NP is a pain syndrome resulting from damage to the peripheral or central nervous system and characterized by spontaneous pain, allodynia, and hyperalgesia(Bouhassira 2019). In addition to the pain caused by itself, NP usually causes physiological and psychological abnormalities such as depression, anxiety, and sleep disturbance, which seriously affect patients' quality of life and health(Cavalli, Mammana, Nicoletti et al. 2019). However, current drugs for NP treatment include anxiolytics, antidepressants, adrenal receptor agonists, opioids, local anesthetics as well as NMDA receptor antagonists, the efficacy of which remains unsatisfactory, and significant side effects limit their use(Szok, Tajti, Nyári et al. 2019). Therefore, it is urgent to find a safe and effective therapeutic drug with few side effects.
Evidence-based clinical studies have shown that although NP is classified as an intractable disease by the World Health Organization (WHO), TCM exhibits several characteristics and advantages in improving the clinical symptoms and improving the quality of life of patients with NP. For example, Yang et al.(Yang, Li, Zou et al. 2021) demonstrated that gallic acid, a TCM extracted from natural plants with anti-inflammatory, analgesic effects, attenuates NP in CCI rats via inhibition of P2X7 receptors and subsequent activation of the TNF-α/STAT3 signaling pathway. In addition, there are other TCM also reported to alleviate NP, for example Divanillyl sulfone(Shao, Xu, Chen et al. 2021), Wu-Tou decoction(Zhu, Xu, Mao et al. 2018), resveratrol(Wang, Shi, Huang et al. 2020), astragalin(Wang, Cai, Wang et al. 2020), etc. SA, a triterpenoid saponin sodium salt, is extracted from the dried fruits of the TCM Solanum, and its main efficacy, including anti inflammation, antioxidation, and restoration of capillary permeability, has been available for half a century since the 1960s. Clinically, SA is mainly used for the treatment of functional abnormalities caused by brain trauma, cerebral ischemia, or cerebral edema. Wang et al.(Wang, Yang, Ju et al. 2016) found that the combined use of AS and mannitol produced satisfactory results in the treatment of early swelling after upper extremity trauma surgery. Fan et al.(Fan, Guo, Xiao et al. 2005) study suggested that AS increased the expression of anti-apoptotic protein Bcl-2 and decreased the expression of protein of Caspase-3, and then protected against reperfusion injury on ischemic brain. Further, SA has also been discovered to exert potent antitumor effects, including breast cancer(Qi, Lv, Meng et al. 2015), liver cancer(Hou, Li, Cui et al. 2019), gastric cancer([Sodium Aescinate Induced Apoptosis of BGC-823 and AGS Cells by Inhibiting JAK-1/STAT-1 Signaling Pathway] 2016). Additionally, a recent study drew our attention. Yang et al.(Liu, Qin, Wu et al. 2020) demonstrated that SA alleviated bone cancer pain-induced pain-related behaviors through the increase of proinflammatory cytokine production as well as microglial activation. This experiment serves as an animal model of NP by applying the CCI model, which has similarities with the characteristics of clinical NP and is widely used by the pain science community. The present experimental study demonstrated that the TCM ingredient SA observably attenuated NP in mice resulting from the CCI model in response to decreased PWT in the ipsilateral paw.
The targets of existing drugs to treat NP are mainly neurons. Studies in multiple models of NP have found that NP is a manifestation of abnormal discharges of dorsal horn neurons caused by peripheral sensation(Lançon, Qu, Navratilova et al. 2021). The common NP therapeutics mainly focus on targeting neuronal functions, but the CNS-related side effects nausea, vomiting, dizziness, etc. caused by these drugs greatly limit the use of these analgesic drugs. Therefore, investigating other mechanisms of the occurrence and development of pain and finding safe and effective means of analgesia become urgent clinical needs. Over the past few decades, there has been increasing research on non-neuronal cells, especially concerning glial cells. Spinal glial cells, mainly microglia, play a crucial role in the development and progression of NP(Tozaki-Saitoh and Tsuda 2019). Microglia are widely distributed in the central nervous system under normal physiological conditions. When the central nerve is injured or ischemia, inflammation occurs, microglia are rapidly activated to change from a resting state to an activated state and constantly activated to proliferate to participate in early pain and inflammation(Tsuda, Koga, Chen et al. 2017). Distinct activation of microglia is evident in multiple models of nervous system pain. Microglial morphology was changed evidently within 24 h of peripheral nerve injury, and microglial activation was markedly increased after 2–3 days of injury(Yi, Liu, Liu et al. 2021). At the same time, activated microglia can release many inflammatory factors, cytokines, proinflammatory factors to produce neuroinflammation ultimately leading to the occurrence of pain(Tsuda 2018). Ye et al.(Ye, Lin, Zhang et al. 2021) suggested that quercetin increased mechanical withdrawal threshold in CCI rats by preventing the activation of microglia and astrocytes. Cheng et al.(Cheng, Chen, Hsu et al. 2021) found that the expression of GFAP (astrocyte marker) and Iba-1 (microglial marker) in the ipsilateral spinal dorsal horn of CCI mice was conspicuously decreased after intraperitoneal administration of loganin. Immunohistochemistry in this study revealed that Iba-1, a marker of microglia, was indeed significantly elevated in the spinal cord of mice after CCI surgery, and that the elevation of Iba-1 was particularly suppressed by multiple intrathecal administrations of SA. Moreover, intrathecal administration of SA also could reverse CCI-induced microglial polarization toward M1 type. Microglia, being able to be activated by different external stimuli, present two phenotypes in which their functional status is quite different from that of surface markers. Among them, the classical activation pathway generates an M1 phenotype, expresses surface markers such as iNOS and CD86, and exerts pro-inflammatory versus neurotoxic effects(El-Deeb, El-Tanbouly, Khattab et al. 2022). However, the selective activation pathway generates an M2 phenotype, expresses surface markers such as Arg-1 and CD206, and exerts anti-inflammatory and neuroprotective effects(Zhou, Ji,Chen 2020). Studies have demonstrated that microglia are significantly activated and polarized toward M1 type in animal models of CCI induction(Wang, Jiang, Li et al. 2021). Yuan et al.(Yuan and Fei 2022) found that lidocaine inhibited M1 microglial polarization (manifested by decreased Iba-1/CD86 positive cells) but promoted M2 microglial polarization (manifested by increased Iba-1/CD206 positive cells) in a CCI-induced NP rat model, which in turn improved NP.
TNF-α, a member of the type Ⅱ transmembrane protein superfamily, is also one of the pro-inflammatory factors that is first upregulated after nerve injury and secondarily upregulated together with other factors(Liu, Zhou, Wang et al. 2017). TNF-α expression was upregulated in peripheral blood, central neurons, microglia, and astrocytes during NP formation(Gao, Bai, Zhou et al. 2020). A previous study found that CCI surgery induced elevated TNF-α production in the lumbar enlargement spinal cord, which peaked on the third day(Zhang, Wang, Zhang et al. 2021). IL-1β and IL-6 are also pro-inflammatory factors that play an important role in NP(Li, Xu,Yang 2017). Intrathecal administration of IL-1β leads to upregulation of inducible NO synthase expression and causes heat pain hypersensitivity in rats(Kanno, Shimizu, Shinoda et al. 2020). In addition, IL-6 can also upregulate substance P and nerve growth factor to sensitize spinal posterior horn neurons to induce hyperalgesia(Li, Zhou, Yang et al. 2022). Studies have shown that following neuroinflammation or nerve injury, microglia are activated and release neural substances and proinflammatory cytokines such as IL-1β, IL-6 and TNF-α, and increase the sensitivity of postsynaptic spinal dorsal horn neurons, ultimately causing hyperalgesia(Yang, Li, Zhang et al. 2020). Our results are consistent with previous studies showing that TNF-α, IL-1β, and IL-6 expressions were prominently upregulated in the spinal cords of mice subjected to CCI surgery, whereas SA intrathecal injection strikingly reduced the elevated pro-inflammatory factors induced by CCI surgery, indicating that SA treatment can ameliorate the secondary damage from neuroinflammation induced by CCI surgery.
MAPKs are an important class of signaling molecules within the cell that, upon activation by different factors, form their respective transduction pathways, which in turn activate different transcription factors to exert different biological effects(Kassouf and Sumara 2020). As mentioned earlier, there are three classical MAPK pathways: JNK, ERK1/2, p38(Kim and Choi 2010). Among them, p38 and JNK signaling pathways are important signals involved in the regulation of inflammatory responses(Li, Zhang, Ge et al. 2019). Studies have found that JNK and p38 phosphorylated protein expression was conspicuously enhanced in the spinal cord of C57BL6 mice with CCI-induced NP, which is accompanied by microglial activation(Riego, Redondo, Leánez et al. 2018). Besides, JNK and p38 pathway activation dependently encouraged TNF-α-induced inflammatory responses(Liu, Guo, Song et al. 2020). In this experimental study, we found that the phosphorylation levels of JNK and p38 in the spinal cord of mice after CCI were conspicuously upregulated compared with those in the sham group, but the total protein level was unchanged, that is, their transcriptional activity was not affected, which was consistent with previous studies. In addition, JNK and p38 phosphorylated levels were obviously downregulated after SA intrathecal injection.