pearson's correlation test, r = .541, P < .01 (n = 11/group). (G) The LPS levels in the sera of ACP patients and healthy control (Ctrl); (H) The TGF‐β1 levels in the sera of ACP patients with/without endotoxemia and Ctrl group. One‐way ANOVA followed by Bonferroni post hoc test, ***P < .001 vs Ctrl, ^### P < .001 vs ACP without endotoxemia. (I) There was a positive correlation between TGF‐β1 and LPS in the sera of ACP patients with endotoxemia. Pearson's correlation test, r = .609, P < .01. (Ctrl: n = 25; ACP without endotoxemia: n = 11; ACP with endotoxemia: n = 18)

Figure 5

Expression of TLR4 and TGF‐β1 was enhanced by LPS in cultured Mφs and PSCs. Immunocytological staining showing the enhanced expression of TLR4 by LPS (100 ng/mL) in cultured Mφs (A) and RP‐2 cells (B) The concentrations of TGF‐β1 were determined by ELISA in the supernatant of cultured Mφs (C) or RP‐2 cells (D) that had been incubated for 24 h in the absence (N/A) or presence of LPS (100 ng/mL). Student's t test, ***P < .001 vs N/A (n = 6/group)

Figure 6

LPS sensitizes PSCs to TGF‐β1 and down‐regulates BAMBI. Col1A1 and α‐SMA mRNA levels (A, B) or Col1 and α‐SMA protein contents (C) in the RP‐2 cells after 12 h and 24 h of stimulation with LPS (100 ng/mL) or TGF‐β1 (10 ng/mL) or in combination were determined by qPCR or Western blot, respectively. One‐way ANOVA followed by Bonferroni post hoc test, *P < .05, ***P < .001 vs N/A; ^### P < .001 vs LPS or TGF‐β1 group. TLR4 mRNA levels (D) or protein contents (E) were determined in the RP‐2 cells that had been incubated for 12 h or 24 h in the absence or presence of LPS (100 ng/mL); BAMBI mRNA levels F or protein contents (G) were determined in the RP‐2 cells after 12 h or 24 h of stimulation with LPS (100 ng/mL); Before treatment, RP‐2 cells were pre‐transfected with either small interfere control RNA (Si‐Ctrl) or small interfere TLR4 RNA (Si‐TLR4) for 24 h; Col1A1 and α‐SMA mRNA levels (H, I) or ERK1/2 and p38 and Smad2/3 phosphorylations (J) were determined in the RP‐2 cells after 12 h or 24 h of stimulation with TGF‐β1 (10 ng/mL); Before treatment, RP‐2 cells were pre‐transfected with either Si‐Ctrl or small interfere BAMBI RNA (Si‐BAMBI) for 24 h. Student's t test, **P < .01, ***P < .001 vs Si‐Ctrl group (n = 6/group)

Figure 7

Down‐regulation of BAMBI by LPS through a MyD88/NF‐kB‐dependent pathway. MyD88 and BAMBI mRNA levels (A, C) or proteins (B, D) in the RP‐2 cells after 12 h and 24 h of stimulation with LPS (100 ng/mL) were determined by qPCR and Western blot, respectively; Western blot showing the expression of pIkBα or IkBα in cytoplasm and NF‐kB p65 or p50 in nucleus of RP‐2 cells after 45 min of LPS stimulation (E, F) For inhibition experiments, RP‐2 cells were pre‐treated for 30 min with NF‐kB inhibitor Bay11(10 μmol/L) or MyD88 inhibitor ST2825 (10 μmol/L); The expression of pSmad2 or pSmad3 or Smad123 in whole cell lysate of RP‐2 cells that had been stimulated with LPS (100 ng/mL) or TGF‐β1 (10 ng/mL) or in combination for 12 h (G). Student's t test, ***P < .001 vs N/A (n = 6/group)