Kyungheechunggan-Tang-01, a New Herbal Medication, Suppresses LPS-Induced Inflammatory Responses through JAK/STAT Signaling Pathway in RAW 264.7 Macrophages

Medicinal plants have been used as alternative therapeutic tools to alleviate inflammatory diseases. The objective of this study was to evaluate anti-inflammatory properties of Kyungheechunggan-tang- (KCT-) 01, KCT-02, and Injinchunggan-tang (IJCGT) as newly developed decoctions containing 3–11 herbs in LPS-induced macrophages. KCT-01 showed the most potent inhibitory effects on LPS-induced NO, PGE2, TNF-α, and IL-6 production among those three herbal formulas. In addition, KCT-01 significantly inhibited LPS-induced iNOS and COX-2 at protein levels and expression of iNOS, COX-2, TNF-α, and IL-6 at mRNA levels. Molecular data revealed that KCT-01 attenuated the activation of JAK/STAT signaling cascade without affecting NF-κB or AP-1 activation. In ear inflammation induced by croton oil, KCT-01 significantly reduced edema, MPO activity, expression levels of iNOS and COX-2, and STAT3 phosphorylation in ear tissues. Taken together, our findings suggest that KCT-01 can downregulate the expression of proinflammatory genes by inhibiting JAK/STAT signaling pathway under inflammatory conditions. This study provides useful data for further exploration and application of KCT-01 as a potential anti-inflammatory medicine.


Introduction
Inflammation is a complicated biological response caused by many harmful stimuli, including pathogens, bacteria, and irritants [1]. It is also a tissue protective reaction of immune cells including macrophages. Initiation of inflammation is triggered by interactions between surface receptors such as Toll-like receptor-(TLR-) 4 and TLR-2 and their ligands, including lipopolysaccharides (LPS) derived from bacteria [2]. When activated by LPS, macrophage as a potent immune diseases, and cancer [4]. Accordingly, materials or compounds that can inhibit these proinflammatory mediators have been considered as potential anti-inflammatory agents.
Janus kinase-(JAK-) signal transducer and activator of transcription (STAT) cascade is a critical inflammatory signaling pathway that mediates immune responses [5]. In particular, STATs have been reported to play a pivotal role in inflammatory signaling cascades triggered by LPS and several cytokines such as interferon gamma (IFN-) [6][7][8]. TLR-4 activation results in phosphorylation of receptorassociated enzymes JAKs known to be activators of STATs [9]. Binding of ligands to their receptors leads to phosphorylation of JAKs, which will induce phosphorylation of STATs that in turn leads to the release of STATs from the receptor complex. Released STATs can form homo-or heterodimers and translocate into the nucleus to regulate transcription of target genes encoding proinflammatory cytokines and inducible enzymes such as inducible nitric oxide synthase [1] and cyclooxygenase-2 (COX-2) [10][11][12]. Since JAK-STAT signaling pathway is involved in the mediation of proinflammatory gene expression, it is important to tightly regulate its activity to prevent inflammatory responses. Viral infection, high consumption of alcohol, fat accumulation, and toxic agent are the main causes of chronic inflammation in the liver [13]. Chronic inflammation causes pathological changes in liver function and, therefore, might progress severe problems such as liver fibrosis and cancer [14]. Thus, controlling inflammatory responses in the liver is desirable in the management of severe hepatic diseases. Many traditional herbal medicines have been revealed to have pharmacological properties against inflammatory injury [15]. Injinchunggan-tang (IJCGT) is a herbal medicine containing 11 medicinal herbs. It has been widely used to treat hepatic diseases in Kyung Hee University Korean Medicine Hospital (Seoul, Republic of Korea) [16][17][18][19][20][21]. Based on IJCGT, we reorganized new herbal medications called Kyungheechunggantang-(KCT-) 01 which contained Artemisia Capillaris Herba, Sanguisorbae Radix, and Curcuma longa Radix. KCT-02 is an extended decoction after adding Rubi Fructus and Salviae Miltiorrhizae Radix to KCT-01 (Table 1). Although hepatoprotective effect of IJCGT and clinical case report have been demonstrated previously, scientific evidence to support the effect of KCT-01 or KCT-02 and their fundamental mechanism of action are currently unclear. Therefore, the objective of the present study was to compare anti-inflammatory effects of KCT-01, KCT-02, and IJCGT. After that, the most potent medicine among the three was selected to unravel the detailed molecular mechanism involved in its anti-inflammatory effect in vitro using LPS-induced RAW 264.7 macrophages and in vivo using croton oil-induced ear edema rat model.
Evidence-Based Complementary and Alternative Medicine 3
For the UPLC-MS analysis, ten milligrams of KCT-01 or IJCGT extract was dissolved in one milliliter of 30% ethanol or 70% ethanol, respectively. Reference standard compounds (1.0 mg/mL) were dissolved in methanol and then mixed into a cocktail solution which was used as reference standards solution. The sample solutions were filtered through a 0.2 m polyvinylidene fluoride syringe filter (Whatman, Maidstone, UK) and reference standards solutions were filtered through a 0.2 m polytetrafluoroethylene syringe filter (Whatman, Maidstone, UK) before injection into UPLC system.

Cell
Culture and Sample Treatment. The RAW 264.7 murine macrophage cell line was purchased from the Korea Cell Line Bank (Seoul, Korea) and cultured in DMEM containing 10% fetal bovine serum, penicillin-streptomycin (100 units/mL) at 37 ∘ C with 5% CO 2 .
Murine bone marrow derived macrophages (BMDMs) were isolated from femur of C57BL/6 mice. Cells in bone marrow were washed several times with cold phosphate buffered saline (PBS). The isolated cells were filtered through sieve mesh, centrifuged, and resuspended in DMEM (supplemented with 10% FBS, 100 units/mL penicillinstreptomycin). The cells were incubated at 2 × 10 6 in Petri dish with 15% L929-conditioned medium in DMEM for 7 days to differentiate into macrophages. The culture medium was added at the 3rd day and replaced at the 6th day of incubation. After being differentiated, the cells were seeded in 24-well culture plates. In all experiments, cells were incubated with samples at various concentrations that was always added 1 h prior to LPS (1 g/mL) treatment for the indicated time.

Animals.
All experiments in the present study were conducted under university guideline of ethical committee for Animal Care and Use of the Kyung Hee University according to an animal protocol (KHUAPS(SE)-16-013). Male C57BL/6 mice (6-8 weeks old) or male Sprague-Dawley (SD) rats (6 weeks old) were purchased from the Orient Bio Inc. (Seongnam-si, Korea) and maintained under constant conditions (temperature: 20 ± 2 ∘ C, humidity: 40-60%, and light/dark cycle: 12 h).
2.5. Cell Viability Assay. Cell viability was evaluated by MTT assay. RAW 264.7 macrophage cells or BMDMs were plated at a density of 2 × 10 5 cells per well in 24-well plates and then treated with samples (KCT-01, KCT-02, or IJCGT) at various concentrations 1 h prior to LPS (1 g/mL) treatment. After 24 h of LPS stimulation, 20 l MTT solution (5 mg/mL) was added to each well, and the cells were further incubated for an additional 4 h. The supernatant was removed and the formazan was resolved with 1 mL/well of DMSO. The optical density was measured at 540 nm by microplate reader.

Nitrite Determination. RAW 264.7 macrophage cells or
BMDMs were plated at 2 × 10 5 cells per well in 24-well plates and then incubated with or without LPS (1 g/mL) in the absence or presence of various concentrations of samples (KCT-01, KCT-02, or IJCGT) at various concentrations for 24 h. Nitrite levels in culture media were determined using the Griess reaction assay and presumed to reflect NO levels. The optical density was measured at 540 nm by microplate reader.
2.7. PGE 2 , TNF-, and IL-6 Assay. RAW 264.7 macrophage cells or BMDMs were plated at 2 × 10 5 cells per well in 24-well plates and then incubated with or without LPS (1 g/mL) in the absence or presence of various concentrations of samples. Dilutions of the cell culture medium were assayed for PGE 2 , TNF-, IL-6, and IL-1 . PGE 2 levels in cell culture medium were determined using a colorimetric competitive enzymelinked immunosorbent assay (ELISA) kit (Enzo Life Science, NY, USA) according to manufacturer's instructions. TNFand IL-6 levels in cell culture medium were quantified using mouse DuoSet kit (R&D Systems, MN, USA) according to manufacturer's instructions.

Protein Extraction and Western Blot
Analysis. RAW 264.7 macrophage cells were seeded in 60 mm 2 dish and incubated for 24 h and then added to KCT-01 1 h prior to LPS (1 g/mL) treatment. The cells were collected by centrifugation and washed three times with PBS. Washed cell pellets were resuspended in protein extraction solution PROPREP (Intron Biotechnology, Seoul, Korea) and then incubated for 30 min at 4 ∘ C. Cell debris was removed by microcentrifugation and supernatants were quickly frozen. The protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories Inc., CA, USA) according to manufacturer's instruction. Proteins were electroblotted onto a PVDF membrane following separation on an 8% or 10% SDS-polyacrylamide gel electrophoresis. The immunoblot was incubated for 1 h with blocking solution (5% skim milk) at room temperature and then incubated overnight with a 1 : 1000 dilution of primary antibody at 4 ∘ C. Blots were washed three times with Tween 20/Tris-buffered saline (T/TBS) and then incubated with a 1 : 2000 dilution of horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc. CA, USA) for 2 h at room temperature. Blots were again washed three times with T/TBS and then developed by enhanced chemiluminescence (Amersham Life Science, IL, USA).

Plasmid, Transient Transfection, and Luciferase Assay.
RAW 264.7 macrophages were cotransfected with pNF-Bluc or pAP-1-luc (Clontech, Shiga, Japan) plasmid plus the phRL-TK plasmid (Promega, WI, USA) using Lipofectamine LTX6 (Invitrogen, CA, USA) as instructed by the manufacturers. After 24 h of transfections, cells were pretreated with KCT-01 for 1 h prior to LPS (1 g/mL) stimulation. After 18 h, each well was washed with cold-PBS and cells were lysed and the luciferase activity was determined using the Promega luciferase assay system (WI, USA).

Ear Edema Animal
Model. Sprague-Dawley (SD) male rats weighing 180-200 g were divided into three groups ( = 6); croton-oil-alone group, KCT-01-50 mg/kg-treated group, and KCT-01-100 mg/kg-treated group. SD male rats were pretreated with KCT-01 (50 or 100 mg/kg, p.o.) and after 1 h, ear edema was induced on inner surface of the right ear by topical application of croton oil (5% solution in 100 L of acetone). The left ear was used as a control and received the same amount of the vehicle (acetone). Two hours after croton oil application, rats were sacrificed using CO 2 , and both ear tissues were collected using 6 mm punching. Ear punch biopsies were immediately measured for thickness to assess ear edema. Ear biopsies were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Ear biopsy sections were stained with hematoxylin and eosin (H&E) at the Seoul Medical Science Institute (SCL Co. Ltd., Seoul, Korea).
The other ear biopsies were immediately frozen (−70 ∘ C) for the determination of myeloperoxidase (MPO) activity, a marker of neutrophil influx into the tissue, and intracellular protein expression including iNOS, COX-2, and STATs. The tissue was thawed and homogenized. The homogenate was then centrifuged at 1500 ×g for 15 min, and the resulting supernatant was assayed for MPO assay and Western blotting.
Results showed that viability of cells was not significantly affected by KCT-01, KCT-02, or IJCGT at concentrations up to 100 g/mL (Figure 3(c)), indicating that their suppressive effects on NO and PGE 2 production were not attributable to their nonspecific cytotoxicity.
for 1 h followed by stimulation with LPS for 24 h. KCT-01, KCT-02, or IJCGT each concentration dependently and significantly suppressed the production of TNF-and IL-6 ( Figure 4). Of these three, KCT-01 showed the most potent inhibition for TNF-(IC 50 : 22.9 g/mL) and IL-6 (IC 50 : 35.5 g/mL) production. Collectively, our results indicate that KCT-01 is a more potent herbal medicine that can suppress proinflammatory mediators compared to KCT-02 or IJCGT (Table 4). Therefore, we selected KCT-01 as the most effective anti-inflammatory agent and conducted further experiments to determine the underlying molecular mechanism involved in its anti-inflammatory effect.

KCT-01 Inhibits Production of NO, PGE 2 , TNF-, and IL-6 in LPS-Induced BMDMs.
To confirm our findings in primary macrophage cells, we examined the inhibitory effect of KCT-01 on production of proinflammatory mediators and cytokines in LPS-induced BMDMs. We found that KCT-01 significantly and concentration dependently inhibited production of NO, PGE 2 , TNF-, and IL-6 in LPS-stimulated BMDMs ( Figure 5), indicating that the anti-inflammatory effect of KCT-01 might not be cell specific responses.

KCT-01 Inhibits Expression Levels of iNOS, COX-2, TNF-
, and IL-6 in LPS-Induced RAW 264.7 Macrophages. Next, we evaluated whether the inhibitory effect of KCT-01 on the production of NO and PGE 2 was related to reduced expression of iNOS and COX-2 by Western blotting and qRT-PCR. As shown in Figure 6  suppressed these upregulations in a concentration-dependent manner. KCT-01 also markedly inhibited LPS-induced iNOS and COX-2 mRNA expression levels ( Figure 6(b)). These findings demonstrate that KCT-01 can downregulate the expression of LPS-induced iNOS and COX-2 and lead to decreased production of NO and PGE 2 . KCT-01 also downregulated TNF-and IL-6 mRNA expression levels in a concentration-dependent manner (Figures 6(c) and 6(d)). Taken together, these results suggest that KCT-01 possesses anti-inflammatory activity by inhibiting expression levels of various LPS-induced proinflammatory mediators.

Effects of KCT-01 on NF-B and AP-1 Activation in LPS-
Induced RAW 264.7 Macrophages. NF-B and AP-1 are key transcriptional factors regulating inflammatory responses mediated by LPS or proinflammatory cytokines [29,30]. Therefore, we explored the effect of KCT-01 on LPS-induced NF-B or AP-1 activity in RAW 264.7 macrophages by luciferase reporter gene assay using pNF-B-luc or pAP-1-luc vector. Our results revealed that KCT-01 did not have any effect on NF-B or AP-1-dependent transcriptional activity (Figure 7). When transcription factors are phosphorylated, these may translocate to the nucleus where they can bind to their consensus DNA binding sites to regulate transcription of target genes [31]. Accordingly, we performed Western blotting to determine the effect of KCT-01 on phosphorylation of p65 (a subunit of NF-B) or c-fos and c-jun (subunits of AP-1). Our results showed that KCT-01 did not have any inhibitory effect on LPS-induced phosphorylation of p65, cfos, and c-jun (data not shown). also affected inflammatory signaling cascades triggered by LPS, IFN-, and other cytokines [7,32]. Since KCT-01 had no effect on NF-B and AP-1 activation, we investigated whether the inhibitory effect of KCT-01 on the expression of proinflammatory mediators was mediated by JAK/STAT signaling pathway. Binding of LPS ligands to their receptors induces phosphorylation of receptor-associated JAKs followed by phosphorylation of STATs [9]. As shown in Figure 8 d) and (e)) Ear tissues were homogenized and lysates were used to determine MPO activity, protein expression, and phosphorylation of iNOS, COX-2, and STAT3. Values are expressed as means ± SD. # < 0.05 versus control; * < 0.05, * * < 0.01, and * * * < 0.001 versus croton oil treated group. at 2 h after LPS stimulation. KCT-01 also significantly and concentration dependently blocked the phosphorylation of JAK1 (Tyr1022/1023) and JAK2 (Tyr1007/1008) at 1 h after LPS challenge (Figure 8(b)). Therefore, KCT-01 potently inhibited JAK1 and JAK2, subsequently downregulating phosphorylation of STAT1 and STAT3.

Effect of KCT-01 on Croton Oil-Induced Ear Edema in Rats.
To determine the anti-inflammatory effect of KCT-01 in vivo, we used a rat model of acute inflammatory ear edema induced by croton oil. As expected, ear thickness increased by croton oil application was reduced by pretreatment with KCT-01. At 50 and 100 mg/kg, KCT-01 decreased ear thickness by 45.00±6.08% and 70.45±8.86%, respectively (Figure 9(a)). To examine histopathological changes during ear edema, cross sections of ear discs were stained with hematoxylin and eosin. Consistent with its inhibitory effect on ear thickness, KCT-01 also inhibited epidermal ear edema after pretreatment 12 Evidence-Based Complementary and Alternative Medicine (Figures 9(b) and 9(c)). Next, neutrophil migration into croton oil-induced ear was indirectly determined using MPO activity assay. As expected, application of croton oil to ear increased MPO production in tissues. However, oral administration of KCT-01 suppressed croton oil-induced MPO production (Figure 9(d)). Consistent with our findings in macrophages, KCT-01 also inhibited iNOS and COX-2 expression levels and STAT3 phosphorylation (Figure 9(e)).

Discussion
Many studies have reported that traditional herbal resources can benefit the management of various diseases, including arthritis [33], atopic dermatitis [34], and hepatic fibrosis [35]. According to classic literature such as well-known herbal medical books "Sang han lon" and "Geum gwe yo lyag," a variety of herbal medications have been used to treat many diseases in Asian countries. In particular, "Geum gwe yo lyag" recommended IJORS (injinoryung-san), a herbal decoction consisting of Artemisia Capillaris Herba, Alismatis Rhizoma, Poria Sclerotium, Atractylodis Macrocephalae Rhizoma, Polyporus Sclerotium, and Cinnamomi Cortex as a typical prescription to treat jaundice. Based on IJORS expelling jaundice, IJCGT is made by omitting Cinnamomi Cortex but adding Sanguisorbae Radix, Rubi Fructus, Glycyrrhizae Radix, Raphani Semen, and Citrus Unshiu Immaturi Pericarpium, and Zingiberis Rhizoma Crudus (Table 1). Currently, IJCGT is mainly applied to treat patients with various liver diseases, such as viral hepatitis, cirrhosis, and hepatocellular carcinoma in Kyung Hee University Korean Medicine Hospital (Seoul, Korea). Among 11 individual medicines, Artemisia Capillaris Herba and Sanguisorbae Radix are regarded as key components of IJCGT for its various pharmacological effects for hepatic diseases, including liver cirrhosis and hepatoma usually caused by chronic inflammation followed by pathological changes in liver function [17,[36][37][38][39][40]. In addition, increasing evidence has revealed that curcumin, an active ingredient of Curcuma longa Radix, possesses antiinflammatory properties [41,42]. Thus, we developed a compressed herbal prescription consisting of Artemisia Capillaris Herba, Sanguisorbae Radix, and Curcuma longa Radix to treat hepatic inflammation. It was named KCT-01. Additionally, KCT-02 was expanded from KCT-01 by adding Rubi Fructus and Salviae Miltiorrhizae Radix to strengthen its antiinflammatory activities [20,43,44]. In the present study, we evaluated anti-inflammatory effects of KCT-01, KCT-02, and IJCGT and elucidated underlying molecular mechanisms of KCT-01 in LPS-induced RAW 264.7 macrophages. Based on their inhibitory potencies on LPS-induced NO, PGE 2 , TNF-, and IL-6 production, we selected KCT-01 to further investigate its anti-inflammatory effects and underlying molecular mechanism in vitro (LPS-induced macrophages) and in vivo (croton oil-induced acute inflammation in ear edema of rat).
LPS, an inflammatory stimulator, can induce various proinflammatory mediators and cytokines. Our data revealed that proinflammatory mediators (NO, PGE 2 ) and cytokines (TNF-, IL-6) were induced by LPS stimulation in RAW 264.7 macrophages. However, KCT-01, KCT-02, and IJCGT attenuated the expression of NO, PGE 2 , TNF-, and IL-6 without causing cytotoxicity. These results indicate that KCT-01, KCT-02, and IJCGT exhibit anti-inflammatory effects on LPS-induced RAW 264.7 macrophages. We selected KCT-01 as the most potent prescription/formulation among the three and elicited molecular mechanisms involved. The inhibitory effect of KCT-01 on the release of NO, PGE 2 , TNF-, and IL-6 was also proved in murine bone marrow derived macrophages. NO and PGE 2 are major proinflammatory mediators produced by enzymes iNOS and COX-2, respectively. These two enzymes are responsible for cell damage and tissue destruction in inflammation [45,46]. Consistently, we observed that KCT-01 also inhibited mRNA expression of iNOS, COX-2, TNF-, and IL-6, indicating that KCT-01 downregulated the expression of inflammatory genes at transcription level.
To further explore the intracellular mechanism underlying the anti-inflammatory effect of KCT-01, we focused on various transcription factors such as NF-B, AP-1, and STATs involved in the regulation of inflammatory genes. Mechanistically, LPS induces TLR4 to activate NF-B, an important transcription factor for iNOS and COX-2 expression in various cells including macrophages [47]. LPS can also activate AP-1 which is stimulated by mitogen-activated protein kinases (MAPKs), including ERK1/2, p38 MAPKs, and JNK, thus enhancing proinflammatory gene expression in macrophages [48][49][50]. However, KCT-01 did not influence NF-B or AP-1 activation. Besides NF-B and AP-1 signaling, some studies have reported that the JAK/STATs signal pathway is crucial for the expression of genes encoding inflammatory enzymes such as iNOS and COX-2 [5,8,9]. The JAK-STATs pathway is also critical for cytokine activated signaling in immune response [51][52][53]. Thus, it is reasonable to speculate that the inhibitory effect of KCT-01 on LPSstimulated inflammatory response might be attributed to its suppression on JAK-STATs signaling pathway. Accordingly, we found that KCT-01 blocked phosphorylation of both JAK and STATs in a concentration-dependent manner which in turn inhibited translocation of STATs to the nucleus to bind to promoters of target genes for their transcription activation [6]. These results demonstrated that KCT-01 could restrain LPS-elevated release of proinflammatory mediators via blocking STAT1 and STAT3 activation.
To verify whether KCT-01 could ameliorate acute inflammatory symptoms in vivo, we used croton oil-induced ear edema rat model. As expected, we found that oral administration of KCT-01 significantly suppressed the swelling of ears and MPO activity in ear tissues, indicating that KCT-01 could inhibit acute inflammation via inhibiting infiltration of inflammatory cells. Consistent with our findings in macrophages, KCT-01 inhibited protein expression of inflammatory mediators (including iNOS and COX-2) and phosphorylation of STAT3 in ear tissues.
Herbal medicine has limitations in practical uses as a drug because it is difficult to standardize herbal medicine or find obvious active components of each formulation. Thus, most herbal medicines are underestimated although they are effective in treating various diseases. As part of our effort to standardize herbal medicine, we analyzed phytochemicals of KCT-01 and IJCGT using chromatographic fingerprint analysis. However, we need to investigate active components of KCT-01. If we can scientifically verify and prove therapeutic effects of this herbal medicine, the value of KCT-01 will be increased.

Conclusions
In conclusion, we found that KCT-01, KCT-02, and IJCGT suppressed inflammatory mediators, with KCT-01 being the most effective one among the three. We also proved that the inhibitory effect of KCT-01 on inflammatory proteins and genes was accompanied by suppression of phosphorylation of JAK1/2 and STAT1/3. Therefore, this study provides evidence that KCT-01 might exhibit anti-inflammatory effect via suppressing JAK/STATs activation in LPS-induced RAW 264.7 macrophages. Such mechanism of action also contributed to the pharmacological potential of KCT-01 in vivo using croton oil-induced ear edema model. Our findings suggest that KCT-01 may have potential as a herbal medicine for treating a variety of inflammatory diseases.

Conflicts of Interest
The authors state that they have no conflicts of interest to declare.