Anti‑inflammatory effects of Perilla frutescens leaf extract on lipopolysaccharide‑stimulated RAW264.7 cells

Huang,Bee‑Piao; Lin,Chun‑Hsiang; Chen,Yi‑Ching; Kao,Shao‑Hsuan

doi:10.3892/mmr.2014.2298

Anti‑inflammatory effects of Perilla frutescens leaf extract on lipopolysaccharide‑stimulated RAW264.7 cells

Authors:
- Bee‑Piao Huang
- Chun‑Hsiang Lin
- Yi‑Ching Chen
- Shao‑Hsuan Kao
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Affiliations: Department of Pathology, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan, R.O.C., Institute of Biochemistry and Biotechnology, Chung Shan Medical University Hospital, Taichung, Taiwan, R.O.C.
Published online on: June 5, 2014 https://doi.org/10.3892/mmr.2014.2298
Pages: 1077-1083

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Abstract

Perilla leaves are widely used in Chinese herbal medicine and in Japanese herbal agents used to treat respiratory diseases. This study aimed to investigate the anti‑inflammatory effects and the underlying mechanisms of Perilla frutescens leaf extract (PLE). Murine macrophage RAW264.7 cells were used as a model. Cell viability and morphological changes were studied by the MTT assay and microscopy. mRNA expression of pro‑inflammatory mediators was assessed by both semi‑quantitative reverse transcription‑polymerase chain reaction (RT‑PCR) and quantitative (q) RT‑PCR. Nitric oxide (NO) and prostaglandin E2 (PGE2) production were analyzed by the Griess test and sandwich enzyme‑linked immunosorbent assay (ELISA), respectively. The activation of kinase cascades was studied by immunoblotting. Our findings showed that PLE slightly affects cell viability, but alleviates LPS‑induced activation of RAW264.7 cells. Furthermore, PLE significantly reduced the LPS‑induced mRNA expression of the interleukin (IL)‑6, IL‑8, tumor necrosis factor‑α (TNF‑α), cyclooxygenase‑2 (COX‑2) and inducible nitric oxide synthase (iNOS), genes in a dose‑dependent manner. In addition, PLE reduced NO production and PGE2 secretion induced by LPS. PLE also inhibited activation of mitogen‑activated protein kinases (MAPKs), increased the cytosolic IκBα level, and reduced the level of nuclear factor (NF)‑κB. Taken together, these findings indicate that PLE significantly decreases the mRNA expression and protein production of pro‑inflammatory mediators, via the inhibition of extracellular‑signal‑regulated kinase (ERK)1/2, c‑Jun N‑terminal kinase (JNK), p38, as well as NF‑κB signaling in RAW264.7 cells stimulated with LPS.

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1	Ueda H and Yamazaki M: Inhibition of tumor necrosis factor-alpha production by orally administering a Perilla leaf extract. Biosci Biotechnol Biochem. 61:1292–1295. 1997. View Article : Google Scholar : PubMed/NCBI
2	Makino T, Ono T, Muso E, Honda G and Sasayama S: Suppressive effects of Perilla frutescens on spontaneous IgA nephropathy in ddY mice. Nephron. 83:40–46. 1999.
3	Ueda H and Yamazaki M: Anti-inflammatory and anti-allergic actions by oral administration of a Perilla leaf extract in mice. Biosci Biotechnol Biochem. 65:1673–1675. 2001. View Article : Google Scholar : PubMed/NCBI
4	Shin TY, Kim SH, Kim SH, et al: Inhibitory effect of mast cell-mediated immediate-type allergic reactions in rats by Perilla frutescens. Immunopharmacol Immunotoxicol. 22:489–500. 2000. View Article : Google Scholar : PubMed/NCBI
5	Hortelano S, Zeini M and Bosca L: Nitric oxide and resolution of inflammation. Methods Enzymol. 359:459–465. 2002. View Article : Google Scholar : PubMed/NCBI
6	Paige JS and Jaffrey SR: Pharmacologic manipulation of nitric oxide signaling: targeting NOS dimerization and protein-protein interactions. Curr Top Med Chem. 7:97–114. 2007. View Article : Google Scholar : PubMed/NCBI
7	Gao YT, Panda SP, Roman LJ, Martasek P, Ishimura Y and Masters BS: Oxygen metabolism by neuronal nitric-oxide synthase. J Biol Chem. 282:7921–7929. 2007. View Article : Google Scholar : PubMed/NCBI
8	Breitbach K, Klocke S, Tschernig T, van Rooijen N, Baumann U and Steinmetz I: Role of inducible nitric oxide synthase and NADPH oxidase in early control of Burkholderia pseudomallei infection in mice. Infect Immun. 74:6300–6309. 2006. View Article : Google Scholar : PubMed/NCBI
9	Cunha IW, Lopes A, Falzoni R and Soares FA: Sarcomas often express constitutive nitric oxide synthases (NOS) but infrequently inducible NOS. Appl Immunohistochem Mol Morphol. 14:404–410. 2006. View Article : Google Scholar : PubMed/NCBI
10	Farley KS, Wang LF, Razavi HM, et al: Effects of macrophage inducible nitric oxide synthase in murine septic lung injury. Am J Physiol Lung Cell Mol Physiol. 290:L1164–L1172. 2006. View Article : Google Scholar : PubMed/NCBI
11	Sacco RE, Waters WR, Rudolph KM and Drew ML: Comparative nitric oxide production by LPS-stimulated monocyte-derived macrophages from Ovis canadensis and Ovis aries. Comp Immunol Microbiol Infect Dis. 29:1–11. 2006. View Article : Google Scholar : PubMed/NCBI
12	Rahman A, Yatsuzuka R, Jiang S, Ueda Y and Kamei C: Involvement of cyclooxygenase-2 in allergic nasal inflammation in rats. Int Immunopharmacol. 6:1736–1742. 2006. View Article : Google Scholar : PubMed/NCBI
13	Geng Y, Zhang B and Lotz M: Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes. J Immunol. 151:6692–6700. 1993.PubMed/NCBI
14	Kim HS, Ye SK, Cho IH, et al: 8-hydroxydeoxyguanosine suppresses NO production and COX-2 activity via Rac1/STATs signaling in LPS-induced brain microglia. Free Radic Biol Med. 41:1392–1403. 2006. View Article : Google Scholar : PubMed/NCBI
15	Park HJ, Kim IT, Won JH, et al: Anti-inflammatory activities of ent-16alphaH,17-hydroxy-kauran-19-oic acid isolated from the roots of Siegesbeckia pubescens are due to the inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages via NF-kappaB inactivation. Eur J Pharmacol. 558:185–193. 2007. View Article : Google Scholar : PubMed/NCBI
16	Nakano H, Shindo M, Sakon S, et al: Differential regulation of IkappaB kinase alpha and beta by two upstream kinases, NF-kappaB-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1. Proc Natl Acad Sci USA. 95:3537–3542. 1998. View Article : Google Scholar
17	Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS and Tannenbaum SR: Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 126:131–138. 1982.
18	Saxena RK, Vallyathan V and Lewis DM: Evidence for lipopolysaccharide-induced differentiation of RAW264.7 murine macrophage cell line into dendritic like cells. J Biosci. 28:129–134. 2003. View Article : Google Scholar : PubMed/NCBI
19	Onuchic AC, Machado CM, Saito RF, Rios FJ, Jancar S and Chammas R: Expression of PAFR as part of a prosurvival response to chemotherapy: a novel target for combination therapy in melanoma. Mediators Inflamm. 2012:1754082012. View Article : Google Scholar : PubMed/NCBI
20	Chen H, Ma F, Hu X, Jin T, Xiong C and Teng X: Elevated COX2 expression and PGE2 production by downregulation of RXRα in senescent macrophages. Biochem Biophys Res Commun. 440:157–162. 2013.PubMed/NCBI
21	Adesso S, Popolo A, Bianco G, et al: The uremic toxin indoxyl sulphate enhances macrophage response to LPS. PloS One. 8:e767782013. View Article : Google Scholar : PubMed/NCBI
22	Hambleton J, Weinstein SL, Lem L and DeFranco AL: Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages. Proc Natl Acad Sci USA. 93:2774–2778. 1996. View Article : Google Scholar : PubMed/NCBI
23	An H, Yu Y, Zhang M, et al: Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells. Immunology. 106:38–45. 2002. View Article : Google Scholar : PubMed/NCBI
24	Wang W, Deng M, Liu X, Ai W, Tang Q and Hu J: TLR4 activation induces nontolerant inflammatory response in endothelial cells. Inflammation. 34:509–518. 2011. View Article : Google Scholar : PubMed/NCBI
25	Tak PP and Firestein GS: NF-kappaB: a key role in inflammatory diseases. J Clin Invest. 107:7–11. 2001. View Article : Google Scholar : PubMed/NCBI
26	Murakami A, Nishizawa T, Egawa K, et al: New class of linoleic acid metabolites biosynthesized by corn and rice lipoxygenases: suppression of proinflammatory mediator expression via attenuation of MAPK- and Akt-, but not PPARgamma-, dependent pathways in stimulated macrophages. Biochem Pharmacol. 70:1330–1342. 2005. View Article : Google Scholar
27	Fujiwara N and Kobayashi K: Macrophages in inflammation. Curr Drug Targets Inflamm Allergy. 4:281–286. 2005. View Article : Google Scholar
28	Heumann D and Roger T: Initial responses to endotoxins and Gram-negative bacteria. Clin Chim Acta. 323:59–72. 2002. View Article : Google Scholar : PubMed/NCBI
29	Desai TR, Leeper NJ, Hynes KL and Gewertz BL: Interleukin-6 causes endothelial barrier dysfunction via the protein kinase C pathway. J Surg Res. 104:118–123. 2002. View Article : Google Scholar : PubMed/NCBI
30	Noda A, Kinoshita K, Sakurai A, Matsumoto T, Mugishima H and Tanjoh K: Hyperglycemia and lipopolysaccharide decrease depression effect of interleukin 8 production by hypothermia: an experimental study with endothelial cells. Intensive Care Med. 34:109–115. 2008. View Article : Google Scholar : PubMed/NCBI
31	Hume DA, Underhill DM, Sweet MJ, Ozinsky AO, Liew FY and Aderem A: Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state. BMC Immunol. 2:112001. View Article : Google Scholar
32	Aggarwal BB, Shishodia S, Ashikawa K and Bharti AC: The role of TNF and its family members in inflammation and cancer: lessons from gene deletion. Curr Drug Targets Inflamm Allergy. 1:327–341. 2002. View Article : Google Scholar : PubMed/NCBI
33	Tsatsanis C, Androulidaki A, Venihaki M and Margioris AN: Signalling networks regulating cyclooxygenase-2. Int J Biochem Cell Biol. 38:1654–1661. 2006. View Article : Google Scholar : PubMed/NCBI
34	Aggarwal BB, Shishodia S, Sandur SK, Pandey MK and Sethi G: Inflammation and cancer: how hot is the link? Biochem Pharmacol. 72:1605–1621. 2006. View Article : Google Scholar : PubMed/NCBI
35	Korhonen R, Lahti A, Kankaanranta H and Moilanen E: Nitric oxide production and signaling in inflammation. Curr Drug Targets Inflamm Allergy. 4:471–479. 2005. View Article : Google Scholar : PubMed/NCBI
36	Geppert TD, Whitehurst CE, Thompson P and Beutler B: Lipopolysaccharide signals activation of tumor necrosis factor biosynthesis through the ras/raf-1/MEK/MAPK pathway. Mol Med. 1:93–103. 1994.PubMed/NCBI
37	Han J, Lee JD, Bibbs L and Ulevitch RJ: A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science. 265:808–811. 1994. View Article : Google Scholar : PubMed/NCBI