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. 2021 Aug 11;7(8):e07759.
doi: 10.1016/j.heliyon.2021.e07759. eCollection 2021 Aug.

The inhibitory effects of Orengedokuto on inducible PGE2 production in BV-2 microglial cells

Yoshika Iwata  1 , Mariko Miyao  1 , Akiko Hirotsu  1 , Kenichiro Tatsumi  1 , Tomonori Matsuyama  2 , Nobuo Uetsuki  1 , Tomoharu Tanaka  1
Affiliations

The inhibitory effects of Orengedokuto on inducible PGE2 production in BV-2 microglial cells

Yoshika Iwata et al. Heliyon. .

Abstract

Background and aim: Reactive microglia has been associated with neuroinflammation caused by the production of proinflammatory molecules such as cytokines, nitric oxide, and prostaglandins. The overexpression of these molecules may provoke neuronal damage that can cause neurodegenerative diseases. A traditional herbal medicine, Orengedokuto (OGT), has been widely used for treating inflammation-related diseases. However, how it influences neuroinflammation remains poorly understood.

Experimental procedure: This study investigated the effects of OGT on inflammatory molecule induction in BV-2 microglial cells using real-time RT-PCR and ELISA. An in vivo confirmation of these effects was then performed in mice.

Results and conclusion: OGT showed dose-dependent inhibition of prostaglandin E2 (PGE2) production in BV-2 cells stimulated with lipopolysaccharide (LPS). To elucidate the mechanism of PGE2 inhibition, we examined cyclooxygenases (COXs) and found that OGT did not suppress COX-1 expression or inhibit LPS-induced COX-2 upregulation at either the transcriptional or translational levels. In addition, OGT did not inhibit COX enzyme activities within the concentration that inhibited PGE2 production, suggesting that the effect of OGT is COX-independent. The inhibitory effects of OGT on PGE2 production in BV-2 cells were experimentally replicated in primary cultured astrocytes and mice brains. OGT can be useful in the treatment of neuroinflammatory diseases by modulating PGE2 expression.

Keywords: Microglia; Neuroinflammation; Orengedokuto; PGE2; p38 MAPK.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
A three-dimensional high-performance liquid chromatography profile of Orengedokuto, provided by Tsumura & Co.
Figure 2
Figure 2
Effects of Orengedokuto (OGT) and its component on PGE2 production in BV-2 cells. BV-2 microglial cells were exposed to OGT (A) or its component, Scutellariae radix (S. radix) (B), Coptidis rhizoma (C. rhizoma) (C), Gardeniae fructus (G. Fructus) (D), Phellodendri cortex (P. Cortex) (E) and berberine (F) for 24 h with lipopolysaccharide (LPS) (1 μg/ml). The level of PGE2 in the cultured media was analyzed by the enzyme immunoassay. Data are shown as mean ± S.D. of three to five independent experiments. ∗∗P < 0.01 versus control, ∗P < 0.05 versus control, N.S., not significant.
Figure 3
Figure 3
Cytotoxic effects of OGT on BV-2 cells. BV-2 cells were treated with OGT (1, 5, and 10 μg/ml) for 24 h. Cell viability and injury were determined using the MTT (A) and LDH (B) assay. The result was shown as the relative ratio of control. Data are shown as mean ± S.D. of three independent experiments; N.S., not significant.
Figure 4
Figure 4
Effects of OGT on COX expression in BV-2 cells. BV-2 cells were exposed to OGT for 24 h with LPS (1 μg/ml). COX-1 (A) and COX-2 (B) mRNA were analyzed using real-time qRT-PCR, normalized to that of 18S rRNA and expressed relative to the mean of control. The data are presented as means ± S.D. of three to five independent experiments. ∗∗P < 0.01 versus control, N.S., not significant. After 24 h of OGT exposure to BV-2 cells, whole cell lysates were analyzed for COX-1 and COX-2 expression by immunoblot assay (C). The figures are representative of at least three independent experiments. Immunoblot quantification of COX-1/β-actin (D) and COX-2/β-actin (E) are shown. ∗P < 0.05 versus control, N.S., not significant. COX enzyme activity affected by OGT was measured with a cyclooxygenase activity assay kit for COX-1 and COX-2 (F). The data are shown as mean ± S.D. of three independent experiments.
Figure 5
Figure 5
Effects of OGT on PGES expression in BV-2 cells. BV-2 cells were exposed to OGT for 24 h with LPS (1 μg/ml). mPGES1 (A), mPGES2 (B), and cPGES (C) mRNA were analyzed using real-time qRT-PCR, normalized to that of 18S rRNA and expressed relative to the mean of control mice. The data are presented as means ± S.D. of three to four independent experiments. ∗∗P < 0.01 versus control, ∗P < 0.05 versus control, N.S., not significant. After 24 h of OGT exposure to BV-2 cells, whole cell lysates were analyzed for cPGES and β-actin expression by immunoblot assay (D). The figures are representative of at least three independent experiments. Immunoblot quantification of cPGES/β-actin are shown in E. N.S., not significant.
Figure 6
Figure 6
Effects of OGT on cPLA-2 in BV-2 cells. After 4 or 24 h of OGT exposure to BV-2 cells, whole cell lysates were analyzed for phospho or total cPLA2 expression by immunoblot assay (A). Immunoblot quantification of phosphor cPLA2/β-actin are shown in B (4 h) and C (24 h). N.S., not significant.
Figure 7
Figure 7
Effects of OGT on MAPKs in BV-2 cells. BV-2 were exposed to LPS (1 μg/ml) with or without OGT for 24 h. Whole cell lysates were analyzed for phosphor-extracellular signal-regulated kinase (p-ERK), ERK, phospho-JNK (p-JNK), JNK, phospho-p38 (p-p38), and p38 MAPK expression by immunoblot assay (A). The figures are representative of at least three independent experiments. Immunoblot quantification of p-p38/p38 MAPK are shown in B. BV-2 microglial cells were exposed to LPS (1 μg/ml) for 24 h with SB203580 (20 μM) or OGT (10 μg/ml) (C–E). The level of PGE2 was analyzed by the enzyme immunoassay. COX-1 (D) and COX-2 (E) mRNA were analyzed using real-time qRT-PCR, normalized to that of 18S rRNA and expressed relative to the mean of control. Data are shown as mean ± S.D. of three independent experiments. ∗∗P < 0.01 versus control, ∗P < 0.05 versus control, N.S., not significant.
Figure 8
Figure 8
Effects of OGT on proinflammatory cytokine expression and activities on NF-κB in BV-2 cells. BV-2 cells were exposed for 24 h to LPS (1 μg/ml) and OGT at the indicated concentrations. Interleukin (IL)-1β (A), IL-6 (B), and tumor necrosis factor-alpha (TNF-α) (C) mRNA were assayed with real-time RT-PCR. Data are presented as mean ± S.D. of three to five independent experiments. The expression levels of IL-1β, IL-6, and TNF-α were normalized to that of 18S and were expressed relative to the control mean. (D) BV-2 cells were exposed to LPS and OGT for 24 h, and nuclear factor-kappa B (NF-κB) transcriptional activity was measured with an Elisa-based kit. The data are shown as mean ± S.D. of three independent experiments. ∗∗P < 0.01 versus control, ∗P < 0.05 versus control, N.S., not significant.
Figure 9
Figure 9
PGE2 inhibitory effects of OGT in primary cultured astrocytes and mice brains. Primary cultured astrocytes were exposed to LPS (1 μg/ml) and OGT. The level of PGE2 was analyzed by enzyme immunoassay (A). The data are shown as mean ± S.D. of three independent experiments. ∗∗P < 0.01 versus control, N.S., not significant. BALB/cCrSlc mice were administered OGT orally 4 g/kg for 4 days; 24 h after LPS (10 mg/kg) injection, mice brains were harvested and PGE2 concentration was quantified (B). Data are presented as means ± S.E. (n = 6).
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