DISCUSSION

Our previous study demonstrated that the expression levels of IL-1β, TNF-α, CCL2, and CXCL1 are significantly increased in the spinal cord dorsal horn under NP conditions, which may in turn cause the activation of microglia and astrocytes [32]. In this study, IL-1β and TNF-α, but not CCL2 and CXCL1, increased the gene expression of NGF, BDNF, GDNF, and artemin in rat glial cells under in vitro inflammatory conditions. On the other hand, Sun et al. reported that CCR5 is upregulated under NP conditions, while the knockout of CCR5 suppresses CCI-induced NP, indicating that CCR5 is directly involved in the generation of NP [16]. In the present study, IL-1β, TNF-α, CCL2, and CXCL1 were found to augment CCR5 expression in glial cells. Thus, these results suggest that IL-1β and TNF-α exhibit both beneficial and adverse actions: neuroprotection due to an increase in NF production and the aggravation of CCR5-mediated inflammation, respectively, in glial cells under NP conditions.

Previous studies have reported that NGF and/or GDNF inducers are therapeutic candidates for support of neuronal survival due to the stimulation of their biosynthesis, which results in the alleviation of NP [56, 57]. Similar to GDNF, artemin has been reported as a promising target for pain control [58]. In addition, it has been shown that NGF upregulates BDNF production through the activation of TrkA [59]. On the other hand, it has been reported that CCR5 plays a substantial role in neurotoxicity in the central nervous system [60]. In addition, NGF/BDNF-heterozygous mice have shown higher expression of CCR5 mRNA but lower gene expression of NGF and BDNF in the brain than wild-type mice [61, 62]. Furthermore, the pharmacological blockade of CCR5 has been reported to be effective in the treatment of NP during the development and maintenance phases. Thus, CCR5 is likely to be a potential therapeutic target for NP [63]. In the present study, WTD and the identified active components Pae and Liq were found to increase the gene expression of NGF, BDNF, GDNF, and artemin but inhibit CCR5 expression in rat glial cells. In addition, the production of NGF and BDNF was augmented by WTD, Pae, and Liq under noninflammatory and inflammatory conditions.

Taken together with results from previous studies showing that WTD is prescribed to

patients with NP in clinics in China [21-23], these findings allow me to speculate that WTD, mostly via Pae and Liq, exerts an anti-NP action by facilitating neuroprotection rather than neuropathy despite inflammatory conditions.

Neurotrophic factors have been reported to exert neuroprotective actions through the activation of the PI3K and PKA signaling pathways, which are required for the regulation of glial cell apoptosis and neuronal differentiation [64-65]. In this study, WTD-, Pae-, and Liq-enhanced gene expression and production of NGF, BDNF, GDNF, and artemin were no longer detectable upon the addition of both LY294002 or H89 to rat glial cells, indicating that a common dual pathway of PI3K and PKA signaling is activated by WTD and its bioactive components to mediated the regulation of NGF, BDNF, GDNF, and artemin expression in rat glial cells. However, neither LY294002 nor H89 influenced WTD-, Pae-, or Liq-downregulated CCR5 gene expression in rat glial cells. Thus, both PI3K and PKA are unlikely to be involved in the agent-mediated suppression of CCR5 expression.

In summary, these results suggest, for the first time, that Pae and Liq are the active components for the WTD-mediated upregulation of NF and downregulation of CCR5 production in rat glial cells under in vitro NP conditions mimicking the SNL animal model.

Furthermore, it is strongly suggested that the regulation of NF expression differs from that of CCR5 expression in rat glial cells treated with WTD, Pae, or Liq in which the PI3K and PKA signaling pathways are involved.

GENERAL DISCUSSION AND CONCLUSION

Our previous study reported that TNF-α, IL-1β, CCL2, and CXCL1 are increased in the spinal cord dorsal horn of SNL mice, in which the progression of NP is enhanced [32].

In Chapter 2, TNF-α and IL-1β were found to induce the gene expression of not only NGF, BDNF, GDNF, and artemin but also CCR5 in rat glial cells. In addition, CCL2 and CXCL1 were found to increase the gene expression of CCR5 in glial cells, while there was no change in the levels of NGF, BDNF, GDNF, or artemin in TNF-α- and IL-1β-treated cells. These findings suggest that chemokines such as CCL2 and CXCL1 are increased in the spinal cord in NP and substantially participate in exacerbating NP symptoms. Furthermore, both IL-1β and TNF-α are likely to be bifunctional modulators that exhibit neuroprotection due to the increase in NF production and the aggravation of CCR5-mediated inflammation in glial cells under NP conditions. Taken together with our previous report showing that WTD treatment clearly decreases the cytokine and chemokine expression in a dose-dependent manner [32], the alteration of cytokine and chemokine levels in the spinal cord may be associated with the WTD attenuation of NP symptoms.

Wu-tou decoction is a classical TCM formula and consists of five plant herbs, which contain a series of components with multiple biological activities [24-27]. Since anti-pain action is the major function of WTD [35], the molecular mechanisms are not well understood. In Chapter 1, WTD effectively attenuated SNL-induced NP in a dose-dependent manner without inducing any side effects in vivo. This finding is supported by clinical reports showing that WTD is widely used in NP patients with a cure rate higher than 80% [21-23]. On the other hand, it has been reported that an NGF inducer is a therapeutic candidate to control neuronal survival by stimulating the biosynthesis of NGF and BDNF, which in turn causes the alleviation of NP [61-62]. In addition, higher expression of CCR5 mRNA but lower gene levels of NGF and BDNF are detectable in the brains of NGF/BDNF-heterozygous mice compared to those in wild-type mice [63], suggesting that CCR5 is a crucial mediator of neurotoxicity in the central nervous system.

As shown in Chapter 2, WTD transcriptionally enhanced the production of NGF and

BDNF but inhibited CCR5 expression in rat glial cells under inflammatory conditions.

Therefore, the anti-pain action of WTD is likely to contribute to a relative increase in NGF and BDNF expression resulting in neuroprotection.

The identification of bioactive compounds from natural products is needed to address the crucial components in therapeutic actions exerted by herbal formulas and accelerate novel natural product-based drug discovery [66]. In this regard, Chapter 1 revealed that Pae and Liq are bioactive compounds of WTD that exhibit analgesic actions and anti-neuroinflammation in NP rats. To date, it has been reported that Pae and Liq exhibit anti-inflammatory, anti-oxidative, antidepressant, and anticancer effects [67-74].

As shown in Chapter 2, Pae and Liq were found to transcriptionally enhance constitutive and IL-1β-increased NGF, BDNF, GDNF, and artemin production in rat glial cells. In addition, the expression of CCR5 was suppressed by both compounds in the IL-1β-treated and untreated cells, and this phenomenon was similar to that observed in the WTD-treated cells. Thus, these results suggest, for the first time, that Pae and Liq are analgesic agents against NP and facilitate neurotrophic reactions.

It has been reported that NFs exert neuroprotective actions by regulating glial cell apoptosis and neuronal differentiation through the activation of PI3K and PKA signaling [65]. As shown in Chapter 2, the WTD-, Pae-, and Liq-enhanced gene levels of NGF, BDNF, GDNF, and artemin were no longer detectable by adding either LY294002 or H89 to rat glial cells. However, neither LY294002 nor H89 influenced WTD-, Pae-, and Liq-downregulated CCR5 gene expression in the cells. Therefore, the common dual pathway consisting of PI3K and PKA signaling may be involved in the control of NF expression but not CCR5 expression, even when rat glial cells are treated with WTD, Pae, and Liq.

Previous reports have shown that activation of CCR5 is crucial for RANTES-induced PI3K/AKT-mediated signaling in RAW264.7 cells [75], and inhibition of PKA downregulates forskolin-induced CCR5 transcription in human bone marrow progenitor cells [76]. The discrepancy in the findings in Chapter 2 and the aforementioned literature may partly be due to the cellular specificity, treatment, and observed parameters. Nonetheless, these results suggest that the regulatory mechanism(s) of CCR5 expression differs from that of NFs in rat

glial cells. Further experiments are needed to investigate the mechanisms underlying the regulatory effects of WTD, Pae, and Liq on CCR5 expression.

In conclusion, these data provide novel evidence that WTD exerts anti-NP actions by predominantly increasing the production of NFs through the activation of PI3K and PKA signaling pathways in rat glial cells. Furthermore, Pae and Liq may function as analgesic candidates in WTD-mediated NP management (Schema 1).

Schema 1 Possible molecular mechanisms of the anti-NP actions of WTD, Pae, and Liq in rat glial cells. WTD, Pae, and Liq augment the expression of NGF, BDNF, GDNF, and artemin by activating PI3K- and PKA-dependent signaling pathways in rat glial cells under neuroinflammatory conditions. In contrast, the expression of CCR5, which is associated with the generation and development of NP, is suppressed by WTD, Pae, and Liq in a PI3K- and PKA-independent pathway(s). These results suggest possible mechanisms by which WTD, Pae, and Liq exhibit anti-NP actions due to the increased levels of neurotrophic factors in glial cells.

ACKNOWLEDGMENTS

First, I want to express my sincere gratitude to my supervisor Professor Takashi Sato for patient guidance, continuous support, and insightful comments for my PhD study and related research.

I also want to thank the faculty in the Department of Biochemistry, Tokyo University of Pharmacy and Life Sciences: Dr. Koji Mizuno, Dr. Noriko Akimoto, and Dr. Hiroaki Sakaue, for their generous help and warm encouragement during my stay in Japan.

In addition, I want to say thanks to Dr. Hideki Hayashi and Prof. Norio Takagi from the Department of Applied Biochemistry, School of Pharmacy, for providing generous help and technical support during my experiments in Japan.

Moreover, I need to say thanks to the students in the Department of Biochemistry, Tokyo University of Pharmacy and Life Sciences, especially Mr. Katsuki Okuyama, for their kind assistance and technical guidance.

I also sincerely thank the China Scholarship Council (201808110263) for supporting my study during my stay in Japan.

Furthermore, I want to show regards to Prof. Na Lin at the China Academy of Chinese Medical Sciences for her kind help and support during my study, life and work, not only in the past seven years but also in the future.

I thank Prof. Yanqiong Zhang at the China Academy of Chinese Medical Sciences for her patient guidance of my study and work.

Last but not least, I appreciate the unrivaled support of my family; their love and understanding always encourage me to improve myself in all aspects.

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