Accepted Manuscript Title: Protective effects of Garcinol against neuropathic painEvidence from in vivo and in vitro studies Authors: Yi-wei Wang, Xiang Zhang, Chun-long Chen, Qing-zhen Liu, Jia-wen Xu, Qing-qing Qian, Wei-yan Li, Yan-ning Qian PII: DOI: Reference:
S0304-3940(17)30225-2 http://dx.doi.org/doi:10.1016/j.neulet.2017.03.015 NSL 32703
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
24-1-2017 1-3-2017 11-3-2017
Please cite this article as: Yi-wei Wang, Xiang Zhang, Chun-long Chen, Qing-zhen Liu, Jia-wen Xu, Qing-qing Qian, Wei-yan Li, Yan-ning Qian, Protective effects of Garcinol against neuropathic pain- Evidence from in vivo and in vitro studies, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2017.03.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised Title: Protective effects of Garcinol against neuropathic pain- Evidence from in vivo and in vitro studies pain Author: Yi-wei Wanga,1, Xiang Zhanga,1, Chun-long Chenb, Qing-zhen Liu, Jiawen Xua, Qing-qing Qiana, Wei-yan Lib, *, Yan-ning Qiana,* a
Department of Anesthesiology, the First Affiliated Hospital of Nanjing Medical
University, Nanjing 210002, P.R. China b
Department of Anesthesiology, Nanjing General Hospital, Nanjing 210002, P. R.
China 1
These authors contributed equally to this work.
*Corresponding
author.
Pro.Yan-ning Qian, Department of Anesthesiology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, P. R. China. E-mail: [email protected] Pro. Wei-yan Li, Department of Anesthesiology, Jinling Hospital, No.305, East Zhongshan Road, Nanjing, P.R. China. E-mails: [email protected]
Revised Highlights •Garcinol can inhibit the lumbar fifth spinal nerve ligation (SNL)induced microglia activation in the spinal cord and ameliorated the neuropathic pain. •Garcinol can attenuate the neuroinflammation induced by SNL in the spinal cord. •
Garcinol
can
inhibit
Lipopolysaccharide(LPS)-simulated
inflammatory response in microglia in vitro. •The protective effects of Garcinol are probably associated with decreasing acetyl-p65 level in the NF-κB pathway, thus downregulating the expression of inflammatory mediators.
Abstract Neuroinflammatory processes have a vital role in the pathogenesis of neuropathic pain. Garcinol, harvested from Garcinia indica, is known to exert potent anti-inflammatory properties. Recent studies have indicated that Garcinol may inhibit activation of nuclear factor-κB (NF-κB) by inhibiting NF-κB/p65 acetylation. These findings prompted us to evaluate the protective effects of Garcinol in the lumbar fifth spinal nerve ligation (SNL)-induced rat model of neuropathic pain and Lipopolysaccharide(LPS)-stimulated primary cultured microglia. In the present study, we found that intrathecal administration of Garcinol significantly attenuated SNL-induced nociceptive behaviors. Garcinol suppressed microglial activation as well as the expression of interleukin (IL)1β, IL-6, inducible nitric oxide synthase (iNOS)/nitric oxide (NO), and cyclooxygenase-2 (COX-2)/ prostaglandin E2 (PGE2) in the spinal cord of SNL rats. It also reduced the nuclear translocation of NF-κB by decreasing acetyl-p65 protein expression. Similarly, in the in vitro study, Garcinol decreased the production of NO/iNOS, PGE2/COX-2, and proinflammatory cytokines in LPS-exposed microglia. Likewise, Garcinol inhibited the NF-κB signaling pathway by downregulating acetylp65 levels in LPS-challenged microglia. Our findings suggest that Garcinol may have protective effects against neuropathic pain that are associated with the inhibition of neuroinflammation in microglia. Therefore, Garcinol could be a promising agent in the treatment of neuropathic pain. Key words: Garcinol, Microglia, Neuropathic pain, Neuroinflammation, Acetylation, NF-κB
1. Introduction Neuropathic pain (NP) refers to “pain initiated or caused by a primary lesion or dysfunction in the nervous system” and possesses the characteristic of spontaneous pain, hyperalgesia, and allodynia [15]. Accumulating evidence supports the theory that neuroinflammation contributes to the development of NP by activating immune responses, in particular by microglia. Highly activated microglial cells express proinflammatory chemokines, such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS)/ nitric oxide (NO), and cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2). These proinflammatory chemokines powerfully modulate excitatory and inhibitory synaptic transmission, leading to central sensitization and enhanced neuropathic pain states [14]. The genes encoding proinflammatory proteins produced in response to microglial activation, such as the proinflammatory cytokines, PGE2/COX-2 and NO/iNOS, are under the transcriptional control of nuclear factor-κB (NF-κB). NF-κB triggers a selfperpetuating process resulting in progressive NP. There are many examples in the literature of small molecules that provide protection against NP by blocking the microglial NF-κB pathway [6, 13]. These reports confirm a central role for the NF-κB pathway in the early stages of NP development. In the nervous system, NF-κB is generally composed of a p50/p65 heterodimer. Normally, the NF-κB heterodimer forms a complex with the inhibitor of κB (IκB), which masks the nuclear localization signal of NF-κB, keeping it in an inactive state within the cytoplasm. The degradation of IκB triggers the release of NF-κB and
promotes its nuclear translocation and the modulation of NF-κB-dependent inflammatory gene expression [16]. Recently, some studies have suggested that degradation of IκB is not sufficient to promote maximal NF-κB activity [2, 19]. Rather, the NF-κB complex must undergo additional post-translational modifications, among which acetylation at the lysine (Lys) 310 residue of NF-κB/p65 plays an important role. P300 possesses a histone acetyltransferase (HAT) enzymatic activity that appears to play a major role in the acetylation of p65[11]. These findings suggest that inhibition of NF-κB acetylation might be a valuable strategy to suppress neuroinflammatory responses in NP, and raise the idea that a therapeutic application for p300 inhibitors could also be found in the neuroinflammation field. Plumbagin [18], anacardic acid [9]and curcumin [22] have been described as an inhibitor of p300, and they were reported to suppress acetylation of p65 and abrogated NF-κB activation induced by different inflammatory stimuli with a consequent reduction in NF-κB-dependent gene expression. Additionally, Zhu et al. [21] showed that curcumin alleviated NP by downregulating p300-regulated gene expression. These results suggest that inhibition of NF-κB acetylation might be a valuable strategy to suppress neuroinflammatory responses in NP. Finally, Garcinol (molecular mass: 602.39), a polyisoprenylated benzophenone derivative of the Garcinia indica fruit rind, has been claimed to be a strong inhibitor of p300. Garcinol has been shown to be a potent inhibitor of the NF-κB activation pathway that leads to the suppression of NF-κB-regulated gene products [5]. Recently, there has been an increased interest in the repurposing of Garcinol as a neuroprotective agent
through targeting iNOS and COX2 in astrocytes [3]. Since NF-κB regulates the expression of a wide variety of genes that are intimately involved in the process of neuroinflammation, inhibition of NF-κB by Garcinol may be an interesting prospect for reducing NP involving the NF-κB signaling pathway. In light of these studies, we evaluated Garcinol for its protective effects in the lumbar fifth spinal nerve ligation (SNL)-induced rat model of neuropathic pain and Lipopolysaccharide (LPS)-exposed primary cultured microglia. 2. Materials and methods Specific surgical and detailed methods are available in the supplementary material section. 3. Results 3.1 Garcinol alleviates thermal hyperalgesia and mechanical allodynia in SNL rats without obvious neurotoxicity Hematoxylin-eosin (HE) staining was used to examine the neurotoxicity of lumbosacral intrathecal Garcinol. The results of HE staining indicate that the spinal sections of all rats demonstrated a normal histological appearance (Figure 1A). Garcinol induced no obvious histopathological changes in the spinal cord. To assess the effects of Garcinol on neuropathic pain, the mechanical withdrawal threshold (MWT) and the thermal withdrawal latency (TWL) were measured on days 0, 1, 3, 5, 7, 9, 11 and 14 after SNL. As illustrated in Figures 1B and 1C, throughout the observation period, there was no obvious difference between the naïve group and sham group. A significant decrease in MWTs and TWLs was observed on post-SNL
days 1 to 14 in the SNL/vehicle group compared with the naïve group, indicating the development of mechanical allodynia and thermal hyperalgesia. When Garcinol was administered intrathecally, the MWTs and TWLs of SNL rats were significantly improved as compared to vehicle administration group. 3.2 Garcinol inhibits the production of IL-1β and IL-6 in the spinal cord after SNL IL-1β and IL-6 are pro-neuroinflammatory cytokines that have been reported to be upregulated in the spinal cord following SNL. Thus, as a first step, an ELISA was utilized to determine changes in IL-1β and IL-6 expression after SNL. The results showed that, compared with that in the naïve group, the levels of IL-1β and IL-6 were notably raised in the SNL group. The elevated expression levels of these cytokines were remarkably attenuated by Garcinol (Figures 2A and 2B). 3.3 Garcinol inhibits NO, iNOS, PGE2 and COX-2 expression in the spinal cord after SNL To obtain further insights into the mechanisms of Garcinol-induced analgesia properties, the levels of pronociceptive factors (NO, iNOS, PGE2 and COX-2) were measured. As expected, SNL resulted in a marked up-regulation of the levels of NO, iNOS, PGE2 and COX-2, and the application of Garcinol dramatically reduced this up-regulation (Figures 3C,3D,3E and 3F). 3.4 Garcinol inhibits microglial activation in the spinal cord after SNL Microglia are the main source of pro-neuroinflammatory mediators in the spinal cord after peripheral tissue damage or nerve injury [14]. Therefore, we predicted that Garcinol could inhibit microglial activation. In order to explore the effects of Garcinol
on microglia activation, immunostaining was used to detect Iba1, a marker for microglia. The result shows the Iba-1-positive cells were more strongly elevated in the SNL group those in the naïve group. Garcinol led to a strong downregulation in Iba-1 immunostaining throughout the spinal cord (Figure 3G). 3.5 Garcinol suppresses the nuclear translocation of NF-κB/p65 and its acetylation in the spinal cord after SNL Increased p65 acetylation is associated with increased activation of NF-κB, leading to increased production of proinflammatory proteins. Garcinol was found to decrease acetylated p65 protein expression level in the NF-κB pathway. Consequently, we next studied the effects of Garcinol on the acetylation and translocation of NF-κB/p65. We first measured the expression of HAT p300, compared with that in the naïve group, elevated immunoreactivity of p300 throughout the spinal cord was demonstrated in the SNL group. P300 immunoreactivity was moderately decreased in the Garcinol-treated SNL rats (Figure 3H). As shown in Figure 3I, the protein expression of acetyl-p65 at Lys310 was obviously increased in the SNL group, while it was suppressed in the Garcinol group. We demonstrated a characteristic spatial mapping of nuclear NF-κB expression in the spinal cord using Western blot. The protein level of nuclear NFκB/p65 in the spinal cord was remarkably higher in the SNL group than in the naïve group, and Garcinol strongly inhibited this elevation (Figure 3J). We also performed double immunofluorescent staining for nuclear NF-κB/p65 and Iba-1. SNL increased the number of nuclear NF-κB/p65 and Iba-1 co-expression cells, which could in turn be significantly inhibited by Garcinol (Figure 3K).
3.6 Garcinol attenuates cytokine production in LPS-treated primary cultured microglial cells without affecting cell viability The cell counting kit-8 assay was performed to evaluate the cytotoxicity of several concentrations of Garcinol and/or LPS toward primary rat microglia. Our results indicate that Garcinol (at a concentration of
S0304-3940(17)30225-2 http://dx.doi.org/doi:10.1016/j.neulet.2017.03.015 NSL 32703
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
24-1-2017 1-3-2017 11-3-2017
Please cite this article as: Yi-wei Wang, Xiang Zhang, Chun-long Chen, Qing-zhen Liu, Jia-wen Xu, Qing-qing Qian, Wei-yan Li, Yan-ning Qian, Protective effects of Garcinol against neuropathic pain- Evidence from in vivo and in vitro studies, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2017.03.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised Title: Protective effects of Garcinol against neuropathic pain- Evidence from in vivo and in vitro studies pain Author: Yi-wei Wanga,1, Xiang Zhanga,1, Chun-long Chenb, Qing-zhen Liu, Jiawen Xua, Qing-qing Qiana, Wei-yan Lib, *, Yan-ning Qiana,* a
Department of Anesthesiology, the First Affiliated Hospital of Nanjing Medical
University, Nanjing 210002, P.R. China b
Department of Anesthesiology, Nanjing General Hospital, Nanjing 210002, P. R.
China 1
These authors contributed equally to this work.
*Corresponding
author.
Pro.Yan-ning Qian, Department of Anesthesiology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, P. R. China. E-mail: [email protected] Pro. Wei-yan Li, Department of Anesthesiology, Jinling Hospital, No.305, East Zhongshan Road, Nanjing, P.R. China. E-mails: [email protected]
Revised Highlights •Garcinol can inhibit the lumbar fifth spinal nerve ligation (SNL)induced microglia activation in the spinal cord and ameliorated the neuropathic pain. •Garcinol can attenuate the neuroinflammation induced by SNL in the spinal cord. •
Garcinol
can
inhibit
Lipopolysaccharide(LPS)-simulated
inflammatory response in microglia in vitro. •The protective effects of Garcinol are probably associated with decreasing acetyl-p65 level in the NF-κB pathway, thus downregulating the expression of inflammatory mediators.
Abstract Neuroinflammatory processes have a vital role in the pathogenesis of neuropathic pain. Garcinol, harvested from Garcinia indica, is known to exert potent anti-inflammatory properties. Recent studies have indicated that Garcinol may inhibit activation of nuclear factor-κB (NF-κB) by inhibiting NF-κB/p65 acetylation. These findings prompted us to evaluate the protective effects of Garcinol in the lumbar fifth spinal nerve ligation (SNL)-induced rat model of neuropathic pain and Lipopolysaccharide(LPS)-stimulated primary cultured microglia. In the present study, we found that intrathecal administration of Garcinol significantly attenuated SNL-induced nociceptive behaviors. Garcinol suppressed microglial activation as well as the expression of interleukin (IL)1β, IL-6, inducible nitric oxide synthase (iNOS)/nitric oxide (NO), and cyclooxygenase-2 (COX-2)/ prostaglandin E2 (PGE2) in the spinal cord of SNL rats. It also reduced the nuclear translocation of NF-κB by decreasing acetyl-p65 protein expression. Similarly, in the in vitro study, Garcinol decreased the production of NO/iNOS, PGE2/COX-2, and proinflammatory cytokines in LPS-exposed microglia. Likewise, Garcinol inhibited the NF-κB signaling pathway by downregulating acetylp65 levels in LPS-challenged microglia. Our findings suggest that Garcinol may have protective effects against neuropathic pain that are associated with the inhibition of neuroinflammation in microglia. Therefore, Garcinol could be a promising agent in the treatment of neuropathic pain. Key words: Garcinol, Microglia, Neuropathic pain, Neuroinflammation, Acetylation, NF-κB
1. Introduction Neuropathic pain (NP) refers to “pain initiated or caused by a primary lesion or dysfunction in the nervous system” and possesses the characteristic of spontaneous pain, hyperalgesia, and allodynia [15]. Accumulating evidence supports the theory that neuroinflammation contributes to the development of NP by activating immune responses, in particular by microglia. Highly activated microglial cells express proinflammatory chemokines, such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS)/ nitric oxide (NO), and cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2). These proinflammatory chemokines powerfully modulate excitatory and inhibitory synaptic transmission, leading to central sensitization and enhanced neuropathic pain states [14]. The genes encoding proinflammatory proteins produced in response to microglial activation, such as the proinflammatory cytokines, PGE2/COX-2 and NO/iNOS, are under the transcriptional control of nuclear factor-κB (NF-κB). NF-κB triggers a selfperpetuating process resulting in progressive NP. There are many examples in the literature of small molecules that provide protection against NP by blocking the microglial NF-κB pathway [6, 13]. These reports confirm a central role for the NF-κB pathway in the early stages of NP development. In the nervous system, NF-κB is generally composed of a p50/p65 heterodimer. Normally, the NF-κB heterodimer forms a complex with the inhibitor of κB (IκB), which masks the nuclear localization signal of NF-κB, keeping it in an inactive state within the cytoplasm. The degradation of IκB triggers the release of NF-κB and
promotes its nuclear translocation and the modulation of NF-κB-dependent inflammatory gene expression [16]. Recently, some studies have suggested that degradation of IκB is not sufficient to promote maximal NF-κB activity [2, 19]. Rather, the NF-κB complex must undergo additional post-translational modifications, among which acetylation at the lysine (Lys) 310 residue of NF-κB/p65 plays an important role. P300 possesses a histone acetyltransferase (HAT) enzymatic activity that appears to play a major role in the acetylation of p65[11]. These findings suggest that inhibition of NF-κB acetylation might be a valuable strategy to suppress neuroinflammatory responses in NP, and raise the idea that a therapeutic application for p300 inhibitors could also be found in the neuroinflammation field. Plumbagin [18], anacardic acid [9]and curcumin [22] have been described as an inhibitor of p300, and they were reported to suppress acetylation of p65 and abrogated NF-κB activation induced by different inflammatory stimuli with a consequent reduction in NF-κB-dependent gene expression. Additionally, Zhu et al. [21] showed that curcumin alleviated NP by downregulating p300-regulated gene expression. These results suggest that inhibition of NF-κB acetylation might be a valuable strategy to suppress neuroinflammatory responses in NP. Finally, Garcinol (molecular mass: 602.39), a polyisoprenylated benzophenone derivative of the Garcinia indica fruit rind, has been claimed to be a strong inhibitor of p300. Garcinol has been shown to be a potent inhibitor of the NF-κB activation pathway that leads to the suppression of NF-κB-regulated gene products [5]. Recently, there has been an increased interest in the repurposing of Garcinol as a neuroprotective agent
through targeting iNOS and COX2 in astrocytes [3]. Since NF-κB regulates the expression of a wide variety of genes that are intimately involved in the process of neuroinflammation, inhibition of NF-κB by Garcinol may be an interesting prospect for reducing NP involving the NF-κB signaling pathway. In light of these studies, we evaluated Garcinol for its protective effects in the lumbar fifth spinal nerve ligation (SNL)-induced rat model of neuropathic pain and Lipopolysaccharide (LPS)-exposed primary cultured microglia. 2. Materials and methods Specific surgical and detailed methods are available in the supplementary material section. 3. Results 3.1 Garcinol alleviates thermal hyperalgesia and mechanical allodynia in SNL rats without obvious neurotoxicity Hematoxylin-eosin (HE) staining was used to examine the neurotoxicity of lumbosacral intrathecal Garcinol. The results of HE staining indicate that the spinal sections of all rats demonstrated a normal histological appearance (Figure 1A). Garcinol induced no obvious histopathological changes in the spinal cord. To assess the effects of Garcinol on neuropathic pain, the mechanical withdrawal threshold (MWT) and the thermal withdrawal latency (TWL) were measured on days 0, 1, 3, 5, 7, 9, 11 and 14 after SNL. As illustrated in Figures 1B and 1C, throughout the observation period, there was no obvious difference between the naïve group and sham group. A significant decrease in MWTs and TWLs was observed on post-SNL
days 1 to 14 in the SNL/vehicle group compared with the naïve group, indicating the development of mechanical allodynia and thermal hyperalgesia. When Garcinol was administered intrathecally, the MWTs and TWLs of SNL rats were significantly improved as compared to vehicle administration group. 3.2 Garcinol inhibits the production of IL-1β and IL-6 in the spinal cord after SNL IL-1β and IL-6 are pro-neuroinflammatory cytokines that have been reported to be upregulated in the spinal cord following SNL. Thus, as a first step, an ELISA was utilized to determine changes in IL-1β and IL-6 expression after SNL. The results showed that, compared with that in the naïve group, the levels of IL-1β and IL-6 were notably raised in the SNL group. The elevated expression levels of these cytokines were remarkably attenuated by Garcinol (Figures 2A and 2B). 3.3 Garcinol inhibits NO, iNOS, PGE2 and COX-2 expression in the spinal cord after SNL To obtain further insights into the mechanisms of Garcinol-induced analgesia properties, the levels of pronociceptive factors (NO, iNOS, PGE2 and COX-2) were measured. As expected, SNL resulted in a marked up-regulation of the levels of NO, iNOS, PGE2 and COX-2, and the application of Garcinol dramatically reduced this up-regulation (Figures 3C,3D,3E and 3F). 3.4 Garcinol inhibits microglial activation in the spinal cord after SNL Microglia are the main source of pro-neuroinflammatory mediators in the spinal cord after peripheral tissue damage or nerve injury [14]. Therefore, we predicted that Garcinol could inhibit microglial activation. In order to explore the effects of Garcinol
on microglia activation, immunostaining was used to detect Iba1, a marker for microglia. The result shows the Iba-1-positive cells were more strongly elevated in the SNL group those in the naïve group. Garcinol led to a strong downregulation in Iba-1 immunostaining throughout the spinal cord (Figure 3G). 3.5 Garcinol suppresses the nuclear translocation of NF-κB/p65 and its acetylation in the spinal cord after SNL Increased p65 acetylation is associated with increased activation of NF-κB, leading to increased production of proinflammatory proteins. Garcinol was found to decrease acetylated p65 protein expression level in the NF-κB pathway. Consequently, we next studied the effects of Garcinol on the acetylation and translocation of NF-κB/p65. We first measured the expression of HAT p300, compared with that in the naïve group, elevated immunoreactivity of p300 throughout the spinal cord was demonstrated in the SNL group. P300 immunoreactivity was moderately decreased in the Garcinol-treated SNL rats (Figure 3H). As shown in Figure 3I, the protein expression of acetyl-p65 at Lys310 was obviously increased in the SNL group, while it was suppressed in the Garcinol group. We demonstrated a characteristic spatial mapping of nuclear NF-κB expression in the spinal cord using Western blot. The protein level of nuclear NFκB/p65 in the spinal cord was remarkably higher in the SNL group than in the naïve group, and Garcinol strongly inhibited this elevation (Figure 3J). We also performed double immunofluorescent staining for nuclear NF-κB/p65 and Iba-1. SNL increased the number of nuclear NF-κB/p65 and Iba-1 co-expression cells, which could in turn be significantly inhibited by Garcinol (Figure 3K).
3.6 Garcinol attenuates cytokine production in LPS-treated primary cultured microglial cells without affecting cell viability The cell counting kit-8 assay was performed to evaluate the cytotoxicity of several concentrations of Garcinol and/or LPS toward primary rat microglia. Our results indicate that Garcinol (at a concentration of
