Mu opioid signaling protects against acute murine intestinal injury in a manner involving Stat3 signaling - PubMed (original) (raw)

Mu opioid signaling protects against acute murine intestinal injury in a manner involving Stat3 signaling

Jason R Goldsmith et al. Am J Pathol. 2011 Aug.

Abstract

Opiates have long been used as analgesics to relieve pain associated with various medical conditions. Here, we evaluated the effect and mechanism of mu opioid signaling on the intestinal wound healing response and assessed downstream pathways known to be protective against intestinal injury. Mice (C57BL/6) were exposed to 3% dextran sodium sulfate (DSS) for 7 days or 4% DSS for 5 days followed by 7 days of water. The mu opioid receptor (MOR)-specific agonist [D-Arg2,Lys4]dermorphin-(1,4)-amide (DALDA) and the antagonist cyprodime were injected s.c. daily for in vivo studies or used for in vitro analysis. We found that MOR activation attenuated DSS-induced histologic and gross intestinal injury and weight loss; diminished Ifng, Tnf, and Il6 mRNA expression; and promoted intestinal healing during recovery. DALDA also enhanced colonocyte proliferation (Ki-67 staining) by 350%. MOR activation increased Stat3 phosphorylation in both DALDA-treated mice and the CMT-93 cell line. Importantly, DALDA-induced colonocyte migration was completely ablated by shStat3 knockdown. Together, this work shows that MOR activation protects against and enhances recovery from DSS-induced intestinal injury. This is associated with an increase in Stat3 activation. Furthermore, Stat3 is required for DALDA-induced colonocyte migration. Consequently, manipulation of MOR signaling may represent a novel means to promote mucosal healing and to maintain intestinal homeostasis after intestinal injury.

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Figures

Figure 1

MOR activation protects against DSS-induced intestinal injury. Mice were given 3% DSS for 7 days concurrent with opioid treatment. DALDA (50 μg/kg), cyprodime (10 mg/kg), or vehicle was injected s.c. daily. All graphs depict mean ± SEM. A: Percentage of weight loss from starting weight. N = 7 for all groups. P < 0.001 × 2-way analysis of variance between treatment groups and over time; _P_ < 0.05 for DALDA-treated versus vehicle-treated at day 7. **B:** Clinical score. _N_ = 7 for all groups. _P_ < 0.01 for DALDA-treated versus vehicle-treated. **C:** Histologic score. _N_ ≥ 5 for all groups. **D:** Crypt damage score. _N_ > 4 for all groups. E: Representative H&E photomicrographs. Scale bar = 100 μm for all sections. F: Fold change Tnf and Il6 distal colonic mRNA expression. N ≥ 4 for all groups. *P < 0.05, **P < 0.01.

Figure 2

MOR activation enhances recovery from DSS-induced intestinal injury. Mice were administered 4% DSS for 5 days, followed by 7 days of water recovery concurrent with treatment: 50 μg/kg DALDA or vehicle was injected s.c. daily. All graphs depict mean ± SEM. A: Percentage of weight loss from starting weight. N > 6 for all groups. B: Representative colonoscopies for each group on the final day of the experiment. C: Clinical score. N = 6 for all groups. D: Histologic score. N > 4 for all groups. E: Crypt damage score. N > 4 for all groups. F: Fold change Tnf, Il6, Il1b, and Ifng mRNA accumulation was determined by RT-PCR. N ≥ 4 for all groups. ns, not significant. *P < 0.05, **P < 0.01.

Figure 3

Enhanced colonocyte proliferation in DALDA-treated mice. All graphs depict mean ± SEM. A: Representative photomicrographs. Scale bar = 100 μm. B: Ki-67 positive cells were counted in 50 crypts across multiple fields of view. N ≥ 4 for all groups except DALDA alone, for which N = 3. *P < 0.05, **P < 0.01.

Figure 4

DALDA enhances Stat3 activation in DSS-induced intestinal injury. Mice were administered 3% DSS + vehicle or 3% DSS + DALDA (50 μg/kg, injected s.c. daily) for 7 days. All graphs depict mean ± SEM. A: Levels of Stat3 phosphorylation (Y705) in distal colonic tissue measured by Western blot analysis. Relative level of pStat3 over total Stat3 was determined by densitometry. Representative of four individual mice. B and C: pStat3 IHC analysis. Representative images of four mice per group. Scale bars = 100 μm in all images. White boxes outline higher magnification images of each section. Bar graph shows pStat3-positive colonocytes per 20 crypts. D: Expression of Stat3-dependent genes (Ptgs2, Myc, Reg3b, Ccnd1) in distal colonic tissue. N ≥ 3 per group. *P < 0.05, **P < 0.01.

Figure 5

DALDA induces Stat3 phosphorylation in CMT-93 cells. A–C: CMT-93 cells were treated for 1 hour with 10 μmol/L DALDA or vehicle. A: Western blot analysis and densitometry compare pStat3 with Stat3 within the same sample. Representative of eight independent experiments. B: pStat3 immunofluorescence staining, representative of triplicate samples, from two independent experiments. C: Activation of Stat3-dependent genes (Ptgs2, Myc, Ccnd1, Reg3b). N ≥ 4 for both groups, in two independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6

DALDA induces ERK1/2 phosphorylation by an opioid-dependent pathway. A: Western blot analysis for pERK1/2 in CMT-93 cells stimulated with 10 μmol/L DALDA for 0 to 60 minutes. Representative of three individual experiments. B: Western blot analysis for pStat3 and pERK1/2 in CMT-93 cells stimulated with DALDA for 1 hour with or without 30-minute pretreatment with 1 mmol/L of the opioid antagonist naloxone. Representative of three individual experiments. C: DALDA induces colonic ERK1/2 phosphorylation in vivo. Western blot analysis for pERK1/2 from distal colonic protein extracts from individual control mice or mice injected with 50 μg/kg DALDA daily s.c. for 7 days.

Figure 7

DALDA enhances colonocyte migration in a Stat3-dependent manner. A: CMT-93 monolayers were scratched and treated with 10 μmol/L DALDA or vehicle. Migration was observed over a 24-hour period. A: Representative photomicrographs of cell migration. Shaded bar shows initial wound area. Scale bar = 100 μm. B: Quantification of migration distance after 8 hours. Mean results of three3 independent experiments; N = 4 for each experiment. C: Reproductive cellular proliferation assays. CMT-93 cells were treated with 10 μmol/L DALDA or vehicle for 8 hours, and direct cell quantification was performed. Mean results of two independent experiments; N = 4 for each experiment. D–F: Cells were infected with a sh_Stat3_ lentivirus to reduce Stat3 expression or control scrambled shRNA (shScramble). D: Western blot analysis representative of three independent experiments show Stat3 knockdown in sh_Stat3_ cells. E: Western blot analyses show reduced DALDA-induced Stat3 phosphorylation in sh_Stat3_-infected CMT-93 cells. Representative of two individual experiments. F: Quantification of migration over an 8-hour period. Representative of two separate experiments; N = 4 per experiment. ns, not significant. *P < 0.05, **P < 0.01.

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Mu opioid signaling protects against acute murine intestinal injury in a manner involving Stat3 signaling - PubMed (original) (raw)