DPN is a critical secondary microvascular complication that adversely affects the quality of life of diabetic patients manifested by augmented nociceptive response and lowered tolerance to painful stimuli (Chang and Yang, 2023; Selvarajah et al., 2019). It commonly involves damage to peripheral nerves from the dorsal root ganglion supplying extremities. At present, there are no therapeutic strategies available to prevent or reverse nerve atrophy partially due to a dearth of understanding of the underlying pathogenic molecular mechanisms of DPN. However, recently natural products showed significant and promising progress in alleviating pain and protecting against nerve degeneration (Resham et al., 2020; Suryavanshi and Kulkarni, 2020). In the present study, we aimed to assess the effects of Ed and the possible molecular mechanism implicated in the STZ-induced DPN model in rats.
STZ in the dose range of 40–70 mg/kg (a single i.p. injection) is commonly used to induce diabetes and its complications in rat models (Furman, 2015). In the contemporary study, STZ administration on day 1 resulted in persistent hyperglycemia in rats. A steep increase in glucose levels was observed after one week of STZ injection. The rats manifested characteristic features of diabetes i.e., polyphagia, polydipsia, and a decline in body weight over the period of 10 weeks. Furthermore, a decrease in circulating insulin levels and an increase in HbA1c was pragmatic at the end of 6th week in rats that were given STZ on day 1. Restrain over HbA1c levels is a major goal in diabetes because HbA1c levels directly correspond to diabetic complications such as DPN. To assess the pancreatic-β cell function and development of IR we determined HOMA-β and HOMA-IR in animals before treatment and after treatment. The data indicated a decline in HOMA-β and an increase in HOMA-IR measured 6 weeks post-STZ administration in rats. The present results are substantiated by previous data that indicated hyperglycemia and a substantial change in HbA1c, feed/water intake, urine volume, and body weight after STZ treatment reminiscent of clinical diabetes (Suryavanshi and Kulkarni, 2020; Pham et al., 2019). The neuropathic pain parameters (mechanical hyperalgesia, thermal hyperalgesia, and mechanical allodynia) measured 6 weeks after STZ injection showed the development of DPN in rats. Treatment with Ed or Gpn for 4 weeks starting after 6 weeks of STZ administration attenuated the hyperglycemia and decreased polyphagia, polydipsia, and loss of body weight against STZ-induced diabetes. Ed or Gpn treatments also improved the circulating insulin levels and ameliorated HOMA-β and attenuated HOMA-IR in rats.
Chronic hyperglycemia exerts multiple impacts leading to inadequate axonal support, vascular insufficiency, and poor autoregulation making extremely long axons susceptible to the distal side and causing demyelination and peripheral nerve injury (Galiero et al., 2023). Glucotoxic mechanisms (i.e., polyol, hexosamine, AMPK/PGC-1α, and Protein kinase C pathways) trigger the generation of reactive oxygen species (ROS), reactive nitrogen species (RNS), and AGEs which involves mitochondria (Pang et al., 2020). Reactive species (ROS and RNS) readily attack cellular macromolecules and compromise cell integrity. In the present study, i.p. injection of STZ on day 1 caused an increase in the biomarkers of oxidative stress and inflammation, measured after 10 weeks, in the sciatic nerve of rats. The data showed an increase in lipid peroxidation (TBARS), nitrosative stress (nitric oxide), and a decline in antioxidants (GSH, GPx, SOD, and catalase) in the sciatic nerve homogenate. Reactive species can directly damage the neuronal lipids (e.g., in myelin sheath) or they might promote attack by macrophages, proteases, and phospholipases. An increase in polyol pathway influx results in GSH deficiency due to the depletion of NADPH which decreases the efficiency to scavenge free radicals. The levels of AGEs were also increased by STZ in the sciatic nerve of rats. AGEs tend to accumulate in the microstructure of the peripheral neurons and induce reactive species via activation of macrophages, NADPH oxidase, and protein kinase C. AGEs cause axon cytoskeletal modifications, demyelination, neurovascular uncoupling, endothelial dysfunction and impairment of regenerative activity in DPN. NF-κB and a number of NF-κB-mediated pro-inflammatory genes are upregulated as a result of interactions between AGE/RAGE. Segmental demyelination is facilitated by the phagocytosis of AGE-modified peripheral nerve myelin by macrophages (Galiero et al., 2023). The current results showed that Ed or Gpn treatment for 4 weeks attenuated oxidative stress and nitrosative stress, and lowered the AGEs levels against STZ-induced DPN in sciatica of rats.
Hyperglycemia-induced pro-inflammatory cytokines such as TNF-α and IL-1β are positively correlated with DPN. In clinical studies, there was a substantial rise in serum TNF-α (Duksal et al., 2016) in patients with DPN relative to controls and diabetic patients lacking DPN. Experimental data revealed that anti-TNF-α therapy can increase nerve conduction velocity and myelination (Xue et al., 2021). Pre-clinical data indicated that an increase in IL-1β expression in the lumbar spinal cord tissues (Chen et al., 2022) and the sciatic nerve (Zhou et al., 2019) is associated with mechanical allodynia and thermal hyperalgesia in DN. IL-1β antagonists may attenuate the pathogenic course of DPN (Hanping et al., 2020). Furthermore, long-term hyperglycemia activates NF-κB via AGE/RAGE interaction and oxidative stress. NF-κB triggers the expression of a diverse set of molecules (e.g., TNF-α, IL-1β, inducible nitric oxide synthase, and cyclooxygenase-2), which cause inflammation, nerve damage, endothelial dysfunction, and micro- and macro-vascular complexities (Sandireddy et al., 2014). The current data suggested an increase in pro-inflammatory markers (TNF-α, IL-1β levels) and NF-κB in the sciatic nerve of rats that were pretreated with STZ. Ed or Gpn attenuated inflammatory molecules including NF-κB against STZ-induced DPN in the sciatic nerve of rats.
Allodynia and hyperalgesia along with deranged biochemical parameters (e.g., oxidative stress and inflammation) are features of STZ-induced DPN (Pham et al., 2019). In the present study, the administration of STZ caused mechanical and thermal hyperalgesia and mechanical allodynia in rats. Treatment with Ed or Gpn for 4 weeks, starting 6 weeks after STZ injection, attenuated STZ-induced hyperalgesia and allodynia in rats. These results indicated that Ed can decrease nociceptive response and improve tolerance to painful stimuli in DPN. The histology of the sciatic nerve also depicted that ED or Gpn attenuated the STZ-induced neural injury.
Recent data indicated that the Wnt/β-catenin pathway contributes to DPN via oxidative stress and vascular aberrations (Nie et al., 2021). Pre-clinical studies suggest that STZ augments the Wnt/β-catenin pathway and inhibition of this pathway can improve pain-linked behavior (e.g., hyperalgesia, allodynia) and nerve functions (e.g., blood flow, conduction velocity) (Resham et al., 2020; Resham and Sharma, 2019). Instigation of the Wnt/β-catenin pathway in the spinal cord is a key attribute of neuropathic pain (Sanabria-de la Torre et al., 2022). Interestingly, in the existing study, the administration of MV (Wnt/β-catenin activator) potentiated the effects of STZ. MV caused a significant rise in glucose levels, feed/water intake, and a decline in the mean b.w. of diabetic animals. MV further decreased the insulin levels and pancreatic-β cell function and enhanced IR in diabetic rats. Oral treatment with Ed (10 mg/kg) attenuated these effects in MV and STZ-treated rats. MV potentiated the pro-oxidant and pro-inflammatory activities of STZ in the sciatic nerve of rats and Ed (10 mg/kg) attenuated these effects in MV and STZ-treated rats.
The search for bioactive molecules effective in ameliorating diabetic complications including pain in diabetic neuropathy has witnessed a great expansion (Rather et al., 2023; Bhargava et al., 2022). This study indicated that Ed might attenuate the pain symptoms in DPN and ameliorate the biochemical indices of oxidative stress and inflammation by a possible control over the Wnt/β-catenin pathway.