The protective role of curcumin nanoparticles on the submandibular salivary gland toxicity induced by methotrexate in male rats

Methotrexate (MTX), previously known as amethopterin, is an analog for folic acid used since 1950 for the treatment of acute childhood leukemia and malignancies. In 1988, it was approved by the Food and Drug Administration to be used in low doses as a medication for adult rheumatoid arthritis and soon became the first line of treatment in juvenile idiopathic arthritis (Hashkes et al., 2014, Wang et al., 2018). Recently, MTX has been prescribed to treat other inflammatory diseases such as psoriasis, vasculitis, and Crohn’s Disease, associated with conventional therapy, especially in pediatric patients (Hashkes et al., 2014). It has been also used to terminate ectopic pregnancy in selected cases to avoid surgical intervention (Barnhart et al., 2001).

Methotrexate acts by interrupting DNA and RNA synthesis through the inhibition of an enzyme called dihydrofolate reductase leading to a deficiency in purines and thymidylate, which are DNA precursors (Barnhart et al., 2001). It also suppresses the mitotic division of cells, therefore preventing cellular proliferation. MTX could also counteract the inflammatory state by inhibiting proinflammatory mediators and cytokines (Khan et al., 2012).

Despite the antineoplastic, anti-inflammatory, and immunosuppressant effects of MTX, it has potential adverse outcomes and may induce toxicity in various organs. Reported undesirable effects of MTX include renal toxicity, hepatotoxicity, and neurotoxicity. Even in low concentrations, MTX affects the bone marrow, the gastrointestinal mucosa and may cause pneumonitis infrequently (Gaies et al., 2012, Wang et al., 2018). Studies also reported the possible occurrence of lymphoproliferative lesions in association with MTX treatment (Niimi et al., 2019). Moreover, animal studies demonstrated its cytotoxic effect on the gonads in addition to the salivary glands; both the parotid and the submandibular glands were affected (Abd El-Fatah et al., 2019, El Emam et al., 2019, Kilinc and Uz, 2021). Hyposalivation was also reported in breast cancer patients on MTX chemotherapy (Jensen et al., 2008). Hence, MTX chemotherapy could trigger salivary gland dysfunction and xerostomia (Jensen et al., 2003).

One of the possible destructive mechanisms of MTX is through the induction of apoptosis as a result of oxidative stress (Elango et al., 2014). It is well known that environmental stress causes apoptosis (Xie et al., 2019). By explaining apoptosis, it is the process of programmed cell death which is controlled by different genes, such as the Bcl2 family, caspase family, C-myc oncogenes, and tumor suppressor gene P53 (Zhao et al., 2021). Not all Bcl2 family members promote apoptosis. One kind inhibits apoptosis, such as Bcl-2, Bcl-XL, Bcl-W, and Mcl-1, while the other promotes apoptosis, such as Bax, Bcl-Xs, Bak, Bik/Nbk, and Bid (Lalier et al., 2022). In the process of apoptosis, Bax induces the release of cytochrome C and different factors inducers of apoptosis (Lee et al., 2020). Bcl2 protein inhibits cytochrome C and the inductor factors to prevent apoptosis (Li et al., 2013).

In mammals there are 18 caspases that have been classified according to their function and structure (Scott & Saleh, 2007). Caspase 3 is a crucial mediator for programmed cell death used in research. Apoptosis occurs in a cascade of steps, starting by the permeabilization of the mitochondrial outer membrane and the release of different factors from cytochrome c, when subjected to an external stress. Then the apoptosmal complex will be formed that triggers the activation of the effector caspase such as caspase 3 to finally induce apoptosis (Julien & Wells, 2017).

Curcumin is a polyphenol compound found in turmeric powder, which is extracted from the rhizome of Curcuma longa Linn. It is widely used in food as a spice, flavoring as well as yellow coloring agent (Basniwal et al., 2011, Moballegh Nasery et al., 2020). Turmeric has been extensively used in Chinese and Indian traditional medicine for various pains and illnesses, since 5000 years, until research revealed that isolated curcumin was responsible for these valuable therapeutic effects (Rahimi et al., 2016).

Studies suggested many pharmacological uses for curcumin such as anticancer, antioxidant, anti-inflammatory, antimicrobial, and anti-human immunodeficiency virus (Anti-HIV) (Chopra et al., 2021, Karthikeyan et al., 2020, Moballegh Nasery et al., 2020). Interestingly, curcumin increases the sensitivity of tumor cells to chemotherapy and radiotherapy and at the same time, it protects normal cells against their cytotoxic effects (Akbari et al., 2020, Rahimi et al., 2016). Other therapeutic applications of curcumin include wound healing, psoriasis, diabetes, obesity, cardiovascular diseases, and Alzheimer’s (Chopra et al., 2021, Karthikeyan et al., 2020, Rahimi et al., 2016). Although the compound is inexpensive and safe, its clinical application showed limitations due to its poor absorption, reduced solubility and bioavailability, rapid metabolism, and excretion (Basniwal et al., 2011). Moreover, high doses, when tested clinically, were associated with side effects such as nausea, diarrhea, headache, and rash (Akbari et al., 2020). Strategies were introduced to improve the properties of curcumin in low doses such as its combination with piperine, turmeric oil, fenugreek, or the use of lipid drug carriers such as liposomes (Tabanelli et al., 2021). The preparation of curcumin nano-formulations is one of the most attractive strategies to overcome its restrictions, by enhancing its solubility, physical stability, and bioavailability thus intensifying its therapeutic effects (Rahimi et al., 2016). Nanotechnology is an emerging science with numerous applications in the food industry, agriculture, drug delivery systems, and specific tissue targeting (Rahimi et al., 2016). Nano-metals (1–100 nm) such as Gold and silver were used as stabilizers, being biocompatible and highly absorbed in plasma. Polymeric nano-stabilizers are also used for the manufacture of curcumin in nanoscale such as Polyethylene glycol (PEG), Poly-(lactic-co-glycolic acid) (PLGA), polyvinyl pyrrolidone (PVP), and polyvinyl alcohol (PVA)(Moballegh Nasery et al., 2020; Rahimi et al., 2016). Polymers prevent the degradation of curcumin in the gastrointestinal tract by trapping its bioactive hydrophobic constituents inside a 3D network (Tabanelli et al., 2021). Chitosan encapsulated nanocurcumin showed enhanced solubility and bioavailability as well as other polysaccharides-based biopolymers like alginate, starch, and cellulose; in addition to biological nanocarriers such as exosomes (Moballegh Nasery et al., 2020, Tabanelli et al., 2021).

Previous animal studies proved that Nanocurcumin could prevent the nephrotoxicity triggered by methotrexate (Abd El-Rahman, 2014). Moreover, hepatotoxicity was also reversed by curcumin in mice and rabbits on methotrexate (Aljebori and Abady, 2018, Khudair and Al-Gareeb, 2021).

No previous studies investigated the possibility of nanocurcumin to prevent the destructive changes caused by methotrexate in the salivary gland tissues. Hence, the present study aimed to investigate the protective role of curcumin PVA-loaded nanoparticles on the toxicity induced by methotrexate in the submandibular salivary glands of albino male rats and exploring the underlying molecular mechanisms by examining Bcl-2 and Caspase-3 immunoexpression.

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