Opioid Use in Murine Model Results in Severe Gastric Pathology that May Be Attenuated by Proton Pump Inhibition
2022; Elsevier BV; Volume: 192; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2022.04.005
ISSN1525-2191
AutoresNillu Ghosh, Kousik Kesh, Sundaram Ramakrishnan, Sabita Roy,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoOpioids 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. 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.1Camilleri M. Opioid-induced constipation: challenges and therapeutic opportunities.Am J Gastroenterol. 2011; 106 (quiz 843): 835-842Crossref PubMed Scopus (191) Google Scholar 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.2Nishimura 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.Gastroenterology. 1986; 91: 1084-1094Abstract Full Text PDF PubMed Scopus (66) Google Scholar 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.3Khansari M. Sohrabi M. Zamani F. The useage of opioids and their adverse effects in gastrointestinal practice: a review.Middle East J Digestive Dis. 2013; 5: 5-16PubMed Google Scholar In addition, opioids have been shown to influence various functions affecting gastric mucosa and the release of various peptides, including gastrin.4Nishi 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.Life Sci. 1987; 41: 1787-1793Crossref PubMed Scopus (11) Google Scholar,5Weigert 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.Regul Pept. 1995; 55: 207-215Crossref PubMed Scopus (10) Google Scholar 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.6da 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.Eur J Pharmacol. 2018; 821: 68-78Crossref PubMed Scopus (27) Google Scholar, 7Chattopadhyay 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.Free Radic Biol Med. 2006; 40: 1397-1408Crossref PubMed Scopus (120) Google Scholar, 8Miller T.A. Protective effects of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms.Am J Phys. 1983; 245: G601-G623PubMed Google Scholar, 9Singh L.P. Mishra A. Saha D. Swarnakar S. Doxycycline blocks gastric ulcer by regulating matrix metalloproteinase-2 activity and oxidative stress.World J Gastroenterol. 2011; 17: 3310Crossref PubMed Scopus (25) Google Scholar, 10Swarnakar S. Paul S. Singh L.P. Reiter R.J. Matrix metalloproteinases in health and disease: regulation by melatonin.J Pineal Res. 2011; 50: 8-20Crossref PubMed Scopus (87) Google Scholar 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.11Esplugues J.V. Barrachina M.D. Esplugues J. Modulation by peripheral opioids of basal and distension-stimulated gastric acid secretion in the rat.Br J Pharmacol. 1992; 106: 33-38Crossref PubMed Scopus (9) Google Scholar,12Watanabe K. Yano S. Minakawa Y. Morphine inhibits the gastric acid secretion stimulated by 2-deoxy-D-glucose via a central mechanism in anesthetized rats.Eur J Pharmacol. 1987; 143: 293-298Crossref PubMed Scopus (14) Google Scholar 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.13Feldman 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.Gastroenterology. 1980; 79: 294-298Abstract Full Text PDF PubMed Scopus (76) Google Scholar,14Konturek 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.Gastroenterology. 1980; 78: 294-300Abstract Full Text PDF PubMed Google Scholar 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.15Wilde M.I. McTavish D. Omeprazole An update of its pharmacology and therapeutic use in acid-related disorders.Drugs. 1994; 48: 91-132Crossref PubMed Scopus (149) Google Scholar 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.16Kedika R.R. Souza R.F. Spechler S.J. Potential anti-inflammatory effects of proton pump inhibitors: a review and discussion of the clinical implications.Dig Dis Sci. 2009; 54: 2312-2317Crossref PubMed Scopus (235) Google Scholar They have been shown to modulate the inflammatory status, reduce oxidative stress, and improve mucosal injury in the small intestine.17Park S.C. Chun H.J. Kang C.D. Sul D. Prevention and management of non-steroidal anti-inflammatory drugs-induced small intestinal injury.World J Gastroenterol. 2011; 17: 4647-4653Crossref PubMed Scopus (48) Google Scholar,18Biswas 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.J Biol Chem. 2003; 278: 10993-11001Abstract Full Text Full Text PDF PubMed Scopus (240) Google ScholarThe 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 MethodsExperimental AnimalsC57BL/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.19Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals.Eighth Edition. National Academies Press, Washington, DC2011Crossref Google ScholarAnimal TreatmentMice were implanted with 25 mg slow-releasing morphine and placebo pellet, as described.20Bryant H.U. Yoburn B.C. Inturrisi C.E. Bernton E.W. Holaday J.W. Morphine-induced immunomodulation is not related to serum morphine concentrations.Eur J Pharmacol. 1988; 149: 165-169Crossref PubMed Scopus (60) Google Scholar 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.20Bryant H.U. Yoburn B.C. Inturrisi C.E. Bernton E.W. Holaday J.W. Morphine-induced immunomodulation is not related to serum morphine concentrations.Eur J Pharmacol. 1988; 149: 165-169Crossref PubMed Scopus (60) Google Scholar 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.HistologyPlacebo-, 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).21Swarnakar 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.J Biol Chem. 2005; 280: 9409-9415Abstract Full Text Full Text PDF PubMed Scopus (212) Google ScholarHistologic Ulcer ScoringHistologic 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.22Farrell 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.J Clin Invest. 2002; 109: 193-204Crossref PubMed Scopus (208) Google ScholarSerum IsolationBlood 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 ExtractionStomachs 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 ZymographyFor 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 1.5123Kleiner D.E. Stetler-Stevenson W.G. Quantitative zymography: detection of picogram quantities of gelatinases.Anal Biochem. 1994; 218: 325-329Crossref PubMed Scopus (816) Google Scholar (NIH; https://imagej.nih.gov/ij, last accessed September 2, 2019).Measurement of MPO ActivityMyeloperoxidase (MPO) activity was measured, as previously described.24Nadatani 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.PloS One. 2013; 8: e80130Crossref PubMed Scopus (32) Google Scholar 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 PCRTotal 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 MucosaThe 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 AssayPlacebo-, 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.25Ghosh 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.Gut Pathogens. 2017; 9: 14Crossref PubMed Scopus (6) Google ScholarMeasurement of Intragastric pHThe 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.26Brenneman 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.PLoS One. 2014; 9: e87411Crossref PubMed Scopus (16) Google ScholarAnalysis of Gastric Acid SecretionThe pylorus ligation model was used to measure acid secretion.27Shay H. Sun D.C. Gruenstein M. A quantitative method for measuring spontaneous gastric secretion in the rat.Gastroenterology. 1954; 26: 906-913Abstract Full Text PDF PubMed Scopus (287) Google Scholar 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 LevelMice 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 TransitGastrointestinal transit was measured, as previously described.28Woting A. Blaut M. Small intestinal permeability and gut-transit time determined with low and high molecular weight fluorescein isothiocyanate-dextrans in C3H mice.Nutrients. 2018; 10: 685Crossref Scopus (65) Google Scholar 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)/100.29Miller M.S. Galligan J.J. Burks T.F. Accurate measurement of intestinal transit in the rat.J Pharmacol Methods. 1981; 6: 211-217Crossref PubMed Scopus (248) Google ScholarGI Protection StudiesOmeprazole 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 ToleranceAnalgesic tolerance was assessed using tail-flick assays as reference.30Ross G.R. Gabra B.H. Dewey W.L. Akbarali H.I. Morphine tolerance in the mouse ileum and colon.J Pharmacol Exp Ther. 2008; 327: 561-572Crossref PubMed Scopus (65) Google Scholar,31Wang 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.Sci Rep. 2018; 8: 3596Crossref PubMed Scopus (106) Google Scholar 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 AnalysisExperimental 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.ResultsMorphine Induces Gastric DamageTo 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.Morphine Up-Regulates the Expression of Inflammatory Cytokines and MMP-9 in Mice Gastric TissueGastric ulcer is associated with an increase in the level of proinflammatory cytokines in the gastric mucosa.6da 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.Eur J Pharmacol. 2018; 821: 68-78Crossref PubMed Scopus (27) Google Scholar,32Slomiany B. Piotrowski J. Slomiany A. Induction of tumor necrosis factor-α and apoptosis in gastric mucosal injury by indomethacin: effect of omeprazole and ebrotidine.Scand J Gastroenterol. 1997; 32: 638-642Crossref PubMed Scopus (79) Google Scholar, 33Watanabe T. Arakawa
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