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Oleanane-triterpenoids from Panax stipuleanatus inhibit NF-κB

In continuation of our research to find biological components from Panax stipuleanatus, four oleanane-type triterpenes (12 to 15) were isolated successively. Fifteen oleanane-type saponins (1 to 15) were evaluated for nuclear factor (NF)-κB activity using a luciferase reporter gene assay in HepG2 cells. Compounds 6 to 11 inhibited NF-κB, with IC50 values between 3.1 to 18.9 μM. The effects on inducible nitric oxide synthase and cyclooxygenase-2 by compounds 8, 10, and 11 were also examined using reverse transcription-polymerase chain reaction. Three compounds (8, 10, and 11) inhibited NF-κB activity by reducing the concentration of inflammatory factors in HepG2 cells.


INTRODUCTION
Nuclear factor (NF)-κB is a dimeric transcription factor that activates the expression of many genes involved in inflammation, e.g., cytokines interleukin (IL)-1β, IL-2 and tumor necrosis factor (TNF)-α, adhesion molecules, and enzymes including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and 5-lipoxygenase [1,2]. Without stimulation, NF-κB is inactive, but it can be activated by extracellular signals such as TNF-α, IL-1, lipopolysaccharide (LPS), UV light, and phorbol esters [3]. In unstimulated cells, NF-κB is retained in the cytoplasm via interaction with its inhibitor, NF of kappa light polypeptide gene enhancer in B-cells inhibitor (IkB). In response to various pro-inflammatory stimuli, IκB is phosphorylated by the IκB kinase complex. This leads to the ubiquitination and subsequent proteasome-mediated degradation of IκB, allowing NF-κB to enter the nucleus [4]. NF-κB is highly activated at the site of inflammation in diverse diseases such as rheumatoid arthritis, atherosclerosis, asthma, inflammatory bowel disease, and Helicobacter pylori-associated gastritis, and it is associated with cancer, cachexia, diabetes, euthyroid sick syndrome, and AIDS. Due to its apparent involvement in a variety of human diseases, NF-κB has been the target of several anti-inflammatory and anti-cancer drugs [5]. In addition, many of the triterpenoids are reported to have antiinflammatory activity by regulating NF-κB [6].
Panax stipuleanatus Tsai et Feng (Araliaceae) is an herb that grows in southeast Yunnan, China and north Vietnam. In China, this plant has been used traditionally as a tonic and in the treatment of bruises, bleeding, and muscular pain [7]. To-date, oleanane-type triterpenoid has been reported as the major component of P. stipuleanatus [8], and stipuleanosides R1 and R2 have been isolated from the methanol extract of this plant [7]. In a previous chemical investigation of this plant, we isolated eleven oleanane-type triterpenoids (1 to 11), and investigated their anti-cancer activity in HL-60 and HCT-116 human cancer cell lines [9]. In continuation of our research into the biological components of P. stipuleanatus, four oleanane-type triterpenes (12 to 15) were isolated successively from the methanol extract (Fig. 1). Their structures were assigned through spectroscopic data analysis using 1 H and 13 C nuclear magnetic resonance (NMR), correlation spectroscopy, heteronuclear multiple spectroscopy, heteronuclear multiple bond correlation, and mass spectroscopy, and were compared with data in the literature. This paper evaluated the effects of compounds 1 to 15 on TNF-α-induced NF-κB activation in HepG2 cells.

General experimental procedures
Preparative HPLC was carried out using a Waters

Plant materials
The rhizomes of P. stipuleanatus were collected in Vietnam in 2007 and taxonomically identified by Dr. Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnam. The reference specimen (VHKC-0044) has been deposited at the Institute of Natural Products Chemistry,Vietnam Academy of Science and Technology, Vietnam.

Cytotoxicity assay
Cell Titer 96 AQUEOUS non-radioactive cell proliferation assay (MTS; Promega, Madison, WI, USA) was used to analyze the effect of compounds on cell viability. Cells were cultured overnight in 96-well plate1×10 4 cells/well). Cell viability was assessed after the addition of compounds at the 10 mM concentrations for 24 h. The number of viable cells was assessed by determination of the A 490nm of the dissolved formazan product after addition of MTS for 30 min as described by the manufacturer (Promega).

Statistical analysis
All results are expressed as the mean±SEM. Data was analyzed by one-factor analysis of variance. Upon observation of a statistically significant effect, the Newman-Keuls test was performed to determine the difference between the groups. A p-value of <0.05 was considered to be significant.

Effect of the compounds 8, 10, and 11 on inducible nitric oxide synthase and cyclooxygenase-2 gene expression through the nuclear factor-κB pathway
To confirm the inhibition of NF-κB activity produced by these compounds in the reporter assay, compounds 8, 10, and 11 were selected to examine the effects on the expression of NF-κB target genes by reverse transcription -Fig. 2. Effect of compounds on tumor necrosis factor (TNF)-a-induced nuclear factor (NF)-kB luciferase reporter activity in HepG2 cell lines.
HepG2 cells transiently transfected with pNF-kB-Luc were pretreated for 1 h with either vehicle dimethyl sulfoxide and compounds, prior to 1 h of treatment with TNF-a (10 ng/mL). Unstimulated HepG2 cells acted as a negative control. Cells were then harvested and luciferase activities were assessed. Results are expressed as relative luciferase activity. Comp, compound.  polymerase chain reaction. In HepG2 cells, treatment with TNFα (10 ng/mL) significantly upregulated mRNA expression levels of NF-κB target genes, COX-2 and iNOS, by approximately 5.2-and 3.5-fold, respectively. The relative levels of mRNAs were assessed by RT-PCR. Expression levels are displayed as the ratio of iNOS and COX-2 signal strength to a reference gene (GAPDH), compensating for variations in the RNA concentrations [14]. Compounds 8, 10, and 11 significantly inhibited the induction of iNOS mRNA by 30-, 5-and 3-fold, and COX-2 mRNA by 17-, 4-, and 2-fold at 10 µM in dosedependent manner, indicating that these compounds attenuated synthesis of these transcripts at the transcriptional level (Fig. 4).
COX-2 and iNOS are two inducible enzymes that are strongly expressed upon stimulation by diverse factors, such as oxidants, inflammatory factors, LPS, and oncogenes [18]. Recent studies have demonstrated that eukaryotic transcription of NF-κB is involved in the regulation of COX-2 and iNOS expression [19]. Accordingly, many substances developed to-date prevent inflammatory damage by either suppressing the activation of iNOS or COX-2 directly, or by inhibiting NF-κB signaling, which regulates iNOS and COX-2 at the transcriptional level [20].
This study demonstrates that compounds 8, 10, and 11 inhibit TNF-α-induced NF-κB promoter activity in a dose-dependent manner by interfering with gene transcription and the expression of two inducible enzymes, iNOS and COX-2, at the transcriptional level, as demonstrated by reduced mRNA levels in TNF-α-treated HepG2 cells. Our data suggest that compounds 8, 10, and 11 isolated from P. stipuleanatus have therapeutic potential as anti-inflammatory, anti-atherosclerotic, and antiarthritic substances. However, elucidation of the detailed inhibitory mechanisms of the TNF-α-induced NF-κB pathway and subsequent decreases in iNOS and COX-2 gene expression with compounds 8, 10, and 11 requires further investigation.
In summary, this study is the first to demonstrate inhibition of NF-κB activity by compounds isolated from P. stipuleanatus. Additionally, all these data support the Fig. 4. Effect of compounds 8, 10, and 11 on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mRNA expression in HepG2 cell lines. HepG2 cells were pretreated in the absence and presence of compounds 8, 10, and 11 for 1 h before tumor necrosis factor (TNF)-α treatment (10 ng/mL), then exposed to TNF-α for 6 h. Total mRNAs were prepared from the cell pellets using Easy-blue reagent. The relative levels of mRNAs were assessed by reverse transcriptase polymerase chain reaction. Expression levels are displayed as the ratio of iNOS and COX-2 signal strength to a reference gene (GAPDH), compensating for variations in the RNA concentrations. Comp, compound.
pharmacological use of P. stipuleanatus, which has been traditionally employed as an herbal medicine for the treatment of inflammatory diseases.