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, 3 (2), e1701

Cannabinoids Reduce Markers of Inflammation and Fibrosis in Pancreatic Stellate Cells

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Cannabinoids Reduce Markers of Inflammation and Fibrosis in Pancreatic Stellate Cells

Christoph W Michalski et al. PLoS One.

Abstract

Background: While cannabinoids have been shown to ameliorate liver fibrosis, their effects in chronic pancreatitis and on pancreatic stellate cells (PSC) are unknown.

Methodology/principal findings: The activity of the endocannabinoid system was evaluated in human chronic pancreatitis (CP) tissues. In vitro, effects of blockade and activation of cannabinoid receptors on pancreatic stellate cells were characterized. In CP, cannabinoid receptors were detected predominantly in areas with inflammatory changes, stellate cells and nerves. Levels of endocannabinoids were decreased compared with normal pancreas. Cannabinoid-receptor-1 antagonism effectuated a small PSC phenotype and a trend toward increased invasiveness. Activation of cannabinoid receptors, however, induced de-activation of PSC and dose-dependently inhibited growth and decreased IL-6 and MCP-1 secretion as well as fibronectin, collagen1 and alphaSMA levels. De-activation of PSC was partially reversible using a combination of cannabinoid-receptor-1 and -2 antagonists. Concomitantly, cannabinoid receptor activation specifically decreased invasiveness of PSC, MMP-2 secretion and led to changes in PSC phenotype accompanied by a reduction of intracellular stress fibres.

Conclusions/significance: Augmentation of the endocannabinoid system via exogenously administered cannabinoid receptor agonists specifically induces a functionally and metabolically quiescent pancreatic stellate cell phenotype and may thus constitute an option to treat inflammation and fibrosis in chronic pancreatitis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cannabinoid receptor 1 in human chronic pancreatitis tissues.
CB1 immunohistochemistry: staining of tubular complexes (A) but immunonegativity of infiltrating mononuclear cells (B); various staining intensities of intrapancreatic nerves (C&D); pancreatic stellate cells in areas of fibrosis (E) or isolated and cultured in vitro (F). Original magnification: x20 (A, B, D), x40 (C), x80 (F). Insets: negative controls. Lower insets (A): CB1 in normal pancreas.
Figure 2
Figure 2. Cannabinoid receptor 2 and endocannabinoid levels in chronic pancreatitis.
Using an anti-CB2 antibody, tubular complexes were strongly stained (A). Invading mononuclear cells were also immunopositive (B), whereas intrapancreatic nerves were mostly unstained or only faintly positive (B). Pancreatic stellate cells were immunopositive for CB2 (C). The endocannabinoids anandamide (AEA) and 1+2-arachidonoylglycerol (1+2-AG) were lower in chronic pancreatitis (AEA: p = 0.14 and 1+2-AG: p = 0.0066; D). Cultured pancreatic stellate cells were faintly CB2-immunopositive (E). Original magnification: x20 (A, B), x40 (C), x80 (F). Insets: negative controls. Lower insets (A): CB2 in normal pancreas.
Figure 3
Figure 3. Effects of cannabinoid receptor antagonism on pancreatic stellate cells.
(A) MTT assays after 48 hours' incubation of PSC with graded concentrations of CB1- and CB2-receptor antagonists AM251 and AM630 (p = n.s. for concentrations of 1.25, 2.5 and 5 µM). (B) LDH in cell culture supernatants and the fraction of apoptotic cells as judged by the GuavaNexin™ test. AM251 significantly increased IL-6 (C) but decreased MCP-1 secretion (D); AM630 had no effect on IL-6 (C) but induced a tendency towards increased MCP-1 levels (D). CB1-/2-receptor antagonism did not affect TGFbeta (E). Data are shown as mean±SEM (*, p<0.05).
Figure 4
Figure 4. Cannabinoid receptor blockade alters synthesis of ECM proteins.
As assessed by immunoblot analysis of cell culture supernatants, AM251 and AM630 (at 2.5 µM) did not affect collagen and fibronection secretion. In PSC cell lysates, alphaSMA levels remain unchanged following CB1-receptor antagonism whereas AM630 induced increased alphaSMA protein levels. White bars: control; black bars: AM251; grey bars: AM630. Data are shown as mean±SEM.
Figure 5
Figure 5. AM251 induces changes in PSC phenotype.
(A) Invasion assays demonstrated a trend toward increased invasiveness induced by AM251. (B) MMP-2 levels were unchanged by antagonist treatment. (C&D) AM251-treated pancreatic stellate cells were smaller and thinner, with a more stretched shape, an increased length, and a loss of intracellular fibres (control PSC: insets and Figure 8C&D). Cells treated with AM630 (E&F) closely resembled the control PSC (see insets C&D and Figure 8C&D). Original magnification: x 40 (C&E), x 80 (D&F). Nuclear stain: DAPI. White bars: control; black bars: AM251; grey bars: AM630. Data are shown as mean±SEM.
Figure 6
Figure 6. Cannabinoid receptor activation on PSC reduces growth, independent of apoptosis or necrosis.
(A) MTT tests of WIN-treated PSC revealed dose-dependent inhibition of growth (p<0.05 at concentrations of 2.5 and 5 µM). (B&C) While AM251 and AM630 alone were not effective, pre-treatment with a combination of AM251 and AM630 blocked WIN-induced growth inhibition (D). The reduction in proliferated cells was neither due to necrosis (unchanged LDH levels) nor to apoptosis (constant low number of apoptotic cells in treated versus control PSC; E). Data are shown as mean±SEM (* p<0.05).
Figure 7
Figure 7. WIN de-activates pancreatic stellate cells.
(A) IL-6 and MCP-1 secretion were significantly reduced by WIN (p = 0.001 and p = 0.0002, respectively), independent of TGFbeta (B; unchanged TGFbeta levels). The reduction in IL-6 and MCP-1 levels was partially reversed by a combination of the CB1-receptor and CB2-receptor antagonists AM251 and AM630 (A). While control PSC secreted significant amounts of fibronectin and collagen 1 (as seen by an intense signal at 220 and 190 kDa, respectively), treatment with WIN reduced the signal at the respective molecular weights (B; immunoblots of cell culture supernatants, pooled from three independent experiments). AlphaSMA levels were also suppressed by WIN (B; immunoblot of PSC cell lysates; gamma-tubulin: equal loading control). A combination of both antagonists AM251 and AM630 partially reversed the suppressive effects elicited by incubation with WIN (B, immunoblots of fibronectin, collagen 1 and alphaSMA). White bars: control; black bars: WIN55,212-2±AM251/AM630. Data are shown as mean±SEM.
Figure 8
Figure 8. Cannabinoid receptor activation reduces invasiveness.
A significant reduction in the number of invaded cells was seen after treatment with WIN (p = 0.0081; A). A near-complete reversal of the reduced invasiveness was seen when the cells were pre-incubated with AM251 or a combination of AM251 and AM630 (A). This was not found with AM630 alone (A). WIN induced a significant decrease in MMP-2 levels (p = 0.026; B) which was partially reversible by a combination of AM251 and AM630 (B). Actin cytoskeleton staining with phalloidin demonstrated that control PSC showed the normal cellular structure (C&D) but that WIN induced a more round and smaller PSC phenotype (E&F). Original magnification: x 40 (C&E), x 80 (D&F). Nuclear stain: DAPI. White bars: control; black bars: WIN55,212-2±AM251/AM630. Data are shown as mean±SEM.

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