In an experimental investigation evaluating the gastrointestinal consequences of oxygen fluctuations resembling obstructive sleep apnea, researchers found that daily administration of melatonin significantly attenuated intestinal mucosal injury and altered circulating oxidative stress markers in male Wistar rats subjected to chronic intermittent hypoxia. The study, conducted by an investigative team from the North China University of Science and Technology and published in Frontiers in Pharmacology, tracked structural and biochemical alterations across two distinct exposure durations. The authors reported that exposure to repeated cycles of low oxygen and reoxygenation produced pronounced tissue disruptions in the small intestine, including mucosal congestion, blunted intestinal villi, and reduced crypt architecture. Concurrently, intraperitoneal melatonin administration before each hypoxic session was associated with preserved villus dimensions and lower circulating markers of mucosal injury and oxidative stress, with the protective associations appearing substantially more prominent after four weeks than after two weeks of continuous daily experimental exposure.

The underlying scientific rationale centered on the systemic reach of chronic intermittent hypoxia, the hallmark pathophysiological perturbation observed in patients suffering from obstructive sleep apnea syndrome. Under typical physiological baselines, a major fraction of total cardiac output is directed through the splanchnic circulation to support gastrointestinal metabolic integrity and epithelial barrier maintenance. Recurrent episodes of deoxygenation followed by rapid reoxygenation can induce hemodynamic stress and trigger vascular redistribution away from the splanchnic bed, creating conditions analogous to localized ischemia and reperfusion. Previous literature has established that chronic inflammatory stimulation and oxidative surges can perturb intestinal microbial equilibrium and foster systemic endotoxemia. Because melatonin functions physiologically as an endogenous pineal neurohormone possessing recognized free-radical-scavenging and antioxidant capabilities, the investigators sought to evaluate whether exogenous melatonin supplementation could provide measurable protection against mucosal remodeling and oxidative stress in an established animal model of hypoxia-reoxygenation injury.

To investigate these dynamics under rigorously controlled conditions, the researchers utilized thirty-six six-week-old male Wistar rats with an initial average body weight of 170 ± 10 grams, housing them under standard laboratory environments with an initial one-week acclimatization phase. Following ethical review and clearance, the animals were randomized into three cohorts of twelve rats each: an ambient normoxic control group, an untreated intermittent hypoxia group, and a melatonin-treated intermittent hypoxia group. To establish a structured temporal evaluation, each cohort was further subdivided into two equal subgroups of six animals designated for terminal evaluations at either two weeks or four weeks. Because terminal assessments required invasive surgical collection of tissue and central venous blood, entirely separate sets of six animals per group were evaluated at the two terminal endpoints, providing an independent cross-sectional assessment of short-term and sustained exposure without repeated harvesting from the same animal subjects.

The experimental hypoxia exposure protocol was executed within an automated environmental chamber connected to high-purity nitrogen and compressed air supplies, operating daily from 9:00 to 16:00 to align with the physiological rest cycle of nocturnal rodents. Animals in the control group were housed in an identical chamber receiving continuous compressed air at a flow rate of 10 liters per minute, maintaining an ambient oxygen fraction of 21% ± 0.5%. For the hypoxia and melatonin cohorts, the programmable system cycled through a 120-second profile comprising three sequential stages: a 30-second hypoxic phase delivering high-purity nitrogen at 10 liters per minute to drive chamber oxygen down to 5% ± 0.5%, a 40-second rapid reoxygenation phase with compressed air at 10 liters per minute, and a final 50-second slow reoxygenation phase with compressed air at 5 liters per minute returning chamber oxygen to approximately 21%. Rats receiving active treatment were injected intraperitoneally with melatonin at 20 mg/kg dissolved in ethanol and diluted in physiological saline to an ethanol concentration below 2% before each daily exposure, while control and untreated hypoxic rats received equivalent volumes of identical vehicle.

Immediately following the final daily cycle at the designated two-week or four-week timeline, terminal surgical procedures were carried out under deep urethane anesthesia confirmed through the complete loss of pedal withdrawal and corneal reflexes. Small intestinal segments located approximately two centimeters proximal to the ileocecal junction were harvested under sterile conditions, formalin-fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histopathological evaluation. Tissue sections were graded by an investigator blinded to group assignments using the Chiu six-point histological scoring system across five representative fields per animal. Furthermore, quantitative morphometric assessments were performed with Image-Pro Plus software, averaging measurements across five well-oriented villi per specimen to document intestinal villus surface area, villus height, villus thickness, and crypt depth, while central venous blood samples were analyzed for serum diamine oxidase, reactive oxygen species, malondialdehyde, and superoxide dismutase activity.

Histological examination revealed that prolonged exposure to intermittent hypoxia caused substantial physical degradation of the intestinal mucosa over time. Tissues from both two-week and four-week control animals maintained intact mucosal architecture, normal epithelial organization, and elongated villi, producing baseline Chiu injury scores of zero. Conversely, rats exposed to intermittent hypoxia without melatonin exhibited villus blunting, structural disruption, and progressive mucosal congestion and edema, yielding Chiu scores of 3.05 ± 0.46 at two weeks and 4.15 ± 0.53 at four weeks. Animals receiving daily melatonin exhibited less severe structural distortion, with Chiu scores averaging 2.88 ± 0.30 at two weeks and 2.40 ± 0.48 at four weeks, which represented statistically significant histological reductions compared to untreated hypoxic animals. These qualitative observations indicated that while pathological alterations were not entirely eradicated by melatonin supplementation, the severity of architectural disorganization within the intestinal lining was measurably mitigated across both intervention periods.

Detailed morphometric measurements corroborated the microscopic findings and revealed distinct time-dependent differences between the treatment cohorts. In untreated hypoxic animals, small intestinal villus dimensions declined markedly compared to controls: at two weeks, mean villus area was 205,375.65 ± 10,677.99 compared to 248,743.79 ± 29,017.76 in controls, villus height decreased to 955.79 ± 121.41 from 1317.20 ± 165.85, villus thickness dropped to 1161.75 ± 146.51 from 1712.99 ± 240.17, and crypt depth fell to 340.00 ± 43.95 from 543.52 ± 127.45. By four weeks, hypoxia caused further deteriorations, with villus area falling to 155,929.90 ± 23,805.46 and height dropping to 774.17 ± 73.13. At the two-week mark, melatonin supplementation yielded no statistically significant differences in morphometric parameters compared with untreated hypoxia. However, after four weeks of daily therapy, melatonin-treated rats exhibited significantly greater villus area at 231,069.31 ± 30,622.49 and height at 1167.17 ± 154.92 than untreated hypoxic rats, demonstrating that structural preservation became prominent only with extended administration.

Analysis of circulating biochemical indicators provided complementary insights into systemic oxidative stress and intestinal integrity. Serum diamine oxidase, an enzyme utilized as a circulating biochemical marker of intestinal mucosal injury, was significantly elevated in untreated hypoxic animals at both two weeks (20.63 ± 4.32 IU/ml) and four weeks (24.16 ± 4.57 IU/ml) relative to corresponding controls (11.96 ± 2.03 IU/ml and 12.89 ± 2.30 IU/ml). At two weeks, melatonin treatment was associated with significantly increased superoxide dismutase antioxidant activity (17.43 ± 0.88 U/ml versus 14.29 ± 0.57 U/ml in the hypoxia group), though reactive oxygen species, malondialdehyde, and diamine oxidase levels did not differ significantly between the two hypoxic groups. By four weeks, however, melatonin-treated animals exhibited a comprehensively improved biochemical profile: reactive oxygen species dropped to 428.56 ± 19.67 IU/ml, malondialdehyde declined to 2.54 ± 0.90 nmol/ml, diamine oxidase decreased to 16.85 ± 3.54 IU/ml, and superoxide dismutase activity remained significantly higher at 18.40 ± 0.58 U/ml compared to untreated hypoxic animals.

In evaluating the observed protective outcomes, the authors highlighted that the endogenous scavenging properties of melatonin and its regulatory effects on endogenous antioxidant defense mechanisms represent plausible contributors to the experimental findings. Superoxide dismutase serves as an essential primary line of enzymatic defense against superoxide radicals, whereas malondialdehyde reflects downstream lipid peroxidation occurring during acute oxidative attacks. The reduction in circulating diamine oxidase at four weeks aligns with histological evidence of preserved villus architecture. Nevertheless, the authors explicitly noted that several alternative molecular mechanisms frequently attributed to melatonin in broader literature—such as the suppression of inflammatory cytokines, modulation of nitric oxide synthase pathways, preservation of mitochondrial membrane potential, or direct inhibition of enterocyte apoptotic signaling—were not assessed in this experiment. Consequently, those candidate pathways remain unverified theoretical explanations rather than established causal mechanisms within the framework of this specific laboratory investigation.

The authors outlined multiple methodological constraints that require cautious interpretation of the findings. The experiment was conducted exclusively in young male Wistar rats, meaning potential sex-dependent differences and variations across other rodent strains or species remain unaddressed. Systemic physiological markers of actual hypoxemia, such as arterial blood gas partial pressures, pulse oximetry saturation profiles, or hypoxia-inducible factor 1-alpha levels, were not recorded, meaning chamber gas control served as an environmental surrogate rather than a direct quantification of internal arterial oxygenation. Furthermore, the protocol evaluated only a single dosage of melatonin at 20 mg/kg, precluding the assessment of dose-response relationships. Most critically, because direct functional assessments of intestinal permeability and molecular examinations of tight-junction complexes such as occludin, claudins, or zonula occludens-1 were not conducted, the study cannot verify functional restoration of the intestinal epithelial barrier. The researchers emphasized that controlled rodent exposures do not capture the chronic medical complexity of clinical sleep apnea, underscoring the need for dedicated translational studies before clinical applications can be considered.

Subject of Research: Protective effects of melatonin on intermittent hypoxia-induced intestinal mucosal injury and oxidative stress in a rat model

Article Title: Protective effect of melatonin on intestinal mucosal injury in intermittent hypoxic rats

Article References: Chen, Q., Zeng, Z., Luo, J., Yuan, X., Guo, A., Zhang, J., Fu, A., & Ge, Y. (2026). Protective effect of melatonin on intestinal mucosal injury in intermittent hypoxic rats. Frontiers in Pharmacology, 17, Article 1982966. https://doi.org/10.3389/fphar.2026.1982966

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1982966

Keywords: melatonin, intermittent hypoxia, intestinal mucosal injury, oxidative stress, Wistar rats, diamine oxidase, superoxide dismutase, malondialdehyde, reactive oxygen species, villus morphology, Chiu score, obstructive sleep apnea, small intestine, histopathology, gastrointestinal pharmacology, crypt depth