Diabetes mellitus (DM) is a chronic condition of multifactorial nature associated with impaired glucose metabolism. Insulin resistance can lead to diabetes mellitus type 2 (T2DM), while type 1 diabetes (T1DM) results from the destruction of insulin-producing pancreatic beta cells (American Diabetes Association, 2020; Atkinson et al., 2014; Katsarou et al., 2017). Over time, long-term effects of high blood glucose levels can lead to micro- and macro-vascular complications such as nephropathy, neuropathy, and retinopathy, and affect cardiovascular functions involving atherosclerosis, and renal failure (Papadopoulou-Marketou et al., 2017; Shi and Vanhoutte, 2017), with negative impacts on the central nervous system (CNS) (Cox et al., 2005; Hamed, 2017).

Impaired brain glucose metabolism compromises transmembrane ion transport, vesicle recycling, and synaptic signaling. Less effective maintenance of transmembrane ion gradients and transmitter release leads to hyperexcitability, excitatory-inhibitory imbalance, and functional impairment of cortical networks, which further compromises the brain's energy efficiency (Yu et al., 2018). These changes are enhanced by disrupted glutamatergic transmission and abnormal astrocyte and oligodendrocyte function as well as impaired autophagy, which, in turn, decreases nutrient recycling, thus contributing to neuroinflammation (Cunnane et al., 2020). Several studies have shown that hyperglycemia induces cognitive damage by increasing neuronal brain inflammation and oxidative stress accompanied by modifications in hippocampal morphology and plasticity (Piątkowska-Chmiel et al., 2022; Spinelli et al., 2019, 2020). Hyperglycemia is also associated with synaptotoxicity (Gaspar et al., 2010), which is a well-established process leading to behavioral alterations, such as memory impairment (Duarte et al., 2009) and mood dysfunction (Duman and Aghajanian, 2012).

Studies have also shown a higher prevalence of diabetes in obsessive-compulsive disorders, anxiety disorders, and depression (Chireh et al., 2019; El-Marasy et al., 2014; Zhu et al., 2020). Some authors have reported that components of the immune system are altered in DM, such as lymphocyte hyperactivity and activation of specific cytokines (DiMeglio et al., 2018; Szablewski and Sulima, 2017). These immunological alterations can lead to increased inflammation, thus causing blood clotting abnormalities, platelet hyperactivity, endothelium dysfunction, which can lead to a higher risk of atherothrombotic events (Hinton et al., 2021; Shruthi et al., 2016).

Vitamin D has several benefits in our metabolism, considered a prohormone, ranging from its role in calcium metabolism and bone formation, to even acting in processes of regulation of insulin metabolism, modulation of autoimmunity, participating in the process of cellular regulation and differentiation (Bhatt et al., 2020; Raygan et al., 2018; Sisley et al., 2016). One of the activation pathways of vitamin D3 in humans is through the canonical pathway, with absorption dependent on UVB light in the B-ring of 7 DHC (7-dehydrocholesterol) in the skin, leading to its opening for the production of pre-vitamin D3 and then after undergoing temperature dependent isomerization produces Vitamin D3 (Bocheva et al., 2021; Slominski et al., 2014; Tripkovic et al., 2012).

Activation of Vit D3 via the canonical pathway is via a series of hydroxylations such as 25-hydroxylase (CYP2R1 or CYP27A1) and hydroxylase (CYP27B1) to produce 1,25(OH)2D3. Both undergo sequential oxidations of their side chains catalyzed by to produce CYP24A1 and then to produce calcitroic acid. However, a new non-canonical pathway occurs D3 activation initiated by CYP11A1 with initial hydroxylation at C20 7,8 or C22 9 and subsequent hydroxylations at C20, C22, C23 and/or C17 7,8,10. The resulting products can be selectively hydroxylated by CYP27A1, CYP24A1, CYP2R1 and/or CYP3A4, with additional hydroxylation at C1a occurring for all intermediates except those having a hydroxyl group at C17 (Slominski et al., 2015, 2020).

Emerging evidence has shown that purinergic signaling, a complex network of receptors and extracellular enzymes responsible for the generation, recognition, and degradation of extracellular ATP and adenosine, plays a central role in several pathophysiological conditions, particularly those associated with the immune system regulation including type 1 and type 2 DM (Fotino et al., 2018; Pereira et al., 2018). High glucose levels can stimulate the release of nucleotides into the extracellular space initiating a purinergic signaling cascade that mediates several functions in the CNS, including neurotransmission, synaptic plasticity, neuroprotection, and neuroinflammation (Abbracchio et al., 2009; Burnstock, 2016; Stefanello et al., 2016; Reichert et al., 2021).

These findings hypothesize that diabetes may also cause modifications in the brain purinergic system, leading to an impairment of the physiological functions activated by ATP through the P2 receptors (Coutinho-Silva et al., 2003; Duarte et al., 2006, 2007). Furthermore, diabetes may trigger a neurodegenerative process, that can be promoted by adenosine metabolism due to its action in specific P1 receptors, including the A1 receptor expressed on brain cells (Pasquini et al., 2021).

Therefore, chronic hyperglycemia in T1DM condition can significantly contribute to the overproduction of pro-oxidant molecules such as lipids, proteins, nucleic acids, and eventually resulting in cell death leading to oxidative stress, a crucial factor in the development and progression of brain disorders (Salim et al., 2010; Reichert et al., 2018).

Interventions to preserve β cells have been made, and several techniques have been assessed for prognosis and diagnosis of chronic diseases. However, wide gaps still exist in understanding T1DM and standardizing clinical care focused on decreasing complications and burdens associated with the disease (Lernmark, 2015). Due to the undesirable side effects of current antidiabetic drugs, new pharmacological-adjuvant and nutraceutical strategies are necessary for the prevention, with lower damage to tissues and organs, mainly brain damage associated with DM1 (Jin et al., 2020).

Metformin as adjuvant therapy to intensive insulin therapy is frequently used in real-life in patients with T2DM. In addition, several short-term studies have suggested a positive effect of metformin on HbA1c and body mass index (BMI) when used in patients with T1DM (Liu et al., 2020). Metformin is prescribed to at least 120 million people worldwide and has an effective and reasonable anti-hyperglycemic property without causing overt hypoglycemia (Vella et al., 2010). It acts on the tyrosine kinase activity of insulin plasma membrane receptors and decreases the resistance of cells to insulin. Furthermore, metformin sharply increases the plasma levels of glucagon-like peptide-1 (GLP1) and decreases hepatic glucose production, mainly by inhibiting gluconeogenesis (Evans et al., 2005; Pusceddu et al., 2016, 2018). There is increasing evidence that vitamin D deficiency may contribute to the onset of diabetes (Stene and Joner, 2003; Grammatiki et al., 2017), once the vitamin D receptor (VDR) polymorphisms have been linked to DM (Rochel et al., 2000). However, animal and human studies have suggested that vitamin D may be a potential modifier of diabetes risk (Kalra and Aggarwal, 2021; Maddaloni et al., 2018; Zella and DeLuca, 2003). It also plays an important role in the disorders of glucose and insulin metabolism (Pittas and Dawson-Hughes, 2010; Pilz et al., 2013). Studies have shown that vitamin D controls gene expression, regulating the Ca2+ and reactive oxygen species at low physiological levels (Neyestani, 2014). It can also contribute to reducing inflammation which helps to control insulin resistance in type 1 and type 2 DM (Cade and Norman, 1986; Mathieu et al., 2005).

Some studies highlight the anti-inflammatory effects of vitamin D3 on P2X7R and ectonucleotidases in different inflammatory conditions (Da Silva et al., 2019) including T1DM (Calgaroto et al., 2014, 2015) and T2DM (Filippelli et al., 2021). Despite the importance of vitamin D in glucose metabolism and homeostasis, few studies have investigated the feasible properties of its combination with other anti-diabetic drugs on DM. Also, the impact of vitamin D on the purinergic signaling during DM and alterations related to CNS is completely unknown. Therefore, the present study aimed to investigate the effects of the treatments with metformin or vitamin D3 on purinergic signaling and oxidative markers in the brain of streptozotocin (STZ)-induced type 1 diabetic rats.