NF-κB and MAPK-Targeted Anti-Inflammatory Activity of Andrographis Paniculata Extract

Andrographis paniculata (Burm.f.) is in the Acanthaceae family. There have been reports on the various medical effects of extracts of A. paniculata and the active ingredient, andrographolide (AG). Previous study confirmed that the standardized A. paniculata extract (APE, ParActin ® ) inhibited the expression of MMPs and inflammatory cytokines (IL-1β and IL-6) in monosodium iodoacetate (MIA)-induced cartilage degradation in rats, and relieved acetic acid-induced writhing responses in mice. To investigate the molecular actions of APE, we treated APE and AG, and examined the level of inflammatory mediators in LPS-treated RAW264.7 cells. APE and AG inhibited NO and PGE 2 production in LPS-treated RAW264.7 macrophages. APE inhibited MAPK pathways (p38, JNK, and ERK) and IκBα phosphorylation, and increased IκBα degradation. The results revealed that anti-inflammatory activity of APE were derived from AG by inhibition of MAPKs and NF-κB pathways.

In a prospective, randomized, and placebo-controlled trial, A. paniculata composition, ParActin ® showed some beneficial effects on osteoarthritis patient symptoms [9]. Furthermore, treatment with A. paniculata showed a reduction in the stiffness score, fatigue, and pain in RA patients [10]. In human osteoarthritic chondrocytes, andrographolide reduces matrix metalloproteinases (MMPs) and inducible nitric oxide synthase (iNOS) [11].
There have been reports on andrographolide's antiinflammatory efficacy in several literatures. The major diterpenoid constituent of A. paniculata, andrographolide has received wide attention of many research groups for its diverse pharmacological properties by inhibition of NF-κB and MAPK pathways [12].

Preparation of APE
The leaf extracts of Andrographis paniculata Nees.
(Acanthaceae) used in the experiment were purchased from HP Ingredients (Bradenton, USA). The dried leaf of A. paniculata was extracted in 75% ethanol solution. The extract was filtered and concentrated under decreased pressure, then, it was spray dried to yield a powder. HPLC result showed that level of andrographolide content was 53% in APE (ParActin ® ).

HPLC Analysis of APE
The chromatographic quantitative analysis, used for APE, was a HPLC analysis system, equipped with a 1200 series HPLC system (Agilent, Palo Alto, USA), as previously described [13].
The chromatic separation was accomplished on C 18 column, 4.6 × 150 mm, 5 µm (Shiseido capcell pak UG120, Shiseido, Yokohama, Japan) at 35℃. The mobile phase composition was 60% of ethanol and 40% of deionized water which was set in isocratic mode and flow rate was 1 mL/min. An aliquot of 10 µL of the sample was injected and the effluent was monitored at 225 nm and the total run time was fixed as 30 min. Data was obtained and analyzed by Agilent Chemstation Software. A total of 1.5 mg of standard compounds was dissolved in 1 mL of methanol, and then diluted to concentrations (μg/mL) of 12, 23, 47, 94, 188 and 375 each.
The obtained coefficients of correlation were 1.000 (y = 12.253x + 15.808). The contents of were quantified by comparing with the standard curve. The 100 and 600 mg of APE were dissolved in 10 mL of DW and dimethyl sulfoxide (DMSO) respectively, and then sonication for 20 min. Additionally, each sample was filtered with a 0.45 μm syringe filter (Waters Corp., Milford, USA). Each sample was diluted for HPLC analysis with DW (from 60,000 to 6,000 ppm) and DMSO (from 10,000 to 100 ppm).

Measurement of NO and PGE 2
RAW264.7 cells seeded at 96 well, 1 × 10 6 /ml, were incubated at 37℃, and 5% CO 2 for 24 h. Cells were treated with different concentrations of APE or AG (0.5-16 µg/mL) and LPS (1 µg/mL) and incubated for 24 h. The inhibitory effect of APE on NO and PGE 2 production was determined by analysis of NO and PGE 2 , using Griess reagent and an enzyme-linked immunosorbent assay kit (R&D systems, Minneapolis, MN, USA), as described previously with some modification [14].

Cytotoxicity
The cytotoxic effects of APE or AG were then evaluated using a conventional MTT assay, as reported previously with some modification [14]. After 24 h of seeding, 5 mg/mL MTT solution was added to RAW264.7 cells and kept at 37℃, 5% CO 2 for 1 h. The supernatant was removed, 200 µL of DMSO was added, and the absorbance was measured at 550 nm.

Western Blot Analysis
The protein expression of the total or phosphorylated forms of p38, ERK, JNK, IκBα, and β-actin was analyzed through Western blot analysis. RAW264.7 cells seeded at 6 well, 1 × 10 6 /ml, were incubated at 37℃, and 5% CO 2 for 24 h. Cells were treated with different concentrations of APE, 2 µg/mL and LPS 1 µg/mL, and incubated for indicated time. The intracellular protein was extracted by RIPA buffer. The protein concentration is measured by bradford assay. The extracted protein was denatured by LDS sample buffer (Thermo Scientific, Massachusetts, USA).
Equal amounts of protein samples were loaded onto SDSpolyacrylamide gel for electrophoresis and the isolated proteins were transferred to PVDF membranes with the Semidry Transfer Cell (Bio-Rad Laboratories, Inc., California, USA) for 7 min at 25 V. Membranes were incubated at room temperature with 3% BSA in TBS-T for 1 h to block non-specific antibody binding. After that, membrane was washed three times with TBS-T and primary antibodies (p38, ERK, JNK, IκBα, and β-actin) were applied to react for 15 h at 4℃. The antibodies were purchased from Cell signaling technology, and Santa Cruz Biotechnology, Inc. The membrane was significance was verified at P < 0.05 level, and measurements were indicated as mean ± standard error of the mean.

Analysis of andrographolide in DW, DMSO solution of the APE and AG standard by HPLC
As shown in Figure 1, it was confirmed that the 6,000 ppm DW and 100 ppm DMSO solution of the APE contained 17.2 and 58.0 ppm AG, respectively. As in a previous study [16], The low solubility of AG on the water is a major limitation of research in cell culture. This limitation shows that the use of organic solvents such as ethanol and DMSO can be an alternative to AG dissolution.

DMSO solution of APE was 3.4 times higher in AG content than DW
solution. This result suggests that DMSO solution was the optimal extraction solvent. Since APE contains about 50% of AG content, the experiment was carried out by preparing the concentration of APE (1 -16 μg/mL) and AG (0.5 -8 μg/mL) to be twice.

Production of Nitric oxide in Macrophages
Nitric oxide (NO) is an important regulatory and effector molecule with various biological functions. [17]. Once iNOS is induced, it produces large amount of NO that profoundly influences and damages cell and tissue function. Large quantity of NO produced by LPS stimulation might play a critical role in LPSinduced tissue damage. NO is a pivotal inflammatory factor which contributes to OA progression by modulation of pro-inflammatory or pro-catabolic factors, including Nos2, Il1b, Mmp13 and Ptgs2.
OA chondrocytes overexpress iNOS and its product, NO, which has been considered as a biomarker for OA [18].
Previous studies have shown that AG inhibits NO production in various in vitro and in vivo models [18,19]. In LPS-induced RAW264.7 cells, APE suppressed the level of NO. APE dissolved in DMSO at different concentrations, the NO production of LPSinduced RAW264.7 cells was dose-dependently decreased by APE at 1-16 μg/mL (IC 50 5.3 μg/mL) (Figure 2A). In LPS-induced RAW264.7 cells, AG suppressed the level of NO in a dose-dependent manner (IC 50 3.1 μg /mL) ( Figure 2B). This is because the content of AG contained in APE is 50%, which is similar to IC 50 , so AG is Our previous study [13], the efficacy was also confirmed in the results of suppressing iNOS expression and NO production in the LPS induction model using APE -water soluble fraction dissolved in water. Through this, it is expected that the inhibitory effect of APE on NO production in this study was revealed by AG, which is contained in APE, suppressing the gene expression of iNOS. Also, inhibition of NO production by AG was confirmed. This result suggested that APE could be a useful complement in the treatment of arthritis symptoms [9,10].

Production of PGE 2 in Macrophages
PGE 2 is synthesized from arachidonic acid by COX-2 and overexpressed after being stimulated by LPS, resulting in sepsisrelated inflammatory symptoms and signs [22]. In addition, PGE 2 has been reported to play a role in releasing pro-inflammatory cytokines, such as TNF-α and IL-6 in many cells [23]. Pain is perceived through activation of endings of nociceptive afferent nerves by pain-producing substances, released from tissue, sensitized by locally produced prostaglandins [24]. Because AG has been reported to have an inhibitory effect on PGE 2 production [5,19,20], PGE 2 expression in LPS-induced RAW264.7 cells was observed.

NO, PGE 2 production and MAPKs activity in various in vitro/in vivo
inflammation models [5].
Because the production of NO and PGE 2 were mainly regulated by MAPKs, we investigated the possible involvement of p38, ERK, and JNK in APE inhibition. Before extracting whole lysates, NO concentration was measured and NO decreases by about 90% after 24 h of APE 2 μg/mL treatment in 6 wells (data not shown).
To determine the inhibitory effects of APE on LPS-activated MAPKs activities, total lysates prepared from RAW264.  Through this, it was confirmed that the extract of APE inhibits the activation of the MAPK pathway. In our previous study [13], APE inhibited the expression of iNOS, COX-2 in an in vitro LPS model, and pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in the blood in rat osteoarthritis (OA) model. Therefore, the MAPK mechanism affects joint damage and bone damage caused by joint inflammation, so it is expected to have a pain relief effect in the arthritis clinical trial of APE [9,10].

Activation of NF-κB in Macrophages
The Most evidence suggests that matrix degradation is achieved through the action of MMPs (MMP-1, 3,8,13). In OA, the degradation of extracellular matrix is driven mainly by these enzymes from the chondrocytes [26].
Previous report has shown that AG inhibited NF-κB and the production of MMP-1, 3, 13 in IL-1β-treated cartilage-derived chondrocytes of osteoarthritis patients [11]. Similarly to this report, several kinds of A. paniculata extracts also inhibited NF-κB pathway in in vivo models [27,28].
To determine the inhibitory effects of APE on LPS-activated NF-κB activities, total lysates prepared from RAW264.7 cells treated with LPS for 15, 30, and 45 min were incubated with APE ( Figure   4A). The phosphorylation of IκBα significantly inhibited by 96% (P<0.01) at 15 min, and 35% (P<0.05) at 45 min after exposure of the cells to APE ( Figure 4B). Due to the suppression of the phosphorylation of IκBα, the degradation of IκBα was suppressed at 30 min. The amount of IκBα proteins increased significantly by 131% (p<0.05) ( Figure 4C). The results are similar to the previous study showing that phosphorylation of IκBα was significantly inhibited when 30 µg/ mL of A. paniculata methanol extract was pretreated for 1 h and then treated with LPS for 15 min [29]. In this study, APE 2 µg/mL was treated at the same time as LPS, and the same effect was shown after 15 min.
In this study, it was confirmed by Western blot that phosphorylation of IκBα decreased and the IκBα protein was degraded by APE. Results are consistent with a mechanism of APE that inhibits the production of IL-1β, IL-6, and TNF-α, proinflammatory cytokines produced by NF-κB gene expression in rat osteoarthritis models and in vitro LPS models. These results point to APE as the anti-inflammatory ingredient that inhibits the expression of iNOS and COX-2 genes [13].
In addition to APE, it supports the effect of protecting joint cartilage by inhibiting MMP-1, 3, 8, 13 in rat osteoarthritis model. Therefore, it affects joint damage and joint cartilage decomposition due to joint inflammation through the NF-κB mechanism. It is expected to alleviate arthritis symptoms in the arthritis clinical trial [9,10].

Conclusion
The study demonstrated that APE suppresses in vitro inflammatory responses in LPS-TLR4-activated macrophages. APE inhibited the expression of NO and PGE 2 increased by LPS in a dose-dependent manner. APE acts as the inhibitor of MAPKs (p38, ERK and JNK) and IκBα which is particularly important in iNOS/ COX-2 expression. AG-containing Andrographis paniculata has been ethnopharmacologically used for a long time and is orally available [18,24]. Based on the results of the previous clinical trials and toxicity tests on A. paniculata, it is expected to be used as a new antiinflammatory material as a substitute for various inflammatory disease treatments, such as osteoarthritis.