Beneficial Effects of Fractions of Nardostachys jatamansi on Lipopolysaccharide-Induced Inflammatory Response

It has been previously shown that Nardostachys jatamansi (NJ) exhibits anti-inflammatory properties against lipopolysaccharide (LPS) challenges. However, the potency of NJ constituents against LPS-induced inflammatory responses has not been examined. In this present study, we determined which NJ extract fractions exhibit inhibitory effects against LPS-induced inflammatory responses. Among the NJ fractions, NJ-1, NJ-3, NJ-4, and NJ-6 inhibited LPS-induced production of NO. The NJ-3, NJ-4, and NJ-6 fractions also inhibited the production of cytokines, such as IL-1β, IL-6, and TNF-α. However, NJ-1, NJ-3, NJ-4, and NJ-6 showed differential inhibitory mechanisms against LPS-induced inflammatory responses. NJ-1, NJ-3, and NJ-4 inhibited LPS-induced activation of c-jun NH2-terminal kinase (JNK) and p38 but did not affect activation of extracellular signal-regulated kinase (ERK) or NF-κB. On the other hand, NJ-6 inhibited activation of MAPKs and NF-κB. In addition, in vivo experiments revealed that administration of NJ-1, NJ-3, NJ-4, and NJ-6 reduced LPS-induced endotoxin shock, with NJ-6 especially showing a marked protective effect. Taken together, these results provide the evidence for the potential of selective NJ fractions against LPS-induced inflammation. Thus, it will be advantageous to further isolate and determine single effective compounds from these potent fractions.


Introduction
Inflammation is a complex immunologic response to harmful stimuli, including various pathogens, irritants, and infections [1]. Infectious agents, such as bacteria and proinflammatory cytokines, can activate macrophages, immune cells that are critically involved in the regulation of innate immunity, through certain receptors [2]. The interaction between Tolllike receptor (TLR)-4 and the ligand lipopolysaccharide (LPS) induces an intracellular signaling cascade that activates the mitogen-activated protein kinase (MAPK) family, extracellular signal-related kinase (ERK), p38, c-jun NH 2 -terminal kinase (JNK), and key proinflammatory transcription factors such as nuclear factor-kappa B (NF-B) [3]. Subsequent to TLR-4-LPS interaction, macrophages release various inflammatory mediators, such as nitric oxide (NO), and proinflammatory cytokines including interleukin (IL)-1 , IL-6, and tumor necrosis factor alpha (TNF-) [4].
Nardostachys jatamansi (NJ) is widely used as a bitter tonic and antispasmodic [5]. It has been reported that the root of NJ contains various sesquiterpenes, including jatamansic acid, and jatamansone, lignans, and neolignans. We previously reported that aqueous extract of NJ is effective in protecting against inflammatory challenges [6][7][8][9][10], particularly against LPS-induced inflammation and endotoxin shock [7]. In particular, one fraction of NJ extract (fraction  4) demonstrated a protective effect against cerulein-induced acute pancreatitis [11]. However, although many of our studies have shown the anti-inflammatory activities of NJ, it is not yet known which particular compound of NJ has the potential to inhibit LPS-induced inflammation. Therefore, to take one step closer to actual bioactive compounds from NJ, we used NJ fractions, which have many bioactive compounds. In this study, we performed in vitro and in vivo analyses to examine whether the fractions of NJ have potential inhibitory effects against LPS-induced inflammation in murine peritoneal macrophages and in an animal model of LPS-induced endotoxin shock. Moreover, to elucidate a potential molecular anti-inflammatory mechanism, we examined the activation of MAPKs and NF-B.  plate reader. The number of NJ fraction-treated viable cells was expressed as a percentage of the control (untreated cells) maintained for the same time period.

Measurement of NO Concentration.
Murine peritoneal macrophages (2 × 10 5 cells/well) were pretreated with NJ fractions for 1 h and then stimulated with LPS (500 ng/mL) for 24 h. To measure NO concentration, 100 L aliquots were removed from conditioned media and incubated with an equal volume of Griess reagent at room temperature for 10 min. The absorbance at 540 nm was then measured.

ELISA.
Murine peritoneal macrophages (1 × 10 6 cells/well) were stimulated with 500 ng/mL of LPS and/or various concentrations of NJ fraction for 24 h. Culture supernatants were collected and stored at −80 ∘ C until use. Cytokine levels in supernatants were determined using a commercial system (R&D Systems) according to the manufacturer's instructions. The ELISA was devised by coating 96-well plates with antibodies specific for IL-1 , IL-6, and TNF-. Coated plates were washed with PBS containing 0.05% Tween-20. All reagents used in this assay were incubated overnight at 4 ∘ C.
Recombinant IL-1 , IL-6, and TNF-were diluted and used as a standard. Serial dilutions starting at 20 ng/mL were used to establish the standard curve. Assay plates were exposed sequentially to biotinylated mouse IL-1 , IL-6, and TNF-, avidin peroxidase, and a substrate solution of 2,2 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing 30% hydrogen peroxide. The plates were read at 405 nm.   a nitrocellulose membrane. The membrane was then blocked with 5% skim milk in PBS-Tween-20 for 2 h at room temperature and incubated overnight with antibodies against phosphorylated ERK 1/2, phosphorylated JNK, phosphorylated p38, and I -B . After washing three times, each blot was incubated with a secondary antibody for 1 h, and immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Amersham, Piscataway, NJ, USA) according to the manufacturer's recommended protocol.

Cytotoxicity of NJ Fractions in Peritoneal Macrophages.
The first approach to study the biological activity of any compound or plant extract is to ensure lack of effect on cellular metabolism. To determine whether fraction of NJ affects cell viability, peritoneal macrophages were incubated for 24 h with varying concentrations of extract ( g/mL), and cell viability was evaluated by MTT assay. NJ-1, NJ-2, NJ-3, and NJ-6 did not demonstrate any significant cytotoxic effects on peritoneal macrophages. However, at a relatively higher dose, NJ-4 and NJ-5 showed significant cytotoxicity ( # < 0.001) (Figure 1). Therefore, because higher levels of NJ-4 (≥100 g/mL) and NJ-5 (≥50 g/mL) could affect cellular metabolism, we excluded data of NJ-4 and NJ-5 at these concentrations.

Effects of NJ Fractions on LPS-Induced NO Production.
Next, to examine the anti-inflammatory effect of NJ fractions, we measured NO production. Murine peritoneal macrophages were pretreated with the indicated concentrations of NJ fractions for 1 h and then stimulated with LPS (500 ng/mL) for 24 h. As shown in Figure 2, LPS stimulation increased NO production. However, pretreatment with NJ-1, NJ-3, NJ-4, and NJ-6 significantly inhibited LPS-induced production of NO but not with NJ-2 and NJ-5 ( Figure 2). on LPS-induced proinflammatory cytokine production, peritoneal macrophages were pretreated with NJ fractions at the indicated concentrations for 1 h and then stimulated with 500 ng/mL of LPS for 24 h. In particular, we treated cells with NJ-1, NJ-3, NJ-4, and NJ-6 fractions, which demonstrated inhibition of NO production. As shown in Figures 3 and 4, protein and mRNA levels of IL-1 , IL-6, and TNF-were increased by LPS stimulation. However, pretreatment with NJ-3, NJ-4, and NJ-6 inhibited the production of IL-1 , IL-6, and TNF-significantly but not with NJ-1 fraction (Figures 3  and 4).

Effects of NJ Fractions on LPS-Induced Endotoxin Shock.
To verify effects of NJ fractions in vivo, we used a model LPSinduced endotoxin shock. NJ fractions (NJ-1, NJ-3, NJ-4, or NJ-6) were injected at the indicated doses. After 3 h, a lethal dose of LPS (37.5 mg/kg) was administered intraperitoneally. The survival rate was recorded every 12 h after LPS treatment. Generally, LPS-injected septic mice died within 48 h. Consistent with in vitro experiments, NJ fractions exhibited strong anti-inflammatory activities in vivo, as shown by significant inhibition of LPS-induced death in the treated mice ( Figure 7). In particular, NJ-4 and NJ-6 showed the most dramatic protection against LPS-induced endotoxin shock; these fractions prevented nearly all harmful effects of LPS (Figures 7(c) and 7(d)).

Discussion
Recently, our laboratory reported the beneficial effects of NJ on various diseases, such as endotoxin shock, pancreatitis, and diabetes [6][7][8][9][10]. Particularly in endotoxin shock, NJ showed a marked preventive effect and strong therapeutic potential against LPS challenge [7]. Although many of our studies have indicated the benefits of NJ on inflammatory diseases, which compounds of NJ could exhibit antiinflammatory properties are unknown. Thus, in this paper, bringing more bright insight to identify the constituent compounds from NJ, we selected and assorted effective fractions that would possess anti-inflammatory potential. Overall, we obtained six fractions from NJ. Using the fourth fraction, we evaluated the protective effects on cerulein-induced acute pancreatitis (AP). However, the sixth fraction remained to be examined for potential to prevent an inflammatory response to stimuli such as LPS. Therefore, in this study, we used six fractions isolated from NJ to examine the anti-inflammatory activity against LPS on murine macrophages.
Generally, sepsis is a life threatening condition caused by the body's response to bacterial products, such as endotoxin, which is often present with severe fever, shock, and respiratory damage [12]. Partially due to this phenomenon, sepsis is often regarded as an uncontrolled inflammatory response [12]. In the context of sepsis, TNF-, IL-1 , IL-6, and IL-8 are the most frequently altered cytokines [13]. When injected into animals, these cytokines recapitulate many clinical and laboratory features of sepsis, supporting the concept that sepsis represents a "cytokine storm. " Thus, regulation of cytokines may prove valuable for the prevention and treatment of sepsis. In this study, just as the total extract of NJ inhibited the production of cytokines [7], fractions 3, 4, and 6 also inhibited the production of cytokines (Figures 3 and 4). These results suggest that the ability of NJ to inhibit cytokine production may originate from fractions 3, 4, and/or 6.
Using the fractions that were found to be effective against LPS, we examined activation of MAPKs and NF-B as possible regulatory mechanisms. Generally, it has been reported that the production of proinflammatory mediators is modulated by the MAPKs and NF-B pathways. Three major groups of MAPKs include the ERK, JNK, and p38, which are all activated by phosphorylation [14][15][16]. NF-B activation occurs mainly through the degradation of I B, thereby leading to subsequent release of NF-B dimers [17]. Subsequently, NF-B dimers translocate from the cytoplasm to the nucleus and activate the transcription of multiple Bdependent target genes [17]. In this study, we examine the phosphorylation of MAPKs and degradation of I -B . NJ-1, NJ-3, and NJ-4 inhibited the phosphorylation and activation of JNK and p38 but not ERK1/2. However, NJ-6 inhibited not only the activation of ERK1/2, JNK, and p38 but also  Endotoxin shock was induced in 6-8-week-old female C57BL/6 mice, as described in Section 2. NJ fractions were administered intraperitoneally to mice at 0.1, 1, or 10 mg/kg 1 h before LPS injection. Then, LPS (37.5 mg/kg) was injected to induce endotoxin shock. The survival rates of endotoxin shock mice were monitored for survival for 120 h. This figure shows data for 10 mice per group.
degradation of I -B . These results suggest that inhibition of NO and cytokine production by NJ fractions is primarily through MAPKs or NF-B pathways.
In this study, we isolated two beneficial fractions of NJ. The first one was NJ-4, which could show similar effects to NJ total extract. NJ-4 inhibited cytokine production and LPS-induced endotoxin shock similar to NJ total extract [7]. Furthermore, the regulatory mechanisms were similar; NJ total extract inhibited only MAPKs, but NJ-4 inhibited the JNK and p38. Secondly, NJ-6 showed dramatic inhibition of LPS-induced inflammatory response. Indeed, NJ-6 inhibited the cytokine production and LPS-induced endotoxin shock dramatically. However, NJ-6 differed from total extract mechanistically; NJ-6 inhibited the degradation of I -B and translocation of NF-B p65, which was not regulated by the NJ total extract. Further isolation of the NJ-4 and NJ-6 fractions could potentially yield a single compound demonstrating inhibitory activity resembling that of NJ total extract.
In conclusion, this study demonstrated which fractions of NJ exhibit inhibitory activity against LPS-induced inflammation. Among the 6 fractions isolated, NJ-1, NJ-3, NJ-4, and NJ-6 showed the inhibition of NO, and NJ-3, NJ-4, and NJ-6 showed the inhibition of cytokines. In addition, NJ-4 and NJ-6 exhibited dramatic prevention against LPS-endotoxin shock. These results suggest that NJ-4 and NJ-6 may be effective and beneficial candidates to ameliorate LPS-induced inflammation.