Combined effect of celecoxib and glucosamine sulfate on inflammatory factors and oxidative stress indicators in patients with knee osteoarthritis

Purpose: To investigate the combined effect of celecoxib and glucosamine sulfate on inflammatory factors and oxidative stress indicators in patients with knee osteoarthritis (KOA). Methods: Patients were randomly assigned to two groups of 60 patients each: control group and study group. The control group received celecoxib at a dose of 200 mg/kg/day, while the study group received glucosamine sulfate (500 mg/kg) in addition to celecoxib, thrice a day. Treatment in both groups lasted 8 weeks. The serum levels of tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), prostaglandin-2 (PGE2), malondialdehyde (MDA), and activity of superoxide dismutase (SOD) were assayed before and after treatment. Visual analogue scale (VAS), osteoarthritis index, Lysholm knee score scale (LKSS), and adverse reactions were also evaluated. Results: After treatment, total effectiveness was significantly higher in the study group (91.33 %) than in control group (71.67 %, p < 0.05). Serum TNF-α, IL-1 and PGE2 were significantly lesser in the glucosamine sulfate-treated patients than in control group (p < 0.05). The activity of SOD was significantly higher in glucosamine sulfate-exposed patients than control patients (p < 0.05). On the other hand, VAS and WOMAC scores were markedly lower in patients given glucosamine sulfate than in control patients (p < 0.05). Conclusion: The combination of celecoxib with glucosamine sulfate effectively reduces immune inflammatory response, oxidative stress damage, and joint pain associated with KOA.


INTRODUCTION
Osteoarthritis (OA) is a degenerative disease of bones and joints which occurs mainly in the knee joint, hip joint, spine and other parts in middleaged and elderly people.It is caused by pain and joint dysfunction.Pain of long duration induces local inflammation of joint soft tissue, aggravates damage to cartilage, and seriously reduces quality of life of sufferers [1].At present, surgery, conservative treatment, and use of antiosteoarthritic drugs are the major treatment options for OA.
Celecoxib is often used as a non-steroidal antiinflammatory analgesic which prevents the synthesis of prostaglandins by inhibiting cyclooxygenase-2 (COX-2).It can effectively relieve pain, but cannot effectively prevent longterm recurrence of the disease [2].Glucosamine sulfate, a naturally occurring amino sugar in the matrix of cartilage, effectively repairs damaged soft tissue and inhibits the recurrence of inflammation [3].Previous studies have shown that a combination of celecoxib with glucosamine sulfate could effectively control inflammatory response and reduce its long-term recurrence [4].
The present study was aimed at investigating the combined effect of celecoxib and glucosamine sulfate on inflammatory factors and oxidative stress indicators in patients with KOA.

Patients and general information
Patients with KOA (120 cases) were recruited over a 1-year period for this study and randomly assigned to two groups of 60 patients each: control group and study group.They consisted of 72 males and 48 females aged 45 to 75 years (mean age = 60.Treatment for both groups lasted 8 weeks during which the patients were not allowed to take other anti-osteoarthritic drugs.They were also advised to avoid excessive use of their knee joints, consumption of spicy food, and cold water baths.
In addition, they were asked to ensure that they kept themselves warm.

Assessment of efficacy
Clinical effectiveness was assessed at the end of 8 weeks of treatment and 3 months after, and classified into four grades: healed, remarkably effective, effective and ineffective.Healed: In this group, there was complete disappearance of clinical symptoms, and joint activity was ≤ 135 o ; remarkably effective: this referred to disappearance of clinical symptoms, absence of pain during walking, occasional pain during activity, and joint activity ≤ 135 o ; effective: marked by slight improvement in clinical symptoms, mild pain during walking, and limited degree of joint activity; ineffective: here, there were no significant improvements in clinical symptoms.The total effectiveness was calculated as shown in Eq 1. TE(%) = {(H+RE+E)/TN} × 100 ……….. (1) where TE is total effectiveness, H is healed, RE is remarkably effective, E is effective and TN is total number of cases.

Determination of inflammatory factors
Joint fluid was taken before and post-treatment, and TNF-α, IL-1 and PGE 2 were assayed in serum using ELISA kits.

Evaluation of oxidative status
Fasting blood (4 mL) was drawn from each patient pre-and post-treatment, into plain sample bottles.The serum samples recovered after centrifugation were refrigerated at -20 °C and used for assay of serum SOD and level of MDA.

Determination of visual analogue scale (VAS), osteoarthritis index, and Lysholm knee score scale (LKSS)
Pain level was scored using VAS (0 to 10 points).The higher the score, the more severe the pain.The osteoarthritis index was scored according to the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC).A total of 24 items were used and assigned a total of 96 points.The higher the score, the more severe the degree of OA.LKSS score consisted of eight questions with scores ranging from 0 to 100.The higher the score, the better the knee function.

Statistical analysis
Continuous variables following a symmetric distribution are presented as mean ± standard deviation (SD), and were analyzed with Student's t-test.Numerical data are presented as frequency (%), and statistical analysis was performed using Chi square.All analyses were carried out with SPSS version 19.0.Statistical significance was fixed at p < 0.05.

Treatment effectiveness
After treatment, the total effectiveness was significantly higher in the observation group (91.33 %) than in control group (71.67 %) (p < 0.05).These results are shown in Table 2.

Inflammatory factors
Before treatment, the levels of TNF-α, IL-1 and PGE 2 were similar between patients in both groups (p > 0.05).However, post-treatment TNFα, IL-1 and PGE 2 were markedly lower in the glucosamine sulfate-treated patients than in control patients (p < 0.05; Table 3).

Activity of SOD and level of MDA
As shown in Table 4, before treatment, SOD activity and MDA level were comparable between patients in both groups (p > 0.05).However, after treatment, the activity of SOD was significant higher in patients treated with glucosamine than in control patients (p < 0.05).However, there was a marked decrease in MDA level in the glucosamine-treated patients, when compared with control patients (p < 0.05).

Joint scores
After treatment, VAS and WOMAC scores in the two groups were markedly reduced, while the LKSS scores were markedly increased, relative to pre-treatment scores (p < 0.05).There were marked decreases in VAS and WOMAC scores, and higher LKSS score in glucosamine-treated patients than in control patients (p < 0.05; Table 5).

Adverse reactions
During treatment, adverse reactions in the observation group (10.00 %) were significantly lower than those in control group (21.67 %, p < 0.05).These results are shown in Table 6.

DISCUSSION
Knee osteoarthritis (KOA), a common form of OA, is caused by degenerative knee disease.Early OA is characterized by painful joints, stiffness, hypertrophy, and limited mobility.In the advanced stage of the disease, loss of joint function is caused by exfoliation of cartilage, necrosis, and bone hyperplasia.This is due to the release of huge amounts of IL-1 and TNF-α in the synovial fluid as a result of phagocytic degradation of synovial cells, which adversely affects the metabolism of chondrocytes [7,8].Patients with KOA are often treated with nonsteroidal anti-inflammatory drugs and cartilage protective agents because of the trauma associated with and high cost of surgery.Studies have shown that celecoxib and glucosamine sulfate alleviate pain and joint swelling in patients with KOA, and exert control effects on advanced cartilage necrosis [9].
In the present study, total effectiveness was significantly higher in the observation group than in control group.VAS and WOMAC scores were significantly lower in the observation group than in control group.However, the corresponding LKSS score was markedly lower in the control group.Incidents of adverse reactions in the observation group were significantly lower than in control group.These results suggest that celecoxib and glucosamine sulfate may be clinically effective as combined treatment of KOA due to the significant reduction in joint pain and improvement of prognosis.The synergistic effect of the two drugs may stimulate synthesis of glycosaminoglycans, increase viscosity of synovial fluid, improve the lubrication function between the joints, inhibit the progression of OA and improve joint function [10].
Studies have shown that OA lesions are associated with varying degrees of synovial inflammation and the release of large amounts of inflammatory factors such as TNF-α and IL-1 in the synovial fluid.The TNF-α stimulates chondrocytes to synthesize inflammatory mediators such as IL-1 and PGE 2 .Prostaglandin-2 (PGE 2 ) increases cell permeability, promotes infiltration of inflammatory cells, causes local tissue edema, and aggravates OA.Therefore, TNF-α, IL-1 and TNF-α play important roles in the pathogenesis of OA [11,12].
In the present investigation, after treatment, TNFα, IL-1 and PGE 2 were markedly reduced in the glucosamine sulfate-exposed patients than in control patients.This suggests that the combination of celecoxib and glucosamine sulfate may effectively inhibit the release of inflammatory factors, block the inflammatory response pathway, and alleviate pain in patients with KOA.It is possible that celecoxib exerts antiinflammatory and analgesic influence through suppression of COX-2 which in turn inhibits the release of PGE 2 .Glucosamine sulfate inhibits the activity of alkaline phospholipase A 2 , and promotes the synthesis of collagen in cartilage matrix [13].These results indicate that the combination of celecoxib and glucosamine sulfate may not only increase anti-inflammatory effects, but may also reduce non-steroidal antiinflammatory drug-induced damage to chondrocytes.
Studies have shown that oxidative stress affects metabolism of collagen in chondrocytes and brings about a decline in normal function of cartilage.As an important antioxidant enzyme, SOD reduces oxidative damage in bone and cartilage of joints by mitigating reactive oxygen species (ROS)-induced cell damage.Malondialdehyde (MDA), one of the most important products of lipid peroxidation, induces apoptosis in chondrocytes, which leads to cartilage atrophy, thereby promoting the development of OA [14].Glucosamine sulfate treatment led to marked increase in SOD activity, and significant decrease in MDA level.This suggests that the combination of celecoxib with glucosamine sulfate effectively inhibits ROSinduced damage to chondrocytes and delays the degeneration of osteoarticular cartilage.Celecoxib potently inhibits COX-2 activity, reduces the generation of oxygen free radicals, and mitigates osteoarthritis-induced oxidative stress damage [15].Glucosamine sulfate directly reduces cartilage degradation, inhibits the production of superoxide free radicals, and promotes cartilage regeneration and repair [16].The combination of the two drugs may reduce chondrocyte damage and strengthen the ability of osteoarthritic patients to resist oxidative stress.

Limitations of the study
This study investigated the combined effects of celecoxib and glucosamine sulfate on inflammatory factors and oxidative stress indicators in patients with knee osteoarthritis.However, the study lasted only eight weeks.Long-term follow-up of patients after combination therapy was not conducted.Therefore, the longterm side effects of the combination therapy were not determined.These aspects require further studies.

CONCLUSION
The results obtained in this study demonstrate that the combination of celecoxib and glucosamine sulfate effectively reduces immune inflammatory response, oxidative stress damage and joint pain associated with KOA.
5 ± 5.8 years), with disease duration ranging from 6 months to 15 years.The clinical scores were: mild (64 cases), moderate (40 cases), and severe (19 cases).Their joints activities ranged from 90 to 146 o .The inclusion criteria were: (1) patients who met the Guidelines for the Diagnosis and Treatment of Osteoarthritis developed by the Chinese Medical Association [5]; (2) patients aged 40 to 80 years; (3) patients whose X-ray results revealed that the affected joints had mild or obvious osteophyte formation; (4) patients who had normal joint spaces; and (5) patients who signed written informed consent with their family members.

Table 3 :
Levels of inflammatory factors (n = 60) # p < 0.05, compared to control group after treatment

Table 6 :
Comparison of adverse reactions between the two groups (n = 60)