Neuropathic pain is clinically characterized by spontaneous pain and an increased response to stimuli. Many clinical pathologies, such as cancer, trauma, and diabetes, can lead to the development of NP(Dreyling, et al.,2016). Unfortunately, NP is usually resistant to standard analgesics, which are further limited by various adverse side effects(Dreyling, et al.,2016; Cavalli, et al.,2019). Therefore, there is a great need to better understand the molecular mechanisms of pain hypersensitivity responses to identify new therapeutic targets and new treatments for patients with NP.
Growth factors and their homologous receptors can promote pain sensitization and have been identified as therapeutic targets for NP (Chang, et al.,2016; Borges, et al.,2021). Geniposide has been confirmed to relieve the expression of inflammatory factors(Cai, et al.,2020). In this study, a CCI rat model was established to further explore the effect of Geniposide on neuralgia in CCI rats through EGFR and explore other potential mechanisms.
Geniposide was found to have the potential to interact with EGFR through molecular docking, and through behavioral experiments, Geniposide treatment was found to increase the MWT and TWL. Pathological results showed that Geniposide reduced the expression of inflammatory factors. The number of DRG neurons was significantly reduced in the CCI group compared to the control group, and CCI significantly increased the expression of p-p38, and the inhibition of p38 may reduce neuronal sensitization(Zhang, et al.,2018). Similarly, the p53-caspase-3 pathway plays a key role in the neural damage of DRG neurons(Gao, et al.,2018). The analgesic effects of Geniposide have been confirmed in several previous studies, including analgesic and anti-inflammatory effects(Gong, et al.,2014). This is consistent with the results of the present study. It was found that upregulation of EGFR neural outgrowth occurred in various pain models(Wang, et al.,2019). The present study found that the therapeutic effect of Geniposide was significantly reversed when further agonists of EGFR were added, suggesting that Geniposide may exert anti-inflammatory and pain threshold enhancing effects through EGFR. The results of the present study for inflammatory factors also reflected the same results. A previous study by Xu et al. found that Geniposide could increase the pain threshold and reduce pain by inhibiting tumor necrosis factor-α (TNF-α) expression in ipsilateral or contralateral DRG cells. Based on the molecular biological mechanisms of nerve injury, Geniposide treatment may be an effective clinical approach for pain relief.
To further investigate the mechanism by which Geniposide alleviates neuralgia in CCI rats, transcriptome analysis was performed on the normal group, the CCI model group and the Geniposide treatment group, and the results showed that the CCI model mainly affected the changes in the functions related to signal transduction and the immune system, which were exactly reversed by Geniposide. In addition, it was shown that one of the fundamental mechanisms by which signal transduction and the immune system function is through Toll-like receptors (TLRs), and Toll-like receptor signaling has also emerged as a major pathway mediating neuronal cell activation after injury and infection/inflammation. It protects and defends the host organism by initiating an inflammatory signaling cascade response in response to tissue injury, while TLRs activation promotes damage brought about by inflammation in sensory neurons, which leads to excessive pain(Lacagnina, et al.,2018). Cell lines overexpressing EGFR have been shown to undergo receptor-mediated apoptosis(Jackson and Ceresa,2017), which shows the same trend as the results of the present study.
Based on the above studies, it is hypothesized that the upregulation of EGFR in microglia in the CCI rat model should promote microglial proliferation, survival and differentiation, which in turn upregulates the inflammatory response and causes increased damage to neuronal cells via inflammatory infiltration, resulting in increased apoptosis of spinal cord cells. In addition, KEGG analysis showed that the Ca2+ signaling pathway was significantly changed in the CCI model group compared to the normal group. Geniposide alleviates NP in CCI rats mainly through the Ca2+ signaling pathway and PI3K/AKT signaling pathway. The Ca2+ signaling pathway is a key signaling pathway for initiating and maintaining NP activity-dependent central sensitization(Liu, et al.,2018). Therefore, the Ca2+ signaling pathway may be a potential target of Geniposide. A study by Zhou et al. found that Geniposide could inhibit influenza through the Ca2+ signaling pathway(Zhou, et al.,2021), suggesting that Geniposide could target the Ca2+ signaling pathway to exert medicinal effects. In addition, Geniposide significantly downregulated the expression of PKC, PKA, CaMK IIα, and CaMK IIδ in CCI rats, which is consistent with the results of Chen et al., who studied rhodopsin to alleviate CCI in rats(Chen, et al.,2021). The results of the present study further confirmed that Geniposide can alleviate the effect on CCI through the Ca2+ signaling pathway, which is a novel finding.
In addition to the Ca2+ signaling pathway, transcriptome analysis showed that Geniposide alleviates chronic NP through the PI3K/AKT signaling pathway. PI3K has been shown to contribute to the establishment and maintenance of NP(Leinders, et al.,2014). Pan et al. found that Geniposide alleviates IL-1β-induced joint inflammation by inhibiting the PI3K/Akt/NF-κB signaling pathway(Pan, et al.,2018), thereby treating degenerative joint diseases, which demonstrates the potential of Geniposide to inhibit PI3K/Akt. In addition, Martin et al(Martin, et al.,2017) showed that EGFR phosphorylation at Y1068 leads to PI3K-Akt signaling, a phenomenon that may be increased within the DRG of various pain models. In conjunction with previous studies on EGFR, the present study continued to investigate the effect of Geniposide on the overall EGFR/PI3K/AKT pathway. Geniposide inhibited the activation of EGFR/PI3K/AKT overall, and the pathological results and western blot results showed the same trend.
In conclusion, Geniposide was able to reduce the expression of inflammatory factors and improve the pain threshold in CCI by inhibiting Ca2+ channel activity and EGFR/PI3K/AKT activity.