Page 11 - H. Cell signaling

9-Hydroxy-Isoegomaketone Inhibits Anti-Inflammatory Activity in LPS-Stimulated RAW 264.7
Macrophages through the Activation of Nrf2 and Induction of HO-1
Hyun Mi Kim¹ ², Bomi Nam¹, Sunil Babu Paudel³, Joo-Won Nam³, Ah-Reum Han¹, Hye Gwang Jeong², Chang Hyun Jin¹ ,*
,
¹ Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si, Jeollabuk-do 56212, Republic of Korea
² College of Pharmacy, Chungnam National University, Daejeon, Chungcheongnam-do 34134, Republic of Korea
³ College of Pharmacy, Yeungnam University, Gyeongsan-si, Gyeongsangnam-do 38541, Republic of Korea

BACKGROUND AIM
A new compound known as 9-hydroxy-isoegomaketone (9-HIK) was isolated from the 9-HIK compound isolated in radiation mutant cultivar has not
extract of a radiation mutant of Perilla frutescens var. crispa using supercritical carbon been reported. Therefore, to confirm the effect of anti-
dioxide. In this study, 9-HIK inhibited the production of inflammatory mediators such as
nitric oxide (NO), interleukin 6 (IL-6), interferon β (IFN-β), and interleukin 1β (IL-1β) inflammatory of 9-HIK obtained from the radiation mutant
production in a dose-dependent manner in LPS-stimulated RAW 264.7 cells. Beside, 9- cultivar P. frutescens, we induced an inflammatory reaction in
HIK increased mRNA expression of heme oxygenase-1 (HO-1) in a dose-dependent RAW 264.7 cells by LPS. We confirmed inflammatory cytokines
manner through the activation nuclear factor erythroid 2-related factor 2 (Nrf2). Also, we in LPS-stimulated RAW 264.7 cells. Besides, we investigated
confirmed using the N-acetylcysteine (NAC) that 9-HIK activated Nrf2 through the
production of ROS in RAW 264.7 cells. Taken together, 9-HIK showed anti-inflammatory whether HO-1 induction by Nrf2 activation inhibits the
activities via ROS/Nrf2 pathway activation. inflammatory response.
METHODS
RAW 264.7 cells were grown in DMEM medium with 10 % FBS and 1 % P/S. Cells were maintained at 37℃ in a incubator containing 5 %
CO₂. RAW 264.7 cells were pretreated with a various concentration of 9-HIK for 24 h. To measured cell viability, we used the EZ-Cytox cell
viability assay kit. To measured LPS-stimulated inflammatory cytokine activity, RAW 264.7 cells were pretreated with a various
concentration of 9-HIK for 2 h and stimulated with LPS 18 h (for production of NO) or LPS 4 h (for mRNA expression of IFN-β, IL-6, and
IL-1β). Furthermore, To measured 9-HIK induced HO-1, RAW 264.7 cells treated 9-HIK in a dose-dependent manner and for the indicated
time. Also, to measured 9-HIK translocated Nrf2, RAW 264.7 cells treated 9-HIK for the indicated time. In addition, RAW 264.7 cells were
treated with NAC, a ROS scavenger, to determine whether 9-HIK-mediated mRNA expression of HO-1 and NO production via ROS
generation.
RESULTS

In this study, cytotoxicity was not shown at 5- Figure 1 Figure 2
20 μM of 9-HIK. To investigated the effect of 15
9-HIK on NO production in LPS-stimulated 10 9-HIK (20 μM)
RAW 264.7 cells, we performed nitric oxide (a)
assay. NO production and the mRNA 5 Nrf2
expression of inflammatory cytokine such as Cytosol
IL-6, IL-1β, and IFN-β was inhibited in a 9- 0 - 5 10 20 β-tubulin
HIK dose-dependent manner. Also, mRNA 9-HIK (20 μΜ) Nrf2
expression of HO-1 increased in a 9-HIK 0 4 8 12 (h) Nuclear
dose-dependent manner. And we confirmed (b) HO-1 Lamin B1
HO-1 mRNA expression and protein 0 1 2 4 (h)
production at different times. The HO-1 β-tubulin
induction was enhanced at 4 h after 9-HIK Figure 3
treated. Furthermore, 9-HIK increased the
Nrf2 protein levels in the nuclear fraction. To (a) (b) 12
investigated whether 9-HIK induced mRNA 10
expression of HO-1 was mediated through the 8
ROS, RAW 264.7 cells were treated with 20 Relative HO-1 mRNA level (HO-1/β-actin) 6
μM of 9-HIK and 5 mM NAC for 4 h. The 9- 4
HIK-induced HO-1 mRNA expression was 2
inhibited by NAC treatment of RAW 264.7 LPS (1 μg/mL) 0 - + - + - +
cells. also, NAC treatment of RAW 264.7 cells 9-HIK (20 μМ) - - + + + +
restored suppressed NO production levels. NAC (5 mМ) - - - - + +

Figure 1. (a) Effect of 9-HIK on mRNA expression and protein level of HO-1 in RAW 264.7 cells.
Figure 2. Effects of 9-HIK on Nrf2 activation in RAW 264.7 cells.
Figure 3. Effects of ROS scavenger on the NO production levels and HO-1 mRNA expression in RAW 264.7 cells.

CONCLUSION REFERENCES

we have demonstrated that 9-HIK 1. BM Nam, YK So, HY Kim, JB Kim, CH Jin
induced HO-1 via ROS/Nrf2 pathway and AR Han, A New Monoterpene from the
Leaves of a Radiation Mutant Cultivar of
activation. Beside, this results proved Perilla frutescens var. crispa with Inhibitory
that the inhibitory effect of 9-HIK on Activity on LPS-Induced NO Production.
Molecules. 2017, 22(9):1471.
LPS-stimulated NO production is 2. CH Jin, HJ Lee, YD Park, DS Choi, DS Kim,
important. SY Kang, KI Seo and IY Jeong, Contact information
Isoegomaketone Inhibits Lipopolysaccharide-
Taken together, the results suggest that Induced Nitric Oxide Production in RAW khm2172@naver.com (H.M.K.)
Macrophages
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9-HIK could be used as a potential Oxygenase-1 Induction and Inhibition of the chjin@kaeri.re.kr (C.H.J.)
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