Acetylsalicylic Acid Combined with Diclofenac Inhibits Cartilage Degradation in Rabbit Models of Osteoarthritis

EXPERIMENTAL AND THERAPEUTIC MEDICINE 12: 2177-2182, 2016

Acetylsalicylic acid combined with diclofenac inhibits cartilage degradation in rabbit models of osteoarthritis

JIANQIANG LIU1,2, CHANGSHUN WU1, DONG WANG1, LAICHENG WANG3 and SHUI SUN1

1Department of Orthopedics, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021; 2Department of Orthopedics, The Fourth People's Hospital of Jinan, Shandong 250033; 3Research Center, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China

Received March 10, 2015; Accepted May 27, 2015

DOI: 10.3892/etm.2016.3560

Abstract. The present study aimed to investigate the effect of abrasion and degradation, osteophyte formation, subchondral different concentrations of acetylsalicylic acid combined with bone remodeling and low‑grade inflammation (1,2). OA is diclofenac on the articular cartilage of a rabbit model of osteo- considered to be induced by several complex interactions and arthritis (OA). A total of 40 New Zealand white rabbits were cross‑talk involving proteoglycan degradation, disruption divided into 5 groups. Group A was a sham‑operated control of the collagen network and progressive erosion of articular group, which was treated with normal saline. Groups B‑E cartilage, which leads to pain, stiffness and chronic physical were OA models and were treated with normal saline and and functional disability (3‑5). Inflammatory factors such acetylsalicylic acid combined with diclofenac at concentrations as inducible nitric oxide synthase, cytokines, interleukin 1β of 5, 10 and 20 mg/kg, respectively. A cartilage macroscopic (IL‑1β) and matrix metalloproteinases (MMPs) are important examination and a pathological observation were performed catabolic factors for the erosion and proteolysis of the extra- to analyze the structure of the articular cartilage in all of the cellular matrix components of the cartilage (6,7). MMPs in treated groups. The nitric oxide (NO) content and interleukin 1β particular are calcium‑dependent, zinc‑containing endopepti- (IL‑1β) levels were detected by an enzyme‑linked immuno- dases that are able to degrade all types of ECM proteins and sorbent assay. In addition, the protein expression of matrix are thus important for erosion and proteolysis. Consequently, metalloproteinase 3 (MMP)‑3 and MMP‑13 were detected by downregulation of the catabolic factors is an important target western blot analysis. The mRNA expression of tissue inhibitor for research in the treatment of OA. of metalloproteinases 1 (TIMP1) was detected by polymerase Currently, pharmacological therapeutic agents for the treat- chain reaction (PCR). The results revealed that different concen- ment of OA primarily include analgesics and non‑steroidal trations of the drugs significantly reduced the scores of cartilago anti‑inflammatory drugs, such as acetylsalicylic acid and articularis, the NO and IL‑1β levels and the protein expression diclofenac (8). Acetylsalicylic acid exerts anti‑inflammatory, of MMP‑3 and MMP‑13. Furthermore, PCR revealed that the analgesic and antipyretic actions and is one of the most widely mRNA expression of TIMP1 was significantly upregulated, and used drugs in the clinical treatment of osteoarthritis (9). the effects increased with increasing drug concentration. Thus, Diclofenac, a non‑steroidal anti‑inflammatory drug, decreases the administration of different concentrations of acetylsalicylic joint stiffness and pain and has been extensively used in the acid combined with diclofenac demonstrates preventive or management of osteoarthritis (10). These two drugs have been therapeutic effects against OA progression. demonstrated to increase bone mineral density, which poten- tially decreases the risk of fracture (11‑13). Introduction In view of the effects of acetylsalicylic acid or diclofenac alone on OA, the protective effects of various doses of acetylsal- Osteoarthritis (OA) is one of the most common degenera- icylic acid combined with diclofenac were presently investigated tive joint diseases in older adults, characterized by cartilage on the standard rabbit anterior cruciate ligament transection (ACLT) model of OA. Furthermore, the production of catabolic factors, including IL‑1β, nitric oxide (NO), MMP‑3 and MMP‑13 were evaluated in order to investigate the mechanism of the combination of the two types of drugs on OA. It was observed Correspondence to: Dr Shui Sun, Department of Orthopedics, that the administration of a larger dose of acetylsalicylic acid Provincial Hospital Affiliated to Shandong University, 324 Jingwu Weiqi Road, Jinan, Shandong 250021, P.R. China in combination with diclofenac would be more effective in the E‑mail: [email protected] progression of cartilage degradation and pathogenesis of OA.

Key words: osteoarthritis, acetylsalicylic acid combined with Materials and methods diclofenac, interleukin 1β, nitric oxide, matrix metalloproteinase Experimental animal model. A total of 40 healthy adult New Zealand white rabbits aged between 4 and 5 months old 2178 LIU et al: ACETYLSALICYLIC ACID WITH DICLOFENAC IN OA

(20 female and 20 male) with a mean weight of 2.5±0.5 kg tube. Detection was performed according to the instructions of were purchased from the Animal Center of Shandong Univer- the NO and rabbit IL‑1β enzyme‑linked immunosorbent assay sity (Shandong, China) and were used in the present study. kit (BD Biosciences, Franklin Lakes, NJ, USA). The optical The study fully complied with the National legislation and the density at 450 nm of this fluid was determined by a DNM‑9606 Guide for the Care and Use of Laboratory Animals issued by microplate reader (Perlong Medical, Beijing, China). the Ministry of Health of the People's Republic of China, and was approved by the local research ethical committees of the MMP‑3 and MMP‑13 protein expression analysis. Western Provicial Hospital Affiliated to Shandong University. blot analysis was used to determine protein expression. In brief, Each rabbit was anesthetized by a marginal ear vein injection ~20 mg femoral cartilage from the rabbits was sectioned and with pentobarbital (30 g/l). A total of 32 rabbits were randomly placed in a homogenizer. Next, 1 ml TRIzol reagent (BD Biosci- selected to induce the OA model. For each of the 32 rabbits, a ences) and 40 µl 10 mmol/l phenylmethanesulfonyl fluoride medial parapatellar incision was made through the skin of the were added to the culture flask and kept in an ice bath for left hind limb knee. Next, ACLT (14‑16) and a complete menis- 10 min. The tissue lysates were aliquoted into Eppendorf tubes cectomy were performed to induce left hind limb knee OA. The and placed in an ice bath for 30 min. The supernatants were other 8 rabbits also underwent a medial parapatellar incision but collected by centrifugation at 12,000 x g for 15 min, and the not an ACLT and meniscectomy. These rabbits were regarded total protein concentrations of the collected supernatants were as the sham‑operation group. Each animal received antibiotic measured by bicinchoninic acid assay. The samples were sepa- prophylaxis with an intramuscular injection of gentamicin rated on a 12% sodium dodecyl sulphate‑polyacrylamide gel by (0.48 g per day) for 5 days following surgery. electrophoresis. Next, the protein bands were transferred to a The 8 sham‑operated rabbits were referred to as group A polyvinylidene fluoride membrane for 1 h at room temperature and the other 32 experimental animals were randomly and were blocked with 5% skimmed milk for 1 h at room temper- assigned to 4 groups (named B, C, D and E). Groups A ature. The membranes were then incubated with rat anti‑MMP‑3 and B were the sham‑operated and OA model control groups, (sc‑1719R, BD Biosciences) and anti‑MMP‑13 (sc‑2119, BD respectively, which were both treated with 10 ml 0.9% normal Biosciences) and rat anti‑glyceraldehyde‑3‑phosphate dehydro- saline; group C was treated with a low dose of acetylsalicylic genase (GAPDH) antibodies (dilution, 1:500; BD Biosciences) acid combined with diclofenac (both with a purity >98% and at 4˚C overnight. Subsequent to washing, the membranes were used at a concentration of 5 mg/kg; Sangon Biotech Co., Ltd., incubated with human, anti‑rabbit secondary antibodies (563‑2; Shanghai, China); group D was treated with a medium dose of dilution, 1:1,000; Zemai Biotech Corporation, Shanghai, China) acetylsalicylic acid combined with diclofenac (both 10 mg/kg) for 1 h at room temperature. Finally, the reaction was visualized and group E was treated with a high dose of acetylsalicylic using an enhanced chemiluminescence detection system (LAS acid combined with diclofenac (both 20 mg/kg). These drugs MINI 4000, GE Healthcare Life Sciences, Little Chalfont, UK). were administered by intraperitoneal injection for 4 weeks. Tissue inhibitor of metalloproteinases 1 (TIMP1) mRNA Macroscopic examination of the cartilago articularis. The expression analysis. The total RNA was extracted from knee articular cartilage tissues were macroscopically scored the femoral cartilage of the rabbit tissue using TRIzol by systems as described previously in the scoring system by reagent (BD Biosciences) according to the manufacturer's Pelletier et al (17), as follows: 0=articular cartilage surface is instructions. Subsequent to measuring the total RNA (total smooth and appears light blue or colorless; 1=articular carti- RNA=OD260 x dilution ratio x 40), RNA was subjected to lage surface is malacic but smooth; 2=articular cartilage tends reverse transcription (RT) into cDNA using a PrimeScript RT to thin and appears like a small fibre bundle; 3=articular carti- reagent kit (Sigma‑Aldrich, St. Louis, MO, USA) according lage appears as a fibrous bundle; 4=articular cartilage appears to the manufacturer's instructions. In brief, a reaction to have wear and tear of the fibrous bundle, accompanied by comprising 2 µl 5X PrimeScript buffer, 0.5 µl PrimeScript subchondral bone exposure and osteosclerosis. RT enzyme mix, 2 µl total RNA and 5 µl RNase‑free dH2O (final volume, 10 µl) was prepared and incubated in a 37˚C Cartilage pathological observation. Following sacrifice by water bath for 15 min, then heated to 85˚C for 15 sec. The air embolism, the left hind limb knee joints of the 40 rabbits TIMP1 primers (5'‑3') were GTCGCATGCTGCGAGTTG were resected and immediately fixed in 4% paraformaldehyde AC and GGGTGGCCAAGAGCCTTGT (IBSBIOA‑156; for 24 h. The tissues were then decalcified in 10% ethylene Sigma‑Aldrich). Quantitative polymerase chain reaction diamine tetraacetic acid decalcifying solution (pH 7.2‑7.5; (qPCR) was performed using SYBR Premix Ex Taq TM II containing 0.01% sodium azide) for 12 weeks and the kit (RR041A; Sigma‑Aldrich). In brief, 2 µl cDNA was mixed decalcifying solution was changed every 3 days. Following with 12.5 µl SYBR Premix Ex Taq, 1 µl forward and 1 µl decalcification, the tissues were embedded in paraffin and reverse primer and 8.5 µl dH2O (to a final volume of 25 µl). cut into 4‑µm‑thick sections for histological evaluation. The The PCR cycling conditions were 40 cycles as follows: 95˚C sections were stained with hematoxylin and eosin, and all of for 5 min (initial denaturation), 95˚C for 20 sec (denaturation), the sections were observed using a TS100 1x70 inverted‑phase 60˚C for 30 sec (annealing) and 72˚C for 20 sec (extension). contrast microscope (Nikon Inc., Melville, NY, USA). This was followed by 71 cycles of: 60‑95˚C for 20 sec with a temperature rise of 0.5˚C for each repeat, which was used Determination content of NO and IL‑1β. The rabbit knee joint for fluorescent signal acquisition. The mRNA levels of the cavity was injected with 1.0 ml 0.9% normal saline. Next, the samples were normalized to the GAPDH mRNA levels as an joint fluid was drained repeatedly and aliquoted into a test internal control; the primers (5'‑3') for this were: ACGTCC EXPERIMENTAL AND THERAPEUTIC MEDICINE 12: 2177-2182, 2016 2179

Figure 1. Rabbit knee articular cartilage scoring, presented as the score (mean ± standard deviation) of each group. Different symbols represent the statistically significant difference between the groups. *P<0.05 vs. group B, #P<0.05 vs. group C, &P<0.01 vs. group D and $P<0.05 vs. group E. (A) Sham‑operated and (B) model controls; (C) low dose (5 mg/kg); (D) medium dose (10 mg/kg) and (E) high dose (20 mg/kg) acetylsalicylic acid combined with diclofenac.

A B C

D E

Figure 2. Representative sections of rabbit knee articular cartilage, demonstrated by hematoxylin and eosin staining. (A) Sham‑operated and (B) model controls; (C) low dose (5 mg/kg); (D) medium dose (10 mg/kg) and (E) high dose (20 mg/kg) acetylsalicylic acid combined with diclofenac.

CATCACGATCCTTC and ACACTCGGATGACGAACT. articularis surfaces were neat without cracks, defects, soft- Finally, 10 µl PCR products were run on a 1% agarose gel by ening and had normal osteophytes). Group B was scored electrophoresis. as 3.9 (cartilago articularis surfaces appeared to have fibre bundle, cracks, defects and softening, and parts of the Statistical analysis. Data are expressed as the mean ± stan- subchondral bones exposed with osteophytes along the edge). dard deviation, and statistical analysis was performed with The scores mentioned were mean scores. After the rabbits SPSS version 13.0 software for Windows (SPSS, Inc., Chicago, were treated with the drugs, cartilago articularis surfaces IL, USA). A pairwise comparison of multiple samples was became smoother with small fibre bundle and capilar cracks. conducted using the Bonferroni test in one way analysis of However, the degree of cartilage wear and osteophyte forma- variance. P<0.05 was considered to indicate a statistically tion was significantly lower compared with the model control significant difference. of group B. The results were as follows: Group C, 3.1, P<0.05; group D, 2.5, P<0.01 and group E, 1.8, P<0.01 (Fig. 1). Results Cartilage pathological observation. In group A, the Macroscopic examination of the cartilago articularis. The sham‑operated control, the articular cartilage surfaces were cartilago articularis of group A was scored as 0 (cartilago neat, translucent, smooth and flexible without cracks, defects, 2180 LIU et al: ACETYLSALICYLIC ACID WITH DICLOFENAC IN OA

Figure 3. Enzyme‑linked immunosorbent assay for determining the NO and IL‑1β content in joint fluid, presented as mean content ± standard deviation of (A) NO and (B) IL‑1β in each group. Different symbols represent a statistically significant difference between the groups.* P<0.05 vs. group B, #P<0.05 vs. group C, &P< 0.01 vs. group D and $P<0.05 vs. group E. (A) Sham‑operated and (B) model controls; (C) low dose (5 mg/kg); (D) medium dose (10 mg/kg) and (E) high dose (20 mg/kg) acetylsalicylic acid combined with diclofenac. NO, nitric oxide; IL‑1β, interleukin 1β.

softening or osteophytes. In the OA model control, group B, the articular cartilage surfaces were rough without normal elasticity, and the surfaces had obvious fibrous bundles, cracks, defects, softening and revealed sections of exposed subchondral bones, with osteophytes along the edge. Following the high dose treatment with acetylsalicylic acid in group E, the articular cartilage surfaces appeared yellow and white and there was a partial loss of normal luster. As a result of the high Figure 4. Western blot analysis for the protein expression of MMP‑3 dose treatment, the surfaces became smoother with a small and MMP‑13, using GAPDH expression level as an internal control. fibrous bundle and capilar cracks. Furthermore, the degree of (A) Sham‑operated and (B) model controls; (C) low dose (5 mg/kg); (D) medium dose (10 mg/kg) and (E) high dose (20 mg/kg) acetylsalicylic cartilage wear and osteophyte formation was lower in group E acid combined with diclofenac. MMP, matrix metalloprotease; GAPDH, compared with group B, and with the low‑ and median‑dose glyceraldehyde‑3‑phosphate dehydrogenase. treatment groups C and E (Fig. 2).

Effects of drugs on the NO and IL‑1β content of joint fluid. The NO (5.97±0.9) and IL‑1β (7.03±0.8) content in group A Effects of drugs on TIMP1 mRNA expression. The TIMP1 were significantly lower (P<0.01) compared with those in mRNA expression levels are shown in Fig. 5. The expression group B (NO, 13.21±1.2; and IL‑1β, 15.43±1.4). Subsequent to levels of TIMP1 in groups D and E significantly increased treating the rabbits with different doses of drugs, the NO and (group D: 0.55±0.02, P<0.05 and group E: 0.73±0.01, P<0.01) IL‑1β content of all the 3 experimental groups significantly compared with group B (0.31±0.00), and these effects were decreased (P<0.05) compared with group B, particularly for enhanced with increasing drug concentration. the high dose treatment of acetylsalicylic acid in group E (NO, 7.32±0.6; and IL‑1β, 8.43±0.4; P<0.01) (Fig. 3). Discussion

Effects of drugs on MMP‑3 and MMP‑13 protein expression. OA is currently considered to cause global organ failure The protein expression levels of MMP‑3 and MMP‑13 were involving all of the tissues of the joint (18). Overall, the treat- highest in group B (Fig. 4). Subsequent to drug treatment, ment of OA is aimed at alleviating pain, swelling and muscle the expression levels of MMP‑3 and MMP‑13 in all 3 groups tightness in order to improve mobility (3). Numerous OA significantly decreased, particularly in the high dose treatment patients have experienced relief of pain and improvement group (group E; P<0.05). in mobility subsequent to acetylsalicylic acid or diclofenac EXPERIMENTAL AND THERAPEUTIC MEDICINE 12: 2177-2182, 2016 2181

Figure 5. Quantitative polymerase chain reaction analysis to demonstrate mRNA expression of tissue inhibitor of metalloproteinases 1, presented as mean expression ± standard deviation. Different symbols represent the statistically significant difference between groups.* P<0.05 vs. group B, #P<0.05 vs. group C, &P<0.01 vs. group D and $P<0.05 vs. group E. (A) Sham‑operated and (B) model controls; (C) low dose (5 mg/kg) (D) medium dose (10 mg/kg) and (E) high dose (20 mg/kg) acetylsalicylic acid combined with diclofenac.The measurements were relative to glyceraldehyde 3‑phosphate dehydrogenase.

administration. In the present study, the effects of different significantly inhibited IL‑1β and NO production, indicating concentrations of acetylsalicylic acid combined with diclof- that the inhibition of the inflammatory mediators may be enac were studied in the OA rabbit model. Acetylsalicylic acid responsible for the anti‑inflammatory effects of acetylsalicylic and diclofenac administered at 3 different concentrations were acid plus diclofenac. observed to reduce the cartilago articularis scores and reduce With regard to the other discussed catabolic factor, MMPs the NO and IL‑1β content. In addition, the protein expression consist of a family of Zn2+‑dependent extracellular enzymes of MMP‑3 and MMP‑13 were downregulated and mRNA that are capable of degrading the extracellular matrix compo- expression of TIMP1 was upregulated. nents, in addition to remodeling normal and pathological In the present study, cartilage macroscopic examination tissue (26,27). Furthermore, MMPs are demonstrated to be and pathological observation were performed to analyze the involved in bone resorption and matrix degradation (28,29). structure of the articular cartilage in all the different groups As an array of proteases, MMPs can break down proteogly- studied. The scoring system (17) is widely used to evaluate cans and type II collagen, which are the main components histological findings of osteoarthritic specimens, and osteo- of the articular cartilage, leading to proteolysis of the carti- phyte formation and cartilage lesions have long been used as lage (26). In particular, MMP‑3 has the ability to degrade observation indexes (19,20). The scoring results demonstrated various components of the cartilage, and MMP‑13 is capable a significant inhibition of degenerative changes in the cartilage of degrading intact type II collagen (27,30). The increased following drug treatment (Fig. 1), which was consistent with amount of proMMPs and MMP production has been observed the histological assessment (Fig. 2). These results may indicate in synovial fluid and joint pathology (31‑33). Previous research that the administration of acetylsalicylic acid combined with revealed that cartilage degradation occurs not only due to the diclofenac has an important effect on the development of carti- increase in MMPs but also as a result of an imbalance between lage degenerative changes. extracellular matrix proteinases and their inhibitors, particu- The proinflammatory cytokine IL‑1β exerts a catabolic larly for MMPs and TIMPs (34). Furthermore, MMPs are effect on chondrocyte metabolism, which subsequently inhibited by specific endogenous TIMPs that are glycoproteins decreases proteoglycan collagen synthesis and increases and inhibit all MMPs by forming high‑affinity complexes aggrecan release by blocking proteases (21). In addition, IL‑1β at a 1:1 ratio (35). Specifically TIMP‑1 inhibits MMP‑3, may activate synovial cells to increase the gene expression of MMP‑9 and MMP‑13 (36). In the present study, the results MMPs that are catabolic factors for the erosion and proteolysis of the western blot analysis demonstrated that the protein of extracellular matrix components of the cartilage (6). IL‑1β expression of MMP‑3 and MMP‑13 was downregulated after also induces synovial cells and chondrocytes to produce other administrating drugs, particularly for the high dose treatment. inflammatory mediators, including IL‑6, IL‑8 and NO (22). Additionally, the results of the PCR analysis revealed that NO is a highly reactive free radical, in addition to a major the mRNA expression of TIMP1 was upregulated following catabolic factor synthesized from L‑arginine by members of drug treatment, however, the effects were enhanced with the the iNOS (23,24). Furthermore, overproduction of NO results increasing concentration. These results indicated a capability in tissue damage and an inflammatory response, which is a of acetylsalicylic acid combined with diclofenac in alleviating key to the pathogenesis of inflammation (25). In the study the destruction of articular cartilage matrix and delaying the by Zhou et al (24) it was suggested that NO led to articular process of osteoarthritis. chondrocyte apoptosis by the inhibition of protein kinase C, In conclusion, the findings of the present study indicate which participated in modulating articular chondrocyte apop- that the administration of acetylsalicylic acid combined with tosis. In the present study, acetylsalicylic acid plus diclofenac diclofenac demonstrate preventive or therapeutic effects on 2182 LIU et al: ACETYLSALICYLIC ACID WITH DICLOFENAC IN OA progressive OA. This result may be achieved by inhibition of 19. 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