Background
Osteoarthritis (OA) is a degenerative joint disease characterized by abrasion of articular cartilage and trabecular bone loss [
1]. The chance of developing the disease increases with age, knee joint lesions, obesity, infectious diseases, and various joint inflammations [
2‐
5]. Several previous studies suggested that abrasion of articular cartilage, subchondral bone sclerosis, and excessive formation of trabecular bone might cause OA [
6‐
8]. Other studies highlighted the importance of prostaglandins in OA treatment [
9]. Prostaglandins are enhanced by stimuli, such as lipopolysaccharide, sodium nitroprusside, or physical injury associated with OA [
10], increased macrophage migration into the synovial fluid, activated nuclear factor (NF)-κB as an inflammatory transcription factor, and both cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) as inflammatory enzymes [
11,
12].
Regarding alleviation of OA, although non-steroidal anti-inflammatory drugs including acetaminophen are mainly prescribed during the early stage of OA, these drugs do not effectively prevent the progression of OA [
13] and there have been some problems with side effects involving the gastrointestinal tract and kidneys. In particular, the use of COX-2 inhibitors is restrictively allowed because of significant side effects on cardiovascular function [
14].
Monosodium iodoacetate (MIA) is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that results in a reduction of glycolysis and causes articular cartilage changes related to the histological and morphological features of OA by impeding integration of the chondral structure and inducing cell death of chondrocytes [
15]. Furthermore, MIA causes a repeated progression of synovial hyperplasia and inflammatory cell infiltration, destroys articular cartilage, and induces bone loss and chondral deformation [
16]. Injection of MIA into the knee joints of rats is considered a suitable model that resembles the phenomena observed in human OA and is thus applied for evaluation of chondroprotective activity [
17].
SHINBARO is a purified herbal formulation containing six traditional medicines,
Ledebouriellae Radix (
Fang Feng),
Achyranthis Radix (
Huai Niu Xi),
Acanthopanacis Cortex (
Wu Jia Pi),
Cibotii Rhizoma (
Gou Ji),
Glycine Semen (
Hei Dou), and
Eucommiae Cortex (
Du Zhong), that has been used to treat inflamed lesions and bone diseases [
18]. Pharmacopuncture is a new form of therapy that involves both herbal medicine administration and acupuncture to manage blood extravasation and improve blood flow. Therefore, pharmacopuncturology is considered to be a medical practice in oriental medicine that involves administration of refined herbal extracts into acupoints [
19]. The anti-inflammatory activity of oral SHINBARO treatment was demonstrated in an adjuvant-induced OA rat model [
18]. However, the anti-arthritic activity of SHINBARO pharmacopuncture and its underlying mechanism have not yet been determined.
This study aimed to investigate the anti-osteoarthritic activities of intra-articular administration of SHINBARO and determine its underlying molecular mechanism in an MIA-induced OA rat model.
Methods
Preparation and composition of SHINBARO
SHINBARO was prepared by Hanpoong Pharmaceutical Co. Ltd. (Jeonju, Republic of Korea). The mixture of six crude drugs,
Ledebouriellae Radix (4.444 g),
Achyranthis Radix (4.444 g),
Acanthopanacis Cortex (4.444 g),
Cibotii Rhizoma (2.778 g),
Glycine Semen (2.778 g), and
Eucommiae Cortex (1.389 g), was powdered and boiled for 3 h in distilled water (1 L). The mixture was then subjected to ultrafiltration through Whatman grade 2 qualitative filter paper (GE Healthcare Life Sciences, Marlborough, MA, USA) to exclude components with molecular weights above 10,000. The resulting filtrate was lyophilized to a powder using a rotary evaporator (Eyela, Miyagi, Japan), and stored at 4 °C until use. SHINBARO was administered intra-articularly at a dose of 2, 10, or 20 mg/kg in saline and orally at a dose of 20 or 200 mg/kg in saline. The same volume of saline was used as a vehicle in control rats. The validation of SHINBARO was performed by high-performance liquid chromatography (Waters™ 600 s controller, 626 pump, temperature control module, in-line degasser, 717 plus autosampler, and 996 photodiode array detector; Waters, Bedford, MA, USA) analysis of each ingredient extract using the following six indicator biological components [
18]: cimifugin for
Ledebouriellae Radix; 20-hydroxyecdysone (0.311–0.312 mg/g) for
Achyranthis Radix; acanthoside D (0.577–0.578 mg/g) for
Acanthopanacis Cortex; onitin-4-O-β-D-glucopyranoside for
Cibotii Rhizoma; genistin (0.0426–0.0427 mg/g) for
Glycine Semen; and geniposide (0.431–0.432 mg/g) for
Eucommiae Cortex. SHINBARO was further standardized for quality control according to the regulations imposed by the Korea Food and Drug Administration.
Animals
Male Sprague–Dawley rats (200–220 g) were obtained from Central Laboratory Animal Inc. (Seoul, Korea), and housed in solid-bottom cages with free access to food and water. The temperature was maintained to 22 ± 2 °C, and a 12-h/12-h light/dark schedule was implemented. Prior to use, the animals were allowed 1 week for acclimatization within the work area environment. All animal experiments were approved by the local Animal Ethics Committee of Seoul National University (Additional file
1), and carried out in accordance with the Institutional Animal Care and Use Committee Guidelines of Seoul National University (Permission Number: SNU-120904-7) and the ARRIVE guideline (Additional files
2 and
3).
MIA-induced OA rat model
Rats were anesthetized with diethyl ether and given a single intra-articular injection of 2.5 mg MIA (Sigma–Aldrich, St. Louis, MO, USA) into the infrapatellar ligament of the right knee [
20]. MIA was dissolved in 0.9 % normal saline and administered in a 25-µL volume. The rats were arbitrarily divided into eight groups containing six rats each. Subsequently, the rats were treated with normal saline (vehicle-treated MIA group), 2, 10, or 20 mg/kg of SHINBARO by intra-articular administration (intra-articular SHINBARO group; IAS group), 20 or 200 mg/kg of SHINBARO by oral administration (oral SHINBARO group; OS group), and 5 mg/kg of diclofenac by oral administration (diclofenac group) once daily for 21 days. Rats treated with normal saline, and not MIA, were used as a control group (Table
1). The SHINBARO concentrations and MIA injection volume were selected based on previous evaluations [
21]. After 21 days of treatment, the animals were euthanized and blood samples were collected for serum isolation. The femurs were dissected and stripped of soft tissue for analysis of the trabecular microarchitecture.
Table 1
Effect of SHINBARO on change in body weight of MIA-induced OA rat model
0th | 305.5 ± 7.9 | 308.1 ± 20.4 | 301.2 ± 17.1 | 305.5 ± 11.8 | 306.2 ± 11.8 | 306.2 ± 14.5 | 306.0 ± 16.2 | 288.0 ± 13.9 |
7th | 352.9 ± 11.6 | 347.6 ± 14.2 | 335.0 ± 14.5 | 352.2 ± 15.0 | 347.2 ± 10.7 | 347.3 ± 11.7 | 351.2 ± 12.7 | 317.8 ± 32.0 |
14th | 384.5 ± 9.5 | 378.6 ± 17.1 | 365.2 ± 16.1 | 387.8 ± 14.1 | 379.3 ± 11.5 | 376.2 ± 11.4 | 387.0 ± 11.7 | 356.0 ± 15.5 |
21st | 411.5 ± 15.2 | 402.0 ± 16.3 | 385.5 ± 19.7 | 411.5 ± 15.5 | 401.3 ± 12.0 | 395.8 ± 17.3 | 407.8 ± 13.1 | 388.5 ± 9.05 |
Morphological analysis of bone loss
The bone microarchitecture of the femur in the region at 0.6–2.1 mm from the growth plate was scanned using a micro-computed tomography (micro-CT) system (SkyScan 1076; SkyScan, Aartselaar, Belgium). The X-ray source was set at a voltage of 50 kV and a current of 200 µA and filtered with a 0.5-mm aluminum filter. The scanning angular rotation was 180° with an angular step of 0.5°. The voxel size was fixed at 8.9 µm. The morphometric indices of the bone region were determined from the micro-CT data on 3D images using CTan software (SkyScan 1076; SkyScan, Aartselaar, Belgium). The following measures characterizing the three-dimensional structure of the trabecular bone were determined: bone volume of interest (BV; µm3); bone volume fraction (bone volume/total volume (BV/TV); %); and mean number of objects per slice (Obj.N). BV indicates the total volume of extracted cartilage from the same parts in each group. The ratio of BV/TV is the segmented trabecular bone volume to total tissue volume. Obj.N implies the connectivity of the specimens, which consisted of several cross-sectional slices of cartilage in a three-dimensional structure.
Histopathological analysis
The right knee joints from the tibia to the distal metatarsal including the tarsal joint were resected and fixed with 10 % neutral-buffered formalin for 24 h at 4 °C. The fixed specimens were decalcified with 20 % formic acid for 3 days and embedded in paraffin. Sections of the tissue specimens were acquired from the paraffin blocks at 5 µm thickness, deparaffinized, and rehydrated in the order of xylene, absolute alcohol, and 50 % alcohol. The rehydrated sections were stained with hematoxylin and eosin (H&E) for observation of morphological changes in the articular tissues and safranin-O fast green (SOFG) for evaluation of the proteoglycan (PG) contents.
Measurement of prostaglandin E2 (PGE2) level
The serum PGE2 levels were measured by enzyme immunoassay (EIA) using a prostaglandin E metabolite EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA). Briefly, aliquots of prepared standard solutions and sample sera were plated in 96-well plates from the PGE2 EIA kit, and a PGE2-acetylcholinesterase (AChE) conjugate (PGE Tracer) and PGE2 monoclonal antibody solution were added and allowed to react at 4 °C for 24 h. Next, Ellman’s reagent, which contains an AChE substrate for color formation, was added to the wells and the optical density was measured at 412 nm (VersaMax ELISA Microplate Reader; Molecular Devices, Sunnyvale, CA, USA). The PGE2 concentrations in the serum samples were determined using a four-parameter logistic equation (logit(B/B0) = ln[B/B0/(1–B/B0)]) by comparisons with the absorbances of the PGE2 standard solutions at several concentrations (8, 16, 31, 62, 125, 250, 500, and 1000 pg/mL).
Measurement of anti-type II collagen antibody level
The serum anti-type II collagen antibody levels were assayed using a rat anti-type I and type II collagen IgG ELISA kit (Chondrex Inc., Redmond, WA, USA) in accordance with the manufacturer’s instructions.
Ex vivo biochemical analysis of inflamed tissues
The right knee joint cartilages of the rats were removed at the end of the treatment period. The tissues were homogenized using Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. The protein levels of inflammatory enzymes (iNOS, COX-2), pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β), and inflammatory mediators (NF-κB, nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor (IκB)) in the cartilaginous tissues were determined by western blot analysis.
Western blot analysis
The proteins in the rat articular cartilage samples were resolved by 6–15 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with blocking buffer, comprising 5 % bovine serum albumin in phosphate-buffered saline containing 0.1 % Tween-20 (PBST), for 1 h at room temperature. After three washes with PBST, the membranes were incubated with primary antibodies against β-actin (1:1000), iNOS (1:1000), COX-2 (1:1000), IL-1β (1:1000), NF-κB (1:500), IκB-α (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TNF-α (1:1000) (Cell Signaling, Danvers, MA, USA) diluted in 2.5 % bovine serum albumin overnight at 4 °C. The membranes were washed three times with PBST and incubated with corresponding secondary antibodies diluted in PBST (1:1000) for 2 h at room temperature. After three washes with PBST, the membranes were visualized with a WEST-ZOL plus Western Blot Detection System (Intron Biotechnology, Sungnam, Korea), and analyzed using an LAS 4000 system (Fuji Film Corp., Tokyo, Japan).
Statistical analysis
All experiments were repeated at least three times. Data were presented as means ± standard derivation for the indicated numbers of independently performed experiments. Statistical significance was assessed by one-way analysis of variance coupled with a Dunnett’s t test. Values of P < 0.05 were considered statistically significant. All statistical analyses were performed using Graph Pad Prism 5.0 for Windows (GraphPad Software, La Jolla, CA, USA).
Discussion
OA is a degenerative knee joint disease that causes intense pain and functional disability [
26]. The disease is classified into two categories: primary or idiopathic arthritis that involves endocrinologic, genetic, and nutritional factors and secondary or subsequent arthritis that is incurred by injury, disorder, and malformation capable of causing damage to the articular cartilage [
27,
28].
Although anti-inflammatory effects of OS treatment were previously reported [
29], the pharmacological effects of IAS treatment, which is already practiced in clinical settings, and its underlying mechanisms of action remain to be determined. Therefore, the present study was conducted to investigate the anti-inflammatory activity of IAS treatment and its underlying mechanisms of action in the MIA-induced OA rat model.
The MIA used in the present study inhibits articular cartilage structural incorporation through suppression of GAPDH activity in the cartilage, leading to histopathological and pathomorphologic changes in the articular cartilage [
30]. SHINBARO prevented the degeneration of the trabecular bone microarchitecture in the distal femur of the rat. IAS treatment also markedly restored bone loss in the MIA-induced OA model (Fig.
1). These data were supported by the protective effect of IAS treatment on the bone morphometric parameters (Fig.
2). In addition, no significant body changes or obvious toxicity were found in the IAS group (Table
1). These results indicated that IAS treatment not only preserves the bone mass, but also recovers the bone microstructural deterioration associated with MIA-induced OA without toxicity.
Healthy cartilage tends to maintain a balance between synthesis and degradation of extracellular matrix (ECM) components, such as PGs and type II collagen. However, the decomposition of the ECM becomes increased in inflamed cartilage, leading to an off-balance of matrix production and damage to the cartilaginous tissues [
31]. The H&E and SOFG staining in the vehicle-treated MIA group revealed significant PG loss and lesion development, and confirmed that the cartilage injury was attenuated by IAS treatment (Fig.
3). In addition, IAS treatment markedly reduced the elevated serum level of anti-type II collagen antibodies associated with MIA in a dose-dependent manner (Fig.
4b). These findings indicate that IAS treatment might delay bone loss through decreased bone turnover.
Major biomarkers for inflammation responses were determined in serum to further confirm the effects of IAS treatment on the regulation of inflammatory responses. PGE
2 acts as an important inflammatory mediator and is a major product of COX activity, which functions pathologically in inflammatory, autoimmune, and neoplastic diseases [
32]. In this study, the serum PGE
2 level was significantly higher in the vehicle-treated MIA group compared with the control group, while IAS treatment inhibited the elevation of PGE
2 production in the MIA-induced OA rat model (Fig.
4a).
Overproduction of PGE
2 is highly associated with overexpression of iNOS and COX-2 in inflammatory responses [
33]. The effects of IAS on iNOS and COX-2 protein expression levels were determined to investigate the mechanism responsible for the inhibition of PGE
2 production mediated by IAS treatment. IAS suppressed the MIA-induced overexpression of iNOS and COX-2 protein levels in a dose-dependent manner (Fig.
5).
We further examined whether IAS treatment regulates the protein expression levels of upstream mediators leading to the production of iNOS and COX-2. NF-κB exists in the cytosol of cells as an inactive heterodimer bound to the inhibitory protein IκB with Rel A (p65), c-Rel, RelB, NF-κB1 (p50/150), and NF-κB2 (p52/100) [
34]. However, NF-κB becomes degraded with IκB sequestration when inflammatory cytokines, TNFs, and lymphotoxins are activated by stimuli, such as mitogens, bacteria, ultraviolet light, oxidants, tetradecanoyl phorbol-13-acetate, ionizing radiation, and phosphatase inhibitors [
35‐
37]. The activated heterodimers of NF-κB subunits p65 and p50 in the cytosol translocate into the nucleus and bind to NF-κB-binding sites in gene promoters or enhancers, thereby inducing the transcription of downstream target genes, such as cytokines, cytokine receptors, cell adhesion molecules, and growth factors. IκB is negatively regulated by phosphorylation on serine residues, which is controlled by two IκB kinases, IKKα and IKKβ [
38]. In the present study, IAS treatment downregulated the protein level of NF-κB and upregulated the protein level of IκB (Fig.
6). These findings suggest that the anti-inflammatory effects of IAS treatment are associated with inhibition of NF-κB activation.
The anti-inflammatory effects of IAS treatment were also observed by measurements of the protein expression levels of pro-inflammatory cytokines, such as TNF-α and IL-1β. These cytokines promote the catabolic processes in OA, causing cartilage degradation. MIA can drive the overexpression of pro-inflammatory cytokines, but the TNF-α and IL-1β levels were effectively reduced by IAS treatment in the MIA-induced OA model (Fig.
6). These data suggest that IAS treatment inhibits inflammatory responses by suppressing the expression of pro-inflammatory cytokines, including TNF-α and IL-1β.
Authors’ contributions
WKK, HJC, JSS, JHL, IHH and SKL conceived and designed the study. WKK, HJC, YP and TJC performed the experiments. WKK, SKL, HJP and JYH wrote and revised the manuscript. All authors read and approved the final manuscript.