Tumor Necrosis Factor-Producing Activity of Wogonin in RAW 264.7 Murine Macrophage Cell Line

• Abstract
• Methods
• Acknowledgements
• References

Abstract

Wogonin from Scutellaria baicalensis, was demonstrated to increase nitric oxide (NO) in the murine macrophage cell line RAW 264.7. It is our aim to investigate the modulatory effect of wogonin on tumor necrosis factor-α (TNF-α) gene expression. By using RAW 264.7 as an in vitro model, the effects of wogonin on inducible nitric oxide synthase (NOS2) and TNF-α gene expression were evaluated by ELISA and reverse transcribed polymerase chain reaction (RT-PCR). Aspirin and H7 were used to determine the possible signal transduction pathways. The results showed that wogonin at the concentration of 10^-5 M and 10^-6 M up-regulated NOS2 gene expression in RAW 264.7 cells. Besides, wogonin up-regulated the gene expression of TNF-α, in terms of TNF-α secretion and transcription, in a dose dependent manner. The fact that aspirin but not H7 blocks the enhancing effect suggests that NF-κB might be involved in wogonin-enhanced TNF-α gene expression. We conclude that a low concentration of wogonin up-regulates NOS2 and TNF-α gene expression through NF-κB pathway.

Tumor necrosis factor-α (TNF-α) is now known to play an important role in a broad spectrum of biological activities, both in inflammatory cascade processes and immune defense mechanisms [1]. A strong association between septic shock and a rise of circulating TNF-α concentration has been reported in both clinical and animal studies [2]. On the other hand, it is generally accepted that TNF-α is necessary for adequate host defense in the treatment of many parasitic infection and neoplastic diseases [3], [4]. Since overproduction of endogenous TNF-α or systemic application of exogenous TNF-α is harmful to the body due its severe toxicity, it is mandatory to further elucidate the regulatory mechanism on TNF-α gene expression.

Wogonin, a pure compound extracted from Scutellaria baicalensis is commonly used in the treatment of fever, infection and jaundice in Eastern countries. Previous studies have shown that wogonin suppresses NOS2 and cyclooxygenase-2 (COX-2) gene expression in LPS-induced mouse macrophage RAW 264.7 cells and protects neuroblastoma HS-SY5Y cells from hydrogen peroxide-induced oxidative stress [5]. In addition to anti-inflammatory and anti-oxidant effects, recent studies suggest that wogonin reduces both the relaxed circular and the linear form of hepatitis B virus (HBV) DNA by suppressing endogenous HBV-DNA polymerase activity [6]. However, the relationship between wogonin, NOS2 and TNF-α gene expression remains unclear.

In contrast to the suppressive effect on NO production in LPS-induced macrophage system, Scutellaria baicalensis has been reported to produce NO as a potent effector molecule against a variety of bacteria and viruses [7]. Some flavonoids are known to have anti-tumor effects and TNF-α is postulated to play an important role in the cytotoxicity of neoplastic cells [8]. Furthermore, several Chinese herbal medicines have recently been screened in our laboratory and a low dose of wogonin has been demonstrated to increase nitric oxide (NO) production in the murine macrophage cell line RAW 264.7. Since TNF-α is a cytokine that ultimately increases the production of NO, it is our aim to investigate the modulatory effect of wogonin on TNF-α gene expression.

Cell viability determined by MTT assay in the presence of wogonin (Fig. [1]) showed that there was little cytotoxic effect except a decreased cell viability (70 - 75 %) at the concentration of 10^-4 M (data not shown). Northern blot analysis showed that wogonin at the concentration of 10^-5 M and 10^-6 M up-regulated the gene expression of NOS2 (data not shown). After RAW 264.7 cells were treated with wogonin for 24 hours, the culture medium was collected for TNF-α measurement. The results demonstrated that wogonin at the concentration of 10^-5 M and 10^-6 M increased the TNF-α level (Fig. [2]). Analyzed by RT-PCR, it was shown that wogonin up-regulated the gene expression of TNF-α and NOS2 in a dose-dependent manner, when cells were treated with wogonin for 4 hours and 24 hours, respectively (Fig. [3]). It was shown that aspirin but not H7 blocked the up-regulated gene expression of TNF-α treated by wogonin (Fig. [4]). The data suggest that wogonin up-regulated TNF-α gene expression is possibly through the NF-κB pathway.

There is consensus that gene expression of NOS2 is regulated by LPS and pro-inflammatory cytokines [2]. Since LPS is a strong inducer for TNF-α production, commercially available wogonin with 99.47 % purity was used in our system to avoid any contamination of LPS in the preparation of drugs or agents. Although some studies showed that wogonin at the concentration of 5 - 100 μM suppressed NO production and NOS2 activity in LPS-activated macrophage system [9], [10], [11] or activated glial cells [12], other studies demonstrated that there was NO producing activity in the same system [13]. In our system, wogonin (10 - 100 μM) suppressed NOS2 gene expression in the LPS-induced mouse macrophage system while wogonin at lower concentration stimulated NO production in macrophage without LPS treatment. Besides, when used in combination with recombinant IFN-γ (r IFN-γ), S. baicalensis induced cooperative NO production in a dose-dependent manner. The synergic effect between rIFN-γ and S. baicalensis was postulated mainly dependent on S. baicalensis-induced TNF-α secretion [13]. We speculated that LPS induced cellular events that might change the biological responses to wogonin treatment. Furthermore, wogonin at a high concentration (100 μM) induced a certain degree of cytotoxicity that might also explain part of wogonin-induced down-regulation of NOS2 gene expression in other series. Nevertheless, the present data that wogonin at low concentration up-regulated TNF-α gene expression in LPS-free RAW 264.7 cells did not contradict the above-mentioned observation.

Many signal transduction pathways such as NF-κB, protein kinase C and IFN-γ regulatory pathway are known to be involved in the regulation of TNF-α gene expression. Previous reports have suggested that salicylate (aspirin) at higher concentrations (> 5 mmol/L) exhibits inhibitory actions on several transcriptional activators, especially NF-κB. Several NF-κB dependent genes, such as those responsible for producing cytokines, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin, are inhibited by salicylate [14]. Salicylate at suprapharmacological concentrations, therefore, may suppress the inflammatory response through its inhibition of monocyte activation and of monocyte and granulocyte interaction with endothelial cells. Our results showed that aspirin but not H7 blocked the up-regulated gene expression of TNF-α in RAW 264.7 cells treated by wogonin. The data suggest that wogonin up-regulated TNF-αgene expression possibly through NF-κB pathway.

In summary, wogonin at low concentration up-regulates NOS2 and TNF-α gene expression, while it down-regulates NOS2 gene expression at a high concentration. The bi-phasic modulation mode might explain the clinical application of the medicinal herb S. baicalensis as an anti-inflammatory and anti-bacterial or anti-tumor agent.

Methods

Mouse macrophage cell line RAW 264.7 (American Type Culture Collection TIB 71) was cultured in DMEM (Dulbecco’s modified Eagle’s medium) at 37 °C, 10 % CO₂, supplemented with 2 mM L-glutamine, 10 % fetal calf serum, NEAA (non-essential amino acid), 100 IU/mL penicillin, 100 μg/mL streptomycin and 0.075 μg/mL fungizone. Lipopolysaccharide (LPS) from E. coli serotype 055:B5 was obtained from Sigma chem. Co. (St. Louis, MO). The cytokines kits related to the gene expression of TNF-α were purchased from Chemicon Co. In order to prevent contamination of LPS on drug administration, pure compound of wogonin (No. 378-85 582, Kishi Da, Inc. Kyoto, Japan) with 99.47 % purity was commercially obtained (Fig. [1]).

Cell viability was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based colorimetric assay [15]. For transcription analysis, total RNA was isolated from 1 × 10⁷ RAW 264.7 cells by using a modified single-step quanidinium thiocyanate method (RNAzol; Cinna/Biotecx) [16]. RNA samples was resolved on a agarose-formaldehyde gel and blotted onto a Hybond-N membrane as recommended by the manufacturer (Amersham Corporation). The NOS2 cDNA was prepared as a template for further hybridization.

TNF-α was also measured by an ELISA kit (R & D system, MN, USA). The modulatory effects of wogonin on NOS2 mRNA were assayed with RT-PCR on cultured cells. Reverse transcription (RT) was performed using a reverse transcription kit. One μg of cellular RNA was used as a template and RT-generated cDNA encoding NOS2, TNF-α and glyceraldehyde-3-phosphate dehydrogenase (G3PDH, internal control) genes were amplified using polymerase chain reaction (PCR). The primers used in this study were NOS2 (762-bp), sense 5′-TAC-TGG-GTC-AAA-GAC-AAG-AGG-CTG-3′; antisense 5′-ATT-GGC-CAG-CTG-CTT-TTG-CAG-GA-3′; TNF-α (297-bp), sense 5′-GGC-AGG-TCT-ACT-TTG-GAG-TCA-TTG-C 3′, antisense 5′-CAG-TGA-ATT-CGG-AAA-GCC-CAT-TTG-AG 3′; G3PDH (983-bp), sense 5′-TGA-AGG-TCG-GTG-TGA-ACG-GAT-TTG-GC-3′, antisense 5′-CAT-GTA-GGC-CAT-GAG-GTC-CAC-CAC- 3′.

Twenty μl of RT-PCR product were determined by 2 % agarose gel electrophoresis containing 0.2 μg/ml ethidium bromide, followed by photography under ultraviolet transillumination. Quantification of RNA transcripts was analyzed according to the method described previously [17]. The PCR results were scanned by an imaging densitometer linked to a personal computer equipped with image analysis software. The size and optic density of the bands produced by the PCR products were quantified using the Kodak digital science 1D image analysis software (EDAS 120, Eastman Kodak Company, Rochester, New York, U.S.A.). For statistical comparison, more than 3 independent samples in each experiment were performed to obtain the ratio of the density of TNF-α to G3PDH mRNA levels, which was expressed as TNF-α/G3PDH ratio. The data in each experimental groups were analyzed and expressed as means ± S.E.M. Significance was determined by using one way ANOVA or Mann-Whitney rank sum test for between-groups comparison.

Acknowledgements

This work was supported by grants from National Science Council, ROC (NSC 88-2314-B-010-062) and Program for Promoting Academic Excellence of Universities Genome-Based Biomedical Research for the 21st Century (89-B-FA22-2-4).

References

• 1 Currier A R, Ziegler M H, Riley M M, Babcock T A, Telbis V P, Carlin J M. Tumor necrosis factor-alpha and lipopolysaccharide enhance interferon-induced antichlamydial indoleamine dioxygenase activity independently.  J Interf Cyto Res. 2000;  20 369-76
  PubMedSearch in Google Scholar
• 2 Murphey E D, Traber D L. Pretreatment with tumor necrosis factor-alpha attenuates arterial hypotension and mortality induced by endotoxin in pigs.  Crit Care Med. 2000;  28 2015-21
  PubMedSearch in Google Scholar
• 3 Daubener W, Remscheid C, Nockemann S, Pilz K, Seghrouchni S, Mackenzie C, Hadding U. Anti-parasitic effector mechanisms in human brain tumor cells: role of interferon-gamma and tumor necrosis factor-alpha.  Eur J Immunol. 1996;  26 487-92
  PubMedSearch in Google Scholar
• 4 Ten Hagen T L, Van Der Veen A H, Nooijen P T, Van Tiel S T, Seynhaeve A L, Eggermont A M. Low-dose tumor necrosis factor-alpha augments antitumor activity of stealth liposomal doxorubicin (DOXIL) in soft tissue sarcoma-bearing rats.  Int J Cancer. 2000;  87 829-37
  CrossrefPubMedSearch in Google Scholar
• 5 Chen Y C, Shen S C, Chen L G, Lee T J, Yang L L. Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide.  Biochem Pharmacol. 2001;  61 1417-27
  CrossrefPubMedSearch in Google Scholar
• 6 Huang R L, Chen C C, Huang H L, Chang C G, Chen C F, Chang C, Hsieh M T. Anti-hepatitis B virus effects of wogonin isolated from Scutellaria baicalensis .  Planta Medica. 2000;  66 694-8
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Gao Z, Huang K, Yang X, Xu H. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi.  Biochim Biophysic Acta. 1999;  1472 643-50
  PubMedSearch in Google Scholar
• 8 Ohtsuka M, Fukuda K, Yano H, Kojiro M. Effects of nine active ingredients in Chinese herbal medicine sho-saiko-to on 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide mutagenicity.  Jap J Cancer Res. 1995;  86 1131-5
  PubMedSearch in Google Scholar
• 9 Chi Y S, Jong H G, Son K H, Chang H W, Kang S S, Kim H P. Effects of naturally occurring prenylated flavonoids on enzymes metabolizing arachidonic acid: cyclooxygenases and lipoxygenases.  Biochem Pharmacol. 2001;  62 185-91
  PubMedSearch in Google Scholar
• 10 Kim H K, Cheon B S, Kim Y H, Kim S Y, Kim H P. Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships.  Biochem Pharmacol. 1999;  58 59-65
  CrossrefPubMedSearch in Google Scholar
• 11 Wakabayashi I. Inhibitory effects of baicalein and wogonin on lipopolysaccharide-induced nitric oxide production in macrophages.  Pharmacol Toxicol . 1999;  84 88-91
  PubMedSearch in Google Scholar
• 12 Kim H, Kim Y S, Kim S Y, Suk K. The plant flavonoid wogonin suppresses death of activated C6 rat glial cells by inhibiting nitric oxide production.  Neurosci Let. 2001;  309 67-71
  CrossrefPubMedSearch in Google Scholar
• 13 Kim H M, Moon E J, Li E, Kim K M, Nam S Y, Chung C K. The nitric oxide-producing activities of Scutellaria baicalensis .  Toxicology. 1999;  135 109-15
  CrossrefPubMedSearch in Google Scholar
• 14 Wu K K. Aspirin and salicylate: An old remedy with a new twist.  Circulation. 2000;  102 2022-3
  PubMedSearch in Google Scholar
• 15 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.  J Immunol Met. 1983;  65 55-63
  CrossrefPubMedSearch in Google Scholar
• 16 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.  Anal Biochem. 1987;  162 156-9
  PubMedSearch in Google Scholar
• 17 Wasser S, Ho J M, Ang H K, Tan C E. Salvia miltiorrhiza reduces experimentally-induced hepatic fibrosis in rats.  J Hepatol. 1998;  29 760-71
  CrossrefPubMedSearch in Google Scholar

Jen-Hwey Chiu MD, Ph. D.

Institute of Traditional Medicine

School of Medicine

National Yang-Ming University

155, Sec. 2, Li-Nong St.

Peitou

Taipei, 112

Taiwan. R.O.C.

Fax: +886-2-28757537

Email: chiujh@ mailsrv.ym.edu.tw

References

• 1 Currier A R, Ziegler M H, Riley M M, Babcock T A, Telbis V P, Carlin J M. Tumor necrosis factor-alpha and lipopolysaccharide enhance interferon-induced antichlamydial indoleamine dioxygenase activity independently.  J Interf Cyto Res. 2000;  20 369-76
  PubMedSearch in Google Scholar
• 2 Murphey E D, Traber D L. Pretreatment with tumor necrosis factor-alpha attenuates arterial hypotension and mortality induced by endotoxin in pigs.  Crit Care Med. 2000;  28 2015-21
  PubMedSearch in Google Scholar
• 3 Daubener W, Remscheid C, Nockemann S, Pilz K, Seghrouchni S, Mackenzie C, Hadding U. Anti-parasitic effector mechanisms in human brain tumor cells: role of interferon-gamma and tumor necrosis factor-alpha.  Eur J Immunol. 1996;  26 487-92
  PubMedSearch in Google Scholar
• 4 Ten Hagen T L, Van Der Veen A H, Nooijen P T, Van Tiel S T, Seynhaeve A L, Eggermont A M. Low-dose tumor necrosis factor-alpha augments antitumor activity of stealth liposomal doxorubicin (DOXIL) in soft tissue sarcoma-bearing rats.  Int J Cancer. 2000;  87 829-37
  CrossrefPubMedSearch in Google Scholar
• 5 Chen Y C, Shen S C, Chen L G, Lee T J, Yang L L. Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide.  Biochem Pharmacol. 2001;  61 1417-27
  CrossrefPubMedSearch in Google Scholar
• 6 Huang R L, Chen C C, Huang H L, Chang C G, Chen C F, Chang C, Hsieh M T. Anti-hepatitis B virus effects of wogonin isolated from Scutellaria baicalensis .  Planta Medica. 2000;  66 694-8
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Gao Z, Huang K, Yang X, Xu H. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi.  Biochim Biophysic Acta. 1999;  1472 643-50
  PubMedSearch in Google Scholar
• 8 Ohtsuka M, Fukuda K, Yano H, Kojiro M. Effects of nine active ingredients in Chinese herbal medicine sho-saiko-to on 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide mutagenicity.  Jap J Cancer Res. 1995;  86 1131-5
  PubMedSearch in Google Scholar
• 9 Chi Y S, Jong H G, Son K H, Chang H W, Kang S S, Kim H P. Effects of naturally occurring prenylated flavonoids on enzymes metabolizing arachidonic acid: cyclooxygenases and lipoxygenases.  Biochem Pharmacol. 2001;  62 185-91
  PubMedSearch in Google Scholar
• 10 Kim H K, Cheon B S, Kim Y H, Kim S Y, Kim H P. Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships.  Biochem Pharmacol. 1999;  58 59-65
  CrossrefPubMedSearch in Google Scholar
• 11 Wakabayashi I. Inhibitory effects of baicalein and wogonin on lipopolysaccharide-induced nitric oxide production in macrophages.  Pharmacol Toxicol . 1999;  84 88-91
  PubMedSearch in Google Scholar
• 12 Kim H, Kim Y S, Kim S Y, Suk K. The plant flavonoid wogonin suppresses death of activated C6 rat glial cells by inhibiting nitric oxide production.  Neurosci Let. 2001;  309 67-71
  CrossrefPubMedSearch in Google Scholar
• 13 Kim H M, Moon E J, Li E, Kim K M, Nam S Y, Chung C K. The nitric oxide-producing activities of Scutellaria baicalensis .  Toxicology. 1999;  135 109-15
  CrossrefPubMedSearch in Google Scholar
• 14 Wu K K. Aspirin and salicylate: An old remedy with a new twist.  Circulation. 2000;  102 2022-3
  PubMedSearch in Google Scholar
• 15 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.  J Immunol Met. 1983;  65 55-63
  CrossrefPubMedSearch in Google Scholar
• 16 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.  Anal Biochem. 1987;  162 156-9
  PubMedSearch in Google Scholar
• 17 Wasser S, Ho J M, Ang H K, Tan C E. Salvia miltiorrhiza reduces experimentally-induced hepatic fibrosis in rats.  J Hepatol. 1998;  29 760-71
  CrossrefPubMedSearch in Google Scholar

Jen-Hwey Chiu MD, Ph. D.

Institute of Traditional Medicine

School of Medicine

National Yang-Ming University

155, Sec. 2, Li-Nong St.

Peitou

Taipei, 112

Taiwan. R.O.C.

Fax: +886-2-28757537

Email: chiujh@ mailsrv.ym.edu.tw