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Neuroprotective effect of 6-paradol in focal cerebral ischemia involves the attenuation of neuroinflammatory responses in activated microglia.

Gaire BP, Kwon OW, Park SH, Chun KH, Kim SY, Shin DY, Choi JW - PLoS ONE (2015)

Bottom Line: Through the initial screening for anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated BV2 microglia, 6-paradol was chosen to be the most effective compound without cytotoxicity.Pretreatment with 6-paradol reduced neuroinflammatory responses in LPS-stimulated BV2 microglia by a concentration-dependent manner, which includes reduced NO production by inhibiting iNOS upregulation and lowered secretion of proinflammatory cytokines (IL-6 and TNF-α).Furthermore, as observed in cultured microglia, 6-paradol administration markedly reduced neuroinflammation in M/R-challenged brains by attenuating microglial activation and reducing the number of cells expressing iNOS and TNF-α, both of which are known to be produced in microglia following M/R challenge.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon, Republic of Korea.

ABSTRACT
Paradols are non-pungent and biotransformed metabolites of shogaols and reduce inflammatory responses as well as oxidative stress as shogaols. Recently, shogaol has been noted to possess therapeutic potential against several central nervous system (CNS) disorders, including cerebral ischemia, by reducing neuroinflammation in microglia. Therefore, paradol could be used to improve neuroinflammation-associated CNS disorders. Here, we synthesized paradol derivatives (2- to 10-paradols). Through the initial screening for anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated BV2 microglia, 6-paradol was chosen to be the most effective compound without cytotoxicity. Pretreatment with 6-paradol reduced neuroinflammatory responses in LPS-stimulated BV2 microglia by a concentration-dependent manner, which includes reduced NO production by inhibiting iNOS upregulation and lowered secretion of proinflammatory cytokines (IL-6 and TNF-α). To pursue whether the beneficial in vitro effects of 6-paradol leads towards in vivo therapeutic effects on transient focal cerebral ischemia characterized by neuroinflammation, we employed middle cerebral artery occlusion (MCAO)/reperfusion (M/R). Administration of 6-paradol immediately after reperfusion significantly reduced brain damage in M/R-challenged mice as assessed by brain infarction, neurological deficit, and neural cell survival and death. Furthermore, as observed in cultured microglia, 6-paradol administration markedly reduced neuroinflammation in M/R-challenged brains by attenuating microglial activation and reducing the number of cells expressing iNOS and TNF-α, both of which are known to be produced in microglia following M/R challenge. Collectively, this study provides evidences that 6-paradol effectively protects brain after cerebral ischemia, likely by attenuating neuroinflammation in microglia, suggesting it as a potential therapeutic agent to treat cerebral ischemia.

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Administration of 6-paradol reduces microglial activation in the post-ischemic brain 3 days after M/R challenge.Mice were challenged with M/R and 6-paradol (10 mg/kg, p.o.) was administered immediately after reperfusion. Microglial activation was assessed 3 days after reperfusion by immunohistochemistry using Iba1 antibody. (A) Representative images of Iba1-immunopositive cells in periischemic (‘P’) and ischemic core (‘C’) regions. Scale bars, 200 μm (top panels) and 50 μm (middle and bottom panels). (B) Quantification of Iba1-immunopositive cells in both regions. n = 6~7 per group. (C) Quantification of morphological changes of Iba1-positive cells in ischemic core regions (from ‘ramified’ into ‘amoeboid’ cells). ***p<0.001, versus vehicle-administered M/R mice (M/R+veh).
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pone.0120203.g006: Administration of 6-paradol reduces microglial activation in the post-ischemic brain 3 days after M/R challenge.Mice were challenged with M/R and 6-paradol (10 mg/kg, p.o.) was administered immediately after reperfusion. Microglial activation was assessed 3 days after reperfusion by immunohistochemistry using Iba1 antibody. (A) Representative images of Iba1-immunopositive cells in periischemic (‘P’) and ischemic core (‘C’) regions. Scale bars, 200 μm (top panels) and 50 μm (middle and bottom panels). (B) Quantification of Iba1-immunopositive cells in both regions. n = 6~7 per group. (C) Quantification of morphological changes of Iba1-positive cells in ischemic core regions (from ‘ramified’ into ‘amoeboid’ cells). ***p<0.001, versus vehicle-administered M/R mice (M/R+veh).

Mentions: To determine whether the neuroprotective effect of 6-paradol is associated with its anti-inflammatory activities in vitro, we assessed microglial activation using immunohistochemical analyses. At first, we examined effects of 6-paradol on well-characterized microglial responses in the different regions 1 and 3 days after M/R as assessed by the increased number of Iba1-immunopositive cells [27,28]. As reported, the number of Iba1-postive cells was markedly increased in both periischemic and ischemic core regions 1 (Fig. 5) and 3 days (Fig. 6A, B) after M/R challenge. Morphological changes of Iba1-positive cells were also obvious in core regions 3 days after M/R challenge (ramified → amoeboid) (Fig. 6A, C). Administration of 6-paradol (10 mg/kg) clearly reduced the number of Iba1-positive cells 1 and 3 days after the challenge (Fig. 5 and Fig. 6A, B). Moreover, 6-paradol dramatically reduced the number of Iba1-postive cells in periischemic regions even after 3 days following M/R challenge (Fig. 6A, B). In ischemic core regions, 6-paradol seemed not to reduce the number of Iba1-positive cells at 3 days following M/R challenge (Fig. 6A, B). But, interestingly, 6-paradol dramatically reversed microglial morphology into ‘ramified’ in ischemic core regions (Fig. 6A, C) despite of no effect on the number of cells bearing Iba1 (Fig. 6A, B). These effects of 6-paradol on microglial responses were further examined by assessing microglial proliferation (Fig. 7 and S4 Fig.). The number of BrdU-positive cells was markedly increased in brains 3 days after M/R and most of BrdU-positive cells also expressed Iba1 (Fig. 7 and S4 Fig.), demonstrating microglial proliferation in the post-ischemic brains. Administration of 6-paradol significantly reduced the number of Iba1/BrdU-double-positive cells (Fig. 7 and S4 Fig.). These data demonstrated that 6-paradol effectively attenuated microglial responses in the post-ischemic brains.


Neuroprotective effect of 6-paradol in focal cerebral ischemia involves the attenuation of neuroinflammatory responses in activated microglia.

Gaire BP, Kwon OW, Park SH, Chun KH, Kim SY, Shin DY, Choi JW - PLoS ONE (2015)

Administration of 6-paradol reduces microglial activation in the post-ischemic brain 3 days after M/R challenge.Mice were challenged with M/R and 6-paradol (10 mg/kg, p.o.) was administered immediately after reperfusion. Microglial activation was assessed 3 days after reperfusion by immunohistochemistry using Iba1 antibody. (A) Representative images of Iba1-immunopositive cells in periischemic (‘P’) and ischemic core (‘C’) regions. Scale bars, 200 μm (top panels) and 50 μm (middle and bottom panels). (B) Quantification of Iba1-immunopositive cells in both regions. n = 6~7 per group. (C) Quantification of morphological changes of Iba1-positive cells in ischemic core regions (from ‘ramified’ into ‘amoeboid’ cells). ***p<0.001, versus vehicle-administered M/R mice (M/R+veh).
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pone.0120203.g006: Administration of 6-paradol reduces microglial activation in the post-ischemic brain 3 days after M/R challenge.Mice were challenged with M/R and 6-paradol (10 mg/kg, p.o.) was administered immediately after reperfusion. Microglial activation was assessed 3 days after reperfusion by immunohistochemistry using Iba1 antibody. (A) Representative images of Iba1-immunopositive cells in periischemic (‘P’) and ischemic core (‘C’) regions. Scale bars, 200 μm (top panels) and 50 μm (middle and bottom panels). (B) Quantification of Iba1-immunopositive cells in both regions. n = 6~7 per group. (C) Quantification of morphological changes of Iba1-positive cells in ischemic core regions (from ‘ramified’ into ‘amoeboid’ cells). ***p<0.001, versus vehicle-administered M/R mice (M/R+veh).
Mentions: To determine whether the neuroprotective effect of 6-paradol is associated with its anti-inflammatory activities in vitro, we assessed microglial activation using immunohistochemical analyses. At first, we examined effects of 6-paradol on well-characterized microglial responses in the different regions 1 and 3 days after M/R as assessed by the increased number of Iba1-immunopositive cells [27,28]. As reported, the number of Iba1-postive cells was markedly increased in both periischemic and ischemic core regions 1 (Fig. 5) and 3 days (Fig. 6A, B) after M/R challenge. Morphological changes of Iba1-positive cells were also obvious in core regions 3 days after M/R challenge (ramified → amoeboid) (Fig. 6A, C). Administration of 6-paradol (10 mg/kg) clearly reduced the number of Iba1-positive cells 1 and 3 days after the challenge (Fig. 5 and Fig. 6A, B). Moreover, 6-paradol dramatically reduced the number of Iba1-postive cells in periischemic regions even after 3 days following M/R challenge (Fig. 6A, B). In ischemic core regions, 6-paradol seemed not to reduce the number of Iba1-positive cells at 3 days following M/R challenge (Fig. 6A, B). But, interestingly, 6-paradol dramatically reversed microglial morphology into ‘ramified’ in ischemic core regions (Fig. 6A, C) despite of no effect on the number of cells bearing Iba1 (Fig. 6A, B). These effects of 6-paradol on microglial responses were further examined by assessing microglial proliferation (Fig. 7 and S4 Fig.). The number of BrdU-positive cells was markedly increased in brains 3 days after M/R and most of BrdU-positive cells also expressed Iba1 (Fig. 7 and S4 Fig.), demonstrating microglial proliferation in the post-ischemic brains. Administration of 6-paradol significantly reduced the number of Iba1/BrdU-double-positive cells (Fig. 7 and S4 Fig.). These data demonstrated that 6-paradol effectively attenuated microglial responses in the post-ischemic brains.

Bottom Line: Through the initial screening for anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated BV2 microglia, 6-paradol was chosen to be the most effective compound without cytotoxicity.Pretreatment with 6-paradol reduced neuroinflammatory responses in LPS-stimulated BV2 microglia by a concentration-dependent manner, which includes reduced NO production by inhibiting iNOS upregulation and lowered secretion of proinflammatory cytokines (IL-6 and TNF-α).Furthermore, as observed in cultured microglia, 6-paradol administration markedly reduced neuroinflammation in M/R-challenged brains by attenuating microglial activation and reducing the number of cells expressing iNOS and TNF-α, both of which are known to be produced in microglia following M/R challenge.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon, Republic of Korea.

ABSTRACT
Paradols are non-pungent and biotransformed metabolites of shogaols and reduce inflammatory responses as well as oxidative stress as shogaols. Recently, shogaol has been noted to possess therapeutic potential against several central nervous system (CNS) disorders, including cerebral ischemia, by reducing neuroinflammation in microglia. Therefore, paradol could be used to improve neuroinflammation-associated CNS disorders. Here, we synthesized paradol derivatives (2- to 10-paradols). Through the initial screening for anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated BV2 microglia, 6-paradol was chosen to be the most effective compound without cytotoxicity. Pretreatment with 6-paradol reduced neuroinflammatory responses in LPS-stimulated BV2 microglia by a concentration-dependent manner, which includes reduced NO production by inhibiting iNOS upregulation and lowered secretion of proinflammatory cytokines (IL-6 and TNF-α). To pursue whether the beneficial in vitro effects of 6-paradol leads towards in vivo therapeutic effects on transient focal cerebral ischemia characterized by neuroinflammation, we employed middle cerebral artery occlusion (MCAO)/reperfusion (M/R). Administration of 6-paradol immediately after reperfusion significantly reduced brain damage in M/R-challenged mice as assessed by brain infarction, neurological deficit, and neural cell survival and death. Furthermore, as observed in cultured microglia, 6-paradol administration markedly reduced neuroinflammation in M/R-challenged brains by attenuating microglial activation and reducing the number of cells expressing iNOS and TNF-α, both of which are known to be produced in microglia following M/R challenge. Collectively, this study provides evidences that 6-paradol effectively protects brain after cerebral ischemia, likely by attenuating neuroinflammation in microglia, suggesting it as a potential therapeutic agent to treat cerebral ischemia.

Show MeSH
Related in: MedlinePlus