Inhibition of TNF-α-mediated NF-κB Transcriptional Activity in HepG2 Cells by Dammarane-type Saponins from Panax ginseng Leaves

Panax ginseng (PG) is a globally utilized medicinal herb. The medicinal effects of PG are primarily attributable to ginsenosides located in the root and leaf. The leaves of PG are known to be rich in various bioactive ginsenosides, and the therapeutic effects of ginseng extract and ginsenosides have been associated with immunomodulatory and anti-inflammatory activities. We examined the effect of PG leaf extract and the isolated ginsenosides, on nuclear factor (NF)-κB transcriptional activity and target gene expression by applying a luciferase assay and reverse transcription polymerase chain reaction in tumor necrosis factor (TNF)-α-treated hepatocarcinoma HepG2 cells. Air-dried PG leaf extract inhibited TNF-α-induced NF-κB transcription activity and NF-κB-dependent cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) gene expression more efficiently than the steamed extract. Of the 10 ginsenosides isolated from PG leaves, Rd and Km most significantly inhibited activity in a dose-dependent manner, with IC50 values of 12.05±0.82 and 8.84±0.99 μM, respectively. Furthermore, the ginsenosides Rd and Km inhibited the TNF-α-induced expression levels of the COX-2 and iNOS gene in HepG2 cells. Air-dried leaf extracts and their chemical components, ginsenoside Rd and Km, are involved in the suppression of TNF-α-induced NF-κB activation and NF-κB-dependent iNOS and COX-2 gene expression. Consequently, air-dried leaf extract from PG, and the purified ginsenosides, have therapeutic potential as anti-inflammatory.


INTRODUCTION
Although inflammation is a basic response to injury or infectious agents, the harmful effects of inflammation cause various chronic diseases including arthritis, fibrosis, and even cancer. Inflammatory responses play important roles during liver diseases in humans and animals. Hepatitis results from inflammation of the liver, characterized by infl ammatory cells present in the organ tissue. Hepatitis can be acute (lasting less than 6 months) or chronic, when it persists longer. Chronic hepatitis is associated with a high risk of liver cancer [1]. At the molecular level, free radicals and aldehydes produced during chronic hepatitis can induce deleterious gene mutations and posttranslational modifi cations in cancer-associated genes [2,3]. Other infl ammatory products, including cytokines, growth factors, and transcription factors such as nuclear factor (NF)-κB, control the expression of cancer genes (e.g., suppressor genes and oncogenes) and key inflammatory enzymes, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 [4-7].
NF-κB is known to play an important role in immune and infl ammatory responses by regulating genes encoding pro-inflammatory cytokines, adhesion molecules, chemokines, growth factors, and inducible enzymes such as COX-2 and iNOS [8,9]. Therefore, NF-κB signaling pathway inhibitors could be applied as novel anti-infl ammatory compounds [10,11].
Panax ginseng (PG) Meyer (Araliaceae), a herbal drug applied in Oriental traditional medicine, has been used as a tonic for the treatment of various diseases [12][13][14]. Ginsenosides are the major active components of the PG. Despite the fact that PG root is considered the most valuable, the leaves contain the highest concentrations of ginsenosides [15][16][17]. Thus, ginseng leaves could function as a supplementary source of pharmacologically active ginsenosides [18,19].
Traditionally, PG root has been air-dried, yielding white ginseng, or steamed at 100°C to yield red ginseng. Steamed ginseng is believed to be more pharmacologically effective than air-dried ginseng. Differences in the biological effects of air-dried and steamed ginseng are attributed to signifi cant ginsenoside changes during the steaming treatment [20]. However, the anti-infl ammatory effects of air-dried and steamed leaves of PG have not been studied.
In our study, the MeOH extract of PG air-dried leaves signifi cantly inhibited NF-κB activity, with an IC 50 value of 39.12±2.79 µg/mL. Here, we characterized the effects of 10 dammarane-type saponins from PG air-dried leaves on tumor necrosis factor (TNF)-α-induced NF-κB activation, as well as iNOS and COX-2 gene expression, in HepG2 cells.

Chemicals and sample preparation
In August 2008, PG leaves were collected in Geumsan, Korea, which is well-known for PG cultivation, and were taxonomically identifi ed. A voucher specimen (CNU 08201) was deposited at the College of Pharmacy, Chungnam National University, Korea. PG air-dried leaves were extracted in MeOH (6.0 L×3, 50°C) and the combined extracts were lyophilized in vacuo. Ten ginsenosides, identifi ed in our previous study, were isolated from PG air-dried leaves, i.e., ginsenosides F 1 , Rb 1 , Rb 2 , Rc, Rd, Re, Rg 1 , Ki, Km, and vinaginsenoside R 4 [16] ( Fig. 1). To compare the anti-infl ammatory activity of airdried and steamed leaves, ginseng leaves were steamed at 120°C for 4 h under 0.15 MPa pressure, without mix-ing, to yield the steamed-leaves sample, which was used for extraction. Pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Luciferase assay
Cells were seeded at 1×10 5 cells per well in a 12-well plate and grown for 24 h. All cells were transfected using lipofectamine LTX (Invitrogen), as described by the manufacturer. Luciferase activity was assayed using an LB 953 Autolumat (EG&G Berthold, Nashua, NH, USA), as described previously [21] and was normalized based on the expression of RSV-β-galatosidase. β-galactosidase activity was assayed colorimetrically. NF-κB-Luc and iNOS-Luc plasmid were kindly provided by Dr. Kyoon E. Kim (Chungnam National University, Daejeon, Korea).

Cytotoxicity assay
An MTS assay (Promega Celltiter 96-Aqueous One Solution Assay; Promega, Madison, WI, USA) was used to analyze the effects of the compounds on cell viability. Cells were cultured overnight in a 96-well plate (~1×10 4 cells/well). Cell viability was assessed after adding the compounds at 10 µM for 24 h. The number of viable cells was determined by 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 ANOVA. Upon observation of a statistically signifi cant effect, the Newman-Keuls test was performed to determine the difference between the groups. A p-value of less than 0.05 was considered to be signifi cant.

Nuclear factor-κB inhibitory effect of Panax ginseng leaf extract in HepG2 cells
To evaluate and compare the anti-infl ammatory activity of ginseng leaves, the nuclear transcription NF-κB cell-reporter system was applied. The anti-infl ammatory activities of air-dried and steamed leaf MeOH extracts were measured using a luciferase assay in HepG2 cells. Extracts of air-dried leaves reduced NF-κB activation by 81% at 100 µg/mL and 43% at 10 μg/mL in a dose-dependent manner. Alternatively the steamed leaf extracts reduced NF-κB activation by 20% at 100 μg/mL (Fig.  2). This suggests that air-dried leaf extracts possess more anti-infl ammatory effects than steamed leaves by inhibiting NF-κB in HepG2 cells.

Eff ect of Panax ginseng leaf extracts on inducible nitric oxide synthase and cyclooxygenase-2 gene expression in HepG2 cells
To evaluate the anti-infl ammatory activity of ginseng leaves which inhibited NF-κB activation in the luciferase assay, the effects of air-dried leaves and steamed leaf extracts on NF-κB target gene expression were evaluated by reverse transcription (RT)-PCR and iNOS promoter luciferase assay. HepG2 cells treated with TNF-α (10 ng/mL) had significantly upregulated iNOS and COX-2 mRNA expression (Fig. 3A) and iNOS promoter regulation (Fig. 3B). Air-dried leaf extracts signifi cantly decreased iNOS and COX-2 mRNA and iNOS promoter activity compared to TNF-α-treated HepG2 cells (Fig.  3). Alternatively the steamed leaf extracts did not significantly inhibit gene expression (Fig. 3A). Therefore, airdried leaves showed potent anti-infl ammatory activity by inhibiting NF-κB activation and iNOS and COX-2 gene expression.

Effect of ginsenosides Rd and Km on inducible nitric oxide synthase and cyclooxygenase-2 gene expression through the nuclear factor-κB pathway
To identify novel NF-κB inhibitors from PG air-dried leaf extracts, 10 dammarane-type ginsenosides were evaluated using the lucifease assay and RT-PCR. Initially, to determine nontoxic concentrations, HepG2 cells were treated with various concentrations (0.1, 1, and 10 μM) of the compounds and cell viability was measured  leaves on tumor necrosis factor (TNF)-α stimulated nuclear factor (NF)-κB transcriptional activity. HepG2 cells transiently transfected with pNF-κB-Luc were pretreated for 1 h with either vehicle (dimethyl sulfoxide) or extracts, then exposed to TNF-α (10 ng/mL) for 1 h. Unstimulated HepG2 cells acted as a negative control. Cells were then harvested and assayed for luciferase activity. Results are expressed as the mean luciferase activity±SEM (n=3). PDTC, pyrrolidine dithiocarbamate. *Signifi cantly different from TNF-α stimulated group (p<0.05).
using an MTS assay. No compounds were signifi cantly cytotoxic at up to 10 μM, indicating that NF-κB inhibition was not due to cell toxicity (Fig. 4A). HepG2 cells were pretreated with various ginsenosides at concentrations ranging from 0.1 to 10 µM for 1 h and then induced with TNF-α for 20 h. Of these ginsenosides, Rd and Km signifi cantly inhibited TNF-α-induced NF-κB transcriptional activity with IC 50 values of 12.05±0.82 and 8.84±0.99 µM, respectively (Table 1 and Fig. 4B), while ginsenosides Rb 1 , Rg 1 , and F 1 reduced NF-κB transcriptional activity with IC 50 values of 27.45±3.69, 28.14±0.28, and 22.36±2.20 µM, respectively. Ginsenoside Rb 2 and vinaginsenoside R 4 weakly inhibited NF-κB transcriptional activity (Table 1). Furthermore, ginsenosides Rc, Re, and Ki had no effects (Table 1). PDTC, as a positive control, potently inhibited NF-κB transcriptional activity with an IC 50 value of 3.35±0.61 µM. Ginsenoside Rd and Km also inhibited expression of COX-2 and iNOS mRNA (Fig. 5A) and iNOS promoter activity in a dose-dependent manner (Fig. 5B). Therefore, ginsenoside Rd and Km showed potent antiinflammatory activity by inhibiting NF-κB activation and iNOS and COX-2 gene expression. h, then exposed to tumor necrosis factor (TNF)-α (10 ng/mL) for 6 h. Total mRNA was extracted from the cell pellets using Easy-Blue reagent. The relative mRNA levels were assessed by reverse transcription polymerase chain reaction. Expression levels are displayed as the ratio of iNOS and COX-2 signal strength to a reference gene (GAPDH) to compensate for varying concentrations of RNA. (B) HepG2 cells transiently transfected with piNOS-Luc were pretreated for 1 h with either vehicle (DMSO) or extracts, then exposed to TNFα (10 ng/mL) for 1 h. Unstimulated HepG2 cells acted as a negative control. Cells were then harvested and assayed for luciferase activity. Results are expressed as the mean luciferase activity±SEM (n=3). PDTC, pyrrolidine dithiocarbamate. *Signifi cantly different from TNFα stimulated group (p<0.05).  Vol. 36, No. 2, 146-152 (2012)

DISCUSSION
This study demonstrated that PG air-dried leaf extract, and its components, Rd and Km, inhibit TNF-α-induced NF-κB promoter activity in HepG2 cells in a dosedependent manner by interfering with gene transcription. Air-dried leaf extract, Rd and Km also inhibited the expression of two inducible enzymes, COX-2 and iNOS, at the transcriptional level, as demonstrated by reduced mRNA levels in TNF-α-treated HepG2 cells. Ginsenoside Rd was recently reported to suppress the inflammatory response by inhibiting expression of iNOS and COX-2 in rats, unexplained the regulatory mechanism [22]. In our study, we report the NF-κB pathway, as a potential mechanism, by ginsenoside Rd in infl ammation. To our knowledge, we are the fi rst to report that air-dried leaf extracts and ginsenoside Km have an inhibitory effect on TNF-α-induced NF-κB activation as well as iNOS and COX-2 expression in HepG2 cells.
Ginseng extracts and ginsenosides were previously shown to inhibit the NF-κB pathway [23]. Saponins and n-butanol extracts of P. notoginseng (PNG) also inhibit the NF-κB pathway in zymosan A-and collagen-induced mice [24]. Flower extracts from PNG attenuate the infarct volume in rat ischemic brain and the inflammatory response of microglia cells [25]. Also, the PG acid polysaccharide extract, ginsan, inhibits the production of interleukin (IL)-1β, IL-6, IL-12, IL-18, and interferonγ and the activation of MAPK, c-Jun N-terminal kinase, and NF-κB pathways in Staphylococcus aureus-infected mice [26,27]. However, the NF-κB inhibitory effect of PG leaf extracts had not been evaluated.
In this study, ginsenosides Rb 1 , Rg 1 , and F 1 reduced NF-κB transcriptional activity and ginsenoside Rb 2 and vinaginsenoside R 4 showed very low inhibitory activities (Table 1). Furthermore, ginsenosides Rc, Re, and Ki had no effects. In corroboration of our results, a study reported that ginsenoside Rb 1 inhibits the expression vascular cell adhesion molecule (VCAM)-1 by inhibiting the TNF-α-induced NF-κB pathway in human umbilical vein endothelial cells [28]. Also, ginsenoside Rg 1 inhibits  iNOS production in mice injected with LPS and VCAM-1 as well as ICAM-1 production in ECV304 human endothelial cells induced by TNF-α [29]. Rb 2 potently inhibited TNF-α production in LPS-induced N9 microglia [30]. Vinaginsenoside R 4 , identifi ed from P. vietnamensis [31], had not previously been characterized. Ginsenoside F 1 protects human HaCat keratinocytes from ultraviolet-B-induced apoptosis [32]. However, the anti-infl ammatory and NF-κB inhibitory effects of ginsenoside F 1 had not been described. This is the fi rst report of vinaginsenoside R 4 and ginsenoside F 1 inhibiting TNF-α-induced NF-κB activation as well as iNOS and COX-2 gene expression in HepG2 cells. Although vinaginsenoside R 4 and ginsenoside F 1 may play the potential anti-infl ammatory compound, further studies are necessary to determine the precise mechanism on TNF-α-mediated NF-κB activation and infl ammation.
In conclusion, we demonstrated that air-dried leaf extracts and their chemical components, ginsenoside Rd and Km, are involved in the suppression of TNFα-induced NF-κB activation; this was shown in a gene reporter assay by a reduction in luciferase activity, which subsequently inhibited iNOS and COX-2 gene expression. Our data demonstrated that air-dried leaf extract from PG and the purifi ed ginsenosides, have therapeutic potential as anti-infl ammatory, anti-atherosclerotic, antiarthritic, anti-oxidative and antiallergic substances. However, detailed mechanisms of TNF-α-induced NF-κB pathway inhibition and subsequent decreases in iNOS and COX-2 gene expression with air-dried leaf extracts from PG and ginsenosides require further investigation.