Chinese Journal of Natural Medicines  2017, Vol. 15Issue (8): 561-575  DOI: 10.3724/SP.J.1009.2017.00561
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Ju-Suk Nam, Supriya Jagga, Ashish Ranjan Sharma, Joon-Hee Lee, Jong Bong Park, Jun-Sub Jung, Sang-Soo Lee. Anti-inflammatory effects of traditional mixed extract of medicinal herbs (MEMH) on monosodium urate crystal-induced gouty arthritis[J]. Chinese Journal of Natural Medicines, 2017, 15(8): 561-575.
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Research funding

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Nos. 2014R1A1A4A03009388 & 2014R1A1A2055560), a grant of the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI12C1265), and Hallym University Research Fund

Corresponding author

Sang-Soo Lee, E-mail:123sslee@gmail.com.

Article history

Received on: 23-Nov.-2016
Available online: 20 Aug., 2017
Anti-inflammatory effects of traditional mixed extract of medicinal herbs (MEMH) on monosodium urate crystal-induced gouty arthritis
Ju-Suk Nam1 , Supriya Jagga1 , Ashish Ranjan Sharma1 , Joon-Hee Lee3 , Jong Bong Park1 , Jun-Sub Jung2 , Sang-Soo Lee1     
1 Institute For Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon, Gangwon-do 24252, Republic of Korea;
2 Institute of Natural Medicine, College of Medicine, Hallym University, Chucheon, Gangwon-do 24252, Republic of Korea;
3 Hana Oriental Clinic, Chucnheon, Gangwon-do 24433, Republic of Korea
[Abstract]: Korean oriental medicine prescription is widely used for the treatment of gouty diseases. In the present study, we investigated anti-inflammatory effects of modified Korean herbal formulation, mixed extract of medicinal herbs (MEMH), and its modulatory effects on inflammatory mediators associated with gouty arthritis. Both in vitro and in vivo studies were carried out to assess the anti-inflammatory efficacy of MEMH on monosodium urate (MSU) crystals-induced gouty inflammation. MSU crystals stimulated human chondrosarcoma cell line, SW1353, and human primary chondrocytes were treated with MEMH in vitro. The expression levels of pro-inflammatory mediators and metalloproteases were analyzed. The effect of MEMH on NFκB signaling pathway in SW1353 cells was examined. Effect of MEMH on the mRNA expression level of pro-inflammatory mediators and chemotactic factor from human monocytic cell line, THP-1, was also analyzed. The probable role of MEMH in the differentiation process of osteoblast like cells, SaOS-2, after MSU treatment was also observed. To investigate the effects of MEMH in vivo, MSU crystals-induced ankle arthritic model was established. Histopathological changes in affected joints and plasma levels of pro-inflammatory mediators (IL-1β and TNFα) were recorded. MEMH inhibited NFκB signaling pathway and COX-2 protein expression in chondrocytes. MSU-induced mRNA expressions of pro-inflammatory mediators and chemotactic cytokines were suppressed by MEMH. In MSU crystals-induced ankle arthritic mouse model, administration of MEMH relieved inflammatory symptoms and decreased the plasma levels of IL-1β and TNFα. The results indicated that MEMH can effectively inhibit the expression of inflammatory mediators in gouty arthritis, demonstrating its potential for treating gouty arthritis.
[Key words]: Gout     Monosodium urate crystals     Inflammation     Chondrocyte     Oriental medicine    
Introduction

Gout is commonly referred as'disease of kings' and is an inflammatory arthropathy associated with hyperuricemia [1]. This rheumatologic condition of chronic gout is characterized by deposition of monosodium urate (MSU) crystals in articular joints, peri-articular tissues and kidney [2]. Accumulation of MSU crystals prompts an intense inflammatory process, followed by pain. Build-up of serum uric acid is one of the main contributors to this disease. As uric acid level rises and exceeds the physiological saturation threshold (approximately 380 μmol·L-1 or 6.8 mg·dL-1) in body tissues, deposition of MSU crystals occurs [3]. An abnormality in handling extreme amount of uric acid, which usually crystalizes in kidney, cause renal diseases, often leading to gout [4]. The metatarsal-phalangeal joint at the base of the big toe is the most commonly affected site. Continued crystallization of MSU crystals may also affect mid-tarsal joints, such as ankles, knees, hands, and wrists [5]. Without appropriate treatment, gout attacks may occur more frequently and can eventually cause permanent distortions and damage to joints.

Joint with MSU crystals tophi undergoes through several alteration in its remodeling and cellular distribution. Recurrent attacks of gouty arthritis result in articular cartilage mineralization. MSU crystals deposited on hyperchoic band of articular cartilage are responsible for catabolic changes, such as intracortical bone erosion, cartilage loss and alterations in chondrocyte phenotype [6-7]. Furthermore, other structural injury such as joint space narrowing together with features of new bone development like bone sclerosis and spur formation are often observed [8]. Indeed, MSU crystals stimulated inflammatory cells, which induce the inflammatory cascade, include fibroblast cells, bone cells, and innate and adaptive immune cells. Interaction of MSU crystal with these complement and resident cells stimulates NFκB, SAPK/JNK and ERK1/2 MAPK signaling dependent expression of pro-inflammatory mediators such as interleukin-1β (IL-1β), tumor necrosis factor (TNFα), interleukin-8 (IL-8/CXCL8), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) [9]. Resultant pro-inflammatory mediators in both the synovial fluid and the synovial membrane trigger the synthesis of matrix metalloproteinases (MMPs) and osteoclast formation, leading to joint destruction [10].

Nowadays, several anti-gout agents, such as allopurinol, are available in market, having ability to control hyperuricemia. Non-steroidal anti-inflammatory drugs, like indomethacin and naproxen, are frequently used to reduce acute inflammation during gout. Nevertheless, these medications are associated with a number of adverse effects, i.e., gastrointestinal toxicity, renal noxiousness, and internal gastrointestinal bleeding, which may limit their clinical uses [11-12]. Therefore, search for better anti-inflammatory and urate-lowering drugs is a necessity. Despite advancement in the application of anti-gout drugs, oriental traditional plant-based medicines have been regularly utilized due to safety profile with high efficacy [13]. These medicines have been practiced for thousands of years in Asian countries to cure skeletal diseases and are known to have effective results. The efficacy of these kinds of drugs has been attributed to cartilage safety and anti-inflammatory properties [13-14]. However, limited information is available for specific mechanism of action for oriental medicine to support their worth [15].

Korean traditional medical literature has been screened, assessed and utilized for several decades for developing therapeutic formulations to counter various diseases [16-17]. Korean medical literature published as "Dongeuibogam" (Treasured Mirror of Eastern Medicine) in the year 1613 has been the source of traditional alternative medicine in North-East Asia [18]. From this literature, we selected Oryeongsan, a formula of five herbal medicines (Alisma orientale Juzepzuk, Polyporus umbellatus Fries, Poria cocos Wolf, Atractylodes macrocephala Koidzumi, and Cinnamomum cassia Presl; Table 1), which has been used in east-Asian traditional medicine for symptoms related with renal diseases [19] and anti-inflam-matory properties [20]. All the five constituent herbs of Oryeongsan are known to enhance diuric and anti-inflam-matory activities. Their activities are summarized in Table 1. In brief, administration of Oryeongsan to rats has been shown to induce diuresis and natriuresis via inhibition of renin-angiotensin-aldosterone system [19]. Anti-inflammatory properties of Oryeongsan were demonstrated in lipopolysaccharide (LPS)-mediated inflammation in the RAW 264.7 macrophage cells by inhibiting NFκB and MAPK signaling pathways. It has been shown that Oryeongsan can suppress the expression of COX-2 and iNOS, NO synthesizing enzymes [20]. To enhance medicinal properties of Oryeongsan, it has been further supplemented with four herbs (Curcuma longa Linn, Schisandra chinensis Baillon, Artemisia capillaris Thunberg, Gardenia jasminoides Ellis), selected from Korean medical literature "Oriental materia medica" based on their anti-nociceptive and anti-inflammatory properties (Table 1) [21]. C. longa, commonly known as turmeric, has been reported to possess anti-inflammatory properties. Extract of turmeric has been shown to inhibit joint inflammation and peri-articular joint destruction in arthritic mice model. A study showed a suppression of NFκB regulated joint inflammation and destruction by suppressing the expression of chemokines, COX-2, and RANKL [22]. Another study showed that turmeric can reduce inflammation and promote the repair parameters of healing process during gingival healing in beagle dogs [23]. Herb, S. chinensis, possesses anti-inf-lammatory properties and has shown significant inhibition of secretion of chemokines like CXCL8 and monocyte chemotactic proetien-1 (MCP-1) in human lung epithelial-derived A549 cells. Administration of S. chinensis extract to LPS-chal-lenged BALB/c mice demonstrated suppressed neutrophil and macrophage infiltrations of lung tissues as well a decrease in the level of IL-6 and TNFα in the bronchoalveloar lavage fluid [24]. A proteoglycan protective effect of S. chinensis was also reported in IL1β-induced matrix degradation in human articular cartilage and chondrocytes [10]. Herb, A. capillaris, owns strong anti-inflammatory properties. It has been shown that administration of A. capillaris extract can protect against alcohol associated hepatic disorder in Sprague-Dawley rats by modulating expression of pro-inflammatory cytokines like TNFα and inflammation inducing transforming growth factor-β (TGF-β) [25]. Another study has revealed anti-hyperalgesic and anti-allodynic activities of A. capillaris in Complete Freund's adjuvant (CFA)-or carrageenan-induced mechanical hyperalgesia model in mice. It has been observed that pretreatment of A. capillaris inhibits NFκB mediated genes (iNOS and COX-2) and reduced plasma level of nitrite, which are involved in pain [26]. In addition, A. capillaris has been shown to inhibit myeloid differentiation primary response gene 88 (MyD88)/ Toll/IL-1 receptor homology domain containing adapter protein (TIRAP) inflammatory signaling in LPS-stimulated RAW 264.7 macrophage cells. A. capillaris treated cells show reduced mRNA and protein levels of iNOS and COX-2. Administration of A. capillaris to CFA-or carrageenan-induced paw edema mice model results in a decrease in nitrite plasma level and TNFα production [27]. A study in carrageenan-induced paw edema mice model has proven anti-inflammatory activities of G. jasminoides by inhibiting vascular permeability induced by acetic acid and production of exudate and nitric oxide [28]. Taken together, scientific evidences for active component of these traditional natural herbs have demonstrated excellent anti-nociceptive and anti-inf-lammatory properties in animal models, as well as cartilage protection and anti-inflammatory properties in several in vitro studies. Indeed, selective individual herbs have demonstrated several medicinal properties, but whether a supplemented formulation of Oryeongsan could affect multifactorial diseases like gout is unclear. Besides, there are no sufficient scientific evidences on their combined effect and possible mechanisms underlying the effectiveness of these plant materials [29-30], especially in relation to molecular events that could explain the exact mechanism for suppression of induced inflammation. Henceforth, on the basis of potential medicinal value offered by each plant, a mixed formulation (Mixed Extract of Medicinal Herbs; MEMH) was prepared and analyzed for the cumulative effect on the inflammation induced by MSU crystals. In addition, the present study was designed to deliver evidence-based scientific data to confirm the anti-inflammatory efficacy of MEMH through in vitro and in vivo experiments.

Table 1 Biological effects of constituents present in MEMH
Methods and Materials Plant materials and MEMH preparation

Nine herbs, as mentioned in Table 1, were obtained from local vender (Kyungdong Market, Seoul, South Korea) and a water-soluble MEMH was prepared. The herbarium voucher specimens, including MPRBP01522 (C. longa), MPRBP00072 (S. chinensis), MPRBP00668 (A. capillaris), MPRBP00627 (G. jasminoides.), MPRBP00817 (A. macrocephala), MPRBP01027 (C. cassia) and WE1103-11 (A. orientale), ME1109-03 (P. umbellatus), and OE0908-2 (P. cocos) were deposited in the Medicinal Plant Resource Bank (MPRB) and Korea Cosmeceutical Material Bank, respectively. Both the Institutes are managed by Korea National Research Resource Center (KNRRC), Republic of Korea. All the herbs were identified and authenticated by Dr. Joon-Hee Lee from Hana Oriental Clinic, Chuncheon, South Korea. To achieve a rationale effect, five contents of Oryeongsan (A. orientale, P umbellatus, P. cocos, A. macrocephala, and C. cassia) were mixed, according to the ratio of 5 : 3 : 3 : 3 : 1 in weight and supplemented with four additional herbs based on their medicinal properties mentioned in Korean medical literature "Oriental Materia Medica" (Table 1). An aqueous extract of the plant mixture was prepared. In brief, MEMH contained a total of 174 g of herbs (A. orientale, 30 g; P. umbellatus, 18 g; P. cocos, 18 g; A. macrocephala, 18 g; C. cassia, 6 g; C. longa, 24 g; S. chinensis, 24 g; A. capillaris, 24 g; and G. jasminoides, 12 g) were soaked in 2 L of cold distilled water overnight and extracted after being heated for 3 h at 100 ℃ on medical heating plate. After boiling, the extract was centrifuged at 3 000 r·min-1 for 10 min. The total aqueous extract was filtered through distilled gauze and concentrated using a rotary evaporator equipped with the water bath (LR4000, Heidolph, Germany) at 40 ± 5 ℃ under reduced pressure for 24 h. Thereafter, the extract was lyophilized (Ilshinbiobase PVTFD10R, Yangju, Korea) and stored at -20 ℃ until used as MEMH. The total lyophilized extract was 9.77 g (yield 17%). The percent yield varied with change in temperature, ranging 15% to 20%. The extract could be dissolved in water up to a concentration of 100 mg·mL-1.

Synthesis of monosodium urate (MSU) crystals

MSU crystals were prepared according to Chen et al. [31] with some modifications. Initially, 100 mg of uric acid was dissolved in 20 mL of distilled water with 60 μL of 10 mol·L-1 NaOH by heating and blending. Next, pH was adjusted to 7.2-7.4 with HCl (1 mol·L-1) at 60 ℃. The solution was kept overnight in constant shaking and then kept at room temperature for 5 d. After 5 days, the mixture was transferred in 15-mL tube and stored at 4 ℃ for 4 d. Needle-like crystals were recovered and suspended by vortex overnight. The crystals were collected after washing twice with 100% ethanol and once with acetone followed by centrifugation at 3 000 r·min-1 for 2 min at 4 ℃. Finally, The MSU crystals were suspended in sterile endotoxin-free phosphate buffered saline (PBS). The crystals obtained were preserved at -20 ℃ after evaporation through heating at 180-200 ℃.

Cell culture

SW1353 cells (ATCC, HTB-94; chondrosarcoma cell line) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco BRL, Darmstadt, Germany) with 10% fetal bovine serum (FBS) (Lonza, Walkersville, USA) supplemented with 1% penicillin/streptomycin (P/S) (Lonza) at final concentrations of 100 IU·mL-1 (P) and 100 μg·mL-1 (S), respectively, in a humidified atmosphere of 5% CO2. SaOS-2 cells (ATCC, HTB-85, human osteosarcoma cell line) were cultured in DMEM supplemented with 10% FBS, containing 1% P/S, in humidified atmosphere 5% CO2 in air at 37 ℃. THP-1 cells (ATCC, TIB-202; human monocytic cell line), were grown in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco BRL) supplemented with 10% FBS, 2 mmol L-glutamine, 4.5 g·L-1 glucose, 10 mmol HEPES, 1 mmol sodium pyruvate, 0.05 mmol 2-mercaptoethanol, and 1% P/S at 37 ℃ and 5% CO2.

Preparation and cultivation of primary chondrocytes

Human primary chondrocytes were cultured as describe previously [32] with some modifications. Human femoral condyles cartilage samples were obtained from healthy patients (58-to 80-year old) undergoing hip replacements surgery. All patients signed a written informed consent form approved by the Hallym University, Chuncheon Sacred Heart Hospital Institutional Review Board (2009-42). Board also reviewed the consent form and protocols to ensure that the investigation was conducted according to the principles expressed in the declaration of Helsinki and permitted this study. Cartilage was dissected in 100-mm dish and washed twice with DMEM containing 10% FBS supplemented with 1% P/S. Hyaluronidase was added into the dish and cartilage was cut into small fragments with sterilized blade. Further, the chopped cartilage pieces were washed twice with serum free DMEM and incubated with protease solution for 1 h at 37 ℃ in humidified atmosphere with 5% CO2. After that, the cartilage samples were again washed twice with serum-free DMEM and digested with Collagenase for about 2 h and 30 min under the same aforementioned conditions. After the completion of incubation time, the suspension was passed through a cell strainer of 70 μmol·L-1 and collected in a 50 mL tube. Next, the collected media were centrifuged at 1500 r·m-1 for 5 min and the pellet was washed by repeating the procedure twice. Initially, the cells pallet was re-suspended with DMEM supplemented with 20% FBS and 1% P/S. After 3 days, the cells were maintained in 10% FBS and 1% P/S DMEM at a density of 6 × 106cells/100-mm dish.

MTT assay

MTT assay is a reliable and extensively used method for evaluating cell viability of cultured cells [33]. Therefore, MTT assay was carried out to estimate the cell viability of SaOS-2, SW1353, and primary chondrocytecells in the present study. It was performed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) based in vitro toxicology assay kit (MTT; Sigma Aldrich, St. Louis, Missouri, USA) as describe in manufacturer's protocol. The MTT (5 mg·mL-1 solution was added to the cell cultures in 96-well plate and incubated at 37 ℃ for 3 h. The supernatant was removed and the insoluble intracellular formazan crystal was dissolved in 200 μL of dimethyl sulfoxide (DMSO). Optical density was measured at 570-nm wavelength. To assess the viability of THP-1 cells, the treated or non-treated cells were washed twice with PBS to remove any residual extract by plate-cen-trifugation. The extract-free cells were suspended in 100 μL of complete RPMI medium with 10% heat inactivated FBS, and MTT assay was performed as above.

Lactate dehydrogenase (LDH) activity assay

LDH assay is a well-established quantitative method for analyzing cytotoxicity in vitro [34]. In the present study, the cytotoxicity was measured with LDH cytotoxicity detection kit (Takara Bio Inc., Shiga, Japan), according to the manufacturer's protocol. In short, the tissue culture medium (10 μL) from individual experimental sample was added to a 96-well plate containing 40 μL of PBS. Thereafter, 50 μL of LDH reagent was added to each well and the plates were incubated for 45 min in dark at 25 ℃. Enzymatic reaction was stopped by the addition of stop solution (50 μL). Absorbance was measured at 492-nm wavelength. Cell lysate of total cells was taken as a positive control.

Alkaline phosphatase (ALP) activity assay

ALP activity was detected as described previously [35]. Briefly, the SaOS-2 cells were cultured at a density of 9 × 104 cells/well in 24-well plates. The cells were washed twice with ice-cold PBS and lysed with 150 μL/well of RIPA buffer. Total cell lysate (20 μL) were mixed with CSPD substrate (100 μL) (Roche, Mannheim, Germany) and incubated for 30 min. The luminescence reading was performed on a luminometer (Glomax, Promega, Sunnyvale, CA, USA). In order to normalize protein concentration, total cell lysate was assayed using a protein assay kit (Bio-Rad, Hercules, CA, USA).

Luciferase assay

The SW1353 cells were plated at a density of 9 × 104 cells/well in 24-well plates, and transfected with Genefectine transfection reagent (Genetrone Biotech Co., Gwangmyeong, Korea), according to the manufacturer's guidelines. The NFκB luciferase reporter (100 ng; Addgene, Cambridge, MA, USA) and Renilla luciferase thymidine kinase construct (Invitrogen, Carlsbad, CA, USA) 50 ng were used to access luciferase activity. With Dual-Luciferase Assay kit (Promega Corp., Madison, WI), luciferase activities Fwere estimated through luminometer (Glomax, Promega, Sunnyvale, CA, USA). Total value of reporter activity of each sample was standardized with Renilla luciferase activity.

RNA isolation and real-time RT-PCR

According to manufacturer's guidelines, total RNA was extracted using TRIzol reagent (Invitrogen, Darmstadt, Germany). The cDNA first strand was formed with 2 μg of total RNA using SuperScript Ⅱ (Invitrogen, Carlsbad, CA, USA). The expressions of genes were determined using one-tenth of the cDNA for each PCR mixture containing EXPRESS SYBR green qPCR Supermix (BioPrince, Seoul, Korea). Real-time PCR was executed using a Rotor-Gene Q (Qiagen, Hilden, Germany). The reaction was completed by 50-cycle amplification at 95 ℃ for 20 sec, at 60 ℃ for 20 sec, and at 72 ℃ for 25 sec. Relative mRNA expression of particular genes was standardized to GAPDH and quantified using the 2-ΔΔCT method. The PCR primers sequences of human are listed in Table 2.

Table 2 Primer sequences for the real-time PCR
Protein extraction and Western blotting analysis

Following stimulation by MSU crystals or MEMH, the cells were immediately washed with ice-cold PBS and incubated for 15 min with ice-cold lysis buffer containing phosphatase and protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). Whole cell lysates were harvested after centrifugation at 13 000 r·min-1 for 15 min to remove debris. The protein concentration was determined by protein assay kit (BioRad) following the manufacturer's protocol. Equal amounts of protein were then loaded onto 10% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bed-ford, Massachusetts, USA). The blots were incubated with appropriate dilutions of primary antibodies, including anti-IkBα (Santa Cruz Biotechnology, CA, USA), anti-phospho p65 (serine 536) (Cell Signaling Technology, Danvers, MA, USA), and anti-COX-2 (Cell Signaling Technology). Anti-β-actin antibody (Santa Cruz Biotechnology) was used as loading control. The blots were washed thrice with TBST (10 mmol·L-1 Tris-HCl, 50 mmol·L-1 NaCl, and 0.25% Tween 20) and incubated with a horseradish peroxidase-conjugated secondary antibody. Finally the bands of interest were visualized by using enhanced chemiluminescence (ECL) reagents (Bionote Inc., Hwaseong, Korea).

MSU crystal-induced ankle arthritis model

MSU crystals (300 μg) suspended in 10 μL of endotoxin-free PBS were injected intra-articularly into the tibiotarsal joint (ankle) of the mice anaesthetized with 2.5% isoflurane [36]. BALB/c Mice were divided to four experimental groups (n = 7/group). All animal experiments were performed according to guidelines and with approval of Animal Experimentation Ethics Committee at Hallym University, South Korea (Permit Number; Hallym R2015-19). MEMH (500 mg·kg-1) was orally administered at different time points (1, 9, and 17 h) after injection of MSU crystals. After 18 h of MSU crystals injection, the mice were sacrificed for evaluation of inflammation parameters. The results are presented as percentage change from baseline diameter.

ELISA

ELISA kit (R & D systems Europe, Abingdon, UK) was used for a quantitative measurement of inflammatory mediators, such as IL-1β and TNFα, according to the manufacturer's recommendations.

Histological analysis

For histopathological evaluation, a portion of ankle tissue was fixed in 2% paraformaldehyde (PFA) solution (Merck, Darmstadt, Western Germany). The paraffin-embedded ankle tissue was sectioned (5-μm thickness) for hematoxylin and eosin (H & E) staining. The stained sections were observed under a microscope at 20 × magnification and photographed under a Ziess AxioCam digital camera (Carl Zeiss, Inc., Thornwood, NY).

Statistical analysis

All the statistical data were analyzed by using Graphpad Prism 5.0 (San Diego, CA, USA) and evaluated by two-tailed Student's t test. P < 0.05 was considered statistically significant.

Results Suppressive effects of MEMH on inflammatory mediators and NFκB signaling activity in MSU crystals-stimulated SW1353 cells

To identify the effects of MEMH on gout related inflammatory mediators resulting from MSU crystals stimulation on the SW1353 cells, the cells were treated with different concentration of MEMH for 24 h after stimulation of MSU crystals (1 mg·mL-1). The SW1353 cells treated with MSU crystals or MEMH or both showed no effect on cell viability or cytotoxicity (Figs. 1A & 1B). Since a dose of 200 μg·mL-1 showed maximum anti-inflammatory effect on SW1353 cells, it was used for performing consecutive experiments (data not shown). Real-time RT-PCR was performed to quantify the expression of pro-inflammatory mediators (IL-1β, TNFα, COX-2, and iNOS) and matrix metalloproteases (MMP-1, MMP-9 and MMP-13) from cDNA samples of the MSU crystals-stimulated SW1353 cells. Real-time RT-PCR analysis showed that MSU crystals stimulated SW1353 cells expressed about 3.5 folds of IL-1β, 4.5 folds of TNFα, 2.5 folds of COX-2, 3 folds of iNOS, 5 folds of MMP-1, 2 folds of MMP-9 and 2.5 folds of MMP-13, compared to the control. Conversely, the treatment of MEMH markedly suppressed the elevated levels of pro-inflammatory mediators as well as matrix metalloproteases. As shown in Figs. 2A-2G, MEMH lowered the expression level of mRNA (about 1.5 folds of IL-1β, 1.5 folds of TNFα, 1 folds of COX-2, 1.5 folds of iNOS, 3 folds of MMP-1, 1 folds of MMP-9, and 1.5 folds of MMP-13). Suppressive effect of MEMH on MSU crystals stimulated release of IL-1β was further confirmed by ELISA (Fig. 2H). MSU crystals have been reported to activate NFκB signaling pathway in articular chondrocytes [37]. While, activation of NFκB signaling pathway has been attributed to induction of inflammatory mediators, like IL-1β, TNFα, COX-2, iNOS and matrix metalloproteases (MMP-1, MMP-9 and MMP-13) in articular chondrocytes [9]. Therefore, to assess whether MEMH could modulate the NFκB signaling pathway in chondrocytes, we evaluated transcriptional activity of NFκB in the SW1353 cells through luciferase assay (Fig. 2I).

Figure 1 Effects of MEMH on cell viability and cytotoxicity of human chondrosarcoma cell line, SW1353, primary chondrocytes and monocytic cell line, THP-1. The cells stimulated with MSU crystals (1 mg·mL-1) were treated with MEMH (100, 200, and 400 μg·mL-1) for 24 h. Cell viability was determined through MTT reduction assay (A, C and E). Cytotoxicity was evaluated through LDH activity assay (B, D, and F).The results are presented as means ± SD of three independent experiments
Figure 2 Effects of MEMH on MSU crystal-induced expression of inflammatory mediators and activity of NFκB signaling in human chondrosarcoma cell line. SW1353 cells were incubated with 1 mg·mL-1 of MSU crystals in the absence or presence of MEMH with concentration of 200 μg·mL-1 for 24 h. Real time RT-PCR was performed with isolated total cellular RNA to analyze the transcript of IL-1β (A), TNFα (B), COX-2 (C), iNOS (D), MMP-1 (E), MMP-9 (F) and MMP-13 (G). GAPDH was used as an internal control. For analysis of NFκB signaling activity, SW1353 cells were transfected with NFκB-luc reporter plasmid and stimulated with MSU crystals treated or untreated with MEMH for 24 h (H). Western blotting was performed to detect the protein expression of cytosolic IκBα, COX-2 and β-actin as control. Cell lysates were collected after treatment of MSU with and without MEMH (I). Amount of IL-1β in supernatant was evaluated by ELISA (J). The results were presented as means ± SD of three independent experiments. *P < 0.05), **P < 0.01 vs control

The SW1353 cells were transfected with NFκB-luc reporter plasmid prior to stimulation with MSU crystals or MSU crystals along with MEMH. Stimulation of MSU crystals induced > 2 folds of luciferase activity in SW1353 cells after 24 h of treatment. Interestingly, co-treatment of MSU crystals with MEMH significantly suppressed NFκB activity induced by MSU crystals (Fig. 2I). In addition, the treatment of MSU also reduced cytosolic stability of IκBα while MEMH restored its stability after 24 h of treatment, confirming activation of NFκB signaling pathway and its subsequent inhibition by MEMH (Fig. 2J). A reduction in phosphorylation of p65 by MEMH in MSU crystals treated samples further confirmed the suppressive role of MEMH on NFκB activity (Fig. 2J). Moreover, induced expression of COX-2 protein was inhibited by the treatment of MEMH in the SW1353 cells, implicating the suppressive role of MEMH in MSU crystals induced inflammation in chondrocytes (Fig. 2J).

Validation of suppressive effect of MEMH on inflammatory mediators in MSU crystals stimulated human primary chondrocytes

Primary chondrocytes isolated from cartilage express all specific markers that are the hallmark of chondrocyte phenotype and are far more responsive to either anabolic or catabolic factors than other established cell lines [38]. Therefore, to verify whether effects of MEMH were also reproducible in more relevant model of cartilage cells, cultured human primary chondrocytes were treated under similar experimental condition as mentioned above in the present study. Alike to SW1353, no effect of MSU crystals or MEMH or both was observed on the cell viability or cytotoxicity of human primary chondrocytes (Figs. 1C & 1D). As depicted in Fig. 3, stimulation of MSU crystals up-regulated the mRNA expression of IL-1β (about 22 folds), TNFα (about 12 folds), COX-2 (about 5.5 folds), iNOS (about 23 folds), MMP-1 (about 2.5 folds), MMP-9 (about 7 folds), and MMP-13 (about 3.5 folds), respectively, compared to the control primary chondrocytes. Induced expression of inflammatory mediators and matrix metalloproteases enzymes from human primary chondrocytes in response to MSU crystals was more pronounced (several folds) than that in SW1353 cells. Identical to the results observed in SW1353 cells, treatment of MEMH exhibited dose dependent effectiveness in inhibiting mRNA expression of pro-inflammatory mediators and matrix metalloproteases. MEMH reduced the mRNA expression level of pro-inflammatory mediators and matrix metalloproteases (about 13 folds of IL-1β, 6 folds of TNFα, 3 folds of COX-2, and 9.5 folds of iNOS, 1.5 folds of MMP-1, 3.5 folds of MMP-9, and 2 folds of MMP-13). Repressive effect of MEMH on MSU crystals stimulated release of IL-1β was further established by ELISA (Fig. 3H). Therefore, these results authenticated our previous results in SW1353 cells treated with MSU crystals along with MEMH and suggested that MEMH may act as a beneficial therapeutic agent for chondroprotection.

Figure 3 Effects of MEMH on MSU crystal-induced expression of inflammatory mediators in human primary chondrocytes. Human primary chondrocytes were incubated with 1 mg·mL-1 of MSU crystals in the absence or presence of MEMH with concentration 200 μg·mL-1 for 24 h. Real time RT-PCR was performed with isolated total cellular RNA, and transcripts were analyzed for IL-1β (A), TNFα (B), COX-2 (C), iNOS (D), MMP-1 (E), MMP-9 (F), and MMP-13 (G). GAPDH was used as an internal control. Release of IL-1β in supernatant was detected by ELISA (H). The results are presented as means ± SD of separate three experiments. *P < 0.05), **P < 0.01 vs control
Suppressive effects of MEMH on inflammatory mediators in MSU crystals stimulated THP-1 cells

During acute joint inflammation caused by MSU crystals, interaction between MSU crystals and articular resident monocytes has been reported. MSU interaction causes activation of these cells, resulting in the secretion of pro-inflammatory mediators, such as IL-1β, TNFα, and COX-2, and chemotactic factors like CXCL8 [39-40]. Henceforth to confirm the ability of MEMH in prevention of inflammatory cascade caused by activation of monocytes, THP-1 cells were stimulated with MSU crystals or co-treated with MEMH for 24 h (Fig. 4). Treatment with MSU crystals or MEMH or both demonstrated no effects on cell viability and cytotoxicity of THP-1 cells (Fig. 1E & F). Thereafter, mRNA was collected and expression level of inflammatory genes from monocytes was determined through Real-time RT-PCR. As shown in Fig. 4, MSU crystals stimulation increased the mRNA level of IL-1β (about 500 folds), TNFα (about 3.5 folds), COX-2 (about 7 folds), and CXCL8 (about 3 folds). However, 200 μg·mL-1 of MEMH significantly decreased the mRNA expression level of pro-inflammatory mediators (about 130 folds of IL-1β, 2 folds of TNFα, 2.5 folds of COX-2, and 1.5 folds of CXCL8). Taken together, MEMH showed anti-inflammatory effects on monocytes stimulated with MSU crystals.

Figure 4 Effects of MEMH on MSU crystal-induced pro-inflammatory mediators and chemotactic factor in human monocytic cell line. THP-1 cells were incubated with 1 mg·mL-1 of MSU crystals in the absence or presence of MEMH of 200 μg·mL-1 for 24 h. Real time RT-PCR was performed with isolated total RNA and Gene expression of IL-1β (A), TNFα (B), COX-2 (C), and CXCL8 (D) were analyzed after MSU crystal stimulation with or without MEMH. GAPDH was used as an internal control. The results are presented as means ± SD of three independent experiments. *P < 0.05 vs control
No significant effect of MEMH on the cytotoxicity and differentiation process of MSU stimulated SaOS-2 cells

During chronic tophaceous gout, it has been observed that tophus infiltration into bone is responsible for the development of bone erosion. Moreover, studies have reported the deposition of MSU crystals within subchondral bone, suggesting presence of MSU crystals in direct contact with bone cells [41]. This direct contact of MSU crystals with osteoblasts have been shown to induce inhibitory effect on osteoblast viability and differentiation process [42]. Therefore, in order to determine any effect of MEMH on osteoblasts, human osteoblast like cell line SaOS-2 cells were treated with MSU crystals with or without MEMH and cell viability were assessed by MTT and LDH assays. Sample incubated with MSU crystals demonstrated reduced number of viable cells, compared to the control. Treatment of MEMH for 48 h at a concentrations range from 100 to 400 μg·mL-1 showed no significant recovery effect on observed cytotoxicity. These results were further confirmed by LDH assay. Additionally, we tried to identify any effect of MEMH on osteogenic activity of SaOS-2 cells. ALP assay was performed as early differentiation parameter for osteoblast on MSU crystals and MEMH treated SaOS-2. Alike to MTT and LDH results, no significant recovery effect of MEMH was observed on ALP activity (Fig. 5).

Figure 5 Effects of MEMH on cell viability and differentiation of human osteosarcoma cell line. Stimulation of SaOS-2cells with MSU crystals (250 μg·mL-1) was followed by the treatment of MEMH with concentration of 100, 200, and 400 μg·mL-1 for 48 h. Cell viability was determined through MTT reduction assay (A), and LDH activity assay (B). ALP assay was performed as an early osteoblast parameter for evaluating osteogenic activity (C). No significant changes were observed after treatment of MEMH to MSU crystals stimulated SaOS-2 cells. The results are presented as means ± SD of three independent experiments
Anti-inflammatory effects of MEMH on MSU Crystal-induced ankle arthritic model

To validate whether the above observed results of MEMH in in vitro systems could be reproduced in vivo, we studied the anti-inflammatory effects of MEMH using an ankle arthritis mouse model. Injection of MSU crystals into the knee joints of the mice (n = 7) for 18 h demonstrated significant swelling of the tarsotibial ankle joints of right hind paws than the untreated mice (Fig 6M). Oral administration of MEMH (500 mg·kg-1) after 1 h of MSU crystal injection followed by twice treatment after 8 h of interval demonstrated significant inhibition in swelling of the hind paw (Figs. 6A-6D and Fig. 6M). Histopathology characters of MSU crystals injected ankle tissue are well characterized for depicting inflammatory symptoms [43-44]. In the current study, histopathological changes were observed after hematoxylin and eosin (H & E) staining of the ankle joints specimen from MSU crystal and MEMH treated mice (Figs. 6E-6H). H & E staining of the MSU crystal injected ankle joints demonstrated acute inflammation and tissue proliferation in the synovial lining of the joints. Synovium hyperplasia and pannus formation with severe leukocyte infiltration were also observed in the MSU crystal treated mice (Fig. 6G). However, MEMH treated ankle joints showed suppressed synovium hyperplasia and reduced tissue proliferation in the synovial lining of the joints (Fig. 6H). Moreover, dermis of hind paw ankle tissue treated with MSU crystal demonstrated fibrinoid necrotic lesions in reticular layer of dermis (Fig. 6K). Also, the treatment with MEMH to gouty model recovered the fibrinoid necrotic lesions induced by MSU crystal injection (Fig. 6L).

Figure 6 Attenuation of inflammation by MEMH in gouty arthritic model. Acute inflammation in ankle joint was assessed by histomorphometric analysis. (A, E, and I): vehicle; PBS injected mice, (B, F, and J): MEMH treatment, (C, G, and K): MSU injection, and (D, H, and L): MSU injection with MEMH treatment. Representative photograph shows modulation of tarsotibial ankle joint swelling of right hind paws (A-D), H & E staining of the ankle joints segital section (E-H) and histological character of dermis of hind paw ankle tissue (I-L). Arrow indicating to symptoms like ankle swelling (C), synovium hyperplasia (G), and fibrinoid necrosis in reticular dermis (K) in MSU injected mice. (M) Effects of MEMH on ankle volume in MSU Crystal-induced ankle arthritic model. Mice were injected with MSU crystals in ankle with or without oral administration of MEMH as described in material and methods. Ankle volume of tarsotibial ankle joint was determined. Plasma level of IL-1β (N) and TNFα (O) were measured by ELISA. Each column represented means ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs control (Bar 100 μm)

To further assess the anti-inflammatory effects of MEMH in the mouse model, the alteration of pro-inflammatory mediators after MSU crystals injection in ankle was analyzed though ELISA. The plasma was collected after 18 h of treatment of MSU crystals and MEMH to determine the levels of IL-1β and TNFα. ELISA results showed that IL-1β and TNFα level were increased in MSU-injected mice in comparison with the control. However, an attenuation of MSU crystals-induced increases in the levels IL-1β (about 60 to 75 pg·mL-1) and TNFα (about 15 to 20 pg·mL-1) was observed after the treatments with MEMH (Figs. 6N and 6O).

Discussion

Crystalized MSU within the joints of individuals, with elevated levels of serum uric acid, triggers inflammatory gouty arthritis. Previous studies have demonstrated that MSU crystals are one of the most potent pro-inflammatory stimuli and can cause activation of pro-inflammatory pathways in articular chondrocytes and synoviocytes [9, 37]. In fact excessive expression of pro-inflammatory cytokines in native cells of joints decides the extent of inflammation present during gout disease [45]. Currently, therapeutic approach for gout treatment involves the prescription of anti-inflammatory agents to get rid of the symptoms along with inhibitors of xanthine oxidase or uricosuric drugs to lower the serum uric acid [46]. Though strategies based on therapeutic drug are generally effective in the treatment of gout but suffer from limitations like adverse side effects and high economic burden to the patients. Traditional formulation of oriental medicine can overcome these hurdles and have been used in Korea for several decades. Oriental medicine formulations are not just a simple mixture of different medicinal herbs, instead these times tested formulations follow empirical rules of the respective traditions [47]. Moreover, it is well established fact that a mixture of various herbal components could respond effectively to the related diseases than each of constituent herb used separately [48]. However, multi-compound formulation is often difficult to explain a scientific rationale for the effectiveness of the various ingredients present in a mixed formula. If a possible action mechanism of these traditionally used herbal formulations put forward to explain their clinical efficacy and safety, these kind of multi-compound herbal formulations could be complemented, even as a part of the modern Western medical system. Therefore, in the present study, the therapeutic properties of MEMH as a Korean oriental medicine formulation were assessed for effectiveness against inflammatory properties for symptom induced in gouty arthritis model, both in in vitro and in vivo systems.

In gouty arthritic joints, deposited MSU crystal comes in direct contact with a number of cells which are responsible for the native homeostasis of joints like chondrocytes, monocytes, osteoblasts, etc. Among these, chondrocytes form the upper layer of the joints and are thought to get affected first by the MSU crystals, present on hyperechoic band. Following interaction with MSU crystals, chondrocytes may contribute to cartilage damage through pathogenic mechanisms such as increased production of degradative enzymes and pro-inflammatory mediators [10]. According to previous studies, exposure of chondrocytes to MSU crystals is responsible for the expression of several pro-inflammatory mediators including IL-1β, TNFα, COX-2, and iNOS [9, 37, 45]. Moreover, several research groups have revealed that MMPs, COX-2, and iNOS are upregulated in chondrocytes after treatment with MSU crystals [42]. In agreements with these studies, our results also demonstrated that exposure of MSU crystals to human chondrosarcoma cell line (SW1353) as well as primary chondrocytes alters the expression level of key pro-inflammatory mediators. An increased mRNA expression of pro-inflammatory mediators like IL-1β, TNFα, COX-2, iNOS, and matrix metalloprotease like MMP-1, MMP-9, and MMP-13 were observed. IL-1β and TNFα play a prominent role in the catabolism of articular cartilage [49]. These mediator cytokines are known to upregulate the production of MMPs and nitric oxide (NO) causing apoptosis of chondrocytes and cartilage breakdown [45, 50]. Excessive production of NO is triggered by up-regulated expression of iNOS by MSU crystals. In turn generation of NO can impair chondrocyte viability, inhibit chondrocyte proteoglycan synthesis, and increase the matrix catabolic activity of MMPs [45]. Also, over expression of COX-2 in articular cartilage is a characteristic feature of inflammatory joint diseases like gouty arthritis and osteoarthritis. In inflamed tissues, COX-2 is responsible for elevated levels of prostaglandin E2 (PGE2) which consecutively results into cartilage degradation, angiogenesis and inflammation [37]. The elevated level of mRNA expression of COX-2 and iNOS by MSU crystals in both SW1353 cells and primary chondrocytes, clearly implicated a role of MSU in initiating inflammation in chondrocytes. Treatment of MEMH was able to ameliorate the induced mRNA expression of inflammatory mediators in both the cell types. These observations clearly showed the chondro-protective effects of MEMH.

Regulation of the gene expression by MSU crystals stimulation, such as IL-1β, TNFα, COX-2, and MMPs, are controlled by activation of the ubiquitous transcription factor NFκB [9, 37, 51]. Therefore, we tried to clarify if there was any possible effect of MEMH on NFκB signaling pathway which thus could explain the recovery effect of MEMH on MSU crystals induced expression of inflammatory mediators. Our results clearly demonstrated that MEMH was able to suppress MSU crystals-induced activation of NFκB activity. In acute gout, deposition of MSU crystals can trigger the ingress of immune cells in synovial joints. Generally, excessive immune cells are absent from the normal joint space and appear only during the development of full-blown acute inflammatory syndrome. Monocyte/macrophage lineages are known to play an important role in host defense and can be directly activated by MSU crystals to induce inflammation [45]. Moreover, infiltration of a vast number of monocytes has often been observed during the MSU crystals induced gouty arthritis [50]. Pro-inflammatory mediators associated with gouty inflammation like IL-1β, TNFα, and CXCL8 have been reported to be induced in macrophages after the exposure of MSU crystals [40, 52]. As discussed earlier, both IL-1β and TNFα are potent pro-inflam-matory cytokines that initiate or amplify the inflammatory response in a particular cell type during acute gout. Chemokine CXCL8 possesses chemotactic activity and can attract other immune cells toward the inflammatory site, intensify the joint destruction event [39]. Therefore, to address whether MEMH could inhibit the induced expression of pro-inflam-matory mediators from MSU crystals stimulated monocytes, we investigated the effects of MEMH on THP-1 cells stimulated with MSU crystals. The results demonstrated that MEMH repressed the gene expression levels of IL-1β, TNFα, COX-2 and CXCL8, proving its anti-inflammatory activity even in THP-1 cells.

Chronic tophaceous gout is usually associated with bone erosion which leads to musculoskeletal disability, due to joint destruction and deformities [41]. Deposition of MSU crystals has been observed in surrounding of subchondral bone, indicating that bone cells have direct interaction with MSU crystals during erosive gout. Recently, it has been reported that MSU crystals contribute to bone erosion in gout through decline of osteoblast viability, function and differentiation as well as elevation of osteoclast formation [42]. Hence, we analyzed the effect of MEMH on the cell viability and on the process of differentiation of osteoblast cells stimulated by MSU crystals. In accordance to Chhana et al., our results also demonstrated a decline in the cell viability as well as ALP activity of SaOS-2 cells treated with MSU crystals, compared to the control [42]. However, the degree of decrease in cell viability and ALP activity observed in our system was far less, compared to the other study at similar concentration of MSU crystals (250 μg.mL-1). Variance in the decrease of cell viability and ALP activity of osteoblasts might be due to the difference in cell type utilized in the studies. Therefore, more detailed studies are required on other osteogenic models to establish the role of MSU crystals on the function of osteoblasts. Additionally, to observe any recovery effect of MEMH on affected osteoblasts by MSU crystals, SaOS-2 cells were analyzed after co-treatment of MSU crystals with MEMH. Results showed no recovery effect on the decreased cell viability and ALP activity of SaOS-2 cells, implicating no survival or bone forming activity of MEMH on osteoblasts (Fig. 5).

In order to validate the anti-inflammatory effect of MEMH, as observed in vitro, we established an ankle gout model in mice. MSU crystals are frequently employed to develop a well-established animal model of gouty arthritis. Consequently, deposition of MSU crystals within the joint cavities promotes acute inflammation. In the present study, MSU crystals injected joints demonstrated edema formation, amplified infiltration of inflammatory cells in synovium, formation of synovium hyperplasia and pannus, tissue proliferation in the synovial lining and fibrinoid necrosis in reticular layer of dermis. MEMH at a dose of 500 mg/kg significantly attenuated the inflammatory symptoms observed after MSU crystal injection in mice ankle. A decrease in paw swelling, reduced infiltration of inflammatory cells in synovium, decline in proliferation of the synovial lining and reduced severity of fibrinoid necrosis in reticular layer of dermis clearly demonstrated the anti-inflammatory activity of MEMH in the ankle gout model. As discussed earlier, MSU crystals can induce the secretion of pro-inflammatory mediators, such as IL-1β and TNFα in chondrocytes, monocytes, neutrophils, and other synovial lining cells. Moreover, IL-1β and TNFα have been implicated in development of acute and chronic gouty arthritis. Therefore, we determined whether administration of MEMH could regulate the secretion of pro-inflammatory cytokines. ELISA results demonstrated that MEMH administration decreased the plasma level of both IL-1β and TNFα induced by MSU injection. Taken together, MEMH exhibited recovery effect of gouty arthritis in vivo, validating its efficacy for anti-inflammatory properties observed in vitro.

Conclusions

The present study demonstrated that Korean traditional formula, MEMH, has anti-inflammatory properties against MSU crystal induced gouty inflammation. Treatment of MEMH repressed the gene expression of pro-inflammatory mediators and MMPs from chondrocytes, induced by MSU crystals. Moreover, MEMH suppressed the activity of NFκB signaling pathway in chondrocytes. The mRNA expression levels of pro-inflammatory mediators and chemotactic cytokine were also ameliorated by MEMH in MSU crystals stimulated THP-1. However, MEMH did not show any recovery effect on the decrease of cell viability and ALP activity, as observed in osteoblasts after the treatment of MSU crystals. In vivo study demonstrated that administration of MEMH could recover the inflammatory histopathological symptoms induced by injection of MSU crystals. Additionally, the plasma levels of both IL-1β and TNFα were decreased after the administration of MEMH in the ankle gout model. Consequently, anti-inflam-matory effects of MEMH observed in in vitro and in vivo studies suggested that MEMH might be a potential agent for the treatment of gouty arthritis. However, more detailed studies evaluating MEMH on the parameters of safety and efficacy would be required to achieve clinical significance.

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