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CLG from Hemp Seed Inhibits LPS-Stimulated Neuroinflammation in BV2 Microglia by Regulating NF-κB and Nrf-2 Pathways

The healthy benefits of hemp (Cannabis sativa L.) seed have often been attributed to its oils and proteins. Recent studies reveal that hemp seed phenylpropionamides could also show various bioactivities. Continuation of our study on hemp seed provided a phenylpropionamide, coumaroylaminobutanol glucopyranoside (CLG). This work investigated the neuroprotective effect of CLG and its underlying mechanism using lipopolysaccharide-induced BV2 microglia. Our study demonstrated that CLG increased…

Published

1 year ago

October 17, 2019

Republished by Plato

. 2019 Sep 26;4(15):16517-16523.

doi: 10.1021/acsomega.9b02168. eCollection 2019 Oct 8.

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Shanshan Wang et al. ACS Omega. 2019.

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Abstract

The healthy benefits of hemp (Cannabis sativa L.) seed have often been attributed to its oils and proteins. Recent studies reveal that hemp seed phenylpropionamides could also show various bioactivities. Continuation of our study on hemp seed provided a phenylpropionamide, coumaroylaminobutanol glucopyranoside (CLG). This work investigated the neuroprotective effect of CLG and its underlying mechanism using lipopolysaccharide-induced BV2 microglia. Our study demonstrated that CLG increased adenosine monophosphate-activated protein kinase (AMPK) expression, suppressed the nuclear factor-kappa B (NF-κB) signaling pathway by inhibiting the phosphorylation of IκBα and NF-κB p65 and decreased proinflammatory cytokine levels in a concentration-dependent manner. Furthermore, CLG reduced the production of cellular reactive oxygen species and stimulated the nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling pathway. Collectively, these results suggested that CLG effectively and simultaneously inhibited inflammatory responses and oxidative stress through the NF-κB and Nrf-2 signaling pathways. AMPK was also involved in the anti-inflammatory effect of CLG. This study provides new insights into the diverse bioactive constituents of hemp seed.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

**Figure 1**

Effect of CLG on the cell viability of
BV2 microglia with or without
LPS stimulation. (a) Structure of CLG. (b) BV2 cells were cotreated
with various concentrations of CLG (5, 10, 15 μM) or RES (10
μM) with or without LPS (100 ng/mL) for 24 h. Cell viability
was determined by the MTT assay. (The data are expressed as the mean
± SD of three experiments.)

**Figure 2**

CLG reduced proinflammatory cytokine production
in LPS-stimulated
BV2 microglia. BV2 cells were pretreated with 5, 10, and 15 μM
CLG for 1 h and then stimulated with LPS (100 ng/mL). After coincubation
for 24 h, the supernatants were collected for the measurement of IL-1β
(a) and IL-6 (b) by ELISA. After coincubation for 6 h, total RNA was
isolated, and relative IL-1β (c) and IL-6 (d) mRNA expression
was measured by RT-PCR. (Data are presented as the mean ± SD
from at least three independent experiments. *p <
0.05, **p < 0.01, and ***p <
0.001 compared with the LPS-treated group; ^###p < 0.001 compared with the control group.)

**Figure 3**

CLG inhibited
TLR4/NF-κB protein expression in LPS-induced
BV2 microglia. BV2 cells were pretreated with CLG for 1 h and stimulated
with LPS. After stimulation for 24 h, cell extracts were prepared
and subjected to western blotting with TLR4 and MyD88 antibodies.
After stimulation for 1 h, cell extracts were prepared and subjected
to western blotting with IκBα, phospho-IκBα,
NF-κB p65, and phospho-NF-κB p65 antibodies. β-Actin
was used as the internal control for normalization. (a) Western blot
bands of TLR4 and MyD88. (b) The density of TLR4 bands was measured,
and their ratio was calculated. (c) Density ratio of MyD88 bands.
(d) Western blot bands of IκBα, phospho-IκBα,
NF-κB p65, and phospho-NF-κB p65. (e–h) Density
ratio of phospho-NF-κB p65, p65, phospho-IκBα, and
IκBα. (The results are presented as the mean ± SD
from at least three independent experiments. *p <
0.05, **p < 0.01, and ***p <
0.001 compared with cells treated with LPS; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared with the control.)

**Figure 4**

CLG reduced cellular ROS generation and promoted Nrf-2/HO-1 expression
in LPS-induced BV2 microglia. BV2 cells were treated with CLG for
1 h prior to LPS stimulation for 24 h. (a) Cellular ROS
levels were measured using DCFH-DA by flow cytometry. (b) Quantification
of the relative % of cells with ROS production. (c) Cell lysates were
collected and analyzed using western blotting for Nrf-2 and HO-1.
(d,e) The density of Nrf-2 and HO-1 bands was measured, and their
ratio was calculated. β-Actin was used as the internal control
for normalization. (The results are presented as the mean ± SD
from at least three independent experiments. **p <
0.01 compared with cells treated with LPS; ^##p < 0.01 and ^###p < 0.001 compared
with the control.)

**Figure 5**

CLG increased AMPK activation in LPS-induced BV2 microglia. BV2
cells were treated with CLG for 1 h prior to LPS stimulation for 24
h. Cell lysates were collected and analyzed using western blotting
for phospho-AMPK and AMPK (a). The density of phospho-AMPK and AMPK
bands was measured, and their ratio was calculated (b). Cells were
pretreated with or without CLG and C–C (1 μM) for 1 h
and then coincubated with LPS for 6 h, total RNA was isolated, and
relative IL-1β (c) and IL-6 (d) mRNA expression was measured
by RT-PCR. β-Actin was used as the internal control. (The results
are presented as the mean ± SD from at least three independent
experiments. *p < 0.05 and ***p < 0.001 compared with cells treated with LPS; ^###p < 0.001 compared with the control.)

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