Methotrexate (MTX) is a folic acid antagonist with both anti-rheumatic and cancer-fighting activities [1]. Despite its clinical effectiveness, a plethora of preclinical and clinical studies report that MTX evokes life-threatening multi-organ concerns such as cardiotoxicity, neurotoxicity [2], nephrotoxicity, and hepatotoxicity [3]. In clinical settings, MTX-mediated renal and neurotoxicity have been reported to negatively affect the quality of life and daily activities of cancer survivors, facts that may jeopardize its avail. The mechanisms implicated in MTX-provoked renal and neurotoxicities are multifactorial and integrated, including oxidative stress, inflammation [4], dysfunction in autophagy flux, and apoptosis [5].

The orchestration between various death/pro-survival signaling trajectories has been acknowledged to intersect to mediate deleterious renal and neuronal impacts following MTX intoxication. Autophagy is a controlled lysosome-degradation mode of cellular death that mops misfolded proteins and damaged mitochondria in order to achieve cell integrity and hemostasis [6].

Dysregulation [7], as well as overstimulation of autophagic flux [8], have been involved in the pathogenesis of various renal and neuronal conditions [9]. However, previous researchs have not acknowledged the impact of autophagy in MTX-provoked hippocampal injury. In the same milieu, reports have revealed the contribution of the 5′ adenosine monophosphate-activated protein kinase (AMPK) and target of the rapamycin (mTOR) nexus in the tuning of autophagy pathway in cadmuim and MTX-provoked testicular [10], renal [11], and neuronal [12] impairments. In this setting, AMPK, a reliable sensor for the deficiency of cellular energy, promotes the autophagy pathway via the attenuation of mTOR complex 1 (mTORC1), a protein that has been documented to modulate autophagic flux [13].

Previous reports have emphasized the role of inflammation in the progression of MTX-evoked renal, hippocampal, and behavioral decrements [14], [15]. Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), which are members of the family of pattern-recognition receptors, are documented to participate in various renal [16], and neuronal abnormalities [17]. Among the listed NLRs, nucleotide-binding oligomerization domain-containing-2 (NOD-2) that mediates inflammatory response via the modulation of various inflammatory signals such as nuclear factor kappa B (NF-κB) [18], and nucleotide-binding domain-like receptor pyrin domain-containing-3 (NLRP3) [19]. The latter has been identified to increase the expression of caspase-1 to generate immense amounts of IL-1β and IL-18 to accelerate neuronal [20], and renal damage [21].

Gap junctions (GJ) are membrane channel proteins that facilitate cell–cell communication to maintain cellular hemostasis and structural integrity [22]. GJ are formed when two connexons or hemichannels are assembled in two adjacent cells to create hydrophilic pores that allow the exchange of various molecules, such as ions, metabolites, and ATP [23]. Each hemichannel consists of six transmembrane proteins called connexins; among these connexins is connexin-43 (Conx-43) [24]. Conx-43 has been documented to play an imperative role in the pathogenesis of various neuronal [25], and renal [26] disorders. In renal disorders, inhibition of Conx-43 markedly preserved renal cells in rodents exposed to cisplatin [27] and high-fat diet [28]. In neuronal disorders, attenuation of Conx-43 markedly reversed sevoflurane-provoked cognitive impairment in rats [29], and protected the dopaminergic neurons in mice exposed to lipopolysaccharide-provoked Parkinson's [30]. In addition, knockout mice of astrocytic Conx-43 notably protected hippocampal neurons and decreased ATP and glutamate release in amyloid beta-evoked Alzheimer’s in mice. Furthermore, increased expression of Conx-43 in renal tissues of diabetic patients [31], and postmortem brains of stroke patients [32] has been documented, suggesting Conx-43 as a promising target in our study. It is worth citing that the role of Conx-43 against MTX-provoked hippocampal and renal injury has not been previously considered.

Boswellic acid (BA) is a pentacyclic triterpenoid pharmacophore molecule isolated from the gum resin of Boswellia species with decent popularity in both modern and traditional medicines [33]. In preclinical studies, BA showed a prominent anti-inflammatory impact against collagen-provoked osteoarthritis [34]in rats. Additionally, BA signified its anti-oxidant and anti-apoptotic character against dextran sulphate sodium (DSS)-induced colitis [35]. Furthermore, it also showed a protective role against cisplatin [36], doxorubicin [37], and cyclophosphamide [38] provoked organ damage in various animal models. In neurodegenerative ailments, BA effectively opposes aluminum chloride-provoked dementia [39], and guards striatum cells against 3-nitropropionic acid-evoked Huntington’s disease [40].

Apigenin (APG), a dietary flavonoid, is found in various vegetables (parsley, onions), fruits (orange), and herbs such as chamomile flower and basil [41]. APG has been acknowledged as a nutraceutical compound with a marked safety profile in rodents and humans [41]. In preclinical settings, APG has shown its marked anti-inflammatory, and anti-apoptotic features against gentamycin [42], cisplatin [43], and cyclophosphamide [44] provoked organs damage. In neurological ailments, APG notably opposes methyl mercury [45], monosodium glutamate [46], and scopolamine [47] provoked neuronal injury. In addition, APG protects hippocampal cells against lipopolysaccharide-[48], and amyloid beta-[49] provoked dementia.

Notwithstanding the abovementioned facts, the current work was tailored to unlock the role of autophagy, NOD-2, and Conx-43 in the pathogenesis of MTX-provoked renal and hippocampal damage. It also extended to reveal the capacity of BA and APG to combat MTX-triggered renal and hippocampal injury, with the emphasis on their modulatory role in some integrating molecular machineries, chiefly NOD-2/NF-κB/NLRP3 cue, autophagy, and Conx-43.

It is noteworthy that the experimental rat model conducted in the current study is a standardized and reproducible model that is widely used to provoke renal [11], and hippocampal damage in rodents [50]. On top of that, it replicates the renal and neuronal adverse reactions that occur in humans following MTX administration [51], [52], [53], proposing this model as a suitable tool to understand the pathogenesis of MTX-provoked renal and neuronal damage, as well as the development of novel therapeutic agents to hamper MTX toxicity.