The Effects of Ozone Therapy on the Expression of IL-8, IL-10, CXCL9 and CXCL11 in Joint Fluid in Patients with Knee Osteoarthritis

Knee joint is one of the common sites of osteoarthritis (OA) with manifestation of localized joint swelling, pain and deformity, often seriously affecting daily work and life. It was found that the activation of Janus kinase / signal transducer and activator of transcription 3 (JAK / STAT3) signal pathway can lead to osteoarthritis. JAK-STAT signal transduction pathway is closely related to painful diabetic peripheral neuropathy and participates in neuropathic pain. Ozone therapy is often used for alleviating pain in neck, shoulder, waist and leg due to its oxidative, anti-inflammatory and analgesic effects. In recent years, ozone therapy is promising in treating knee osteoarthritis, which can significantly reduce the pain and swelling of the joints. The mechanism of ozone for knee osteoarthritis remains unclear and might associated with inhibition of inflammatory factors and matrix metalloproteinase activity, and intervention of free radical metabolism. In this study, the efficacy of ozone in the treatment of OA and its effect on inflammatory factors were evaluated by knee joint cavity ozone injection and detecting the expression of inflammatory factors IL-8, IL-10, CXCL9 and CXCL11 in joint fluid.


INTRODUCTION
Osteoarthritis (OA) is a kind of joint degenerative disease, which is characterized by local joint swelling, pain and deformity. It often occurs in the weight-bearing joints of lower limbs, especially the knee joint, and often seriously affects daily work and life. The main pathological changes were apoptosis and destruction of bone cells, synovitis, and then subchondral bone hyperplasia. Knee osteoarthritis is a common disease in the elderly [1,2]. The World Health Organization reported that the prevalence of OA in the elderly aged 65 ~ 70 is close to 62%, and nearly 70% in the population over 70. With the advent of China's aging society, the treatment of OA will become an urgent problem in geriatrics in China.
Medical trioxide has strong oxidation ability and anti-inflammatory and analgesic effects [3]. It is mainly used for anti-inflammatory and analgesic treatment of neck, shoulder, waist and leg pain. In recent years, medical trioxide has achieved good clinical results in the treatment of knee osteoarthritis, which can significantly reduce the pain and swelling of patients' joints. The exact target of medical trioxide in the treatment of knee osteoarthritis is not clear, which may be related to the inhibition of inflammatory factors, matrix metalloproteinase activity and intervention of free radical metabolism.

Study Population
According to the diagnostic criteria for knee OA in the "Osteoarthritis Diagnosis and Treatment Guidelines (2018 edition)" [4], 20 patients with advanced knee osteoarthritis participated our study from January 2019 to March 2020 at Second People's Hospital of Foshan. All patients received no drugs or other treatments within 2 weeks before outpatient visiting. There was neither skin broken or infection around the knee joint, nor do patients have previous cardiopulmonary disease, obvious impairment of liver and kidney function, history of infectious diseases and autoimmune diseases, or history of drug allergies. Patients with ISOA (index of severity for knee joint osteoarthritis) score over 14 were excluded.

Interventions
The patients were placed in spine position with both legs straightened. Expose the affected knee joint, disinfecting and draping as usual. Local anesthesia was performed with 1% lidocaine hydrochloride 2mL, then puncture the joint cavity under ultrasound guide. Firstly, 4mL of effusion was extracted and storage in -70 ° C refrigerator. After that, a 20mL gas mixture of air and ozone were injected into the joint cavity. Ozone was prepared by ozone therapy device (Ozomed® Smartline, Kastner-Praxisbedar, German) with concentration of 30 μg/mL. Finally, puncture point was bandaged by sterile patch. The above procedure was repeated after 7d for each patient.

Statistical Analysis
All data were expressed as mean±SD and analyzed using SPSS version 17.0 (SPSS Inc.). VAS scores at all time points were compared using one-way ANOVA. ISOA scores and inflammatory factor expressions before and after therapy wad analyzed by paired t-test. P<0.05 was considered to indicate a statistically significant difference.

Study Population
20 patients with advanced knee osteoarthritis were finally involved. There were 6 males and 14 females at the age of 65±8, whose course of disease were 41±5 months. All patients received intraarticular ozone injection and no complications such as infections or hemorrhage occurred.

VAS and ISOA Scores
All patients suffered mild to severe pain before therapy whose VAS score was 8.3±1.0.

DISCUSSION AND CONCLUSION
KOA is a common disease in geriatric patients with mainly pathological changes of apoptosis and destruction of bone cells, synovitis, and subsequent subchondral osteoporosis. Treatment of OA will become urgent as the coming of China's aging society. OA is an inflammatory disease induced by various of inflammatory mediators [5][6][7][8]. Laboratory tests showed that the serum C-reactive protein, IL-6, IL-8, CXCL9, and CXCL11 levels in OA patients were higher than normal, suggesting that inflammatory response was involved in the pathogenesis of OA. Synovitis can occur in both early and advanced stage of knee osteoarthritis and is associated with joint pain, degeneration, and defects. Inflammatory cytokines such as IL-6, TNF-a, IL-1 and JAK-STAT are all associated with the occurrence of OA. TNF-a plays an important role in matrix degradation and bone destruction in osteoarthritis, and it induces the production of other cytokines such as IL-6 and matrix metalloproteinase. Studies have shown that IL-1β is associated with pain and the severity of radiological manifestations of joint structures in OA patients. Intraarticular injection of IL-1β receptor antagonists can improve synovial inflammation and hyperplasia, reduce the level of high-sensitivity C-reactive protein, and facilitate the treatment of OA.
As a strong oxidant, ozone can eliminate free radicals in the joints of KOA patients and improve the environment of joint cavity, thus it is often used for the treating chronic pain. Studies have shown that ozone could reduce the expression of inflammatory factors in the joint fluid of KOA patients and decrease inflammatory responses [1,9]. Our study found that intra-articular injection of ozone could relieve the pain of KOA patients, improve joint function, and reduce VAS and ISOA scores, suggesting that intra-articular injection of ozone is effective in relieving pain and improving joint function of KOA patients. ELSA assay of IL-8, IL-10, CXCL9, CXCL11 showed that ozone can reduce the content of inflammatory factors IL-8, CXCL9 and CXCL11 in the joint fluid of KOA patients, increase the content of antiinflammatory factor IL-10, suggesting that ozone might alleviate the pain and improve joint function of KOA patients by regulating inflammatory response.
Meanwhile, Attention should be paid for very severe KOA patients as ozone might be ineffective or even deteriorate the conditions due to its strong oxidizing property. Dose and frequency should also be cautious to reduce adverse effects. In summary, intra-articular ozone injection could alleviate the pain of KOA patients and the mechanism may associated with decreasing of inflammatory factors and promoting of anti-inflammatory factors in joint fluid.

ETHICAL APPROVAL
As per international standard or university standard written ethical approval has been collected and preserved by the author(s).

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).