Diabetic peripheral neuropathy (DPN) is a debilitating complication affecting up to 60% of patients with types I and II diabetes mellitus (DM) (Standl et al., 2019). It is characterized by various peripheral nerve abnormalities and dysfunctions due to chronic hyperglycemia that contributes to hyper- or hyposensitizations of the peripheral organs (Freeman et al., 2016). These include apparent endoneurial edema of the sciatic and sural nerves and significant reductions in motor nerve conduction velocity observed in patients with DM and in experimental models (Jakobsen 1978; Giannini and Dyck, 1994). The latter is largely due to irregular nerve phenotypes, including total endoneurial lipid concentration reduction, myelinated fiber loss, and increased frequencies of denatured Schwann cells and regenerating fibers (Brown et al., 1979; Giannini and Dyck 1994; Cermenati et al., 2012). Voltage-gated sodium channels (NaVs) are critical for the initiation and propagation of action potentials and are essential for the transmission of noxious stimuli in the nociceptive neurons. Peripheral nerve damage, inflammation, and metabolic diseases alter the expression and function of these NaVs, leading to increases in neuronal excitability and pain. The NaV subunits −1.3, −1.7, and 1.8 are well known for their distinctively altered expression and repriming profiles in injured neurons and STZ-induced diabetic rats (Cheng et al., 2014; Bennett et al., 2019). The normally slow-activating and repriming NaV-1.7 exhibited significantly higher expression and similar nerve injury-induced inflammatory pain and hypersensitivity in cases of sciatic nerve axotomy, (Rush et al., 2007; Dib-Hajj et al., 2017; Bennett et al., 2019). Distinct elevations in NaV-1.3 were also observed in both the small and large DRG neurons of rat models with spinal nerve contusion, sciatic nerve axotomy, and type I DM (Bennett et al., 2019). Modulation of the peripheral NaVs expression and activity is a promising avenue for neuropathic pain management.

Cilostazol is a selective phosphodiesterase (PDE)-3 inhibitor that has demonstrated antiplatelet, antithrombotic, vasodilatory, and anti-proliferative effects on smooth muscle cells in vivo (Chapman and Goa 2003; Asal and Wojciak 2017). It was originally recommended for treating intermittent claudication, but has also emerged as an option for treating other vascular dysfunctions (Donnelly 2002; Thompson et al., 2002; Chapman and Goa 2003). Recent studies demonstrated that oral cilostazol improved the overall vascular health of hindlimb ischemia in mice by increasing the levels of endothelial progenitor cells in the circulation, as well as those of granulocyte colony-stimulating factor and vascular endothelial growth factor in the ischemic muscles (Biscetti et al., 2013). Although cilostazol does not appear to affect the blood glucose levels in people with DM, it is effective in modulating the levels of albuminuria and hyperglycaemia-induced metabolic abnormalities (Matsumoto et al., 2008; Tang et al., 2013). Additionally, cilostazol effectively manages cellular inflammation and oxidative stress, provides renoprotection, and reduces the coronary heart disease risk in DM (Agrawal et al., 2007; Wang et al., 2008; Lee et al., 2020). In an in vitro study, cilostazol demonstrated the ability to protect human endothelial cells against lipopolysaccharide-induced apoptosis via the ERK-1/2 and p38 MAPK-dependent pathways (Lim et al., 2008). In 2007, Iwama et al. (2007) reported that oral cilostazol effectively reduced the levels of neuronal injury and subsequent retinal injury in rats with cerebral ischemia by inhibiting ischemia-induced interactions between the leukocytes and endothelial cells (Iwama et al., 2007). Koyanagi et al. reported that cilostazol could suppress Schwann cell dedifferentiation by promoting cyclic AMP (cAMP) signaling via PDE inhibition, suggesting its potential in ameliorating chemotherapy-induced peripheral neuropathy (Koyanagi et al., 2021).

DPN is a major chronic complication of DM. Its pathogenesis mainly involves chronic glucose toxicity and nerve ischemia. Apart from its positive effects on vascular and endothelial abnormalities, information on the benefits of cilostazol on peripheral nerve function remains limited. The action of cilostazol on the peripheral nerve includes increased nerve blood flow and restoration of nerve Na⁺/K⁺-ATPase activity, leading to improved sciatic-tibial nerve conduction velocity and amelioration of DPN (Kihara et al., 1995; Naka 1995). In addition, the neuroprotective effects of cilostazol include the ability to promote axonal regeneration in the sciatic nerves of DM rats (Yamamoto et al., 1998). This study aimed to identify effective oral cilostazol dosages for preventing the development of DPN via various pathways including regulating the peripheral sodium channels, glial cell activation in the spinal cord, and alleviation of diabetic neuropathic pain.

The present study revealed that consistent daily cilostazol administration for 6 weeks influenced glial cell expression in SDH, and NaVs in DRGs, to approach approximately normal levels and provide an analgesic effect. Importantly, low-dose (10 mg) cilostazol was sufficient to suppress the aberrant excitability of SDH glial cells and alleviate neuropathic pain, although high-dose (100 mg) cilostazol was necessary to restore the expression of NaVs in the DRG. Our results indicate that cilostazol has vasodilatory, anti-inflammatory, and renoprotective actions, and can be used as an alternative agent in treating diabetic neuropathy with neuropathic pain.

The variable etiology of peripheral neuropathic pain in diabetic neuropathy is characterized by chronic hyperglycemia. Recent knowledge of the mechanisms causing DPN and the generation of neuropathic pain in DM remains incomplete. Sodium channels are critical determinants of sensory neuronal excitability, associated neuropathic pain signals, and peripheral neuropathy (Cardoso and Lewis 2018). Patients with genetic variants of sodium channels are at risk of neuropathic pain during the development of diabetes (Lauria et al., 2014). Our current and previous studies demonstrated significant increases in the levels of NaV-1.1, −1.2, −1.3, −1.6, and −1.7, and reduction in NaV-1.8 protein expression in DRG neurons in diabetic rats (Cheng et al., 2014). Similar findings have demonstrated that dysregulated sodium channels are associated with DRG hypersensitivity and DPN (Faber et al., 2012a; Faber et al., 2012b; Yang et al., 2016). Particularly, variants in the genes encoding for the NaV-1.7, −1.8, and −1.9 sodium channel subunits have been discovered in patients with small-fiber neuropathy and can lead to the development of pain (Lauria et al., 2014; Sopacua et al., 2019).

Glial cells, most notably astrocytes and microglia, which cooperate to promote and preserve neuronal health, play important roles in regulating the activity of neuronal networks in the brain. The activation of microglia secondary to neuroinflammatory processes contributes to the development and pain signaling through the activation of p38 MAPK or by expressing P2X4 receptors in microglia (Tsuda et al., 2003; Cheng et al., 2014). In our previous and present studies, the expression of microglia increased, and a positive correlation between mechanical allodynia, NaV-1.3, and microglial activation was observed in STZ-induced diabetic rats (Cheng et al., 2014). Furthermore, microglial activation is mediated by the phosphorylation of p-38 mitogen-activated protein kinase (Cheng et al., 2014).

Astrocytes are vulnerable to hypoxia under acidic conditions in diabetes. A previous study reported that hyperglycemic ischemia caused astrocyte activation in the early stage but astrocyte death in the late stage with enhanced free radical production (Muranyi et al., 2006). Another report stated that STZ leads to the activation of microglia and astrocytes in the DRG and spinal cord (Barragán-Iglesias et al., 2018; Tawfik et al., 2018). However, Zhang et al. addressed the decreased astrocyte expression in the spinal cord of diabetic rats (Cheng et al., 2014; Zhang et al., 2018). GFAP activation was reduced in the first 2 months and then returned to the level found in control rats at the third and 6 months in STZ-induced diabetic rats (Cheng et al., 2014). An interesting study by Liao reported that a db/db type 2 diabetes mouse model that displayed obvious mechanical allodynia was associated with the activation of spinal astrocytes but not microglia by the “Astrocyte-IL-1β-NMDAR-Neuron” pathway (Liao et al., 2011). In brief, increased activation of microglia in the spinal cord of diabetic rats has been consistently reported; however, the activation status of astrocytes in the spinal cord in STZ-induced diabetes was diverse. Further studies are needed to clarify the role of astrocytes in DPN.

Apart from facilitating good glycemic control, tricyclic antidepressants, serotonin-noradrenaline reuptake inhibitors, and anticonvulsants are recommended as first-line drugs for DPN to reduce pain; however, these treatments remain inadequate (Khdour 2020). Voltage-gated sodium channels are another therapeutic target for the treatment of painful diabetic neuropathy (Knezevic et al., 2020). Sodium channel blockers have been investigated for years as a potential treatment for chronic pain (Zuliani et al., 2010; Urru et al., 2020). Drugs from natural products (Chopra et al., 2010; Tiwari and Chopra 2011) or traditional Chinese Medicine (Bai et al., 2019) with therapeutic potential on diabetic complications were also reported. Cilostazol at 30 mg/kg/day improves Na⁺/K⁺-ATPase activity, while cilostazol at 10 mg/kg/day increases intracellular cAMP levels in the peripheral nerves of diabetic rats (Naka 1995). These studies suggest that cilostazol may have potential in the treatment of diabetic neuropathy. The efficacy of cilostazol in the treatment of DPN has not been proven in humans; however, participants in a human clinical trial with DM with critical limb ischemia administered cilostazol for more than 3 months achieved significantly decreased amputation and mortality rates and had significantly better outcomes (Lee et al., 2020). Cilostazol 100 mg/d was effective in improving walking speed in patients with DM with neuropathy; however, no significant deterioration or improvement in motor and sensory nerve conduction parameters was observed (Rosales et al., 2011). In our present diabetic neuropathy animal study, cilostazol 100 mg/kg/day demonstrated significant improvements in aberrant NaV expression in the DRG. Based on our results, we suggest that cilostazol may be an alternative to restore sodium channels for treating neuropathic pain in diabetes-induced sodium channel dysfunction. The cellular mechanisms of cilostazol in microglia are poorly understood. According to published results, cilostazol suppresses the NF-kappa B, ERK, and JNK signaling pathways to inhibit pro-inflammatory cytokines such as TNF-alpha production in activated microglia (Yoshikawa et al., 1999; Jung et al., 2010). Our results indicated that cilostazol suppressed the levels of activated microglial cells and ameliorated the decreased astrocyte expression levels in the SDH. Cilostazol may act as an anti-inflammatory agent by downregulating diabetes-induced microglial activation and suppressing mechanical allodynia. Although improvements in DM-induced allodynia were observed at the lowest cilostazol dosage, our study demonstrated that consistent, high-dose oral cilostazol administration could not only ameliorate the symptoms of mechanical allodynia in STZ-induced DM rats, but could also reduce levels of changes in the key NaV proteins in the DRG. These results indicated that the glial cells are more sensitive to the effect of cilostazol than are neurons; in other words, cilostazol exerts a cell-based dosage disparity effect in the nervous system (Table 1).

In conclusion, we confirmed that treatment with cilostazol may attenuate diabetic neuropathic pain by inhibiting glial cell dysregulation and NaVs malfunction in the SDH and DRG, respectively. This pilot study provides the foundation for an animal study to clarify alternative targets of cilostazol for pain modulation in DNP.