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Low Dose Naltrexone Reduces In Vitro Endoplasmic Reticulum Stress and Stimulates Wound Healing in Intestinal Epithelial Cells

Low Dose Naltrexone Reduces In Vitro Endoplasmic Reticulum Stress and Stimulates Wound Healing in Intestinal Epithelial Cells
Publication Type
Research Type
Reported as
Clinical Trial
May 01, 2016
Fuhler, G., Lie, M., Dimitrijevic, P., Van Der Woude, C.J., and Peppelenbosch, M.
Erasmus MC University Medical Centre

BACKGROUND: Naltrexone (NTX), a μ-opioid receptor (MOR) antagonist, up regulates MOR and its ligands when used at low doses. Stimulation of MOR in mouse models alleviates intestinal inflammation, and treatment of IBD patients with low-dose NTX can induce remission and mucosal healing. Although NTX reduces immunocyte proliferation and cytokine production, it is unclear whether a direct action of low-dose naltrexone (LDN) on intestinal epithelial cells (IEC) can contribute to its beneficial effects in IBD. In this study, we aimed to investigate the in-vitro effect of NTX on IECs, and confirm these results in IBD patients treated with low-dose NTX.

METHODS: In total, 40 patients (43% male, median age 40y, IQR 28–52 years) were treated with 5 mg NTX/day. Response was seen in 23 patients (13 CD; 10 UC), with a median duration of 2 months (IQR 2–3 months). Serum was obtained from 7 patients before NTX and 3 months into treatment. Formalin-fixed paraffin-embedded (FFPE) biopsies were obtained from 12 patients before treatment and 5 patients 3 months into treatment. MOR expression in IECs (Caco2 and HCT116) was determined by rt-polymerase chain reaction (PCR), IEC cell viability was determined by MTT assay, and wound healing was determined by scratch wound assays. Cytokine levels in supernatants from IECs and patient sera were determined by ELISA. The effect of NTX on endoplasmic reticulum (ER) stress was determined in IECs by Western blot analysis of GRP78 expression and by immunohistochemistry on FFPE slides from low-dose NTX treated patients.

RESULTS: IEC express MOR mRNA, allowing for direct NTX interaction. IEC viability was not affected by NTX at concentrations up to 100 μg/ml. At 1 μg/ml, NTX significantly improved cell migration into scratch wounds compared with control (330 ± 27 vs 140 ± 19 pixels, p = 0.0021 for HCT116, 470 ± 16 vs 248 ± 19, p < 0.001 for Caco2). Although no TNFα production could be detected in IEC supernatants, neither basal nor E. Coli-induced IL8 secretion were affected by NTX. Likewise, no modulation of TNFα or IL8 levels upon NTX was observed in patient sera. Mucosal ER stress is known to play an important role in IBD, and could be induced in IECs upon treatment with either E. Coli (DH5α) or Tunicamycin (2μM). Interestingly, NTX significantly reduced this ER stress. High levels of ER stress were observed in the inflamed intestinal interstitium and crypts from IBD patients before low-dose NTX treatment, and decreased upon NTX treatment (1.14 ± 0.5 vs 0.8 ± 0.5 and 0.9 ± 0.4 vs 0.7 ± 0.6) although statistical significance was not reached because of low patient numbers. However, in 3 paired samples, NTX treatment reduced interstitial ER stress.

CONCLUSION: NTX may have a beneficial effect in the treatment of IBD by directly stimulating epithelial wound healing and reducing intestinal ER stress.