Neurochem Res (2015) 40:1655–1660 DOI 10.1007/s11064-015-1646-6
ORIGINAL PAPER
Protocatechuic Acid Inhibits Inflammatory Responses in LPSStimulated BV2 Microglia via NF-jB and MAPKs Signaling Pathways Huan-yu Wang1 • Hong Wang1 • Jin-huan Wang1 • Qiong Wang1 Quan-feng Ma1 • Yi-yang Chen1
•
Received: 9 April 2015 / Revised: 11 June 2015 / Accepted: 19 June 2015 / Published online: 2 July 2015 Ó Springer Science+Business Media New York 2015
Abstract Protocatechuic acid (PA), a major metabolite of anthocyanins, has been reported to possess antioxidant and anti-inflammatory activities. However, the effects of PA on LPS-induced inflammatory responses in microglia have not been reported. The aim of this study was to investigate the anti-inflammatory effects and molecular mechanisms of PA on LPS-stimulated BV2 microglia. The production of inflammatory mediators TNF-a, IL-6, IL-1b, and PGE2 were detected by ELISA. TLR4, NF-jB and MAPKs activation were detected by western blotting. Our results demonstrated that PA dose-dependently inhibited LPS-induced TNF-a, IL6, IL-1b, and PGE2 production. In addition, PA suppressed LPS-induced TLR4 expression, NF-jB and MAPKs activation, which resulted in the inhibition of inflammatory mediators. In conclusion, these results suggested that PA exhibited anti-inflammatory effects on LPS-stimulated BV2 microglia and the mechanisms were involved in the inhibition of TLR4mediated NF-jB and MAPKs signaling pathways. Keywords Protocatechuic acid LPS Inflammatory mediators NF-jB MAPKs
Introduction Microglia are the resident immune cells in the brain which play an important role in host defense and tissue repair in brain [1]. Microglia can be activated by various factors
such as infection and injury [2]. LPS, the main component of Gram-negative bacteria, has been reported to be one of the most potent stimuli for microglia activation [3–5]. Stimulating of microglia by LPS induces NF-jB and MAPKs (JNK, P38, ERK) activation, which regulates the release of inflammatory mediators TNF-a, IL-6, PGE2, and IL-1b [6, 7]. Overproduction of these inflammatory cytokines induce several inflammatory and neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [8]. Previous studies showed that reduction of these inflammatory mediators could attenuate the severity of these diseases [9]. Protocatechuic acid (PA), a major metabolite of anthocyanins, has been found to possess antioxidant, anti-inflammatory and anti-tumor activities [10, 11]. PA was found to inhibit TNF-a, IL-1b, and COX-2 expression in LPS-stimulated RAW264.7 cells [12] and inhibit human dendritic cell function activation in vitro [13]. Moreover, previous reports showed that PA attenuated LPS-induced acute lung injury and protected against sepsis in mice [14, 15]. In addition, PA has been reported to inhibit LPS-induced rat hepatic damage [16]. However, the effects of PA on LPS-stimulated BV2 microglia remain unclear. The aim of this study was to investigate the anti-inflammatory effects and mechanisms of PA on LPS-stimulated BV2 microglia.
Materials and Methods & Hong Wang [email protected] 1
Tianjin Cerebral Vascular and Neural Degenerative Disease Key Laboratory, Tianjin Huanhu Hospital, Tianjin 300060, China
Materials LPS (Escherichia coli O55:B5) and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO, USA). Protocatechuic
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acid was purchased from National Institutes for Food and Drug Control (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits of PGE2, TNF-a, IL-6 and IL-1b were purchased from R&D Systems (Minneapolis, MN, USA). Rabbit anti-human TLR4, NF-jB p65, IjBa, ERK, JNK, p38, and b-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). All other reagents were of analytical grade.
protein were separated on 10 % SDS–polyacrylamide gel. The proteins were transferred onto PVDF membranes and blocked in 5 % skim milk for 1 h at room temperature. Then the membrane was incubated with primary antibodies overnight at 4 °C. The membrane was incubated with a horseradish peroxidase-(HRP)-conjugated secondary antibody for 2 h at room temperature.
Cell Culture
All values are expressed as mean ± SEM. Differences between mean values of normally distributed data were analyzed using one-way ANOVA (Dunnett’s t test) and two-tailed Student’s t test. Statistical significance was accepted P \ 0.05 or P \ 0.01.
Murine BV2 microglia cells were purchased from the American Type Culture Collection, (Manassas, VA, USA). Cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 5 % FBS, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C in a humidified incubator under 5 % CO2.
Statistical Analysis
Results
MTT Assay
Effects of PA on Cell Viability
The effect of PA on cell viability was determined by MTT assay. Briefly, BV2 microglia cells were seeded in 96 well plates and treated with various concentrations of PA for 24 h. 20 lL MTT (5 mg/mL) was added to each well, and the cells were further incubated for an additional 4 h. The resulting formazan crystals were dissolved in DMSO (150 lL/well). Absorbance was determined at 540 nm.
The effects of PA on the viability of BV2 microglia cells were detected by MTT assay. BV2 microglia were cultured with the indicated concentrations of PA for 24 h in the absence or presence of LPS. As shown in Fig. 1, the result showed that PA (5, 10, 20 lM) did not affect the cell viabilities of BV2 microglia cells. Hence, PA (5, 10, 20 lM) exerted no significant cytotoxicity on BV-2 microglial cells.
Cytokine Assays
PA Inhibits LPS-Induced TNF-a, IL-1ß, IL-6, and PGE2 Production
BV2 cells were pretreated with PA 1 h before LPS administration. 24 h later, the culture supernatant were collected. The levels of TNF-a, IL-6, and IL-1b in the culture supernatant were determined using a commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.
To investigate the anti-inflammatory effects of PA, the effects of PA on TNF-a, IL-1ß, IL-6 and PGE2 production
Measurement of PGE2 BV2 cells were pretreated with the indicated concentrations of PA 1 h prior to LPS stimulation for 24 h. PGE2 in the culture medium was measured by ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Western Blot Analysis BV2 cells were pretreated with the indicated concentrations of PA 1 h prior to LPS stimulation for 30 min. Total proteins were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo). Protein concentrations were determined by BCA protein assay kit. Equal amounts of
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Fig. 1 Effects of PA on the cell viability of BV2 microglial cells. Cells were cultured with different concentrations of PA (5, 10, 20 lM) in the absence or presence of 0.5 lg/mL LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the mean ± SEM of three independent experiments
Neurochem Res (2015) 40:1655–1660
were analyzed at 24 h after LPS challenge by ELISA. As shown in Fig. 2, inflammatory mediators TNF-a, IL-1ß, IL-6 and PGE2 production increased significantly after LPS challenge. However, PA inhibited TNF-a, IL-1ß, IL-6 and PGE2 production induced by LPS in a dose-dependent manner (P \ 0.01).
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cytokines gene expression [18]. To further elucidate the mechanisms of PA, the effects of PA on MAPKs activation, which are upstream signaling molecules in inflammatory responses, were detected in this study. The results showed that pretreatment with PA inhibited LPS-induced MAPKs (p38, JNK and ERK) activation in a dose-dependent manner (Fig. 4).
PA Inhibits LPS-Induced TLR4 Expression and NF-jB Activation
Discussion To further elucidate the mechanisms of PA, the effects of PA on LPS-induced TLR4 expression and NF-jB activation were detected Western blot analysis. As shown in Fig. 3, LPS induced NF-jB P65 and IjBa phosphorylation. PA inhibited LPS-induced NF-jB P65 and IjBa phosphorylation in a dose-dependent manner (Fig. 3). In addition, the results showed that PA significantly inhibited LPS-induced TLR4 expression (Fig. 3).
It is well known that MAPKs (p38, JNK and ERK) play crucial roles in the inflammatory response [17]. MAPKs is an important pathway in mediating pro-inflammatory
Microglia constitute a unique population of immune cells in the CNS and play critical roles in host innate immune response [19]. Activated microglia produce numerous proinflammatory cytokines, which are thought to play important roles in the pathogenesis of neurodegenerative diseases [20]. The pharmacologic regulation of microglial activity is useful in the treatment of neurodegenerative diseases. PA, a major metabolite of anthocyanins, has been reported to have anti-inflammatory effect. In this study, the anti-inflammatory effects of PA on LPS-stimulated BV2 microglial cells were detected. The results showed that PA inhibited LPS-induced microglia activation by suppressing inflammatory cytokines production.
Fig. 2 Effects of PA on LPS-induced TNF-a, IL-1ß, IL-6 and PGE2 production. Cells were treated with PA (5, 10, 20 lM) for 1 h and stimulated with LPS (0.5 lg/mL) for 24 h. The production of TNF-a, IL-1ß, IL-6 and PGE2 were measured by ELISA. The data presented
are the mean ± SEM of three independent experiments and differences between mean values were assessed by Students’s t test. # P \ 0.05 versus control group; *P \ 0.05, **P \ 0.01 versus LPS group
PA Inhibits LPS-Induced MAPKs Activation
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Fig. 3 PA inhibits LPS-induced TLR4 expression and NF-jB activation. Cells were treated with PA (5, 10, 20 lM) for 1 h and stimulated with LPS (0.5 lg/mL) for 30 min. Protein samples were analyzed by western blotting. The values presented are the
mean ± SEM of three independent experiments and differences between mean values were assessed by Students’s t test. #P \ 0.05 versus control group; *P \ 0.05, **P \ 0.01 versus LPS group
Microglia can be activated by various factors. LPS is one of the most important factors that induces microglia activation [21]. Stimulating of microglia by LPS could induce the production of inflammatory mediators including TNF-a, IL-1ß, IL-6 and PGE2 [22]. Meanwhile, an increasing number of studies have shown that inflammatory mediators TNF-a, IL-1ß, IL-6 and PGE2 were involved in the pathogenesis of neurodegenerative disorders, such as Parkinson’s disease (PD) and Alzheimer’s disease [6, 21]. Elevated TNF-a, IL-1ß, IL-6 and PGE2 levels were observed in patients with neurodegenerative disorders [23]. Previous studies showed that reduction of these inflammatory mediators could attenuate the severity of these diseases [24, 25]. Thus, agents with the ability to inhibit inflammatory cytokines expression are potentially
beneficial in the treatment of neurodegenerative diseases. In this study, the effects of PA on inflammatory mediators production were detected. The results showed that PA inhibited LPS-induced inflammatory mediators TNF-a, IL-1ß, IL-6 and PGE2 in a dose-dependent manner. NF-jB has been reported to play an important role in the regulation of inflammatory cytokines production [26, 27]. Normally, NF-jB is sequestered in the cytoplasm and combined with its inhibitors IjB. Once stimulating by LPS, NF-jB p65 translocates from the cytoplasm to the nucleus [28]. In the nucleus, NF-jB induces the transcription of inflammatory mediators such as TNF-a, IL-1ß, IL-6 and PGE2 [29–31]. MAPKs are a group of signaling molecules that also play critical roles in regulation of inflammatory mediators production [32–34]. To investigate the
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Fig. 4 PA inhibits LPS-induced MAPKs activation. Cells were treated with PA (5, 10, 20 lM) for 1 h and stimulated with LPS (0.5 lg/mL) for 30 min. Protein samples were analyzed by western blotting. The values presented are the mean ± SEM of three
independent experiments and differences between mean values were assessed by Students’s t test. #P \ 0.05 versus control group; *P \ 0.05, **P \ 0.01 versus LPS group
mechanisms of PA on the inhibition of inflammatory mediators production in microglia, the effects of PA on LPS-induced NF-jB and MAPKs activation were detected. Our results showed that PA significantly suppressed LPSinduced NF-jB and MAPKs activation in BV2 microglial cells. TLR4 is the main receptor of LPS. Activation of TLR4 by LPS leads to the activation of NF-jB and MAPKs. Therefore, we detected the effects of PA on LPSinduced TLR4 expression. The results showed that PA inhibited LPS-induced TLR4 expression in a dose-dependent manner. These results suggested that PA exhibited its anti-inflammatory effects by inhibiting TLR4 signaling pathway. In conclusion, this study demonstrated that PA inhibited LPS-induced inflammatory mediators production by suppressing TLR4-mediated NF-jB and MAPKs activation in BV2 microglial cells.
Acknowledgments This study was supported by Grants from the National Key disciplines Fund of the Ministry of Health of the People’s Republic of China, the Foundation of Tianjin Science and Technology Committee (12ZCDZSY17700, 14JCZDJC35600), National Key Technology Support Program 2014BAI04B02. Conflict of interest All authors declare that they have no conflict of interest.
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ORIGINAL PAPER
Protocatechuic Acid Inhibits Inflammatory Responses in LPSStimulated BV2 Microglia via NF-jB and MAPKs Signaling Pathways Huan-yu Wang1 • Hong Wang1 • Jin-huan Wang1 • Qiong Wang1 Quan-feng Ma1 • Yi-yang Chen1
•
Received: 9 April 2015 / Revised: 11 June 2015 / Accepted: 19 June 2015 / Published online: 2 July 2015 Ó Springer Science+Business Media New York 2015
Abstract Protocatechuic acid (PA), a major metabolite of anthocyanins, has been reported to possess antioxidant and anti-inflammatory activities. However, the effects of PA on LPS-induced inflammatory responses in microglia have not been reported. The aim of this study was to investigate the anti-inflammatory effects and molecular mechanisms of PA on LPS-stimulated BV2 microglia. The production of inflammatory mediators TNF-a, IL-6, IL-1b, and PGE2 were detected by ELISA. TLR4, NF-jB and MAPKs activation were detected by western blotting. Our results demonstrated that PA dose-dependently inhibited LPS-induced TNF-a, IL6, IL-1b, and PGE2 production. In addition, PA suppressed LPS-induced TLR4 expression, NF-jB and MAPKs activation, which resulted in the inhibition of inflammatory mediators. In conclusion, these results suggested that PA exhibited anti-inflammatory effects on LPS-stimulated BV2 microglia and the mechanisms were involved in the inhibition of TLR4mediated NF-jB and MAPKs signaling pathways. Keywords Protocatechuic acid LPS Inflammatory mediators NF-jB MAPKs
Introduction Microglia are the resident immune cells in the brain which play an important role in host defense and tissue repair in brain [1]. Microglia can be activated by various factors
such as infection and injury [2]. LPS, the main component of Gram-negative bacteria, has been reported to be one of the most potent stimuli for microglia activation [3–5]. Stimulating of microglia by LPS induces NF-jB and MAPKs (JNK, P38, ERK) activation, which regulates the release of inflammatory mediators TNF-a, IL-6, PGE2, and IL-1b [6, 7]. Overproduction of these inflammatory cytokines induce several inflammatory and neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [8]. Previous studies showed that reduction of these inflammatory mediators could attenuate the severity of these diseases [9]. Protocatechuic acid (PA), a major metabolite of anthocyanins, has been found to possess antioxidant, anti-inflammatory and anti-tumor activities [10, 11]. PA was found to inhibit TNF-a, IL-1b, and COX-2 expression in LPS-stimulated RAW264.7 cells [12] and inhibit human dendritic cell function activation in vitro [13]. Moreover, previous reports showed that PA attenuated LPS-induced acute lung injury and protected against sepsis in mice [14, 15]. In addition, PA has been reported to inhibit LPS-induced rat hepatic damage [16]. However, the effects of PA on LPS-stimulated BV2 microglia remain unclear. The aim of this study was to investigate the anti-inflammatory effects and mechanisms of PA on LPS-stimulated BV2 microglia.
Materials and Methods & Hong Wang [email protected] 1
Tianjin Cerebral Vascular and Neural Degenerative Disease Key Laboratory, Tianjin Huanhu Hospital, Tianjin 300060, China
Materials LPS (Escherichia coli O55:B5) and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO, USA). Protocatechuic
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acid was purchased from National Institutes for Food and Drug Control (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits of PGE2, TNF-a, IL-6 and IL-1b were purchased from R&D Systems (Minneapolis, MN, USA). Rabbit anti-human TLR4, NF-jB p65, IjBa, ERK, JNK, p38, and b-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). All other reagents were of analytical grade.
protein were separated on 10 % SDS–polyacrylamide gel. The proteins were transferred onto PVDF membranes and blocked in 5 % skim milk for 1 h at room temperature. Then the membrane was incubated with primary antibodies overnight at 4 °C. The membrane was incubated with a horseradish peroxidase-(HRP)-conjugated secondary antibody for 2 h at room temperature.
Cell Culture
All values are expressed as mean ± SEM. Differences between mean values of normally distributed data were analyzed using one-way ANOVA (Dunnett’s t test) and two-tailed Student’s t test. Statistical significance was accepted P \ 0.05 or P \ 0.01.
Murine BV2 microglia cells were purchased from the American Type Culture Collection, (Manassas, VA, USA). Cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 5 % FBS, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C in a humidified incubator under 5 % CO2.
Statistical Analysis
Results
MTT Assay
Effects of PA on Cell Viability
The effect of PA on cell viability was determined by MTT assay. Briefly, BV2 microglia cells were seeded in 96 well plates and treated with various concentrations of PA for 24 h. 20 lL MTT (5 mg/mL) was added to each well, and the cells were further incubated for an additional 4 h. The resulting formazan crystals were dissolved in DMSO (150 lL/well). Absorbance was determined at 540 nm.
The effects of PA on the viability of BV2 microglia cells were detected by MTT assay. BV2 microglia were cultured with the indicated concentrations of PA for 24 h in the absence or presence of LPS. As shown in Fig. 1, the result showed that PA (5, 10, 20 lM) did not affect the cell viabilities of BV2 microglia cells. Hence, PA (5, 10, 20 lM) exerted no significant cytotoxicity on BV-2 microglial cells.
Cytokine Assays
PA Inhibits LPS-Induced TNF-a, IL-1ß, IL-6, and PGE2 Production
BV2 cells were pretreated with PA 1 h before LPS administration. 24 h later, the culture supernatant were collected. The levels of TNF-a, IL-6, and IL-1b in the culture supernatant were determined using a commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.
To investigate the anti-inflammatory effects of PA, the effects of PA on TNF-a, IL-1ß, IL-6 and PGE2 production
Measurement of PGE2 BV2 cells were pretreated with the indicated concentrations of PA 1 h prior to LPS stimulation for 24 h. PGE2 in the culture medium was measured by ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Western Blot Analysis BV2 cells were pretreated with the indicated concentrations of PA 1 h prior to LPS stimulation for 30 min. Total proteins were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo). Protein concentrations were determined by BCA protein assay kit. Equal amounts of
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Fig. 1 Effects of PA on the cell viability of BV2 microglial cells. Cells were cultured with different concentrations of PA (5, 10, 20 lM) in the absence or presence of 0.5 lg/mL LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the mean ± SEM of three independent experiments
Neurochem Res (2015) 40:1655–1660
were analyzed at 24 h after LPS challenge by ELISA. As shown in Fig. 2, inflammatory mediators TNF-a, IL-1ß, IL-6 and PGE2 production increased significantly after LPS challenge. However, PA inhibited TNF-a, IL-1ß, IL-6 and PGE2 production induced by LPS in a dose-dependent manner (P \ 0.01).
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cytokines gene expression [18]. To further elucidate the mechanisms of PA, the effects of PA on MAPKs activation, which are upstream signaling molecules in inflammatory responses, were detected in this study. The results showed that pretreatment with PA inhibited LPS-induced MAPKs (p38, JNK and ERK) activation in a dose-dependent manner (Fig. 4).
PA Inhibits LPS-Induced TLR4 Expression and NF-jB Activation
Discussion To further elucidate the mechanisms of PA, the effects of PA on LPS-induced TLR4 expression and NF-jB activation were detected Western blot analysis. As shown in Fig. 3, LPS induced NF-jB P65 and IjBa phosphorylation. PA inhibited LPS-induced NF-jB P65 and IjBa phosphorylation in a dose-dependent manner (Fig. 3). In addition, the results showed that PA significantly inhibited LPS-induced TLR4 expression (Fig. 3).
It is well known that MAPKs (p38, JNK and ERK) play crucial roles in the inflammatory response [17]. MAPKs is an important pathway in mediating pro-inflammatory
Microglia constitute a unique population of immune cells in the CNS and play critical roles in host innate immune response [19]. Activated microglia produce numerous proinflammatory cytokines, which are thought to play important roles in the pathogenesis of neurodegenerative diseases [20]. The pharmacologic regulation of microglial activity is useful in the treatment of neurodegenerative diseases. PA, a major metabolite of anthocyanins, has been reported to have anti-inflammatory effect. In this study, the anti-inflammatory effects of PA on LPS-stimulated BV2 microglial cells were detected. The results showed that PA inhibited LPS-induced microglia activation by suppressing inflammatory cytokines production.
Fig. 2 Effects of PA on LPS-induced TNF-a, IL-1ß, IL-6 and PGE2 production. Cells were treated with PA (5, 10, 20 lM) for 1 h and stimulated with LPS (0.5 lg/mL) for 24 h. The production of TNF-a, IL-1ß, IL-6 and PGE2 were measured by ELISA. The data presented
are the mean ± SEM of three independent experiments and differences between mean values were assessed by Students’s t test. # P \ 0.05 versus control group; *P \ 0.05, **P \ 0.01 versus LPS group
PA Inhibits LPS-Induced MAPKs Activation
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Fig. 3 PA inhibits LPS-induced TLR4 expression and NF-jB activation. Cells were treated with PA (5, 10, 20 lM) for 1 h and stimulated with LPS (0.5 lg/mL) for 30 min. Protein samples were analyzed by western blotting. The values presented are the
mean ± SEM of three independent experiments and differences between mean values were assessed by Students’s t test. #P \ 0.05 versus control group; *P \ 0.05, **P \ 0.01 versus LPS group
Microglia can be activated by various factors. LPS is one of the most important factors that induces microglia activation [21]. Stimulating of microglia by LPS could induce the production of inflammatory mediators including TNF-a, IL-1ß, IL-6 and PGE2 [22]. Meanwhile, an increasing number of studies have shown that inflammatory mediators TNF-a, IL-1ß, IL-6 and PGE2 were involved in the pathogenesis of neurodegenerative disorders, such as Parkinson’s disease (PD) and Alzheimer’s disease [6, 21]. Elevated TNF-a, IL-1ß, IL-6 and PGE2 levels were observed in patients with neurodegenerative disorders [23]. Previous studies showed that reduction of these inflammatory mediators could attenuate the severity of these diseases [24, 25]. Thus, agents with the ability to inhibit inflammatory cytokines expression are potentially
beneficial in the treatment of neurodegenerative diseases. In this study, the effects of PA on inflammatory mediators production were detected. The results showed that PA inhibited LPS-induced inflammatory mediators TNF-a, IL-1ß, IL-6 and PGE2 in a dose-dependent manner. NF-jB has been reported to play an important role in the regulation of inflammatory cytokines production [26, 27]. Normally, NF-jB is sequestered in the cytoplasm and combined with its inhibitors IjB. Once stimulating by LPS, NF-jB p65 translocates from the cytoplasm to the nucleus [28]. In the nucleus, NF-jB induces the transcription of inflammatory mediators such as TNF-a, IL-1ß, IL-6 and PGE2 [29–31]. MAPKs are a group of signaling molecules that also play critical roles in regulation of inflammatory mediators production [32–34]. To investigate the
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Fig. 4 PA inhibits LPS-induced MAPKs activation. Cells were treated with PA (5, 10, 20 lM) for 1 h and stimulated with LPS (0.5 lg/mL) for 30 min. Protein samples were analyzed by western blotting. The values presented are the mean ± SEM of three
independent experiments and differences between mean values were assessed by Students’s t test. #P \ 0.05 versus control group; *P \ 0.05, **P \ 0.01 versus LPS group
mechanisms of PA on the inhibition of inflammatory mediators production in microglia, the effects of PA on LPS-induced NF-jB and MAPKs activation were detected. Our results showed that PA significantly suppressed LPSinduced NF-jB and MAPKs activation in BV2 microglial cells. TLR4 is the main receptor of LPS. Activation of TLR4 by LPS leads to the activation of NF-jB and MAPKs. Therefore, we detected the effects of PA on LPSinduced TLR4 expression. The results showed that PA inhibited LPS-induced TLR4 expression in a dose-dependent manner. These results suggested that PA exhibited its anti-inflammatory effects by inhibiting TLR4 signaling pathway. In conclusion, this study demonstrated that PA inhibited LPS-induced inflammatory mediators production by suppressing TLR4-mediated NF-jB and MAPKs activation in BV2 microglial cells.
Acknowledgments This study was supported by Grants from the National Key disciplines Fund of the Ministry of Health of the People’s Republic of China, the Foundation of Tianjin Science and Technology Committee (12ZCDZSY17700, 14JCZDJC35600), National Key Technology Support Program 2014BAI04B02. Conflict of interest All authors declare that they have no conflict of interest.
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