Osteoarthritis (OA) is one of the long-term chronic degenerative bone joint diseases that affects the aged population over 65 [
1]. Generally, OA patients are diagnosed with damaged cartilage, inflamed synovium, and eroded chondrocytes, which trigger pain and physical distress [
2]. Arthritic pain is predominantly caused by the degeneration of cartilage in joints by inflammation, and when the cartilage is seriously damaged bones can collide with each other causing unbearable pain and physical hardship [
3]. The involvement of inflammatory mediators with symptoms such as pain, swelling, and stiffness of the joint is well documented. In OA patients, inflammatory cytokines, which cause the erosion of cartilage and subchondral bone are found in the synovial fluid [
4]. Two major complaints that OA patients generally have are pain and synovial inflammation. Therefore the primary goals of the current OA therapies are to lower pain and inflammation. [
5]. Although the available OA treatments, including non-steroidal and steroidal drugs, have proven efficacies in alleviating pain and inflammation, the long-term uses of these drugs have severe health consequences such as cardiovascular, gastro-intestinal, and renal dysfunctions [
6]. Thus, a more effective medicine with fewer side effects has to be developed for the treatment of osteoarthritis.
Natural health products are being increasingly popular for being safe and easily available [
7]. Traditional Korean medicines have proven efficacies against several inflammatory diseases, including arthritis [
8]. Aucklandia lappa DC. is known for its medicinal properties, such as enhancing the circulation of qi for relieving pain and soothing the stomach, and has been used traditionally as a natural analgesic [
9]. Previous reports suggest that A. lappa possesses anti-inflammatory [
10,
11], analgesic [
12], anticancer [
13], and gastroprotective [
14] effects. The various biological activities of A. lappa are caused by its major active compounds: costunolide, dehydrocostus lactone, dihydrocostunolide, costuslactone, α-costol, saussurea lactone and costuslactone [
15]. Earlier studies claim that costunolide showed anti-inflammatory properties in lipopolysaccharide (LPS), which induced the macrophages through the regulation of NF-kB and heat shock protein pathway [
16,
17]. However, no study has investigated the potential activities of A. lappa for OA treatment. The present research has investigated the therapeutic effects of A. lappa against OA using (monosodium-iodoacetate) MIA and acetic acid-induced rodent models.
Monosodium-iodoacetate (MIA) is famously used to produce much of the pain behaviors and the pathophysiological features of OA in animals [
18,
19,
20]. When injected into knee joints, MIA disarrays the chondrocyte metabolism and induces inflammation and inflammatory symptoms, such as cartilage and subchondral bone erosion, the cardinal symptoms of OA [
18]. Writhing response induced with acetic acid is widely regarded as the simulation of peripheral pain in animals where the inflammatory pain can be quantitatively measured [
19]. The mouse macrophage cell line, RAW264.7, is popularly used to study the cellular responses to inflammation. Upon activation with LPS, RAW264 macrophages activate inflammatory pathways and secrete several inflammatory intermediaries, as such TNF-α, COX-2, IL-1β, iNOS, and IL-6 [
20]. This study has evaluated the anti-nociceptive and anti-inflammatory effects of A. lappa against OA in MIA animal model, acetic acid-induced animal model, and LPS-activated RAW264.7 cells.
2. Materials and Methods
2.1. Plant Material
The dried root of A. lappa DC. used in the experiment was procured from Epulip Pharmaceutical Co., Ltd., (Seoul, Korea). It was identified by Prof. Donghun Lee, Dept. of Herbal pharmacology, Col. of Korean Medicine, Gachon University, and the voucher specimen number was deposited as 18060301.
2.2. HPLC Analysis of A. lappa Extract
A. lappa was extracted using a reflux apparatus (distilled water, 3 h at 100 °C). The extracted solution was filtered and condensed using a low-pressure evaporator. A. lappa extract had a yield of 44.69% after freeze-drying under −80 °C. Chromatographic analysis of A. lappa was conducted with a HPLC connected using a 1260 InfinityⅡ HPLC-system (Agilent, Pal Alto, CA, USA). For chromatic separation, EclipseXDB C18 column (4.6 × 250 mm, 5 µm, Agilent) was used at 35 °C. A total of 100 mg of the specimen was diluted in 10 mL of 50% methanol and sonicated for 10 min. Samples were filtered with a syringe filter (Waters Corp., Milford, MA, USA) of 0.45 μm. The mobile phase composition was 0.1% phosphoric acid (A) and acetonitrile (B) and the column was eluted as follows: 0–60 min, 0%; 60–65 min, 100%; 65–67 min, 100%; 67–72 min, 0% solvent B with a flow rate of 1.0 mL/min. The effluent was observed at 210 nm using an injection volume of 10 μL. The analysis was performed in triplicate.
2.3. Animal Housing and Management
Male Sprague–Dawley (SD) rats aged 5 weeks and male ICR mice aged 6 weeks were purchased from Samtako Bio Korea (Gyeonggi-do, Korea). Animals were kept in a room using constant temperature (22 ± 2 °C) and humidity (55 ± 10%) and a light/dark cycle of 12/12 h. The animals were familiarized with the condition for more than a week before the experiment started. Animals had an ad libitum supply of feed and water. The current ethical rules for animal care and handling at Gachon University (GIACUC-R2019003) were strictly followed in all animal experimental procedures. The study was designed investigator-blinded and parallel trial. We followed the euthanasia method according to the guidelines of the Animal Experimental Ethics Committee.
2.4. MIA Injection and Treatment
Rats were randomly separated into 4 groups, namely sham, control, indomethacin, and A. lappa. Being anesthetized with 2% isofluorane O2 mixture, the rats were injected using 50 μL of MIA (40 mg/m; Sigma-Aldrich, St. Louis, MO, USA) intra-articularly into the knee joints to lead to experimental OA. The treatments were conducted as below: control and sham groups were maintained only with AIN-93G basic diet. Only, indomethacin group was provided with indomethacin (3 mg/kg) incorporated into AIN-93G diet and A. lappa 300 mg/kg group was assigned to AIN-93G diet supplemented with A. lappa (300 mg/kg). The treatments were continued for 24 days since the day of OA induction at the rate of 15–17 g per 190–210 g body weight on a daily basis.
2.5. Weight Bearing Measurement
After OA induction, weight-bearing capacity measurement of hind limbs of the rats was performed with the incapacitance-MeterTester600 (IITC Life Science, Woodland Hills, CA, USA) as scheduled. The weight distribution on hind limbs was calculated: weight bearing capacity (%)
