Exploring the effects of Citrus aurantifolia fruits peel essential oil, cultivated in Al-Hasa, in Methotrexate induced kidney and hepatic toxicities: Network Pharmacology, Molecular Docking, and in vivo Experiments.

ETHNOPHARMACOLOGICAL RELEVANCE: Ancient and medieval sources, particularly from Arabic and Mediterranean traditions, reported Citrus aurantifolia (Lime) as antidotes against poison, tonics for digestion, antibacterial and immunomodulatory effects. Moreover, its traditional use as a diuretic and antitoxic agent supports its ethnomedicinal relevance in managing renal ailments such as urolithiasis and toxin-induced nephropathies. Methotrexate (MTX) is consumed in several types of carcinomas. Still, numerous studies have stated MTX side effects, including kidney and liver damage.

AIM OF THE STUDY: The purpose of the current study is to elucidate the defending strategies of Citrus aurantifolia (Christm.) Swingle (Rutaceae) essential oil (CAEO) in the amelioration of MTX induced hepatic and kidney toxicities focusing on the Nrf2/HO-1 and JAK2/STAT3/NF-κB pathways.

MATERIALS AND METHODS: CAEO was extracted and its composition was investigated. After the acclimation period, rats were allocated into five groups. Normal and CAEO groups in which animals were given saline or CAEO orally (10 days). MTX group in which rat were given MTX (20 mg/kg ip, once on 4th day). CAEO-50/MTX and CAEO-100/MTX groups in which animals received oral administration of CAEO 50 and 100 mg/kg for 10 days and MTX (20 mg/kg ip, on 4th day). Histopathological, liver and kidney functions, Cytochrome c release and mitochondrial swelling assessment. Oxidative and nitrosative stress, Lipid peroxidation, Inflammatory mediators and apoptosis markers assessment. Gene expression of JAK2/STAT3/NF-κB and Nrf2/HO-1/NQO1were measured.

RESULTS: CAEO showed a monoterpene-dominant profile. Integrated analysis of docking scores suggested that a small group of abundant monoterpenes, supported by selected sesquiterpenes, drives the predicted interaction with JAK2, with comparatively weaker binding to the Keap1-related target. The network pharmacology analysis of CAEO ingredients specified JAK2/STAT3 signaling as one of the key mechanistic insights into how CAEO might counteract MTX-induced organ toxicity. Co-administering CAEO with MTX decreased hepatic and renal function tests caused by MTX and improved liver and kidney architecture. CAEO with MTX increased GST, GSH-Px, GSH-R and SOD and diminished NO and MDA levels. CAEO effectively reserved the MTX associated cytochrome c release from mitochondria and lessened mitochondrial swelling. CAEO instigated the gene expression and levels of Nrf2, HO-1, NQO1 while reduced the gene and protein expressions of JAK2, STAT3 and NF-κB. CAEO reduced inflammatory markers (TNF-α, MPO and ICAM), apoptotic markers (caspase 3 and 9).

CONCLUSION: CAEO considerably alleviated the oxidative and nitrosative stress, mitochondrial dysfunction, inflammatory and apoptosis responses caused by MTX. This shielding effect may be due to the decline in JAK2/STAT3/NF-κB and the triggering in Nrf2/HO-1/NQO1 pathways.