This study was designed to evaluate the pharmacological effects of Vaccinium bracteatum Thunb. methanol extract (VBME) on microglial activation and to identify the underlying mechanisms of action of these effects. The anti-inflammatory properties of VBME were studied using lipopolysaccharide (LPS)-s...
This study was designed to evaluate the pharmacological effects of Vaccinium bracteatum Thunb. methanol extract (VBME) on microglial activation and to identify the underlying mechanisms of action of these effects. The anti-inflammatory properties of VBME were studied using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We measured the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin $E_2$ ($PGE_2$), tumor necrosis factor-alpha (TNF-${\alpha}$), interleukin-1 beta (IL-$1{\beta}$), and interleukin-6 (IL-6) as inflammatory parameters. We also examined the effect of VBME on intracellular reactive oxygen species (ROS) production and the activity of nuclear factor-kappa B p65 (NF-${\kappa}B$ p65). VBME significantly inhibited LPS-induced production of NO and $PGE_2$ and LPS-mediated upregulation of iNOS and COX-2 expression in a dose-dependent manner; importantly, VBME was not cytotoxic. VBME also significantly reduced the generation of the pro-inflammatory cytokines TNF-${\alpha}$, IL-$1{\beta}$, and IL-6. In addition, VBME significantly dampened intracellular ROS production and suppressed NF-${\kappa}B$ p65 translocation by blocking $I{\kappa}B-{\alpha}$ phosphorylation and degradation in LPS-stimulated BV2 cells. Our findings indicate that VBME inhibits the production of inflammatory mediators in BV-2 microglial cells by suppressing NF-${\kappa}B$ signaling. Thus, VBME may be useful in the treatment of neurodegenerative diseases due to its ability to inhibit inflammatory mediator production in activated BV-2 microglial cells.
This study was designed to evaluate the pharmacological effects of Vaccinium bracteatum Thunb. methanol extract (VBME) on microglial activation and to identify the underlying mechanisms of action of these effects. The anti-inflammatory properties of VBME were studied using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We measured the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin $E_2$ ($PGE_2$), tumor necrosis factor-alpha (TNF-${\alpha}$), interleukin-1 beta (IL-$1{\beta}$), and interleukin-6 (IL-6) as inflammatory parameters. We also examined the effect of VBME on intracellular reactive oxygen species (ROS) production and the activity of nuclear factor-kappa B p65 (NF-${\kappa}B$ p65). VBME significantly inhibited LPS-induced production of NO and $PGE_2$ and LPS-mediated upregulation of iNOS and COX-2 expression in a dose-dependent manner; importantly, VBME was not cytotoxic. VBME also significantly reduced the generation of the pro-inflammatory cytokines TNF-${\alpha}$, IL-$1{\beta}$, and IL-6. In addition, VBME significantly dampened intracellular ROS production and suppressed NF-${\kappa}B$ p65 translocation by blocking $I{\kappa}B-{\alpha}$ phosphorylation and degradation in LPS-stimulated BV2 cells. Our findings indicate that VBME inhibits the production of inflammatory mediators in BV-2 microglial cells by suppressing NF-${\kappa}B$ signaling. Thus, VBME may be useful in the treatment of neurodegenerative diseases due to its ability to inhibit inflammatory mediator production in activated BV-2 microglial cells.
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문제 정의
Indeed, the detailed molecular mechanisms underlying the effects of VBME on neuro-inflammation have not yet been investigated. Therefore, in this study we evaluated the pharmacological effects of VBME on microglial activation. We also identified the underlying mechanism of action by investigating the phosphorylation status of MAPKs, PI3K/Akt, and GSK-3β, in addition to assessing nuclear factor-kappa B p65 (NF-κB p65) activation.
제안 방법
However, un- regulated phosphorylation of MAPKs, PI3K/Akt, and GSK-3β signaling proteins results in excessive production of inflammatory molecules, which has adverse consequences. Therefore, we performed further experiments to test our hypothesis that VBME tightly regulates the expression of MAPKs to induce anti-inflammatory effects in LPS-stimulated microglial cells. Our results indicated that VBME is a potent inhibitor of phosphorylation of JNK, ERK 1/2 MAPKs, PI3K/Akt, and GSK-3β.
001). To further investigate the effect of VBME on LPSinduced intracellular ROS accumulation, we visualized DCFHDA staining on a fluorescence microscope. Microphotographs of DCFH-DA staining revealed excessive intracellular ROS accumulation after LPS stimulation (Fig.
대상 데이터
The aboveground parts of VB were collected in Yunnan Province, China, in September 2012. A voucher specimen was deposited in the herbarium of the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, Republic of Korea). VB was obtained from the International Biological Ma terial Research Center (IBMRC) of KRIBB.
Ammonium pyrrolidinedithiocarbamate (PDTC), 2,7’-dichlorofluorescin diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), Hoechst 33258, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipopolysaccharide (LPS; Escherichia coli, 026:B6), poly-D-lysine, quercetin, and anti-β-actin antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
데이터처리
, San Diego, CA, USA) and are expressed as means ± SEMs. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by the Newman-Keuls test. Statistical significance was set at p<0.
성능/효과
Our results indicated that VBME significantly inhibited LPS-induced intracellular ROS production in BV-2 microglial cells, suggesting a possible mechanism for the inhibitory effects of VBME on MAPKs/PI3K/Akt/GSK-3β phosphorylation and NF-κB activation.
The LPS-mediated increases in phosphorylation of JNK, ERK 1/2, PI3K/Akt, and GSK-3β were significantly inhibited by VBME in a dosedependent manner (p<0.01 and p<0.001), whereas the phos phorylation of p38 was not significantly affected.
후속연구
Therefore, quercetin might act either independently or synergistically by regulating multiple signaling pathways to block neuro-inflammation. However, the precise concentrations of these compounds in VBME extract that are required to reach the brain to produce beneficial effects remain unknown and will require further research.
Although we did not evaluate whether VBME inhibits inflammation-related neuronal damage in vivo, we demonstrated that VBME exerts anti-inflammatory activity in BV-2 microglial cells. Therefore, further studies are needed to elucidate the precise activities of VBME and its major compounds in vivo. We are currently working to isolate other active components of VBME using an activity-guided fractionation approach.
To further test the therapeutic potential of VBME for treating neurodegenerative diseases such as AD and PD, the neuroprotective effects of VBME should be further investigated in in vivo models. Although we did not evaluate whether VBME inhibits inflammation-related neuronal damage in vivo, we demonstrated that VBME exerts anti-inflammatory activity in BV-2 microglial cells.
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