論文の公開元へPharmacological Mechanisms of Wu-tou Decoction against Neuropathic Pain
概要
INTRODUCTION
Neuropathic pain (NP) results from lesions or diseases of the somatosensory system, with the major features of mechanical allodynia and thermal hyperalgesia. As a debilitating disease, NP diminishes people’s health and decreases their quality of life. NP encompasses various diseases, such as peripheral diabetic neuropathy, fibromyalgia, postherpetic neuralgia, cancer chemotherapy-induced NP, and acute and chronic pain, with the total prevalence ranging from 6.9% to 10% in the general population, which continues to increase with the growing number of brain injury, diabetes, and cancer cases. To date, the primary means for conservative clinical treatment of NP is pharmacotherapy. According to the guidelines for NP treatment, the major first-line drugs consist of tricyclic antidepressants, anticonvulsants and serotonin-noradrenaline uptake inhibitors such as pregabalin (PGB), gabapentin, and duloxetine. However, due to limited clinical efficacy, side effects and inadequate responses to drug treatments, only 30-40% of NP patients are relieved of less than 50% of their pain intensity. Thus, an increasing number of NP patients are seeking pain-relieving drugs used in traditional Chinese medicine (TCM).
Previous reports have shown that glial cells, mainly astrocytes and microglia, are activated along with an increase in cell number and neurotrophic factor (NF) secretion when NP conditions are mimicked in several classic animal models, such as SNL, partial sciatic nerve ligation, and chronic constriction injury. NFs have been reported to play important roles in the survival, growth, and maintenance of discrete populations of neurons in neurodegenerative disorders and spinal cord injury. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been reported to induce the development of normal functional properties of sensory neurons, which results in an increase in their responsiveness to peripheral stimuli. In addition, glial cell line- derived neurotrophic factor (GDNF) has been reported to play a neuroprotective role in mature organisms that may promote neuroregeneration and recovery from neurological insults. Furthermore, artemin, a member of the GDNF family, modifies injury-induced neuropathic changes in the nervous system. On the other hand, C-C chemokine receptor type 5 (CCR5) upregulation has been identified in a mouse model of chronic constriction injury (CCI), which mimics NP conditions. In addition, knockout of CCR5 has been reported to suppress CCI-induced NP. Therefore, NFs such as NGF, BDNF, GDNF, artemin, and CCR5 may be regarded as potential new candidates for NP therapy.
According to TCM theory, NP is categorized as arthromyodynia. There is plenty of evidence to prove that TCM has a profound curative effect on NP with an abundance of available sources and few side effects. As a classic TCM formula, Wu-tou decoction (WTD) has been extensively used in the clinical treatment of NP, including chronic inflammatory pain, trigeminal neuralgia, and cancer-induced pain, with a total cure rate of approximately 80%. However, it remains unclear how WTD controls NP, what bioactive component(s) of WTD are involved in NP treatment, and whether WTD regulates the expression of NFs and CCR-5 under NP conditions.
In the present study, I demonstrate that WTD attenuates the severity of NP symptoms by improving mechanical allodynia and thermal hypersensitivity through the inhibition of neuroinflammation in spinal nerve ligation (SNL) rats in a dose-dependent manner. In addition, I found that paeoniflorin (Pae) and liquiritin (Liq) were analgesic candidates for WTD-mediated anti-NP actions in SNL rats (Chapter 1). Furthermore, WTD, Pae, and Liq transcriptionally enhanced the expression of NGF, BDNF, GDNF, and artemin in tumor necrosis factor α (TNF-α)- and interleukin 1β (IL-1β)-treated glial cells through PI3K- and PKA-dependent pathways. In contrast, TNF-α- and IL- 1β-increased CCR5 expression was suppressed by WTD, Pae, and Liq in a PI3K- and PKA-independent manner (Chapter 2). Thus, these results provide novel evidence that WTD exerts anti-NP actions by predominantly increasing the production of neurotrophic factors through the PI3K and PKA signaling pathways in glial cells. Moreover, Pae and Liq are likely to act as analgesic candidates in WTD-mediated NP management.
METHODS
The anti-pain effect of WTD was evaluated by detecting mechanical hypersensitivity and cold hypersensitivity in SNL rats. Cell species in the spinal cord of SNL rats were characterized by immunofluorescent staining with antibodies against ionized calcium-binding adapter molecule 1 (IBA 1 ) and glial fibrillary acidic protein (GFAP). The bioactive compounds of WTD were investigated by HPLC, and absorption distribution metabolism excretion (ADME) evaluation was performed using ACD/Percepta software 5.07. An in vitro experiment was performed in primary rat glial cells. Cell viability was measured using CellTiter-Blue in WTD-, Pae-, and Liq-treated cells. The production and gene expression of NGF, BDNF, GDNF, artemin, and CCR5 were evaluated by RT-PCR and ELISA.
RESULTS
CHAPTER 1 Analgesic Effect of Wu-tou Decoction against Neuropathic Pain1)
1.1 WTD attenuated SNL- induced mechanical allodynia and cold hyperalgesia
To determine whether WTD attenuates SNL- induced NP, WTD at dose of 15 g/ kg was orally administered once per day for 3 consecutive days, and then, behavioral assessment was performed 1 h after each drug t reatment. When SNL markedly reduced the 50 % paw withdrawal threshold ( PWT) compared with the control ( Con) group, all t reatments, such as WTD- Low, - Middle, and – High, and PGB, attenuated mechanical allodynia, which lasted for at least 2 h, with an optimal inhibitory effect 1 h post- WTD administration ( Fig. 1 A). Thus, 1 h post- WTD administration was selected as the optimal t ime point for further experiments. On the other hand, since the reaction time was increased in SNL rats exposed to cold stimulus using acetone, t reatment with WTD at middle and high doses and PGB markedly decreased the reaction t ime ( Fig. 1 B). In addition, there was no difference between WTD and PGB in the attenuation of the 50 % PWT and the decrease in reaction time to the cold stimulus.
1.2 Astrocytes and microglia tended to increase in the spinal cord of rats Since GFAP and IBA 1 have been reported to be cell markers of astrocytes and microglia, respectively, the expression of GFAP and IBA 1 in spinal cord tissues tended to be increased by SNL. These results indicated an increase in astrocytes and microglial cells in the spinal cord under NP conditions. Based on these findings, an in vitro experiment using primary rat glial cells was performed to clarify the molecular mechanism(s) of WTD- induced analgesic action in Chapter 2.
1.3 Identification and pharmacological characterization of paeoniflorin and liquiritin as bioactive compounds in WTD for use in NP treatment
In the present study, 77 compounds were identified in WTD, and f ive s ingle herbs comprise WTD. Forty- three of the 77 compounds were identified as candidate bioactive compounds with potential druggability by ADME evaluation in silico . In addition, Pae and Liq, which are known to be effective for NP control, were found among these compounds. Furthermore, LC- MS analysis revealed that the contents of Pae and Liq in lyophilized WTD powder were 9225 .0 and 3740 .6 ng/ mg, respectively. On the other hand, when s imilar in vivo experiments using SNL rats were performed by administering Pae or Liq and the combination of Pae and Liq ( Pae+Liq), all t reatments were found to attenuate mechanical allodynia. In addition, the inhibitory effect of Pae+Liq was s imilar to that of WTD but was remarkable compared to the single use of Pae or Liq. Furthermore, the time to reach the maximum plasma concentration of Pae and Liq after the administration of Pae+Liq was 5 min, which was less than that of WTD (120 min and 30 min for Pae and Liq, respectively). Moreover, the maximum plasma concentrations of Pae (668 ng/mL) and Liq (17.1 ng/mL) in rats were approximately 4.6-fold and 1.3-fold higher than that of WTD (Pae and Liq at 144 ng/mL and 13.3 ng/mL, respectively).
CHAPTER 2 Pharmacological Mechanisms of the Anti-NP Actions of WTD, Pae, and Liq in Primary Rat Glial Cells 2, 3)
2.1 Regulation of NGF, BDNF, GDNF, artemin, and CCR5 expression by WTD, Pae, and Liq in primary rat glial cells
WTD, Pae, and Liq were found to augment the m RNA expression of NGF, BDNF, GDNF, and artemin in a dose- dependent manner (Fig. 2 A). In addition, IL- 1 β- augmented NGF, BDNF, GDNF, and artemin expression was enhanced by WTD,
Pae, and Liq in rat glial cells in a dose dependent manner (Fig. 2 A). Furthermore, the transcriptional augmentation of NGF and BDNF expression led to an increase in their protein levels.
In contrast, both constitutive and IL- 1 β- augmented CCR 5 mRNA expression was suppressed by WTD, Pae, and Liq in a dose- dependent manner (Fig. 2 B). Therefore, these results suggested that WTD, Pae, and Liq t ranscriptionally augmented the production of NFs and reduced CCR5 expression in rat glial cells.
2.2 Differential involvement of the PI3 K and PKA pathway in the WTD-, Pae-, and Liq- regulated expression of NFs and CCR 5 in primary rat glial cells
WTD-, Pae-, and Liq- augmented NGF, BDNF, GDNF, and artemin gene expression was inhibited by adding potent inhibitors against phosphoinositide 3- kinase (PI3K) and protein kinase A (PKA) (LY 294002 and H 89 , respectively) in a dose- dependent manner. In contrast, neither LY 294002 nor H 89 influenced the WTD-, Pae-, and Liq- decreased levels of CCR 5 m RNA. Therefore, these results suggested that the PI3 K and PKA pathways were required for the augmentation of NGF, BDNF, GDNF, and artemin gene expression but not the decrease in CCR5 mRNA expression by WTD, Pae, and Liq in rat glial cells.
CONCLUSION
The present study demonstrated that WTD effectively attenuated SNL-induced NP without any side effects in a dose-dependent manner, and Pae and Liq were identified as major bioactive components with effective anti-NP effects in SNL rats. In addition, WTD, Pae, and Liq were found to transcriptionally enhance constitutive and IL-1β-increased NGF, BDNF, GDNF, and artemin production through PI3K- and PKA- dependent pathways in rat glial cells. In contrast, the expression of CCR5 was suppressed by WTD, Pae, and Liq through PI3K and PKA-independent pathways in IL- 1β-treated and untreated cells. Thus, these results 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).
この論文で使われている画像
