Andrographis paniculata Extract Relieves Pain and Inﬂammation in Monosodium Iodoacetate-Induced Osteoarthritis and Acetic Acid-Induced Writhing in Animal Models

: Osteoarthritis (OA), being the most prominent degenerative joint disease is a ﬀ ecting millions of elderly people worldwide. Although Andrographis paniculata is an ethnic medicine with a long history of being used as analgesic agent, no study using a monosodium iodoacetate (MIA) model has investigated its potential activities against OA. In this study, experimental OA was induced in rats with a knee injection of MIA, which represents the pathological characteristics of OA in humans. A. paniculata extract (APE) substantially reversed the loss of hind limb weight-bearing and the cartilage damage resulted from the OA induction in rats. Additionally, the levels of serum pro-inﬂammatory cytokines, such as IL-1 β , IL-6, and TNF- α as well as the concentration of matrix metalloproteinases, including MMP-1, MMP-3, MMP-8, and MMP-13 were decreased by APE administration. Acetic acid-induced writhing responses in mice which quantitatively measure pain were signiﬁcantly reduced by APE. In vitro, APE inhibited the generation of NO and downregulated the expression of IL-1 β , IL-6, COX-2, and iNOS in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The above results suggest the potential use APE as a therapeutic agent against OA.


Introduction
Osteoarthritis (OA) is the leading degenerative joint disease with an incidence of more than 50% among people aged 65 or older throughout the world [1]. The common characteristics of OA include the erosion of cartilage, synovial swelling, and a disruption in chondrocyte metabolism which cause pain and immobility [2,3]. Arthritic pain is the direct result of the cartilage damage in the joint caused by inflammation; and in the absence of joint cartilage, bones can rub against each other, which causes severe pain and immobility [4,5]. The involvement of inflammatory mediators in osteoarthritic inflammation and pain is now well-known. Inflammatory cytokines are secreted primarily by the synovial chondrocytes and can be found in the synovial fluids of OA patients [6]. As pain and synovial inflammation are two of the major complaints of OA patients, the available treatments for OA are designed mainly to alleviate pain and inflammation [7,8]. Currently, non-steroidal anti-inflammatory drugs (NSAIDS) and steroids are being used as a first-line therapy against OA to control pain and inflammation [9]. However, their prolonged use in elderly is associated with adverse health consequences, such as gastro-intestinal, cardiovascular, and urinary disorders [10,11]. An effective medicine with less or no side effects is immensely needed for the treatment of OA. As a consequence, natural products are receiving tremendous attention for OA treatment.
(Applied Biosystems, Foster City, USA). Then, 4 µg of cDNA was amplified using 10 µg of PowerUp TM SYBR TM Green Master mix (Applied Biosystems, Foster City, USA) and forward/reverse primers (Macrogen, Seoul, Korea). The conditions of PCR progress were to be repeated, as follows: 15 s of reacting at 95 • C and 60 s of reacting at 63 • C by 40 cycles after 10 min of pre-incubation step at 95 • C. The sequences of the primers used in the experiment are shown in Table 1. Table 1. Sequences of mRNA primers used in knee cartilage tissues.

Western Blot Analysis for Knee Cartilage Tissue
The protein expression of MMP-1 (#10371-2-AP 1:500), MMP-3 (#SC-21732 1:500), MMP-8 (#SC-514803 1:500), MMP-13 (#18165-1-Ap 1:500), and β-actin (SC-47778 1:1000) was analyzed through Western blot analysis. Extraction of total protein in OA induced knee cartilage tissues was performed using RIPA buffer (Cell Signaling Technology Inc., Danvers, USA) and cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Sigma, USA) with Tissue Grinder Moter (Korea Ace Co Ltd., Seoul, Korea). Equal amounts of protein samples were loaded onto SDS-polyacrylamide gel for electrophoresis and the isolated proteins were transferred to PVDF membranes with the Semidry Transfer Cell (Bio-Rad Laboratories, Inc., California, USA) for 1 h at 15 V. Membranes were incubated at room temperature with 5% skim milk in TBS-T for an hour to block non-specific antibody binding. After that, membrane was washed three times with TBS-T and primary antibodies (MMP-1, MMP-3, MMP-8, MMP-13, and β-actin) were applied to react for 24 h at 4 • C. The antibodies were purchased from Proteintech Group, Inc., and Santa Cruz Biotechnology, Inc. The membrane was probed using secondary antibody at room temperature for 2 h and then reacted using D-Plus™ ECL Femto System (Dongin, Seoul, Korea) solution. The Western blot image was identified with Azure 280 (Azure Biosystems, Dublin, USA).

Measurement of Acetic Acid-Induced Writhing
ICR mice were divided into five groups (n = 8 per group) and administrated with APE (200, 600, and 1000 mg/kg), control (distilled water), and ibuprofen 200 mg/kg (Sigma, St. Louis, USA). Then, 30 min after oral administration, 0.7% acetic acid was injected in peritoneal cavity at 10 mL/kg, and after 10 min writhing responses were recorded. A twist reaction was composed of a contraction of the abdominal wall and a turn of the pelvis following the swelling of the hind limbs. A significant decrease in writhing response in the administered group compared with the control group was considered as pain response.

Measurement of NO and Cytotoxicity
RAW264.7 cells seeded at 5 × 10 5 /well, were incubated at 37 • C, and 5% CO 2 for 24 h. Cells were treated with different concentrations of APE (10-1000 µg/mL) and LPS (1 µg/mL) and incubated for 24 h. After mixing the culture supernatant with Griess reagent (1:1), the amount of nitric oxide (NO) in the mixture was recorded at an absorbance of 540 nm. Cell toxicity was recorded using MTT assay. After 24 h of seeding, 5 mg/mL MTT solution was included to RAW264.7 cells and kept at 37 • C, 5% CO 2 for 1 h. The supernatant was removed, 100 µL DMSO was included, stored for 10 min, and the absorbance was recorded at 540 nm. The RAW2647 cell line was purchased from Korean Cell Line Bank (Seoul, Korea). All cell experiments were performed three times with n = 3.
2.11. Real-Time Quantitative PCR Analysis of LPS-Stimulated RAW264.7 Cells Cells were cultured with different concentrations of APE, 1 µg/mL of dexamethasone, and 1 µg/mL LPS for 24 h. RNA extraction was performed with QIAzol Lysis reagent (Qiagen Ltd., Manchester, UK). After cDNA synthesis with using Reverse Transcription Kit (R&D Systems, Minneapolis, USA), Real-Time Quantitative PCR was performed with StepOnePlus real-time PCR system (Applied Biosystems, USA). Power SYBR ® Green PCR Master mix (Applied Biosystems, Foster City, USA) was used to amplify the 4 µg of cDNA with forward/reverse primers (Bioneer, Daejeon, Korea). The conditions of PCR progress were to be repeated, as follows: 15 s of reacting at 95 • C, and 60 s of reacting at 60 • C by 40 cycles after 10 min of pre-incubation step at 95 • C. The sequences of primers used in the experiment are shown in Table 2. Table 2. Sequences of mRNA primers used in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.

Statistics
Statistical analysis was performed using GraphPad Prism ® 5.0 (GraphPad Software, San Diego, USA) with two-sided one-way ANOVA and Dunnett's post hoc test. The significance was verified at p < 0.05, and measurements were indicated as mean ± standard error of the mean.

HPLC Analysis
Quantification of andrographolide, 14-deoxyandrographolide, and neoandrographolide in APE was carried out through HPLC analysis. APE contained 485.42 mg/g of andrographolide, 131.31 mg/g of 14-deoxyandrographolide, and 26.26 mg/g of neoandrographolide. Three dimensional HPLC chromatogram and the structures of the constituent compounds are shown in Figure 1.

Effects on Weight-Bearing Distribution in MIA Model
The weight-bearing ratios were recorded during 24 days after OA induction. As shown in Figure 2A, the weight-bearing distribution in the control (MIA) group was significantly reduced on day 3 and continued to be lower afterwards in contrast with the sham group. On the other hand, APE administration resulted in the significant increase in the weight-bearing of MIA rats. Particularly, the increase of weight-bearing by 300 APE was comparable to that of indomethacin ( Figure 2B).

Effects on Weight-Bearing Distribution in MIA Model
The weight-bearing ratios were recorded during 24 days after OA induction. As shown in Figure  2A, the weight-bearing distribution in the control (MIA) group was significantly reduced on day 3 and continued to be lower afterwards in contrast with the sham group. On the other hand, APE administration resulted in the significant increase in the weight-bearing of MIA rats. Particularly, the increase of weight-bearing by 300 APE was comparable to that of indomethacin ( Figure 2B).

Effects on Weight-Bearing Distribution in MIA Model
The weight-bearing ratios were recorded during 24 days after OA induction. As shown in Figure  2A, the weight-bearing distribution in the control (MIA) group was significantly reduced on day 3 and continued to be lower afterwards in contrast with the sham group. On the other hand, APE administration resulted in the significant increase in the weight-bearing of MIA rats. Particularly, the increase of weight-bearing by 300 APE was comparable to that of indomethacin ( Figure 2B).

Preventing Effects on Knee Joint Damage in MIA Model
The representative images of the knee joints in each experimental group show that APE prevented the cartilage erosion triggered by MIA injection. As illustrated in Figure 3, the joint cartilage of the sham rat was in a lustrous, smooth state; while the cartilage of the control rat was precisely less polished and rougher with damages in some areas. The erosion of cartilage induced with MIA was significantly prevented and recovered in APE and indomethacin administered rats. Noticeably, the preventing and recovery of cartilage erosion by APE was similar with that by indomethacin. MIA rats on 0, 3, 7, 10, 14, 17, 21, 24 days with 100 and 300 mg/kg Andrographis paniculata extract or 3 mg/kg indomethacin treatment and (B) area under the curve (AUC) were recorded using incapacitance meter tester. ### p < 0.001 vs. sham, ** p < 0.01 vs. control *** p < 0.001 vs. control.

Preventing Effects on Knee Joint Damage in MIA Model
The representative images of the knee joints in each experimental group show that APE prevented the cartilage erosion triggered by MIA injection. As illustrated in Figure 3, the joint cartilage of the sham rat was in a lustrous, smooth state; while the cartilage of the control rat was precisely less polished and rougher with damages in some areas. The erosion of cartilage induced with MIA was significantly prevented and recovered in APE and indomethacin administered rats. Noticeably, the preventing and recovery of cartilage erosion by APE was similar with that by indomethacin.

Effects on Inflammatory Cytokines in MIA-Induced Rat Model
The levels of serum inflammatory cytokines were recorded after isolating serum from blood collected from each experimental group. APE administrated rats had a significant reduction in the concentration of IL-1β, IL-6, and TNF-α in comparison with the control group in a dose-pendent manner. Interestingly, 300 APE reduced the cytokine levels similar to the indomethacin group ( Figure  4).

Effects on Inflammatory Cytokines in MIA-Induced Rat Model
The levels of serum inflammatory cytokines were recorded after isolating serum from blood collected from each experimental group. APE administrated rats had a significant reduction in the concentration of IL-1β, IL-6, and TNF-α in comparison with the control group in a dose-pendent manner. Interestingly, 300 APE reduced the cytokine levels similar to the indomethacin group ( Figure 4).

Effects on Matrix Metalloproteinases Responses in Knee Joint Cartilage Tissue
The measurement of the mRNA levels of MMP-1, MMP-3, MMP-8, and MMP-13 in the rats revealed that APE administration significantly reduced the MMP-1, MMP-3, MMP-8, and MMP-13 levels in knee joint cartilage tissue compared to the control rats ( Figure

Effects on Matrix Metalloproteinases Responses in Knee Joint Cartilage Tissue
The measurement of the mRNA levels of MMP-1, MMP-3, MMP-8, and MMP-13 in the rats revealed that APE administration significantly reduced the MMP-1, MMP-3, MMP-8, and MMP-13 levels in knee joint cartilage tissue compared to the control rats ( Figure 5A-D). Noticeably, 300 APE rats had lower levels of all four MMPs than the indomethacin group. Western blot analysis also demonstrated APE's downregulating effects on MMP-1, MMP-3, MMP-8, and MMP-13 in MIA rats in a dose-dependent manner ( Figure 5E-I).

Effect on Acetic Acid-Induced Writhing Responses
The analgesic effects of APE were investigated against the writhing responses in mice induced with acetic acid. The average writhing number in the control group for 10 min was recorded as 100%. APE administration resulted in the reduction in the number of writhing compared to the control. Rats fed with 1000 mg/kg of APE had the average writhing number of 67.22%, which was close to that of the ibuprofen group (58.09%). This result shows the analgesic effects of APE against peripheral pain ( Figure 6).

Anti-Inflammatory Effects in LPS-Stimulated RAW264.7 Cells
In LPS-stimulated RAW264.7 cells, APE suppressed the anti-inflammatory responses by reducing the level of NO and the expression of IL-1β, IL-6, iNOS, TNF-α, and COX-2. In cell viability assay, no apparent cytotoxicity by APE was found with up to 300 µg/mL ( Figure 7A). The NO generation of LPS-stimulated RAW264.7 cells was dose-dependently decreased by APE at 300 µg/mL, the reduction of NO was more than 60% compared to the control ( Figure 7B). APE suppressed the expression of IL-1β, iNOS, and COX-2 in a dose-dependent manner (Figure 7C,F,G). The reduction of these cytokines by 300 µg/mL APE was comparable to dexamethasone (1 µg/mL).

Effect on Acetic Acid-Induced Writhing Responses
The analgesic effects of APE were investigated against the writhing responses in mice induced with acetic acid. The average writhing number in the control group for 10 min was recorded as 100%. APE administration resulted in the reduction in the number of writhing compared to the control. Rats fed with 1000 mg/kg of APE had the average writhing number of 67.22%, which was close to that of the ibuprofen group (58.09%). This result shows the analgesic effects of APE against peripheral pain ( Figure 6).

Effect on Acetic Acid-Induced Writhing Responses
The analgesic effects of APE were investigated against the writhing responses in mice induced with acetic acid. The average writhing number in the control group for 10 min was recorded as 100%. APE administration resulted in the reduction in the number of writhing compared to the control. Rats fed with 1000 mg/kg of APE had the average writhing number of 67.22%, which was close to that of the ibuprofen group (58.09%). This result shows the analgesic effects of APE against peripheral pain ( Figure 6).

Anti-Inflammatory Effects in LPS-Stimulated RAW264.7 Cells
In LPS-stimulated RAW264.7 cells, APE suppressed the anti-inflammatory responses by reducing the level of NO and the expression of IL-1β, IL-6, iNOS, TNF-α, and COX-2. In cell viability assay, no apparent cytotoxicity by APE was found with up to 300 µg/mL ( Figure 7A). The NO generation of LPS-stimulated RAW264.7 cells was dose-dependently decreased by APE at 300 µg/mL, the reduction of NO was more than 60% compared to the control ( Figure 7B). APE suppressed the expression of IL-1β, iNOS, and COX-2 in a dose-dependent manner (Figure 7C,F,G). The reduction of these cytokines by 300 µg/mL APE was comparable to dexamethasone (1 µg/mL).

Anti-Inflammatory Effects in LPS-Stimulated RAW264.7 Cells
In LPS-stimulated RAW264.7 cells, APE suppressed the anti-inflammatory responses by reducing the level of NO and the expression of IL-1β, IL-6, iNOS, TNF-α, and COX-2. In cell viability assay, no apparent cytotoxicity by APE was found with up to 300 µg/mL ( Figure 7A). The NO generation of LPS-stimulated RAW264.7 cells was dose-dependently decreased by APE at 300 µg/mL, the reduction of NO was more than 60% compared to the control ( Figure 7B). APE suppressed the expression of IL-1β, iNOS, and COX-2 in a dose-dependent manner (Figure 7C,F,G). The reduction of these cytokines by 300 µg/mL APE was comparable to dexamethasone (1 µg/mL).

Discussion
The study demonstrated that APE improved weight-bearing capacity of MIA rats and decreased acetic acid-induced writhing responses in mice. APE also suppressed IL-1β, IL-6, and MMPs in MIA rats and IL-1β, iNOS, COX-2 in LPS-induced RAW264.7 cells. Furthermore, APE administration prevented the MIA-induced cartilage degradation in rats.
Previous studies have shown that APE can decrease paw edema and the expressions of inflammatory cytokines in Freund's adjuvant or collagen injected rats which mimic the human pathophysiology of rheumatoid arthritis and most of them were conducted with samples of its active compounds, andrographolide [36][37][38]. Even though APE has been reported to improve osteoarthritic pain in OA patients [28], no studies were performed for APE using MIA animal model targeting OA, to our knowledge. While a number of surgical and chemically induced arthritis models are available, the MIA model is one of the most accepted systems for studying OA, as it can produce much of the pain related behaviors as well as the patho-physiological characteristics that are found in human OA, including synovial inflammation, cartilage degeneration, and the erosion of subchondral bone [39]. The incapacitance test that measures the hind limb's weight-bearing capacity is a representative behavioral test frequently used in joint pain studies including MIA-induced OA model, based on the fact that the degree of weight-bearing on hind limbs decreases as the animal feels pain [40,41]. The weight-bearing capacity of the rats fed with APE was superior to the control rats, and was comparable with that of indomethacin treated rats. The improved weight load on the hind limbs by APE in the rats translates a pain-relieving effect in the osteoarthritic knees, which is the primary goal of current OA treatments. These results suggest that APE have analgesic effects against OA pain.
OA patients are often diagnosed with increased concentrations of inflammatory cytokines, including IL-4, IL-10, and IL-13 that are spontaneously released by synovial membrane and cartilage [42,43]. The anti-inflammatory properties of any therapeutic agent includes the decreased production of IL-1β, TNF-α, and MMPs, upregulation of IL-1Rα and TIMP-1, and the inhibition of PGE2 release [44,45]. Pro-inflammatory cytokines play active roles in the pathogenesis of OA by promoting synovial inflammation and the cartilage erosion [46,47]. Particularly, IL-1β is regarded as a key mediator in the inflammatory process in OA [48] and MMPs induced by IL-1β degrades extracellular matrix components, which plays significant roles in the pathogenesis of OA [49]. IL-1β initiates an initial inflammatory reaction, elevates the production of MMPs that impaired chondrocyte in OA, and interrupts the proteoglycan and collagen, which constitute the knee cartilage [50,51]. We conducted an experiment on MMPs that have critical function in causing OA. MMP-1, MMP-8, and

Discussion
The study demonstrated that APE improved weight-bearing capacity of MIA rats and decreased acetic acid-induced writhing responses in mice. APE also suppressed IL-1β, IL-6, and MMPs in MIA rats and IL-1β, iNOS, COX-2 in LPS-induced RAW264.7 cells. Furthermore, APE administration prevented the MIA-induced cartilage degradation in rats.
Previous studies have shown that APE can decrease paw edema and the expressions of inflammatory cytokines in Freund's adjuvant or collagen injected rats which mimic the human pathophysiology of rheumatoid arthritis and most of them were conducted with samples of its active compounds, andrographolide [36][37][38]. Even though APE has been reported to improve osteoarthritic pain in OA patients [28], no studies were performed for APE using MIA animal model targeting OA, to our knowledge. While a number of surgical and chemically induced arthritis models are available, the MIA model is one of the most accepted systems for studying OA, as it can produce much of the pain related behaviors as well as the patho-physiological characteristics that are found in human OA, including synovial inflammation, cartilage degeneration, and the erosion of subchondral bone [39]. The incapacitance test that measures the hind limb's weight-bearing capacity is a representative behavioral test frequently used in joint pain studies including MIA-induced OA model, based on the fact that the degree of weight-bearing on hind limbs decreases as the animal feels pain [40,41]. The weight-bearing capacity of the rats fed with APE was superior to the control rats, and was comparable with that of indomethacin treated rats. The improved weight load on the hind limbs by APE in the rats translates a pain-relieving effect in the osteoarthritic knees, which is the primary goal of current OA treatments. These results suggest that APE have analgesic effects against OA pain.
OA patients are often diagnosed with increased concentrations of inflammatory cytokines, including IL-4, IL-10, and IL-13 that are spontaneously released by synovial membrane and cartilage [42,43]. The anti-inflammatory properties of any therapeutic agent includes the decreased production of IL-1β, TNF-α, and MMPs, upregulation of IL-1Rα and TIMP-1, and the inhibition of PGE 2 release [44,45]. Pro-inflammatory cytokines play active roles in the pathogenesis of OA by promoting synovial inflammation and the cartilage erosion [46,47]. Particularly, IL-1β is regarded as a key mediator in the inflammatory process in OA [48] and MMPs induced by IL-1β degrades extracellular matrix components, which plays significant roles in the pathogenesis of OA [49]. IL-1β initiates an initial inflammatory reaction, elevates the production of MMPs that impaired chondrocyte in OA, and interrupts the proteoglycan and collagen, which constitute the knee cartilage [50,51].
We conducted an experiment on MMPs that have critical function in causing OA. MMP-1, MMP-8, and MMP-13 are the main collagenases that are distributed in synovial membrane and in synovial fluid [52]. MMP-3 is an enzyme called stromelysin, which plays an important role in joint substrate degradation and has a significant impact on other collagenase activations [53]. This study found a significant increase in IL-1β in serum and MMPs in knee cartilage tissue following MIA injection in rats, while APE administration resulted in a dramatic decrease in serum IL-1β and significant decline in MMP-1, MMP-3, MMP-8, and MMP-13 in knee cartilage tissues. Since the excessive production of IL-1β and MMPs enhances OA progression and hinders weight-bearing functions by promoting synovial inflammation, cartilage damage, and the release of prostaglandins, the reduction of IL-1β and MMPs by APE implicates the improvement in weight-bearing capacities by inhibiting the inflammatory responses and cartilage loss.
The loss of cartilage is one of the prominent indicators of OA and the cartilage erosion observed in MIA rats resemble the pathologic features of human OA [54,55]. Research evidence suggests that MIA causes the breakdown of proteoglycan matrix of the cartilage and induces joint impairment in rodents with characteristics similar to those of human OA [56]. The representative photos also showed a remarkable improvement in the cartilage tissue of MIA rats by APE. The degradation of cartilage in arthritic knees is commonly caused by pro-inflammatory cytokines and mediators. Our results showed that APE prevented the articular cartilage degradation in OA.
While central inputs are clearly involved in OA pain, clinical evidence suggests that the pain in OA patients is largely driven by peripheral inputs active in the affected joints since joint replacement, which eliminates peripheral pain inputs or the application of peripheral antinociceptors can suppress OA pain in the majority of the cases [57]. APE has been previously reported to alleviate pain in acetic acid-induced writhing and tail clip models [58]. In order to evaluate the analgesic effects of APE against peripheral pain, a writhing test was conducted. The injection of acetic acid into the peritoneal cavity causes the production of prostaglandins, such as PGE 2 and PGF 2α in the peritoneal fluid of the mice [59]. In this study, APE suppressed the acetic acid-induced writhing response in mice; noticeably, at 1000 mg/kg APE lowered the frequency of writhing similar as the ibuprofen group. When injected into the peritoneal cavity, acetic acid induces pain by releasing pain mediators, such as PGE 2 and PGF 2α , which leads to writhing response by releasing inflammatory pain mediators [60]. Hence, the substantial reduction of writhing response in acetic acid-induced mice by APE indicates its high efficacy in suppressing peripheral pain. This result suggests that the peripheral analgesic effect of APE contributes to the pain-relief by APE in the MIA-induced OA.
Macrophages are immune cells, which upon activation with LPS produce inflammatory cytokines and mediators [61]. This study investigated whether APE could suppress inflammatory responses using LPS-stimulated RAW264.7 macrophages. APE decreased the production of NO and the levels of IL-1β, IL-6, iNOS, and COX-2 dose-dependently. Andrographolide, a representative diterpene of APE, has been reported to reduce the production of NO and inhibit releasing IL-1β in LPS-stimulated macrophage [62,63]. The excessive amounts of COX-2 and iNOS in joint induce the production of pro-inflammatory cytokines and cause the destruction of cartilage, swelling, and pain [64,65]. IL-1β is known as the most important pro-inflammatory cytokine in OA, which stimulates the production of COX-2, iNOS, IL-6, and MMPs [66,67]. The present study demonstrated that APE inhibited IL-1β and other pro-inflammatory cytokines and mediators, and therefore could be a useful therapeutic agent against OA inflammation.
Andrographolide is well known as an active ingredient in A. panniculata, and other minor diterpenes 14-deoxyandrographolide, neoandrographolide, isoandrographolide, and 14-deoxy-11,12didehydroandrographide are also known to have anti-inflammatory, anti-atherosclerotic, and immunomodulatory effects [68][69][70][71]. In this study, the most contained and the representative active ingredient, andrographolide, was set as a standard compound and the effect of the extract was observed, but it is also necessary to evaluate the effect of these minor diterpenes in a future study to fully understand the effects of A. paninculata.
In summary, this study indicated that APE could relieve OA pain and reverse the cartilage degeneration in knee joints by suppressing the inflammatory responses. Notably, the pain-relieving effects of APE measured by weight-bearing in MIA rats were as close as indomethacin. The acetic acid-induced writhing responses were substantially reduced by APE. These analgesic effects were accompanied with protection of knee joints and reduced expressions of pro-inflammatory cytokines. These results demonstrate that APE could be a therapeutic candidate for relieving pain and inflammation in OA patients.