Investigating the Therapeutic Effects of Caffeine-Naringenin Combination on the Repair of Intestinal Permeability and Composition of Gut Microbiota in Obese Rats Under Long-Term High-Fat Diet Publisher



Dehghani A ; Mirzaei A ; Malakoutikhah M ; Karimi P ; Zadhoush F ; Ghasemi M
Source: Naunyn-Schmiedeberg's Archives of Pharmacology Published:2026

Abstract

Obesity-related metabolic dysfunction is closely associated with gut dysbiosis, impaired intestinal barrier function, and endotoxemia, which can induce oxidative stress and liver injury along the gut–liver axis. In this context, caffeine and naringenin were chosen for their potential to address liver injury. Caffeine, a natural methylxanthine, has shown liver-protective properties by modulating lipid metabolism and enhancing antioxidant capacity. Naringenin, a citrus flavonoid, is known for its anti-inflammatory and antioxidant effects and its ability to improve liver function by reducing oxidative stress. This study investigates whether their combination can provide additive or synergistic protection in obese rats sustained on a long-term high-fat diet (HFD). Thirty-five adult male Wistar rats were randomized (n = 7/group) to: control (standard diet), HFD, HFD + caffeine, HFD + naringenin, or HFD + caffeine + naringenin. Obesity was induced by 10 weeks of HFD (≈60% kcal from fat). Treatments were then administered by oral gavage for 6 weeks (caffeine 50 mg/kg/day; naringenin 12.5 mg/kg/day; combination at the same doses). Body weight, BMI/Lee index, lipid profile (TC, TG, LDL, HDL), liver enzymes (AST, ALT, ALP, GGT), systemic oxidative stress markers (MDA, SOD, TAC), and serum LPS (a surrogate of intestinal permeability) were measured. Gut microbiota profiling was done using 16S rRNA V3–V4 Illumina sequencing with standard bioinformatics (e.g., QIIME2). Colon histopathology was evaluated using H&E-stained sections. HFD increased body weight and the Lee index vs. controls, while the combination group showed the largest reductions in both indices. HFD induced dyslipidemia (elevated TC, TG, LDL, and decreased HDL). Caffeine or naringenin alone partially improved lipids, while the combination most effectively normalized them. Microbiota analysis revealed HFD-associated dysbiosis. Monotherapies improved it, and the combination produced the most pronounced eubiotic profile. Intestinal permeability was improved. Serum LPS was significantly elevated in HFD and was reduced by caffeine and, most robustly, by the combination. Systemic redox status improved. HFD altered antioxidant defenses, and treatments increased TAC/SOD, with the combination yielding the most consistent restoration of antioxidant capacity. MDA changes were consistent with attenuated lipid peroxidation under combination therapy. Liver injury markers (AST, ALT, ALP, and GGT) were also ameliorated by the combination. Histopathology of ileum/colon showed HFD-induced inflammatory/architectural damage and a treatment-associated restoration of mucosal structure by the combination. This study demonstrates that the combination of caffeine and naringenin offers a multi-targeted approach to mitigate obesity-related intestinal permeability, microbiota dysbiosis, and liver injury under long-term high-fat diet conditions. The synergistic effects of these compounds not only provide a promising therapeutic strategy for improving metabolic health and liver function but also offer the advantage of minimal side effects, making them a safe and effective option for managing obesity-related complications. © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2026.