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Opioid Use in Murine Model Results in Severe Gastric Pathology that May Be Attenuated by Proton Pump Inhibition

      Opioids are the gold standard for chronic and acute pain management; however, their consequence on gastric function is relatively understudied. Opioid users have a higher incidence of gastric dysfunction, worse quality of life, increased hospitalizations, and increased use of antiemetic and pain modulator medications. The current study shows that morphine treatment in the murine model results in greater disruption of gastric epithelial cell morphology, increased gastric cell apoptosis, elevated inflammatory cytokines, and matrix metallopeptidase-9 secretion. Morphine treatment also increases gastric acid secretion and causes delays in gastric emptying. Moreover, morphine treatment causes an increase in systemic IL-6 level, which plays an important role in morphine-induced delayed gastric emptying and gastric damage. IL-6 knockout mice show a significant level of reduction in morphine-induced gastric delaying, acid retention, and gastric damage. Thus, morphine-mediated gastric damage is a consequence of the accumulation of acid in the stomach due to increased gastric acid secretion and delayed gastric emptying. Treatment with a proton pump inhibitor resulted in a significant reduction in morphine-induced gastric inflammation, gastric delaying, and improved morphine tolerance. Hence, these studies attribute morphine-mediated induction in gastric acidity and inflammatory cytokines as drivers for morphine-associated gastric pathology and show the therapeutic use of proton pump inhibitors as an inexpensive approach for clinical management of morphine-associated pathophysiology and analgesic tolerance.
      Pain therapy is an important challenge in the global community. Opioids are effective analgesics for cancer and are increasingly used for noncancer and postoperative pain management. However, the use of morphine to alleviate pain also results in undesired adverse effects, such as addiction, analgesic tolerance, and immunosuppression. Clinical dose of opioids in pain management often causes undesirable gastrointestinal (GI) adverse effects such as nausea, vomiting, and reduced gastrointestinal movement known as opioid-induced bowel dysfunction.
      • Camilleri M.
      Opioid-induced constipation: challenges and therapeutic opportunities.
      Reduced GI transit leads to bloating, idiopathic constipation, and subsequently gastroesophageal reflux disease. The presence of three discrete opioid receptors (μ, δ, and κ) on gastric smooth muscle cells has been reported by Nishimura et al.
      • Nishimura E.
      • Buchan A.M.
      • McIntosh C.H.
      Autoradiographic localization of mu- and delta-type opioid receptors in the gastrointestinal tract of the rat and guinea pig.
      Endogenous opioids have been shown to play an important role in the control of gastrointestinal tract motility. Opioid peptides have effects on several gastrointestinal functions, including motility, acid secretion, and intestinal electrolyte and fluid transport.
      • Khansari M.
      • Sohrabi M.
      • Zamani F.
      The useage of opioids and their adverse effects in gastrointestinal practice: a review.
      In addition, opioids have been shown to influence various functions affecting gastric mucosa and the release of various peptides, including gastrin.
      • Nishi S.
      • Seino Y.
      • Kitano N.
      • Seno M.
      • Tsuji K.
      • Kurose T.
      • Taminato T.
      • Tsuda K.
      • Yanaihara C.
      • Yanaihara N.
      Effects of naloxone on basal and vagus nerve-induced secretions of GRP, gastrin, and somatostatin from the isolated perfused rat stomach.
      ,
      • Weigert N.
      • Schäffler A.
      • Reichenberger J.
      • Madaus S.
      • Classen M.
      • Schusdziarra V.
      Effect of endogenous opioids on vagally induced release of gastrin, somatostatin and bombesin-like immunoreactivity from the perfused rat stomach.
      However, the effect of opioids prescribed for moderate pain management on gastric acid secretion, gastric emptying, and associated gastric pathology has not been investigated in detail.
      Gastric ulcer occurs through a sequential multifaceted step, including abnormal acid secretion, reactive oxygen species generation, prostaglandin inhibition, and extracellular matrix degradation.
      • da Silva D.M.
      • Martins J.L.R.
      • de Oliveira D.R.
      • Florentino I.F.
      • da Silva D.P.B.
      • dos Santos F.C.A.
      • Costa E.A.
      Effect of allantoin on experimentally induced gastric ulcers: pathways of gastroprotection.
      • Chattopadhyay I.
      • Bandyopadhyay U.
      • Biswas K.
      • Maity P.
      • Banerjee R.K.
      Indomethacin inactivates gastric peroxidase to induce reactive-oxygen-mediated gastric mucosal injury and curcumin protects it by preventing peroxidase inactivation and scavenging reactive oxygen.
      • Miller T.A.
      Protective effects of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms.
      • Singh L.P.
      • Mishra A.
      • Saha D.
      • Swarnakar S.
      Doxycycline blocks gastric ulcer by regulating matrix metalloproteinase-2 activity and oxidative stress.
      • Swarnakar S.
      • Paul S.
      • Singh L.P.
      • Reiter R.J.
      Matrix metalloproteinases in health and disease: regulation by melatonin.
      Literature with regard to the effects of morphine on gastric acid secretion and gastric ulceration is controversial and inconclusive. The underlying mechanism associated with morphine-related gastric inflammation is also not known. Several conflicting reports exist regarding the effect of opioids on gastric acid secretion.
      • Esplugues J.V.
      • Barrachina M.D.
      • Esplugues J.
      Modulation by peripheral opioids of basal and distension-stimulated gastric acid secretion in the rat.
      ,
      • Watanabe K.
      • Yano S.
      • Minakawa Y.
      Morphine inhibits the gastric acid secretion stimulated by 2-deoxy-D-glucose via a central mechanism in anesthetized rats.
      Various opioids can alter gastric secretion by modulating the function of different types of opioid receptors in multiple anatomic regions, with different effects in different species. In humans and dogs, enkephalins and their stable analogs, which bind to δ receptors, either inhibit or stimulate gastric acid secretion, whereas morphine has been shown to stimulate gastric acid secretion in dogs.
      • Feldman M.
      • Walsh J.H.
      • Taylor I.L.
      Effect of naloxone and morphine on gastric acid secretion and on serum gastrin and pancreatic polypeptide concentrations in humans.
      ,
      • Konturek S.J.
      • Tasler J.
      • Cieszkowski M.
      • Mikoś E.
      • Coy D.H.
      • Schally A.V.
      Comparison of methionine-enkephalin and morphine in the stimulation of gastric acid secretion in the dog.
      In addition, opioid antagonist naloxone has been shown to reduce gastric acid secretion in humans. Opioids have been shown to inhibit gastric emptying. Opioid-mediated inhibition of gastric emptying is mediated by both central and peripheral mechanisms. Dysregulation in gastric acid secretion and delaying in gastric emptying may be one of the initial causes of gastric acid retention and thereby gastric inflammation.
      Proton pump inhibitors (PPIs), such as omeprazole, are highly effective inhibitors of acid secretion and have been shown to prevent gastric ulcer and reflux esophagitis.
      • Wilde M.I.
      • McTavish D.
      • Omeprazole
      An update of its pharmacology and therapeutic use in acid-related disorders.
      In combination with antibiotics, PPIs are also an integral part of therapy for Helicobacter pylori–induced gastric ulcer. Besides the acid-suppressing effects, PPIs also exert anti-inflammatory effects, which make these drugs more effective.
      • Kedika R.R.
      • Souza R.F.
      • Spechler S.J.
      Potential anti-inflammatory effects of proton pump inhibitors: a review and discussion of the clinical implications.
      They have been shown to modulate the inflammatory status, reduce oxidative stress, and improve mucosal injury in the small intestine.
      • Park S.C.
      • Chun H.J.
      • Kang C.D.
      • Sul D.
      Prevention and management of non-steroidal anti-inflammatory drugs-induced small intestinal injury.
      ,
      • Biswas K.
      • Bandyopadhyay U.
      • Chattopadhyay I.
      • Varadaraj A.
      • Ali E.
      • Banerjee R.K.
      A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical.
      The present study aims to investigate the consequence of morphine use on gastric damage and to understand the underlying mechanism driving morphine-induced gastric ulceration. The goal of this study is to identify novel or existing strategies that could be exploited pharmacologically to overcome morphine-mediated delayed gastric emptying and may potentially be beneficial to overcome long-term adverse effects of morphine.

      Materials and Methods

      Experimental Animals

      C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were male and aged 8 to 10 weeks. Food and tap water were available ad libitum. All mice were housed three to five per cage and maintained on a 12-hour light/dark cycle, in a constant temperature (22 ± 1°C) and 50% humidity. All procedures were approved by the University of Miami Institutional Animal Care and Use Committee. All procedures were conducted in line with the guidelines set forth by the NIH Guide for the Care and Use of Laboratory Animals.
      Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council
      Guide for the Care and Use of Laboratory Animals.

      Animal Treatment

      Mice were implanted with 25 mg slow-releasing morphine and placebo pellet, as described.
      • Bryant H.U.
      • Yoburn B.C.
      • Inturrisi C.E.
      • Bernton E.W.
      • Holaday J.W.
      Morphine-induced immunomodulation is not related to serum morphine concentrations.
      The 25-mg morphine pellet maintains a plasma level of morphine in the range of 0.6 to 0.2 μg/mL (range observed in opioid abusers and patients on opioids for moderate to severe pain). Furthermore, this model is commonly used in the study of opiate dependence and addiction.
      • Bryant H.U.
      • Yoburn B.C.
      • Inturrisi C.E.
      • Bernton E.W.
      • Holaday J.W.
      Morphine-induced immunomodulation is not related to serum morphine concentrations.
      Briefly, placebo or morphine pellets or 30-mg naloxone pellets (NIH/National Institute on Drug Abuse, Bethesda, MD) were inserted in a small pocket generated by a small skin incision on the animal's dorsal side; incisions were closed using surgical wound clips (9-mm stainless steel; Stoelting, Wooddale, IL). In another set of experiments, mice were injected with either saline or morphine (15 mg/kg) and were sacrificed at 30-, 60-, or 120-minute time points.

      Histology

      Placebo-, morphine-, or omeprazole-treated mouse stomachs were sectioned for histologic studies. The tissue samples were fixed in 10% formalin and embedded in paraffin. The sections (5 μm thick) were cut using a microtome and stained with hematoxylin and eosin. Slides were assessed using a microscope (Leica Microsystems, Wetzlar, Germany) at original magnification 10 × 10 and processed in Adobe Photoshop (San Jose, CA).
      • Swarnakar S.
      • Ganguly K.
      • Kundu P.
      • Banerjee A.
      • Maity P.
      • Sharma A.V.
      Curcumin regulates expression and activity of matrix metalloproteinases 9 and 2 during prevention and healing of indomethacin-induced gastric ulcer.

      Histologic Ulcer Scoring

      Histologic evaluation of the severity of ulceration and the degree of associated inflammation was performed by an investigator (K.K.) blinded to the group using a validated scoring system. The damage score was assigned on the basis of the following scale: 0 indicates normal; 1, edema and/or vacuolation, but minimal changes in crypt architecture; 2, epithelial disruption; and 3, erosion extending to the muscularis mucosae. The inflammatory scoring system was established after reviewing all slides to assess the range of inflammation, and the following scores were then assigned as follows: 0 indicates normal; 1, minimal inflammatory cells; 2, moderate number of inflammatory cells; and 3, large number of inflammatory cells. Ulcer damage and inflammation scores were calculated after morphine treatment at 24 and 48 hours. Results are expressed as the mean damage and inflammation score ± SEM.
      • Farrell J.J.
      • Taupin D.
      • Koh T.J.
      • Chen D.
      • Zhao C.-M.
      • Podolsky D.K.
      • Wang T.C.
      TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury.

      Serum Isolation

      Blood samples were isolated from mice by puncturing the heart, followed by incubation for 30 minutes at room temperature. Serum was isolated from the clotted blood by low centrifugation. The serum sample was mixed with protease inhibitor mixture and stored at −80°C. An equal volume of serum was used for enzyme-linked immunosorbent assay.

      Tissue Extraction

      Stomachs of placebo-, morphine-, or omeprazole-treated mice were suspended in lysis buffer (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1% Triton X-100, and protease inhibitors) minced, and centrifuged at 12,000 × g for 15 minutes to obtain tissue extracts. Extracts were preserved at −80°C for future studies.

      Gelatin Zymography

      For assay of matrix metallopeptidase (MMP)-9 activities, tissue extracts were electrophoresed in an 8% SDS polyacrylamide gel containing 1 mg/mL gelatin (Sigma, St. Louis, MO), under nonreducing conditions. The gels were washed twice in 2.5% Triton X-100 (Sigma) and then incubated in calcium assay buffer (40 mmol/L Tris-HCl, pH 7.4, 0.2 mol/L NaCl, and 10 mmol/L CaCl2) for18 hours at 37°C. Gels were stained with 0.1% Coomassie Blue, followed by destaining. The zones of gelatinolytic activity came as negative staining. Standard MMP-9 was purchased from Abcam (Cambridge, UK). Quantification of zymographic bands was done using ImageJ software version 1.51
      • Kleiner D.E.
      • Stetler-Stevenson W.G.
      Quantitative zymography: detection of picogram quantities of gelatinases.
      (NIH; https://imagej.nih.gov/ij, last accessed September 2, 2019).

      Measurement of MPO Activity

      Myeloperoxidase (MPO) activity was measured, as previously described.
      • Nadatani Y.
      • Watanabe T.
      • Tanigawa T.
      • Ohkawa F.
      • Takeda S.
      • Higashimori A.
      • Sogawa M.
      • Yamagami H.
      • Shiba M.
      • Watanabe K.
      • Tominaga K.
      • Fujiwara Y.
      • Takeuchi K.
      • Arakawa T.
      High-mobility group box 1 inhibits gastric ulcer healing through Toll-like receptor 4 and receptor for advanced glycation end products.
      Briefly, the gastric tissues were homogenized in 50 mmol/L potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma Chemical Co., St. Louis, MO). Suspensions were centrifuged, and MPO activity in the resulting supernatant was measured with a spectrophotometer. One unit of MPO activity was defined as the amount of enzyme that degraded 1 μmol peroxide/minute at 25°C. The results are expressed as units per gram of gastric tissue.

      Real-Time PCR

      Total cellular RNA from mice gastric tissue was extracted using TRIzol (Invitrogen, Waltham, MA), and cDNA was synthesized with the M-MLV Reverse Transcription Kit (Promega, Madison, WI). Primers for IL-6, IL-1β, tumor necrosis factor (TNF)-α, and 18S ribosomal RNA were purchased from Invitrogen. Quantitative real-time PCR was performed using LightCycler 480 SYBR Green I Master (Roche Diagnostics, Meylan, France). All samples were run in triplicate. The 18-second ribosomal RNA expression was used to normalize the relative mRNA expressions. Primer sequences were as follows: 18S, forward primer 5′-GTAACCCGTTGAACCCCATT-3′ and reverse primer 5′-CCATCCAATCGGTAGTAGCG-3′; IL-6, forward primer 5′-TGGCTAAGGACCAAGACCATCCAA-3′ and reverse primer 5′-AACGCACTAGGTTTGCCGAGTAGA-3′; TNF-α, forward primer 5′-CCTCCCTCTCATCAGTTCTATGG-3′ and reverse primer 5′-CGTGGGCTACAGGCTTG-TC-3′; and IL-1β, forward primer 5′-GGCAGGCAGTATCACTCATT-3′ and reverse primer 5′-AAGGTGCTCATGTCCTCATC-3′.

      TUNEL Assay to Measure Apoptotic Cells in the Gastric Mucosa

      The tissue samples were fixed in 10% formalin for 48 hours, dehydrated in ascending alcohol series, and embedded in paraffin wax. Paraffin-embedded sections were cut into slices (4 μm thick) and were fixed to glass slides. Terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) assay was performed by using a commercial reagent kit (apoptosis detection kit; Abcam). Briefly, the sections were deparaffinized, rehydrated, and digested with Proteinase K and then labeled with TUNEL reaction mixture (biotin labeled that catalyzes the addition of biotin-labeled deoxynucleotides), followed by incubation with streptavidin–horseradish peroxidase conjugate. Positive controls where tissue sections were treated with DNase I and negative controls where TdT was substituted with water were included. The signal was detected using 3,3′-diaminobenzidine substrate, and sections were counterstained with Methyl Green. Tissue sections were screened for positive nuclei under a light microscope. Data from all fields were pooled to obtain the apoptotic index and are presented as the percentage of TUNEL-positive cells in the overall cell population, manually counted in 10 randomly selected fields.

      Measurement of Cytokines in Mice Gastric Tissue by Enzyme-Linked Immunosorbent Assay

      Placebo-, morphine-, and omeprazole-treated mice gastric tissues were homogenized in 1 mL sterile phosphate-buffered saline and centrifuged. The supernatants were analyzed for TNF-α, IL-1α, and IL-6 using sandwich enzyme-linked immunosorbent assay kits, according to the manufacturer’s instruction (Thermo Fisher Scientific, Vienna, Austria). Total protein was measured by the Lowry method. The cytokine concentrations in gastric tissue extracts were expressed as pg/mg.
      • Ghosh N.
      • Ghosh P.
      • Kesh K.
      • Mukhopadhyay A.K.
      • Swarnakar S.
      Attenuation of Helicobacter pylori-induced gastric inflammation by prior cag(-) strain (AM1) infection in C57BL/6 mice.

      Measurement of Intragastric pH

      The gastric pH was measured in the gastric lumen, as previously described. Briefly, the pH electrode (micro pH combination electrode; Sigma-Aldrich, St. Louis, MO) was inserted into the lumen to measure the pH of the gastric content without touching the mucosa.
      • Brenneman K.E.
      • Willingham C.
      • Kilbourne J.A.
      • Curtiss 3rd, R.
      • Roland K.L.
      A low gastric pH mouse model to evaluate live attenuated bacterial vaccines.

      Analysis of Gastric Acid Secretion

      The pylorus ligation model was used to measure acid secretion.
      • Shay H.
      • Sun D.C.
      • Gruenstein M.
      A quantitative method for measuring spontaneous gastric secretion in the rat.
      Mice were fasted overnight with free access to water. The pylorus of the mice was ligated under ketamine/xylazine anesthesia to trap gastric juice in the stomach. After the operation, mice were injected with morphine (15 mg/kg). After the mice woke up from anesthesia, they were housed in cages without food or water for 3 hours. Three hours later, mice were sacrificed. Gastric juice was collected, the volume was measured, and concentrations of acid were determined manually by titration with 0.1N NaOH and a pH meter. The acid contents were expressed as mEq/L.

      Measurement of Serum IL-6 Level

      Mice were fasted overnight with free access to water. Mice were injected with morphine (15 mg/kg) and were sacrificed at 30-, 60-, or 120-minute time points. Blood was immediately collected by cardiac puncture and centrifuged at 1000 × g for 5 minutes. Serum was collected and frozen at −20°C. Frozen serum samples were used for enzyme-linked immunosorbent assay of IL-6 (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions.

      Determination of Gastrointestinal Transit

      Gastrointestinal transit was measured, as previously described.
      • Woting A.
      • Blaut M.
      Small intestinal permeability and gut-transit time determined with low and high molecular weight fluorescein isothiocyanate-dextrans in C3H mice.
      Briefly, mice were fasted for 6 hours and administered 15 mg/kg morphine (intravenously). After 3 hours, animals were orally administered 100 μL of 0.5% (w/v) fluorescein isothiocyanate (FITC)–dextran (Sigma Chemical Co.) in physiological saline using a feeding tube. After 30-minute administration of FITC-dextran, the entire GI tract was isolated and separated into the following segments: stomach and six equally sized segments of the small intestine. The fractionated GI tract was collected in 1 mL of phosphate-buffered saline to extract the contents of the lumen and then centrifuged at 1500 × g, 4°C, for 15 minutes. The supernatant was centrifuged at 12,000 × g for 10 minutes, and the fluorescent intensity of the obtained supernatant was measured under the following conditions: excitation at 485 nm and fluorescence at 535 nm. The geometric center value of the distribution, an index of GI motility, was then calculated by the following calculation formula: geometric center = Σ (% of total fluorescent signal per segment × segment number)/100.
      • Miller M.S.
      • Galligan J.J.
      • Burks T.F.
      Accurate measurement of intestinal transit in the rat.

      GI Protection Studies

      Omeprazole was purchased from Sigma-Aldrich. Mice were fasted overnight with free access to water, and omeprazole was dissolved in distilled water and administered orally at a dose of 40 mg/kg body weight, 1 hour before morphine pellet, to test the gastroprotective role of omeprazole.

      Experiment for Determining Effects of Omeprazole on Morphine Tolerance

      Analgesic tolerance was assessed using tail-flick assays as reference.
      • Ross G.R.
      • Gabra B.H.
      • Dewey W.L.
      • Akbarali H.I.
      Morphine tolerance in the mouse ileum and colon.
      ,
      • Wang F.
      • Meng J.
      • Zhang L.
      • Johnson T.
      • Chen C.
      • Roy S.
      Morphine induces changes in the gut microbiome and metabolome in a morphine dependence model.
      Briefly, mice were intraperitoneally injected with saline or 15 mg/kg morphine twice daily for 8 days at 12-hour intervals. Omeprazole was administered orally at a dose of 40 mg/kg once a day. Behavioral assessment was performed before and 30 minutes after saline or morphine administration in the morning. Only the phosphate-buffered saline group and omeprazole group served as control groups. Withdrawal latencies of the tail from a radiant heat source were measured by tail flick. Voltage to the light source was adjusted to achieve baseline latency between 2 and 3 seconds. The cutoff time is 10 seconds to avoid tissue damage. The mice were placed on the tail-flick assay for 5 minutes for habituation every day for 2 days before the behavior test. Every day, the averages of each two measurements before and after morphine injection were recorded as a baseline and then the response to morphine antinociceptive effect was recorded. Tail-flick analgesic responses were calculated as the percentage of the maximum possible effect (%MPE). %MPE = (post-drug latency – predrug latency)/(cutoff – predrug latency) × 100% for tail-flick analgesia.

      Statistical Analysis

      Experimental data were analyzed using Prism 6 (GraphPad Software, San Diego, CA). Parametric data were compared using t-test (two tail). For multiple-group comparison, data were analyzed by analysis of variance one-way analysis, followed by Bonferroni correction or two-way analysis, followed by the Tukey multiple comparison method. To ensure reproducibility, all experiments were repeated at least three times. All results were considered statistically significant if P < 0.05.

      Results

      Morphine Induces Gastric Damage

      To investigate the effect of morphine on gastric inflammation, mice were pelleted with 25 mg morphine or placebo for 24 and 48 hours. Macroscopic observation of the whole stomach shows that morphine causes dramatic gastric bloating and a threefold increase in stomach volume at both 24 and 48 hours compared with placebo-treated mice (Figure 1, A and B ). Further detailed observation of mice stomach reveals that there was an accumulation of blood and significant discoloration of the stomach. The presence of heme in the small intestine was confirmed using a hemoccult test strip, showing bleeding in the upper gastrointestinal tract (Supplemental Figure S1F). Histologic images show the denudation of surface epithelium, disruption of the gastric pit, and infiltration of inflammatory cells in the gastric mucosa in morphine-treated mice (Figure 1B and Supplemental Figure S1A). The use of the opioid receptor antagonist, naloxone, reduced gastric bloating and the associated increase in stomach volume (Figure 1, A and B). Naloxone pretreatment in mice also reduced morphine-induced gastric epithelial damage and recruitment of infiltrating neutrophils (Figure 1C). The effect of morphine on gastric pathology was analyzed in greater depth using a formerly validated ulcer scoring system that measures both depth of ulcer injury and level of associated inflammation. There was a significant increase of damage score and inflammation score found in morphine group compared with placebo at both 24- and 48-hour time points (Figure 1, E and F, and Supplemental Figure S1, C and D). Because gastric ulcer is characterized by an increased rate of apoptosis, the number of apoptotic cells in placebo- and morphine-treated gastric tissue was measured using TUNEL assay. There was an increase in the number of TUNEL-positive cells in the gastric mucosa of morphine-treated mice. The opioid receptor antagonist naloxone antagonized the effects of morphine and decreased the rate of apoptosis in mice gastric tissue (Figure 1, D and G, and Supplemental Figure S1, B and E). Furthermore, MPO activity also increased in gastric tissue after morphine treatment (Figure 1H). Altogether, these data suggested that morphine use results in significant gastric pathology and is mediated by the classic opioid receptor because the effects were antagonized by naloxone.
      Figure thumbnail gr1
      Figure 1A and B: Morphine induces gastric inflammation: macroscopic view of the mice stomach at 24 hours (A) and 48 hours (B). C: Representative hematoxylin and eosin–stained sections of different groups of mouse gastric tissue at 24 hours. D: Apoptosis in mice gastric tissues was detected by terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) assay. E and F: Damage score (E) and inflammation score (F) were measured. G: The average number of TUNEL-positive cells. H: Myeloperoxidase (MPO) activity in mice gastric tissue. Data were analyzed by t-test (two tail). Experiment was repeated three times. Gastric epithelial disruption and apoptotic cells are shown by the black arrows. Data are represented as means ± SD (EH). n = 6 in each group (AD and H). ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bars: 70 μm (C); 50 μm (D). Original magnification, ×20 (D). Nal, naloxone.

      Morphine Up-Regulates the Expression of Inflammatory Cytokines and MMP-9 in Mice Gastric Tissue

      Gastric ulcer is associated with an increase in the level of proinflammatory cytokines in the gastric mucosa.
      • da Silva D.M.
      • Martins J.L.R.
      • de Oliveira D.R.
      • Florentino I.F.
      • da Silva D.P.B.
      • dos Santos F.C.A.
      • Costa E.A.
      Effect of allantoin on experimentally induced gastric ulcers: pathways of gastroprotection.
      ,
      • Slomiany B.
      • Piotrowski J.
      • Slomiany A.
      Induction of tumor necrosis factor-α and apoptosis in gastric mucosal injury by indomethacin: effect of omeprazole and ebrotidine.
      • Watanabe T.
      • Arakawa T.
      • Fukuda T.
      • Higuchi K.
      • Kobayashi K.
      Role of neutrophils in a rat model of gastric ulcer recurrence caused by interleukin-1 beta.
      • Aziz R.S.
      • Siddiqua A.
      • Shahzad M.
      • Shabbir A.
      • Naseem N.
      Oxyresveratrol ameliorates ethanol-induced gastric ulcer via downregulation of IL-6, TNF-α, NF-κB, and COX-2 levels, and upregulation of TFF-2 levels.
      Therefore, the mRNA levels of proinflammatory cytokines IL-6, TNF-α, and IL-1β in the gastric tissues of mice treated with 25 mg slow-release morphine were measured. Morphine treatment significantly increased gastric expression of TNF-α (Figure 2A), IL-1β (Figure 2B), and IL-6 (Figure 2C), compared with control at both 24 and 48 hours. Naloxone treatment significantly reduced the expression of morphine-induced inflammatory cytokines TNF-α, IL-1β, and IL-6 (Figure 2, A–C). MMPs are responsible for the degradation of a variety of extracellular matrix molecules, and their up-regulation often coincides with increased expression of inflammatory cytokines in various pathologic conditions. Therefore, the activity of MMP-9 protein in mice gastric tissue was measured. Morphine induced a significant up-regulation of MMP-9 activity in mouse gastric mucosa both at 24 hours (80-fold) and at 48 hours (90-fold) compared with that in control. However, naloxone treatment reduced MMP-9 activity (Figure 2, D and E). Altogether, these data showed that morphine modulates gastric expression of proinflammatory cytokines and the matrix metallopeptidase, MMP-9, in a naloxone-reversal manner, implicating the involvement of μ-opioid receptors in morphine-induced gastric damage.
      Figure thumbnail gr2
      Figure 2A–C: Morphine induces the up-regulation of inflammatory molecules in mice gastric tissues: expression of inflammatory cytokines tumor necrosis factor (TNF)-α (A), IL-1β (B), and IL-6 (C) in different groups of mouse gastric tissues was measured by real-time PCR. Expression levels were normalized to 18S. Tissue homogenates of gastric tissues of different groups of mice were subjected to substrate zymography, and matrix metallopeptidase (MMP)-9 activity was measured. D: Representative zymogram of MMP-9. E: Histographic representation of MMP-9 activity, as measured by ImageJ software (version 1.51). Values were obtained from above zymogram and two other zymograms from independent experiments. Data were analyzed by t-test (two tail). The experiment was repeated three times. Data are represented as means ± SD (AC and E). n = 6 in each group (AC). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Nal, naloxone.

      Morphine Increases Basal Level of Gastric Acid Secretion and Induces Gastric Delaying

      Gastric acid plays an important role in many types of gastric injuries.
      • Chattopadhyay I.
      • Bandyopadhyay U.
      • Biswas K.
      • Maity P.
      • Banerjee R.K.
      Indomethacin inactivates gastric peroxidase to induce reactive-oxygen-mediated gastric mucosal injury and curcumin protects it by preventing peroxidase inactivation and scavenging reactive oxygen.
      ,
      • Miller T.A.
      Protective effects of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms.
      ,
      • Watanabe T.
      • Higuchi K.
      • Tominaga K.
      • Fujiwara Y.
      • Arakawa T.
      Acid regulates inflammatory response in a rat model of induction of gastric ulcer recurrence by interleukin 1beta.
      To investigate whether gastric acid is involved in morphine-mediated gastric damage, the pH of the gastric content after morphine treatment was measured. Groups of mice were pelleted with slow-release 25-mg morphine pellet subcutaneously for 24 and 48 hours, and the pH of the gastric content was measured by inserting the pH electrode directly inside the stomach of the mice. Surprisingly, the pH of the gastric content of morphine-treated mice was found significantly lower compared with that of placebo-treated mice at both 24 and 48 hours. To confirm the role of opioid receptors in morphine-induced pH change, naloxone was implanted along with morphine. The results suggested that naloxone antagonized morphine-induced decrease in pH change. No significant difference in pH of the gastric content between placebo and naloxone-treated mice was observed (Figure 3A).
      Figure thumbnail gr3
      Figure 3A: Morphine-induced gastric inflammation is associated with an increase in the basal level of acid secretion and delayed gastric emptying: C57BL/6 mice were pelleted with 25-mg morphine pellet with or without naloxone (Nal) for 24 hours, and pH of the stomach content was measured by inserting a micro pH electrode inside the stomach. B: In another set of experiments, overnight fasted C57BL/6 mice were injected with morphine (15 mg/kg) or saline for 3 hours, and real-time measurement of gastric emptying throughout the gastrointestinal tract after injection of fluorescein isothiocyanate (FITC)–dextran was performed. C: FITC concentration in the mice stomach. Overnight fasted C57BL/6 mice were injected with morphine (15 mg/kg) or saline for 3 hours after gastric pyloric ligation. D: Total acid of the stomach content was measured. Data were analyzed by t-test (two tail). The experiment was repeated three times. Data are represented as means ± SD (AD). n = 6 (A and D), n = 5 (B and C) in each group. ∗∗P < 0.01, ∗∗∗P < 0.001.
      To investigate the effect of morphine treatment in gastric delaying, GI transit was measured using FITC-dextran after 3 hours of morphine treatment. The results show that, in morphine-treated mice, FITC-dextran was distributed primarily in the stomach and proximal small intestine. In contrast, in control mice, FITC-dextran was primarily distributed in the distal small intestine (Figure 3B). These results clearly indicate that morphine inhibits upper GI motility (Figure 3C). They also indicate that morphine has an inhibitory action on gastric emptying, and the delayed GI transit induced by morphine is caused, at least partly, by delayed gastric emptying.
      Gastric pyloric ligation experiment was performed to determine whether reducton in gastric pH in morphine-treated mice was due to the direct effect of morphine in the regulation of acid secretion or a consequence of the accumulation of acid because of delayed gastric emptying. The pylorus region of mice was ligated in both saline- and morphine-treated mice. Morphine treatment caused a significant augmentation of gastric acid secretion compared with saline treatment after 3 hours (Figure 3D) in pylorus-ligated condition. This result suggests that a decrease in gastric pH in morphine-treated mice is a consequence of elevated gastric acid secretion and delayed gastric emptying.

      Involvement of IL-6 in Morphine-Mediated Delayed Gastric Emptying

      To investigate the molecular mechanism associated with morphine-induced gastric delaying, the serum level of IL-6 was measured and compared. Animals were treated with either saline or morphine, sacrificed at 30-, 60-, or 120-minute time point, and basal IL-6 levels were measured. The results show a sustained increase in serum IL-6 levels up to 60-minute time point, after which its level declined (Figure 4A). A time-dependent decrease of the gastric pH was observed at 30, 60, or 120 minutes after morphine treatment (Figure 4B). Taken together, these findings show that an acute increase in circulating IL-6 after morphine treatment is associated with delayed gastric emptying.
      Figure thumbnail gr4
      Figure 4Role of IL-6 in morphine-induced delayed gastric emptying: C57BL/6 mice were injected with either saline or morphine (15 mg/kg) and were sacrificed at 30, 60, or 120 minutes. A: Serum IL-6 level was measured by enzyme-linked immunosorbent assay, and results are expressed as pg/mL protein. B: In the same experiment, the pH of the stomach content was measured by inserting micro pH electrode inside the stomach. To show gastric emptying, in vivo imaging was performed in overnight fasted wild-type (WT) and IL-6 knockout (KO) mice gavaged with fluorescein isothiocyanate–dextran. C and D: Representative images of fluorescence distribution in the gastrointestinal tract (C) and summary of fluorescent signal distribution in the stomach (D). IL-6 KO and WT mice were pelleted with 25 mg morphine or placebo pellet for 24 hours. E: The pH of the gastric content was measured. F: Histology of the IL-6 KO mice gastric tissue pelleted with 25 mg morphine and placebo for 24 hours. Data were analyzed by t-test (two tail). The experiment was repeated three times. Data are represented as means ± SD (A, B, D, and E). n = 6 (A and B), n = 5 (CF) in each group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars = 70 μm (F).

      Morphine-Mediated Delayed Gastric Emptying and Gastric Damage Are Attenuated in IL-6 KO Mice

      To further evaluate the role of IL-6 in morphine-induced delayed gastric emptying, morphine-treated IL-6 knockout (KO)/wild-type mice were gavaged with FITC-dextran and subjected to in vivo imaging. A significant decrease in FITC retention in the stomach of the IL-6 KO morphine mice was noted compared with that in wild-type morphine mice (Figure 4, C and D). Moreover, gastric content pH of morphine-treated IL-6 KO mice was found significantly higher compared with that in wild-type morphine mice (Figure 4E). Next, to investigate whether the delay in gastric emptying and associated gastric acid accumulation resulted in gastric damage, the histology of morphine-treated IL-6 KO mice along with wild-type morphine mice was compared. Gastric inflammation in morphine-implanted IL-6 KO mice (Figure 4F) was found significantly attenuated. Hence, accumulation of gastric acid due to gastric delaying results in gastric damage.

      Morphine-Induced Gastric Pathology Is Protected by PPI (Omeprazole) through Modulation of Gastric pH and IL-6

      To evaluate the gastroprotective effect of omeprazole on morphine-induced gastric damage, macroscopic and histologic examination of mice gastric tissue was performed. Macroscopic observation revealed that omeprazole significantly reduces the gastric bloating and volume of morphine-treated mice. The stomach morphology and omeprazole-treated mice appeared similar to placebo-treated mice (Figure 5A). Hematoxylin and eosin staining showed that pretreatment of omeprazole ameliorated the morphine-induced pathologic changes in the stomach, which include reduced edema, decreased infiltration of inflammatory cells to the submucosal layer, and the destruction of the gastric epithelium compared with the morphine group (Figure 5C). A significant decrease in both gastric damage and inflammation score was seen in the omeprazole-pretreated morphine group compared with the morphine group (Figure 5, E and F). The number of TUNEL-positive cells in the gastric mucosa was also markedly decreased in the omeprazole-treated morphine group compared with the saline-treated morphine group (Figure 5, D and G). Levels of the various proinflammatory cytokines such as TNF-α and IL-6 were also notably decreased after omeprazole pretreatment (Figure 5, H and I). To determine whether omeprazole pretreatment affects the acid secretion, pH of the mice gastric content was measured using a pH electrode. In the omeprazole-treated group, the gastric pH was found significantly higher than morphine alone group (Figure 5B). These data support the conclusion that gastric pH changes may be associated with morphine-induced gastric damage. To observe the effect of omeprazole on morphine-mediated delayed gastric emptying, in vivo imaging was performed. C57BL/6 mice were treated with either morphine or morphine with omeprazole for 24 hours, and mice were fasted for 6 hours before being gavaged with FITC-dextran. In vivo imaging was performed after 30 minutes of FITC-dextran gavaging. A reduced FITC signal was observed in the stomach of omeprazole pretreated morphine mice compared with that in morphine-treated mice (Figure 5J). This result suggests that omeprazole pretreatment attenuated morphine-induced delaying of gastric emptying.
      Figure thumbnail gr5
      Figure 5Effect of omeprazole (Omez) in morphine-induced gastric pathology: C57BL/6 mice were gavaged with 40 mg/kg omeprazole or phosphate-buffered saline before 25 mg morphine or placebo pelleting for 24 hours. A: Macroscopic views of the stomach of mice. B: The pH of the gastric content was measured. C: Histology of mice gastric tissue. Apoptosis in gastric tissues of mice was detected by terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) assay. D: Nucleus stained by substrate diaminobenzidine represents apoptosis occurrence. Gastric epithelial disruption and apoptotic cells are shown by the black arrows. E and F: Gastric ulcer damage score (E) and inflammation score (F). G: The average number of apoptosis cells in gastric mucosa was measured randomly from five different fields. H and I: Levels of the inflammatory cytokines tumor necrosis factor (TNF)-α (H) and IL-6 (I) in mice gastric tissue homogenate were measured by enzyme-linked immunosorbent assay. To show gastric emptying after omeprazole treatment, C57BL/6 mice were treated with either morphine or morphine with omeprazole for 24 hours, and mice were fasted for 6 hours before gavage with fluorescein isothiocyanate–dextran. Fluorescence distribution of gastrointestinal (GI) tract was measured by in vivo imaging. J: Representative images of fluorescent signal distribution in the GI tract. Data were analyzed by t-test (two tail). The experiment was repeated three times. Data are represented as means ± SD (B and EI). n = 7 in each group (AD); n = 6 in each group (HJ). ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bars: 70 μm (C); 50 μm (D). Original magnification, ×20 (D).

      Omeprazole Attenuates Morphine Analgesic Tolerance

      Previous reports indicate that chronic morphine treatment leads to analgesic tolerance in mice.
      • Wang F.
      • Meng J.
      • Zhang L.
      • Johnson T.
      • Chen C.
      • Roy S.
      Morphine induces changes in the gut microbiome and metabolome in a morphine dependence model.
      ,
      • Zhang L.
      • Meng J.
      • Ban Y.
      • Jalodia R.
      • Chupikova I.
      • Fernandez I.
      • Brito N.
      • Sharma U.
      • Abreu M.T.
      • Ramakrishnan S.
      • Roy S.
      Morphine tolerance is attenuated in germfree mice and reversed by probiotics, implicating the role of gut microbiome.
      Besides the gastroprotective role of omeprazole on morphine-induced gastric damage, the study investigated whether omeprazole modulated the analgesic effect of morphine. Towards this end, withdrawal latencies of the tail from a radiant heat source were measured by tail flick. Mice were treated with saline, morphine, or both omeprazole and morphine for 8 days. Surprisingly, mice from morphine-treated group pretreated with omeprazole showed significant attenuation of morphine analgesic tolerance compared with morphine-alone treated mice (Figure 6, A and B). Mice that received repeated doses of morphine presented 50% MPE between day 5 and day 6. In contrast, 50% MPE occurred between day 7 and day 8 in morphine mice pretreated with omeprazole. The omeprazole pretreated morphine mice showed significantly attenuated analgesic tolerance, with 45% MPE at day 8 compared with 0% MPE in morphine-alone mice (Figure 6A). These data clearly show that pretreatment with omeprazole before morphine administration prolongs the efficacy of morphine’s analgesic effect. On the basis of these results, we propose that omeprazole is not only protective against morphine-induced gastric damage but also attenuates morphine development of analgesic tolerance.
      Figure thumbnail gr6
      Figure 6Effect of omeprazole (Omez) on morphine tolerance: morphine analgesic tolerance with omeprazole was determined by mouse reaction to heat. A: Analgesic tolerance, interpreted as the percentage of maximum possible effect (MPE%), was determined by tail-flick analgesic test. Mice were gavaged with omeprazole (40 mg/kg) once a day and intraperitoneally injected with saline or 15 mg/kg morphine twice daily for 8 days with 12-hour intervals. Behavioral assessment was performed before and 30 minutes after saline or morphine administration in the morning. B: Daily antinociceptive behavior of morphine with or without omeprazole throughout an 8-day chronic morphine schedule (15 mg/kg, intraperitoneally, twice daily). Data were analyzed by one-way analysis of variance, followed by Bonferroni multiple comparisons test. C: Histology of mice gastric tissue. Gastric epithelial disruption is shown by the black arrows. D and E: Gastric ulcer damage score (D) and inflammation score. The experiment was repeated three times. Data are represented as means ± SD (A, B, D, and E). n = 6 in each group (AC). ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bars = 70 μm (C).
      Next, the protective effect of omeprazole on chronic morphine-induced GI pathophysiology was investigated. Histologic examination of mouse gastric tissues revealed that chronic morphine treatment caused mucous depletion and loss of continuity of surface epithelium, along with distortion and erosion of surface epithelial cells in morphine-treated mice. Glandular atrophy and infiltration of inflammatory cells were also detected in the gastric tissues of morphine-treated mice. A significant increase in damage score and inflammation score was detected in the morphine group. Mucosal damage by morphine was rescued by omeprazole. Omeprazole-alone treated group showed no significant gastric epithelial cell damage (Figure 6C).

      Discussion

      Opioids are the most effective drugs for pain management. However, the chronic use of opioids is associated with significant comorbidities, including gastrointestinal problems. Because of the lack of a better alternative, morphine is still considered one of the best pain management drugs clinically.
      • Pergolizzi J.
      • Böger R.H.
      • Budd K.
      • Dahan A.
      • Erdine S.
      • Hans G.
      • Kress H.G.
      • Langford R.
      • Likar R.
      • Raffa R.B.
      • Sacerdote P.
      Opioids and the management of chronic severe pain in the elderly: consensus statement of an International Expert Panel with focus on the six clinically most often used World Health Organization Step III opioids (buprenorphine, fentanyl, hydromorphone, methadone, morphine, oxycodone).
      Several groups of researchers, including our laboratory, have been actively working for a considerable time on understanding the phenomenon and deciphering the mechanism underlying the gastrointestinal adverse effects of morphine.
      • Wang F.
      • Roy S.
      Gut homeostasis, microbial dysbiosis, and opioids.
      Endogenous opioids play an important role in the control of gastric motility and blood flow and in the ability of the gastric mucosa to withstand exogenous challenges.
      • Holzer P.
      Opioid receptors in the gastrointestinal tract.
      • Pique J.M.
      • Esplugues J.V.
      • Whittle B.J.
      Influence of morphine or capsaicin pretreatment on rat gastric microcirculatory response to PAF.
      • Esplugues J.V.
      • Whittle B.J.
      Morphine potentiation of ethanol-induced gastric mucosal damage in the rat: role of local sensory afferent neurons.
      However, the effect of exogenous opioids on gastroenterological functions under both physiological and pathophysiological circumstances has not been studied in depth.
      The current study investigated the effect of morphine in gastric inflammation and the underlying mechanisms associated with morphine-induced gastric damage. It also revealed the gastroprotective effects of the PPI omeprazole on morphine-induced gastric damage. It showed that morphine treatment resulted in significant disruption of the gastric mucosal cells with a reduced glandular region and an increased number of apoptotic cells, which are characteristic features of gastric damage (Figure 1). Morphine-induced mucosal damage and its antagonism by naloxone suggested that this effect was brought about by the actions of classic opiate receptors. Previous studies indicate that administration of opioids, including morphine, augments gastric mucosal injury in a variety of experimental models,
      • Esplugues J.V.
      • Whittle B.J.
      Morphine potentiation of ethanol-induced gastric mucosal damage in the rat: role of local sensory afferent neurons.
      • Playford R.J.
      • Vesey D.A.
      • Haldane S.
      • Alison M.R.
      • Calam J.
      Dose-dependent effects of fentanyl on indomethacin-induced gastric damage.
      • Esplugues J.V.
      • Whittle B.J.
      • Moncada S.
      Modulation by opioids and by afferent sensory ndomet of prostanoid protection of the rat gastric mucosa.
      such as morphine-augmented mucosal damage induced by indomethacin administration or stress.
      • Gyires K.
      • Fürst S.
      • Farczádi E.
      • Márton A.
      Morphine potentiates the gastroulcerogenic effect of ndomethacin in rats.
      ,
      • Till M.
      • Gáti T.
      • Rábai K.
      • Szombath D.
      • Székely J.I.
      Effect of [D-Met2,Pro5]enkephalinamide on gastric ulceration and transmucosal potential difference.
      In contrast, contradictory findings have shown that morphine can reduce mucosal damage induced by some stress and alcohol-induced gastric damage.
      • Gyires K.
      Morphine inhibits the ethanol-induced gastric damage in rats.
      However, the mechanisms underlying these differing results are not clear, and hence more studies are needed to resolve these contradictory findings of the effects of opioids on gastric physiology.
      IL-6, IL-1β, and TNF-α are the key cytokines involved in many inflammatory diseases, including gastric inflammation.
      • Aziz R.S.
      • Siddiqua A.
      • Shahzad M.
      • Shabbir A.
      • Naseem N.
      Oxyresveratrol ameliorates ethanol-induced gastric ulcer via downregulation of IL-6, TNF-α, NF-κB, and COX-2 levels, and upregulation of TFF-2 levels.
      ,
      • Kany S.
      • Vollrath J.T.
      • Relja B.
      Cytokines in inflammatory disease.
      Herein, an increase in expression of IL-6, IL-1β, and TNF-α was detected in the gastric tissue of morphine-treated mice (Figure 2). Furthermore, gastric mucosal damage is directly associated with extracellular matrix degradation, in which MMPs play a crucial role.
      • Swarnakar S.
      • Paul S.
      • Singh L.P.
      • Reiter R.J.
      Matrix metalloproteinases in health and disease: regulation by melatonin.
      The current study showed that MMP-9 activity was significantly increased in the mice gastric mucosa after morphine treatment (Figure 2). Herein, MMP-9 was selected because prior observations in humans and mice have indicated the direct association of MMP-9 in early phases of gastric ulcer.
      • Swarnakar S.
      • Ganguly K.
      • Kundu P.
      • Banerjee A.
      • Maity P.
      • Sharma A.V.
      Curcumin regulates expression and activity of matrix metalloproteinases 9 and 2 during prevention and healing of indomethacin-induced gastric ulcer.
      ,
      • Ganguly K.
      • Swarnakar S.
      Induction of matrix metalloproteinase-9 and -3 in nonsteroidal anti-inflammatory drug-induced acute gastric ulcers in mice: regulation by melatonin.
      To unravel the underlying mechanism of morphine-induced gastric damage, the pH of mice stomach after morphine treatment was measured because gastric acid plays an important role in many types of gastric injuries.
      • da Silva D.M.
      • Martins J.L.R.
      • de Oliveira D.R.
      • Florentino I.F.
      • da Silva D.P.B.
      • dos Santos F.C.A.
      • Costa E.A.
      Effect of allantoin on experimentally induced gastric ulcers: pathways of gastroprotection.
      • Chattopadhyay I.
      • Bandyopadhyay U.
      • Biswas K.
      • Maity P.
      • Banerjee R.K.
      Indomethacin inactivates gastric peroxidase to induce reactive-oxygen-mediated gastric mucosal injury and curcumin protects it by preventing peroxidase inactivation and scavenging reactive oxygen.
      • Miller T.A.
      Protective effects of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms.
      Watanabe et al
      • Watanabe T.
      • Higuchi K.
      • Tominaga K.
      • Fujiwara Y.
      • Arakawa T.
      Acid regulates inflammatory response in a rat model of induction of gastric ulcer recurrence by interleukin 1beta.
      reported that gastric acid is required for the recurrence of gastric ulcers, through stimulating the inflammatory process in scarred mucosa. A significant increase in gastric acid secretion in the stomach of morphine-treated mice implies that gastric acid might play a role in morphine-induced gastric damage (Figure 3). Gastric pylorus ligation was performed to investigate whether low pH in the stomach occurred due to morphine-stimulated gastric acid secretion. Interestingly, morphine treatment causes significant augmentation of gastric acid secretion after pylorus ligation (Figure 3). This observation is in line with the finding of Esplugues et al
      • Esplugues J.V.
      • Barrachina M.D.
      • Esplugues J.
      Modulation by peripheral opioids of basal and distension-stimulated gastric acid secretion in the rat.
      of a dose-dependent increase in the basal level of acid secretion by morphine. In contrast, Watanabe et al
      • Watanabe K.
      • Yano S.
      • Minakawa Y.
      Morphine inhibits the gastric acid secretion stimulated by 2-deoxy-D-glucose via a central mechanism in anesthetized rats.
      reported that morphine suppressed the 2-deoxy-D-glucose (2-DG)–induced gastric acid secretion in rats. Morphine antagonist naloxone has been shown to decrease basal and meal-stimulated gastric acid secretion in humans, which implies opioid receptor involvement in gastric acid secretion. Secretion of H+ by gastric parietal cells is mainly regulated by histamine acting at H2 histamine receptors, by acetylcholine acting primarily at M1 muscarinic cholinergic receptors, and by gastrin acting at gastrin receptors. Several studies have shown that opioids can stimulate mast cell–mediated histamine release directly without a specific immunologic mechanism.
      • Rosow C.E.
      • Moss J.
      • Philbin D.M.
      • Savarese J.J.
      Histamine release during morphine and fentanyl anesthesia.
      It is plausible that morphine-mediated induction in gastric acid secretion may be regulated by histamine.
      Opioid analgesic drugs cause delayed gastric emptying, thereby increasing the retention time of acid in the stomach, and that may be one reason for morphine-mediated gastric damage. Therefore, this study measured morphine-induced gastric emptying by in vivo imaging, and the data suggest that morphine does indeed cause a delay in gastric emptying (Figure 3).
      Both central and peripheral mechanisms are involved in opioid-mediated inhibition of gastric emptying, but their relative contributions are uncertain.
      • Manara L.
      • Bianchetti A.
      The central and peripheral influences of opioids on gastrointestinal propulsion.
      Opioid receptors are also found at high concentrations in the gastric antrum.
      • Holtsman M.
      • Fishman S.M.
      Chapter 10 – opioid receptors.
      Hence, it is relevant to study the peripheral mechanism of gastric emptying in this context. The involvement of cytokines in the regulation of opioid-induced delaying of gastric emptying is an emerging field of research.
      • Lang Lehrskov L.
      • Lyngbaek M.P.
      • Soederlund L.
      • Legaard G.E.
      • Ehses J.A.
      • Heywood S.E.
      • Wewer Albrechtsen N.J.
      • Holst J.J.
      • Karstoft K.
      • Pedersen B.K.
      • Ellingsgaard H.
      Interleukin-6 delays gastric emptying in humans with direct effects on glycemic control.
      Measuring the cytokine level in morphine-treated mice serum showed a significant up-regulation of IL-6 in the serum after acute treatment with morphine (Figure 4). Stimulation of central opioid receptors results in the elevation of plasma IL-6 level through autonomic activation, specifically of the adrenal cortex.
      • Houghtling R.A.
      • Bayer B.M.
      Rapid elevation of plasma interleukin-6 by morphine is dependent on autonomic stimulation of adrenal gland.
      We hypothesized that elevated levels of IL-6 might be involved in the morphine-mediated delay in gastric emptying. Acutely elevated IL-6 delays gastric emptying in response to exercise.
      • Lang Lehrskov L.
      • Lyngbaek M.P.
      • Soederlund L.
      • Legaard G.E.
      • Ehses J.A.
      • Heywood S.E.
      • Wewer Albrechtsen N.J.
      • Holst J.J.
      • Karstoft K.
      • Pedersen B.K.
      • Ellingsgaard H.
      Interleukin-6 delays gastric emptying in humans with direct effects on glycemic control.
      To establish the involvement of IL-6 in the morphine-mediated delay of gastric emptying, IL-6 KO mice model was used. Delayed gastric emptying was attenuated in morphine-treated IL-6 KO mice. The pH of the stomach content in morphine-treated IL-6 KO mice was similar to the placebo group, which clearly implicates the role of IL-6 in the morphine-mediated delay of gastric emptying and acid retention. Interestingly, no gastric inflammation in morphine-treated IL-6 KO mice was detected (Figure 4). Hence, we report, for the first time, that an acute increase in IL-6 after morphine treatment causes a delay in gastric emptying, leading to the accumulation of acid and thereby resulting in gastric inflammation.
      Omeprazole (PPI) is widely used to prevent gastric damage and is believed to offer its antiulcer activity through acid suppression, scavenging of .OH radical, and blocking apoptotic cell death.
      • Wilde M.I.
      • McTavish D.
      • Omeprazole
      An update of its pharmacology and therapeutic use in acid-related disorders.
      ,
      • Biswas K.
      • Bandyopadhyay U.
      • Chattopadhyay I.
      • Varadaraj A.
      • Ali E.
      • Banerjee R.K.
      A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical.
      Cumulative evidence suggests that omeprazole can also suppress the production of inflammatory cytokines and act as an anti-inflammatory molecule.
      • Kedika R.R.
      • Souza R.F.
      • Spechler S.J.
      Potential anti-inflammatory effects of proton pump inhibitors: a review and discussion of the clinical implications.
      However, the effects of omeprazole on morphine-induced gastric inflammation have never been investigated. The current results demonstrated, for the first time, that pretreatment with omeprazole reduces morphine-induced gastric damage (Figure 5). Omeprazole pretreatment also significantly reduced inflammatory mediator (TNF-α and IL-6) levels and prevented morphine-induced gastric acid secretion, gastric delay, and gastric inflammation. Hence, co-administration of omeprazole with morphine provides gastroprotection by blocking gastric acid secretion and directly reducing inflammatory mediators.
      The current study raised an important concern about whether the gastroprotective effect of omeprazole in any way compromises the analgesic effect of morphine. Interestingly, omeprazole pretreatment caused a significant attenuation in morphine-induced analgesic tolerance (Figure 6). Morphine-induced gut dysbiosis leads to gut barrier disruption and bacterial translocation, resulting in the activation of proinflammatory cytokines that drive morphine tolerance.
      • Zhang L.
      • Meng J.
      • Ban Y.
      • Jalodia R.
      • Chupikova I.
      • Fernandez I.
      • Brito N.
      • Sharma U.
      • Abreu M.T.
      • Ramakrishnan S.
      • Roy S.
      Morphine tolerance is attenuated in germfree mice and reversed by probiotics, implicating the role of gut microbiome.
      Based on the findings of the current study, we hypothesize that omeprazole breaks this vicious cycle of chronic morphine tolerance by reducing the level of proinflammatory cytokines. Thus, omeprazole not only prevents morphine-induced gastric damage but also attenuates morphine analgesic tolerance.
      In conclusion, morphine-induced gastric damage occurs due to increased gastric acid secretion and delayed gastric emptying. Inflammatory cytokine IL-6 is involved in morphine-induced delay in gastric emptying, leading to accumulation of acid and thereby resulting in gastric inflammation (Figure 7). Omeprazole prevents morphine-induced gastric damage by regulating acid secretion and inflammation. The current studies have clear clinical implications and suggest that omeprazole treatment at the time of morphine administration is a promising, safe, and inexpensive approach for reducing morphine-induced GI pathology, attenuating morphine analgesic tolerance, and prolonging its efficacy as an analgesic agent.
      Figure thumbnail gr7
      Figure 7Schematic diagram of morphine-induced delayed gastric emptying and gastric inflammation: morphine treatment increases gastric acid secretion and IL-6–mediated delayed gastric emptying, which leads to accumulation of acid in the stomach, thereby resulting in gastric inflammation. Omeprazole prevents morphine-induced gastric damage by regulating acid secretion and inflammation.

      Acknowledgments

      We thank Dr. Anthony Ferrantella for help with the pylorus ligation experiment; and Dr. Valerie Gramling at the writing center of University of Miami for assistance with grammar.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1A: Morphine induces gastric inflammation at 48 hours: representative hematoxylin and eosin–stained sections of placebo, morphine, naloxone (Nal), and Nal + morphine–treated mouse gastric tissue. B: Apoptosis in gastric tissues of mice was detected by terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) assay. C and D: Gastric ulcer damage score (C) and inflammation score (D). E: The average number of TUNEL-positive cells. The average number of apoptotic cells in gastric mucosa was measured randomly from five different fields/mm2 area in each group from three different slides. F: Hemoccult test strip, showing presence of heme in the small intestine, confirming bleeding in the gastrointestinal tract. Data were analyzed by t-test (two tail). The experiments were repeated three times. The black arrows show gastric epithelial damage. Data are represented as means ± SD (CE). n = 6 in each group (A and B). ∗∗∗P < 0.001. Scale bars: 70 μm (A); 50 μm (B). Original magnification, ×20 (B).

      Author Contributions

      S.Ro. obtained funding and supervised the study; N.G., S.Ra., and S.Ro. conceived and designed the experiments; N.G. and K.K. performed the experiments; N.G., S.Ra., and S.Ro. analyzed the data; and N.G., K.K., and S.Ro. wrote the manuscript.

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