Riboflavin, also known as vitamin B₂, consists of a group of water-soluble organic compounds that play a role in a wide range of cellular, tissue-growth, and developmental functions (Altun and Kurutas, 2016; Schwechheimer et al., 2016; Saedisomeolia and Ashoori, 2018). Moreover, it is found in food, including eggs, green vegetables, milk and other dairy products, meat, mushrooms, and almonds, and is used as a dietary supplement. Its deficiency (also called ariboflavinosis) results in some skin lesions and inflammations, such as stomatitis, cheilosis, oily scaly skin rashes, and itchy, watery eyes (Schmelz et al., 1997). In particular, riboflavin has been demonstrated to have anti-inflammatory, anti-nociceptive, anti-hyperalgesic, and anti-allodynic effects in inflammatory and neuropathic pain models (Franca et al., 2001; Granados-Soto et al., 2004; Bertollo et al., 2006), as well as antioxidant, anti-aging (Zou et al., 2017), and anticancer properties (Naseem et al., 2015). Recently, it was reported that riboflavin reduces hyperalgesia and inflammation in rats (Bertollo et al., 2006), which might involve the inhibition of synthesis and/or action of inflammatory mediators. However, in the formalin test, only the second phase of the formalin-induced pain response was inhibited (Franca et al., 2001; Granados-Soto et al., 2004). Although some of the analgesic effects of riboflavin on pain are known, its effect on itching due to skin problems is not yet known.

Itch (known as pruritus) and pain are the unpleasant sensations that frequently provoke scratching or are associated with actual or potential tissue damage, respectively. Both are also mediated by primary sensory neurons, such as the dorsal root ganglion (DRG) or the trigeminal ganglion (Ikoma et al., 2006). Itch is exclusively sensed as an irritation in the skin that causes a desire to scratch, whereas pain can be experienced almost all over the body (Yosipovitch et al., 2003). Itch is also a symptom of many common diseases, including diabetes mellitus, atopic dermatitis, and inflammatory, metabolic, neurologic, thyroid, and psychiatric diseases (Stander et al., 2003). People with chronic itch experience a decreased quality of life because of psychological disturbances often associated with the condition, including sleeplessness, anxiety, and depression (Dhand and Aminoff, 2014). Several substances are known to act as mediators and signaling pathway molecules during the pathogenesis of chronic itch (Song et al., 2018); however, the mechanisms involved remain unclear.

Histamine is one of the best-known mediators of cutaneous, pro-inflammatory conditions such as cutaneous mastocytosis, insect-bite reactions, urticaria, and drug rashes, all of which can cause itch (Moore et al., 2018). It is stored in basophilic leukocytes and mast cells and its release is triggered by a variety of itch stimuli, exciting a subset of unmyelinated C-fiber mechano-insensitive primary afferent neurons via the histamine-dependent (histaminergic) signaling pathway (Jian et al., 2016). Furthermore, the itch is often divided into histamine-dependent and histamine-independent subtypes (Ji et al., 2018). Histamine receptors are the members of G-protein-coupled receptors (GPCRs), and four subtypes (H1–H4) have been identified (Huang and Thurmond, 2008; Shim and Oh, 2008). More specifically, several studies have found that histamine H1 and H4 receptors can be detected on DRG neurons and are involved in histamine-induced itch (Han et al., 2006). Histamine in the skin is mainly supplied through the degranulation of mast cells. Tryptase secreted from the activated mast cells induces an itching sensation by activation of proteinase-activated receptor 2 (PAR2) in C-nerve fibers. Meanwhile, another intracellular mechanism is an increase in the excitability of cells through the activation of transient receptor potential vanilloid 1 (TRPV1) after the histamine that is released from mast cells binds to the histamine receptor present at the end of the C-nerve fiber, causing itching. Considering the results of the studies to date, the mechanism through which histamine-dependent itching activates TRPV1 has only been partially elucidated, suggesting that there is a possibility of developing antipruritic drugs targeting this marker.

TRPV1 is a nonselective cation channel that belongs to the family of transient receptor potential (TRP) channels and has been implicated in the mediation of pain and itch (Moore et al., 2018). This protein is modulated by GPCR signaling (Jian et al., 2016), and most itch stimuli activate GPCRs and trigger an itch sensation by activating TRP channels, including TRPV1 (Shim et al., 2007). Recent studies have demonstrated that the histamine H1 receptor (H1R) also activates TRPV1 via the production of 12-hydroxyeicosatetraenoic acid, a downstream metabolite of the PLA2 and lipoxygenase pathway in sensory neurons of the somatic nervous system (Shim et al., 2007). H1R is related to G_q/G₁₁ and activates phospholipase Cβ3 (PLCβ3), resulting in increased intracellular Ca²⁺ in DRG neurons via TRPV1 (Han et al., 2006). As with H1R, H4R also induces an increase in Ca²⁺ via the intracellular PLC signaling pathway and TRPV1, but not TRPA1 (Kim et al., 2004). In cultured sensory neurons, the highly selective TRPV1 antagonist capsazepine was found to significantly inhibit activation of the histamine H4R (Jian et al., 2016). In addition, TRPV1^−/− mice exhibit significantly attenuated scratching behavior after pro-itch stimulation (Costa et al., 2008). Therefore, TRPV1 is likely to play an important role in the induction of itch downstream of signaling through the activation of H1R or H4R and might thus be involved in the mechanism underlying the histamine-induced itch response. In the present study, regarding the effect of riboflavin on itch, we sought to assess its effects in vivo and in vitro based on behavioral and neurotransmission outcomes, respectively. We found that riboflavin inhibited histamine-induced currents via TRPV1 in small-sized DRG neurons and had an antipruritic effect in a mouse model of histamine-induced itch.

Itch is mainly mediated by unmyelinated, slow conducting C-fibers, which are found at the dermal–epidermal junction, with free nerve endings that extend into the epidermis (Schmelz et al., 1997; Mcneil and Dong, 2012; Cho et al., 2016). GPCRs or ion channels can initiate itch signals (Kremer et al., 2014) via proteases such as tryptase and neuropeptides, such as substance P, prostaglandins, and histamines (Mollanazar et al., 2016). Under pathological conditions such as nerve injury and tissue damage, the sensation of itch develops into a chronic condition resulting from excess peripheral firing or the attenuation of central inhibition of the itch-pathway neurons (Steinhoff et al., 2018). However, the diagnosis of chronic itch is challenging because different types (focal/widespread, peripheral/central) exist and there are few available treatment options (Lee et al., 2016). Currently, available strategies include treating or preventing the causal disease, such as diabetes or herpes zoster, and topical or systemic medication that attenuates excess neuronal firing (Luo et al., 2015). Unfortunately, the efficacy of these treatments is limited. Thus, it is necessary to develop more effective anti-pruritic medications for therapeutic applications to acute or chronic itch.

Histamine is known as the primary endogenous itch-inducing substance and is predominant in mast cells and basophile granulocytes (Mollanazar et al., 2016). Although antihistamines are often used for the treatment of itch (Yosipovitch and Bernhard, 2013), effective anti-pruritic therapeutics are still lacking for many types of itch (Song et al., 2018). In recent decades, great progress has been made toward revealing the molecular and cellular mechanisms for (acute) itch (Mishra and Hoon, 2013). Primary DRG sensory neurons are responsible for transmitting itch signals to the spinal cord dorsal horn and then projecting them to the brain (Han and Dong, 2014). Histamine binds H1 and/or H4 receptors to activate PLC3 and TRPV1 on free nerve terminals in the skin to elicit the itch sensation, which requires TRPV1-expressing C-fibers in the periphery (Imamachi et al., 2009). Therefore, an intracellular signaling pathway might converge on TRPV1 to mediate histamine itch response, although TRPA1-dependent and non-TRPV1–TRPA1-dependent itch pathways also exist (Luo et al., 2015).

TRPV1, a nonselective cation channel expressed predominantly in primary small-sized sensory neurons, is activated by capsaicin and acid or by temperatures >43°C (Caterina et al., 1997; Zygmunt et al., 1999). Further, TRPV1 sensitization induced by inflammatory mediators might contribute nociceptor hyperexcitability and neuropathic pain after spinal-cord injury (Wu et al., 2013). However, unlike the well-recognized role of TRPV1 in pain, its function in itch sensation has only recently been established (Luo et al., 2015). TRPV1-null mice show significantly attenuated itch behavior in response to the intradermal injection of histamine (Shim et al., 2007; Imamachi et al., 2009). In addition, histamine can activate TRPV1 channels expressed in HEK293 cells, and TRPV1 mediates the histamine-induced excitation of DRG neurons (Shim et al., 2007). The direct activation of TRPV1 induces pain, whereas the GPCR-mediated indirect activation of TRPV1 produces itch. Therefore, it appears that TRPV1 is differentially involved in pain and itch (Luo et al., 2015).

Vitamins are essential nutrients because they perform hundreds of roles in the body. Imbalances in vitamin absorption and deficiency can protect against or cause several diseases, respectively. In a previous study, some vitamins were used as diagnostic factors in chronic pruritus (Rajagopalan et al., 2017). One member of the vitamin B family, riboflavin, is also an essential component of normal cellular function and metabolism, acting to ameliorate neuroinflammation, glutamate excitotoxicity, mitochondrial dysfunction, and oxidative stress, which are involved in a wide array of diseases, such as migraine, anemia, cancer, diabetes mellitus, hypertension, Parkinson’s disease, and other neurological disorders (Mcnulty et al., 2006; Zhou et al., 2008; Long et al., 2012; Hwang, 2013; Dey and Bishayi, 2016; Marashly and Bohlega, 2017). Thus, a number of recent studies have highlighted the cellular processes and biological effects associated with riboflavin (Jaeger and Bosch, 2016; Schwechheimer et al., 2016; Manole et al., 2017; Naghashpour et al., 2017; Udhayabanu et al., 2017; Saedisomeolia and Ashoori, 2018). In particular, riboflavin has been shown to induce anti-nociceptive and anti-inflammatory effects in multiple experimental models of acute pain and inflammation (Franca et al., 2001; Granados-Soto et al., 2004; Bertollo et al., 2006) and has been used to treat patients with painful conditions, such as migraine (Boehnke et al., 2004), carpal tunnel syndrome (Folkers et al., 1984), diabetic polyneuropathy (Jorg et al., 1988), and premenstrual tension (Wyatt et al., 1999). In addition, one report found that the synthesis of FAD decreased in patients with amyotrophic lateral sclerosis (Lin et al., 2009). Although there are distinct subpopulations of sensory neurons and spinal circuits involved in the transduction of pain and itch stimuli, they are closely related because they are both initiated by the activation of primary sensory neurons and involve some of the same ion channels (Sun et al., 2009; Mishra and Hoon, 2013; Han and Dong, 2014). Therefore, despite the beneficial analgesic effects of riboflavin, few studies have assessed its ability to regulate anti-itch mechanisms. Thus, it would be expected that this compound would regulate the itch response. Recent reports have shown that the oral application of riboflavin reduces inflammation, thermal hyperalgesia, and formalin-induced nociception, but not tactile allodynia and motor activity based on the rotarod test in mice (Granados-Soto et al., 2004; Bertollo et al., 2006). Oral medication is the main treatment option for patients with widespread chronic itch and for those with a focal itch that is refractory to topical treatment (Mirzoyev and Davis, 2013; Maciel et al., 2014; Matsuda et al., 2016). Our results demonstrated that the oral administration of riboflavin reduced histamine-induced animal scratching (Figure 1A), although it did not change animal motor activity (Figure 1B). Further, as histamine-sensitive C-fibers cover the largest innervation area in the lower leg, the peripheral chronic itch can spread to the innervation site of damaged nerves or nerve roots (Schmelz, 2015). The primary cause of chronic pain appears to be focal neuronal discharge caused by the spontaneous or excess firing of a damaged cutaneous C-fiber after injury, scarring, or neuropathy (Sene et al., 2013). Therefore, we found that riboflavin blocked the high-frequency bursting discharge and action potentials based on histamine-sensitive single C-fiber ex vivo recordings (Figure 2). Riboflavin is the precursor of two active coenzyme forms in the body, FMN and FAD (Saedisomeolia and Ashoori, 2018). Riboflavin is converted to FAD through an enzymatic reaction whereby FAD synthetase adds an AMP to FMN (Schwechheimer et al., 2016; Thakur et al., 2017). These act as electron carriers in a number of redox reactions involved in energy production and in numerous metabolic pathways (Thakur et al., 2017). AMP is a well-known intermediary substance formed during the creation of energy in the form of adenosine triphosphate from food and was reported to reduce the duration of pain due to shingles and prevent the development of postherpetic neuralgia (Bednarczuk, 1986). In addition, endogenous adenosine can act as a pain suppressor, and adenosine is generated from AMP. To determine whether riboflavin or its coenzymes FMN and/or FAD have any effects on itch, we examined the effects of riboflavin, FMN, and FAD on capsaicin-induced inward currents and histamine-induced scratching in vitro and in vivo, respectively. Interestingly, we found that riboflavin and FMN attenuated histamine-induced itch behavior (Figures 1C,D, 4D) and that FMN inhibited capsaicin-induced currents in cultured mouse DRG neurons and TRPV1-transfected HEK293 cells, but that FAD did not have these effects (Figures 4, 5). This result indicates that FMN and FAD might differentially function in anti-nociception or anti-pruritus responses via a TRPV1-dependent pathway.

Taken together, our findings suggest that riboflavin levels, as well as FMN and FAD levels, are differentially related to histamine-induced itch and thermal-hyperalgesia responses via distinct mechanisms. Although many distinct compounds including chloroquine, β-alanine, serotonin, endothelin-1, and also those found in cowhage, induce itching/scratching behaviors and intracellular signals via distinct histamine-independent molecular pathways (Mcneil and Dong, 2012), our data demonstrated that histamine-induced scratching behaviors in mice are specifically inhibited by riboflavin. These results indicate that the anti-itching effect of riboflavin depends primarily on histamine-dependent pathways and not another pro-itch mechanism.

In summary, we have shown in this study that riboflavin has anti-pruritic effects on histamine-induced scratching behaviors, which at least in part suppresses itching behaviors. In peripheral sensory neurons, TRPV1 is further involved in the riboflavin-mediated inhibition of histamine-dependent itch. Given this, we suggest that riboflavin might serve as a novel anti-itch treatment for histamine-dependent itch.

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.

The animal study and all experimental methods were reviewed and approved by the Experimental Animal Ethics Committee of Hanyang University.

KL, YIC, and S-TI performed electrophysiological experiments and analyzed the data. H-KL and J-ZI performed behavioral tests. KL, S-MH, and YHK drafted the manuscript. SJJ and C-KP conceived and supervised the project. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.