Diabetes mellitus, a collection of metabolic diseases related to chronic inflammation, is characterized by hyperglycemia and markedly contributes to mortality and morbidity (American Diabetes Association, 2011; Li et al., 2017). Based on the different etiologies underlying the disease, diabetes is classified into four categories, including 1) type 1 diabetes, which results from an insulin-synthesis deficiency of beta cells out of autoimmune disorders; 2) type 2 diabetes, which is due to cellular insulin-resistance activities; 3) gestational diabetes, which indicates the presence of glucose intolerance during pregnancy; and 4) the fourth group, which involves other types of diabetes (Deshpande et al., 2008; American Diabetes Association, 2011). Among the distinctive types of diabetes, the vast majority contribute to long-term organic damage and potentially provoke other serious diseases, in which diabetic cardiac autonomic neuropathy (DCAN) is a common and quite devastating consequence (Vinik and Ziegler, 2007; American Diabetes Association, 2011).

Diabetic cardiac autonomic neuropathy (DCAN), is defined as an “impairment of cardiovascular autonomic control in patients with established diabetes after excluding other causes” (Spallone et al., 2011; Dimitropoulos et al., 2014; Agashe and Petak, 2018). As one of the most common diseases that is strongly related to diabetes, DCAN frequently manifests in one-fourth of patients with type 1 diabetes and one-third of patients with type 2 diabetes (Vinik and Ziegler, 2007). In addition to its high prevalence, there are multiple devastating complications of DCAN, such as silent myocardial ischemia (MI), all of which immensely influence the normal life of patients (Kahn et al., 1986; Vinik and Ziegler, 2007; Fedorowski et al., 2010; Balcıoğlu and Muderrisoglu, 2015; Agashe and Petak, 2018). Behind the clinical manifestations and complications, there is a complicated pathogenesis for DCAN. In the pathological case of DCAN, both the diabetes-induced diffused damage and the disorder of functional balance between cardiac sympathetic and parasympathetic nervous systems significantly evoke DCAN (Vinik et al., 2013). From previous studies, during DCAN progression, sophisticated interactions among glycemic regulation, blood pressure, and neuronal death contribute to the destruction of autonomic nerve fibers. As a result, the autonomic nerve fibers that innervate the cardiovascular system are damaged, consequently resulting in dysfunctional clinical manifestations (Vinik and Ziegler, 2007; Pop-Busui, 2010; Agashe and Petak, 2018). Within the complex interactions, the vast majority of hypotheses have suggested that hyperglycemia acts as the primary cause that triggers self-damaging reactions (Pop-Busui, 2010; Vinik et al., 2013; Dimitropoulos et al., 2014; Agashe and Petak, 2018). After the initiation of self-damaging reactions, all mitochondrion-derived oxidative stress, diabetes-associated DNA damage and increased specific inflammatory cytokines (such as interleukin 6, and C-reactive protein) contribute to microvascular abnormalities. Consequently, defects in neuronal blood supply promote impairments in peripheral nerves, eventually resulting in neural dysfunction and death (The Diabetes Control and Complications Trial Research Group, 1998; Ramasamy et al., 2005; Vinik and Ziegler, 2007; Pop-Busui, 2010; Vinik et al., 2013; Dimitropoulos et al., 2014; Balcıoğlu and Muderrisoglu, 2015; Agashe and Petak, 2018).

While monitoring the progression of DCAN, the superior cervical ganglion (SCG) should never be neglected. The superior cervical ganglion (SCG), as the largest ganglion that exists on both sides of the neck, communicates with the vagus nerve and glossopharyngeal nerve (Fazliogullari et al., 2016; Mitsuoka et al., 2017). Investigators have found that the SCG innervates the myocardium, by which it can participate in cardiac excitatory transmission and the regulation of cardiac physiological activities (Fazliogullari et al., 2016; Zou et al., 2018). Furthermore, it is well understood that SCG can integrate information from extrinsic and intrinsic stimulation, such as irritation derived from DCAN, indicating SCG plays an indispensable role in the progression of DCAN (Zou et al., 2018).

The complicated regulatory role of SCG is determined by sophisticated constituents (Hanani, 2010; Zou et al., 2018). SCG not only consists of neurons but is also sheathed by glial cells, especially satellite glial cells (SGCs) (Hanani, 2010; Zou et al., 2018). There are two principal functional significances for SGCs. On the one hand, SGCs harbor the ability to express immunological molecules such as interleukin-6, suggesting that SGCs are essential in inflammatory processes and are relevant to DCAN pathogenesis (Hanani, 2010). On the other hand, complex communications among SGCs and enclosed neurons are established by gap junctions. The specialized gap junctions render metabolites and ions transported smoothly between cells, achieving efficient intercellular connection. Therefore, SGCs in the SCG may play an important role in DCAN progression after the activation of SGCs by receptors.

One of the most significant receptor families expressed on the surface of SGCs are P2 receptors, which are classified into inotropic ligand-gated receptors (P2X receptors) and metabotropic G-protein coupled receptors (P2Y receptors) (Kobayashi et al., 2006; Villa et al., 2010). The P2Y₁₄ receptor is a novel member of the metabolic receptor family (Kobayashi et al., 2006; Villa et al., 2010). The P2Y₁₄ receptor is essentially expressed in glial cells (such as SGCs) and inflammatory cells (such as neutrophils). It can be activated by uridine diphosphates (UDPs) and UDP-sugars and responds accurately to extracellular nucleotides, exerting striking effects in two main aspects (Cho et al., 2014; Lin et al., 2019a). First, the P2Y₁₄ receptor mediates the inflammatory responses to cellular damage and tissue injury (Cho et al., 2014). Second, the P2Y₁₄ receptor is involved in inflammatory cytokine release, indirectly assisting immune reactions (Lin et al., 2019b). Therefore, the P2Y₁₄ receptor, which participates in inflammatory responses and immune reactions, is a critical element in the pathogenesis of DCAN and may be one target for DCAN prognosis and treatment.

During the process of finding effective medicines to extend the survival of individuals with diabetes, naringin was discovered and widely studied. Naringin, a traditional Chinese herbal medicine, is an important flavonoid extracted from Citrus plants and harbors diverse functions, including anti-inflammation, antioxidant stress, and the amelioration of metabolic syndromes (Chen et al., 2016; Li et al., 2017). First, anti-inflammatory naringin can effectively diminish the secretion of diabetes-associated inflammatory molecules, such as interleukin-6 (IL-6) and TNF-α, both of which contribute to insulin resistance and hyperglycemia (Hotamisligil et al., 1993; Dandona et al., 2004; Krogh-Madsen et al., 2006; Alam et al., 2014; Chen et al., 2016). Furthermore, the antioxidant naringin not only has a strong potential to scavenge free radicals, such as diabetes-induced reactive oxygen species (ROS) (Guimarães et al., 2010; Chen et al., 2016) but also enhances the activity of antioxidant enzymes. Both can effectively alleviate free radical damage and ameliorate diabetes (Jeon et al., 2002; Ali and El Kader, 2004; Punithavathi et al., 2008). Moreover, naringin can mitigate mitochondrial dysfunction by regaining mitochondrial homeostasis (Chen et al., 2016).

Previous studies have shown that iron accumulation occurs in type 2 diabetes (Sha et al., 2021), and numerous reactive species are involved in DCAN, both of which are reminiscent of reactive lipid species (RLS) as well as ferroptosis. Ferroptosis is an iron-dependent cell death driven by the accumulation of lipid peroxide and is regarded as a one novel form of necrosis (Dodson et al., 2019). In the case of ferroptosis, free iron accumulation (resulting from an altered iron transport system or breakdown of iron-containing proteins) can initiate the overwhelming lipid peroxidation reaction, a process by which ROS and reactive nitrogen species (RNS) react with polyunsaturated fatty acids (PUFAs) in membranes to generate lipid peroxides (Dodson et al., 2019). Normally, there are several mechanisms to defend against the oxidative stress, including 1) the glutathione pathway as aided by glutathione peroxidase 4 (GPX4); 2) the NADPH-FSP1-CoQ10 ferroptosis surveillance pathway; and 3) the tetrahydrobiopterin (GCH1/BH4) pathway (Yang et al., 2014; Doll et al., 2019; Kraft et al., 2020). They are all precisely controlled, and various regulators are involved, such as nuclear factor erythroid 2-related Factor 2 (NRF2), by which intracellular homeostasis of reactive species is maintained and cells survive (Yang et al., 2014; Doll et al., 2019; Kraft et al., 2020). Nevertheless, when dysfunction occurs, homeostasis is destroyed, and ferroptosis is initiated, resulting in cell death.

On the one hand, the role of SGCs involved in DCAN has been tested, and the P2Y₁₄ receptor is endowed with essential modulatory effects (Hanani, 2010). On the other hand, naringin harbors both anti-inflammation and antioxidant effects, and can function on specific receptors to suppress glial cell expression (Ahmed et al., 2019). However, not only does the detailed naringin effects on DCAN require more clarification, but also the sophisticated signaling pathways affected by naringin require further study.

In this experiment, we further studied the effects of naringin on DCAN as well as an associated mechanism of naringin for P2Y₁₄ receptors in SGCs. Here, blood pressure (BP), heart rate (HR), and heart rate variability (HRV) were measured, sympathetic nerve discharge (SND) was recorded, and concentrations of ROS were detected, all of which helped us assess cardiac and nerve injury as well as ferroptosis-based DCAN. Additionally, Western blotting and triple-labeled immunofluorescence were used to evaluate the expression of P2Y₁₄, IL-1β, NRF2, and GPX4. The experiment attampts to assist the vast majority of individuals suffering from the devastating DCAN by means of exploring a novel medicine target, the P2Y₁₄ receptor, and taking advantage of a new therapy based on naringin effects, which are of great significance for further research on DCAN and the establishment of a basis for better treatment of DCAN.

Within the pathological progression of DCAN, there are three prominent issues involved, namely 1) diabetes-driven SGC activation, 2) subsequent autonomic nerve damage (neuropathy), and 3) eventual cardiac damage. Naringin can effectively relieve DCAN by interacting with P2Y₁₄ and then rescuing this pathological progression.

1) Diabetes-driven SGC activation via the P2Y₁₄ receptor causes cardiac damage, which can be mitigated by P2Y₁₄-targeted naringin.

Due to diabetes, abundant nucleotides are released from stressed cells, such as damaged neuronal soma in the nervous system, after which the exported nucleotides work as a “danger signal” to “talk” with other cells via purinergic receptor activation (Hochhauser et al., 2013; Lazarowski and Harden, 2015). Two categories of purinergic receptors can be activated, namely, ionotropic receptor (P2X receptor) and metabotropic GPCR (P2Y receptor), both of which are significant in the regulation of neuronal activities and interactions between neurons and glial cells (Calvert et al., 2004; Kobayashi et al., 2013; Rivera et al., 2016). Among the various purinergic receptors, the P2Y₁₄ receptor is a novel metabotropic GPCR that can be activated by extracellular uridine diphosphates (UDPs) and UDP-sugar conjugates (Dobolyi et al., 2011; Curet and Watters, 2018). The P2Y₁₄ receptor is broadly distributed in glial cells (such as SGCs, astrocytes, and microglia), where it plays significant roles in inflammatory and immunological processes (Barrett et al., 2013; Kinoshita et al., 2013; Lazarowski and Harden, 2015; Lin et al., 2019b). In our research, the localization of the P2Y₁₄ receptor in the SCG was reidentified by double immunofluorescence labeling. The expression level of P2Y₁₄ was consistent with that of GFAP, a biomarker of satellite glial cell (SGC) activation (Feldman-Goriachnik and Hanani, 2021), indicating that the P2Y₁₄ receptor plays an essential role in the activation of SGCs. Compared with normal rats, the P2Y₁₄ receptor was highly expressed and SGC cells were overtly activated in the case of diabetes. Furthermore, there was overtly elevated expressions of the P2Y₁₄ receptor in diabetic rats compared with that of normal rats, and treatment with either P2Y₁₄ shRNA or naringin effectively diminished the overexpression in diabetic rats, indicating that the naringin can effectively relieve DCAN by downregulating the expression of the P2Y₁₄ receptor in the SCG.

After extracellular UDP and UDP-sugar binding to P2Y₁₄ receptors, natively coupled with G_i/o heterotrimers, P2Y₁₄ receptors are activated and trigger potent suppression of adenylyl cyclase G_i/o (Carter et al., 2009; Fricks et al., 2009; Kobayashi et al., 2013; Lin et al., 2019b). Subsequently, downstream signaling pathways, such as the MAPK pathway, are modulated, consequently influencing cardiovascular activities and possibly causing cardiac damage (Fricks et al., 2009; Hochhauser et al., 2013; Lazarowski and Harden, 2015; Lin et al., 2019b). In our experiments, the results of HR, BP (involving SBP, DBP, and MBP) and cardiac histological structure suggest the presence of cardiac abnormalities in the diabetic model, which were prominently mitigated after treatment with either P2Y₁₄ shRNA or naringin. Therefore, it was suggested that naringin could clearly rescue cardiac damage in DCAN by targeting the P2Y₁₄ receptor.

2) After SGC activation, autonomic nerve damage follows and plays a significant role in the progression of DCAN, which can be partially rescued by P2Y₁₄-targeted naringin.

After the release of uridine nucleotides, the activation of the P2Y₁₄ receptor brings about autonomic nerve damage and consequently causes neuropathy. In our research, spectral analysis of HRV acts as an effective way to understand the alterations of autonomic nerves. Spectral analysis of HRV not only showed that both sympathetic and parasympathetic nerve activities were impaired in subjects with diabetes but also confirmed that the balance between sympathetic and parasympathetic nerve activities is destroyed in diabetic conditions. However, the abnormally increased LF/HF ratio can be partially restored by treatment with either P2Y₁₄ shRNA or naringin. Furthermore, SND also revealed abnormal nerve discharge activities in diabetic rats, which can return to normal in part after treatment with either P2Y₁₄ shRNA or P2Y₁₄-targeted naringin. Both of these results indicate that the P2Y₁₄ receptor is involved in DCAN and that diabetes-induced nerve damage can be reversed by naringin-targeting P2Y₁₄ receptors.

Within the whole process, satellite glial cells (SGCs) in the SCG can be activated and respond to environmental changes, consequently influencing nearby SGCs and neurons (Hanani, 2005; Hanani, 2010). First, activated SGCs induce the elevated expression of inflammatory cytokines, such as IL-1β, to mediate immunological responses and influence the excitability of surrounding neurons (Zhang et al., 2007; Ji et al., 2013; Ramesh et al., 2013; Lazarowski and Harden, 2015; Magni et al., 2015; Zhang et al., 2017; Wang et al., 2018; Lin et al., 2019b). Our research demonstrated that compared with that of the control group, the expression of IL-1β was evidently increased in activated SGCs (DM group), while there was a noteworthy reduction in the expression after either P2Y₁₄ shRNA or naringin treatment. This suggests that overexpressed P2Y₁₄ can play a role in DCAN by boosting the secretion of various inflammatory cytokines (such as IL-1β) in SGCs and that naringin can relieve the progression of DCAN by reducing their secretion from SGCs. Second, activated SGCs can also trigger the formation of gap junctions between SGCs as well as between SGCs and neurons, which can assist rapid transmission of chemical signals and thus directly modulate neuronal excitability (Warwick and Hanani, 2013; Lin et al., 2019b; Spray et al., 2019). Gap junctions among SGCs can join separated SGCs into functional networks, and gap junctions between SGCs and neurons can mediate neuron-glial bidirectional chemical signaling (Gu et al., 2010; Suadicani et al., 2010; Rozanski et al., 2012; Magni et al., 2015; Spray et al., 2019).

Both the release of inflammatory factors from SGCs and the formation of gap junctions between nerve cells give rise to autonomic nerve injury and neuropathy (Román-Pintos et al., 2016).

3) The NRF2/GPX4 pathway involved in ferroptosis may be one of the most prominent mechanisms involved in DCAN, and P2Y₁₄-targeted naringin may alleviate DCAN by enhancing the NRF2/GPX4 pathway and suppressing the ferroptosis process.

There is abundant evidence suggesting that the ferroptosis-relevant NRF2/GPX4 pathway may play an indispensable role in nerve damage progression. In that case, the activities of antioxidant enzymes are overtly diminished, promoting the production of free radical (such as ROS) (Román-Pintos et al., 2016; de M Bandeira et al., 2013). Glutathione peroxidase 4 (GPX4), a potent antioxidant enzyme capable of removing lipid peroxides, may be involved in the pathological process (Liu and Wang, 2019). Furthermore, GPX4 can be directly or indirectly regulated by NRF2, a distinguished transcription factor that plays an important role in antioxidation. The activation of NRF2 can effectively protect against oxidative injuries in various pathological conditions, such as inflammatory conditions (Lin et al., 2019b; Song and Long, 2020). In our research, significantly elevated ROS content was detected in diabetic conditions, in which the expression levels of NRF2 and GPX4 were both diminished, suggesting reduced antioxidative capacity and an activated ferroptosis process. However, these issues were rescued by treatment with either P2Y₁₄ shRNA or P2Y₁₄-targeted naringin, demonstrating that ferroptosis may become involved in DCAN progression via the P2Y₁₄ receptor and that P2Y₁₄-targeted naringin can mitigate DCAN by boosting the NRF2/GPX4 pathway, eliminating ROS and thus inhibiting the ferroptosis process.

In our results, there are several issues that require more attention. First, double bands were shown in the Western blotting image of NRF2, which was also observed by other scientists and published in the research paper entitled “Prevention of Carcinogen-Induced Oral Cancer by Sulforaphane” (Bauman et al., 2016). We think NRF2 is involved in abundant interactions with various molecules, such as KEAP1 molecules and NRF2 inducers, by which it can sensitively regulate the expression of cytoprotective proteins, such as GPX4 (Tonelli et al., 2018; Baird and Yamamoto, 2020). Therefore, the band of NRF2 will be slightly affected and will vary in different cases of oxidative stress, thus several bands may be shown. It was also a surprising phenomenon that the level of ROS (a general indicator of ferroptosis) in the DM + P2Y₁₄ shRNA group and DM + naringin group was even lower than that in the control group, which was also similarly demonstrated in another paper entitled “Inhibition of ferroptosis by upregulating NRF2 delayed the progression of diabetic nephropathy” (Li et al., 2021). In this paper, it was suggested that compared with those in the control group, the levels of MDA and iron (the other indicators of ferroptosis) were extremely diminished after the rescue of ferroptosis by fenofibrate. There are many possibilities and hypotheses based on these results, and we suppose that naringin can affect various pathways of ferroptosis and interact with several receptors in SCG to achieve a powerful anti-inflammatory effect; thus, the ROS level becomes even lower than that in the control group. Additional research is required to test the hypotheses mentioned above.

The paper has some limitations. One limitation in our research is the lack of real-time experiments to accurately assess the interaction between naringin and the P2Y₁₄ receptor, although molecular docking provided some evidence. One paper entitled “Fragment screening by SPR and advanced application to GPCRs” gave us some insight (Shepherd et al., 2014). The interaction can be measured by detecting the change in refractive index in real time at the surface interface where a binding event occurs between the receptors and drugs, by which detailed information of the binding, including drug affinity, kinetics and thermodynamics can be deeply understood (Shepherd et al., 2014). Moreover, we primarily explored the ferroptosis involved in DCAN by several key and preliminary experiments, but more scientific research is required to help us better understand it.

In conclusion, diabetes can trigger SGC activation mediated by the P2Y₁₄ receptor under oxidative stress conditions, followed by autonomic neuropathy and cardiac damage. Significantly, P2Y₁₄ is prominently overexpressed, inflammatory factors (such as IL-1β) are massively released and gap junctions between nerve cells are formed in diabetic cases, all of which facilitate DCAN, consequently causing cardiac damage. Our research also demonstrated that the GPX4/NRF2 pathway was suppressed, and thus, ROS accumulated in the case of DCAN. However, naringin, a traditional Chinese medicine, that targets P2Y₁₄ receptors, can not only diminish the expression of the P2Y₁₄ receptor and inflammatory factors but also restore the function of the antioxidant GPX4/NRF2 pathway, thus effectively relieving DCAN.

Our research preliminarily demonstrated the potent alleviative effect of naringin on DCAN as well as one possible mechanism, which may provide a promising perspective for DCAN treatment in the future.