6-Shogaol attenuates LPS-induced inflammation in BV2 microglia cells by activating PPAR-γ

6-Shogaol, a pungent agent isolated from Zingiber officinale Roscoe, has been known to have anti-tumor and anti-inflammatory effects. However, the anti-inflammatory effects and biological mechanism of 6-Shogaol in LPS-activated BV2 microglia remains largely unknown. In this study, we evaluated the anti-inflammatory effects of 6-Shogaol in LPS-activated BV2 microglia. 6-Shogaol was administrated 1 h before LPS treatment. The production of inflammatory mediators were detected by ELISA. The expression of NF-κB and PPAR-γ were detected by western blot analysis. Our results revealed that 6-Shogaol inhibited LPS-induced TNF-α, IL-1β, IL-6, and PGE2 production in a concentration dependent manner. Furthermore, 6-Shogaol inhibited LPS-induced NF-κB activation by inhibiting phosphorylation and nuclear translocation of NF-κB p65. In addition, 6-Shogaol could increase the expression of PPAR-γ. Moreover, inhibition of PPAR-γ by GW9662 could prevent the inhibition of 6-Shogaol on LPS-induced inflammatory mediator production. In conclusion, 6-Shogaol inhibits LPS-induced inflammation by activating PPAR-γ.


INTRODUCTION
The incidence of neurodegenerative disease, particularly Parkinson disease (PD) and Alzheimer's disease, increased markedly in the last decades [1,2]. Microglia, the major immune cells in the brain, plays a key role in host defence response to injury or infectious agents [3]. Microglia is exquisitely sensitive to brain injury and disease [4]. Overactivation of microglia leads to the production of inflammatory mediators which plays a critical role in the development of neuroinflammation [5,6]. Neuroinflammation has recently been implicated as an important mechanism responsible for the pathological processes of neurodegenerative diseases [7,8]. Therefore, the identification of agents to inhibit neuroinflammation might be an effective approach for the treatment of neurodegenerative diseases.
6-Shogaol, a pungent agent from Zingiber officinale Roscoe, has been reported to have anti-tumor and antiinflammatory effects. 6-Shogaol has been reported to protect against LPS-induced acute lung injury in mice. Also, 6-Shogaol was found to attenuate neuroinflammation and cognitive deficits in animal models of dementia [9]. Furthermore, 6-Shogaol has been reported to inhibit LPSinduced iNOS and COX-2 expression in macrophages [10]. In addition, studies showed that 6-Shogaol could protect against LPS-induced toxicity in murine astrocytes [11]. However, whether 6-Shogaol could inhibit LPSinduced anti-inflammatory response in activated microglial cells remains unclear. In the present study, we evaluated the anti-inflammatory effects of 6-Shogaol in LPS-stimulated BV2 microglia.

Effects of 6-Shogaol on cell viability
To test whether 6-Shogaol has cytotoxicity on BV2 microglia, MTT assay were used in this study. The results showed that 6-Shogaol had no cytotoxicity on BV2 Research Paper microglia at the concentration of 0 to 20 μg/mL ( Figure 1). Therefore, 6-Shogaol (5, 10, 20 μg/mL) were used in the following experiments.

6-Shogaol inhibited LPS-induced NF-κB activation
NF-κB has been known to be involved in the regulation of inflammatory mediators. To investigate the anti-inflammatory mechanism of 6-Shogaol, LPS-induced NF-κB activation were detected in the present study. The results showed that LPS significantly up-regulated the phosphorylation levels of NF-κB p65 and IκBα. Pretreatment of 6-Shogaol concentration dependently inhibited LPS-induced NF-κB p65 phosphorylation and IκBα phosphorylation and degradation ( Figure 3).

Effects of 6-Shogaol on PPAR-γ expression
Previous studies showed that activation PPAR-γ could inhibit LPS-induced NF-κB activation. Thus, we detected whether 6-Shogaol could up-regulated the expression of PPAR-γ. As shown in Figure 4, 6-Shogaol increased the expression of PPAR-γ in a concentration dependent manner ( Figure 4).

GW9662 prevented the anti-inflammatory effects of 6-Shogaol
To further evaluate the anti-inflammatory mechanism of 6-Shogaol, PPAR-γ was blocked by its inhibitor GW9662. As shown in Figure 5, our results indicated that the inhibition of 6-Shogaol on TNF-α, IL-1ß, IL-6 and PGE 2 production were prevented by GW9662. These results suggested that 6-Shogaol exhibited anti-inflammatory effects in BV2 microglia by activating PPAR-γ.

DISCUSSION
Microglia has been known to play an important role in neurodegenerative diseases [12]. Increasing evidences suggested that controlling the activation of microglia may have protective effects against neurodegenerative diseases [13]. In this study, the results showed that 6-Shogaol inhibited LPS-induced microglia activation by activating PPAR-γ.
Microglia, the prime effector cells in the brain, plays a critical role in immune defense and inflammatory responses [14]. However, overactivation of microglia could lead to the pathological process of neurodegenerative diseases [15]. LPS has the ability to induce microglia activation, which lead to the release of inflammatory mediators [12,16]. These inflammatory mediators, such as TNF-α, IL-1ß, IL-6 and PGE 2 , play an important role in the pathological process of neurodegenerative diseases [17]. In the present study, our results showed that 6-Shogaol significantly inhibited LPS-induced inflammatory mediators production in BV2 microglia. The results indicated that 6-Shogaol exhibited anti-inflammatory effects in BV2 microglia.
It has been reported that NF-κB played a critical role in neuroinflammation [18]. LPS could induce NF-κB activation and inflammatory cytokines release [19].
In conclusion, our results demonstrated that 6-Shogaol suppressed LPS-induced inflammatory mediators production by activating PPAR-γ, which subsequently inhibited LPSinduced NF-κB activation. 6-Shogaol might be an effective agent for the treatment of neurodegenerative diseases.

Cell culture
Murine BV2 microglia cells were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). The cells were cultured in DMEM with 5% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were treated with 6-Shogaol 1 h before LPS treatment.

Cell viability
For determination of cell viability, MTT assay was applied in this study. BV2 microglia was incubated with 6-Shogaol alone and with LPS for 18 h. Then, the cells were treated with MTT for 4 h and the formazan formed was dissolved with DMSO (150 μl/well). The optical density was determined at 570 nm using a Bio-Rad spectrophotometer.

ELISA assay
24 h after LPS treatment, the levels of TNF-α, IL-1β, IL-6, and PGE2 in culture media were tested using commercially available ELISA kits (BioLegend, San Diego, CA). The assay was performed following the instructions provided by the manufacturers.

Western blot analysis
The cells were lysed using RIAP lysis buffer and the concentration was measured by BCA method. Equal amount of protein was resolved using 12% SDS-polyacrylamide gel. The proteins were transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk and incubated with primary antibodies and HRP-conjugated goat anti-rabbit IgG. The proteins were tested using the chemiluminescence detection system (Amersham, Berkshire, UK). Finally, the bands were analyzed using ImageJ software.

Statistical analysis
Data were presented as means ± SEM. Statistical comparison of the data were analyzed by one-way ANOVA with post-test Neuman-Keuls. A p value < 0.05 was considered as significant.