Efficacy And Mechanism Of Using Different Concentrations Ozone To Treat Rheumatoid Arthritis Rats

BackgroundRheumatoid arthritis (RA) is a chronic systemic disease, primarily of the joints, marked by inflammatory changes in the synovial membranes and articular structures. Etiology is unknown, but autoimmune mechanisms have been implicated. The incidence of RA is approximately 0.4% in China and around 0.5 to 1.0 percent in the world. It's mainly lesions occurred in synovial, can affect articular cartilage, ligaments, tendons and body tissue, Leading to the joint swelling and pain, followed by cartilage destruction, joint space narrowing. Without proper treatment early,70% rate of joint destruction Within 3 years, leading to joint deformity and disability. As the high incidence of disease and disability, the disease has seriously harmed people's quality of life.The cause of RA remains unclear, Most scholars believe that RA is genetic, immune, infection, hormone levels, diet, living conditions and other factors working together caused an autoimmune disease. Genetic factors contribute to the body of the RA susceptibility, infectious may induce diseases. At the same time the patient's immune dysfunction, endocrine abnormalities, living environment, physical activity and metabolic abnormalities, abnormal neuroendocrine-immune function and other factors are closely related with the pathogenesis of RA. Therefore, RA may be the infectious agent directly or indirectly stimulate susceptible individuals, and T cells, B cells, fibroblasts, synovial cells, and include tumor necrosis factor-α(TNF-α), interleukin-1 (IL-1), IL-6, IL-2, fibroblast growth factor (FGF), platelet growth factor (PGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and other cytokines, participate in the joint and the body lesion of RA.RA pathogenesis has not been yet completely clear. Therefore, the lack of specificity treatment in clinical, only symptomatic treatment, and to some extent control and prevention of joint destruction and loss of function and improve quality of life of RA patients. RA common treatments include drug therapy, autologous hematopoietic stem cell transplantation, gene therapy, immune purification, advanced surgical treatment and other adjuvant therapy.Non-steroidal anti-inflammatory drugs, slow-acting antirheumatic drugs, corticosteroids, biological agents and other drugs can be used for drug treatment of RA. Non-steroidal anti-inflammatory drugs are widely used in clinical. It can effectively reduce the pain symptoms, but long-term use prone to gastrointestinal symptoms and renal toxicity damage. Because of its inhibition of prostaglandin E2 (PGE2) production, may lead to a variety of increased expressions of inflammatory cytokines, and promote intra-articular synovial fibroblast-like cell proliferation, more serious damage to the joints. Slow-acting antirheumatic drugs including improving the condition of the anti-rheumatic drugs and immunosuppressive drugs, need long-term medication. However, most of these drugs have serious side effects, often make patients had discontinued during the treatment period, so it is difficult to achieve the desired effect. Glucocorticoids can use to treat RA with systemic or local joint injection. Long-term systemic use of corticosteroids can cause significant side effects and the formation of dependence, and improper use may appear opportunistic infections, aseptic bone necrosis and other serious complications. Sometimes those hazards are greater than RA itself. Local injection can effectively inhibit the inflammatory response in joints of patients to reduce their pain, but it's not frequent injections, otherwise will have some damage effect to cartilage. Biological agents, including cell adhesion molecules and receptors for biological agents, and chemokine receptors for biological agents, biological agents for cell function, cellular factors for biological agents, often used in the treatment of refractory RA. However, the current biological agents are only the design for a target, its long-term efficacy remains to be seen. With the clinical application of research, found that long-term use of biological agents may cause serious infection or activation of latent tuberculosis, and its expensive. These have limited the clinical application of biological agents.The surgical treatments of RA include removal of invasive or open the way the synovial lesions and joint replacement. Surgical treatment of RA has a significant effect, but with a certain degree of risk, coupled with expensive. Therefore, often used to treat other treatments ineffective late RA. Autologous stem cell transplantation, gene therapy and immune purification have been used in clinical treatment, and achieved certain results, but its exact effect remains to be seen. Ozone (O3) treatment is the new methods of treatment. The used of O3 treatment of RA can be treated, to some extent, overcome these deficiencies.O3 for the treatment of joint disease has been getting the exact effect, but its application focus on the treatment of osteoarthritis. There are a few hospitals using of O3 to treat rheumatoid arthritis. However, the effect of O3 treating rheumatoid arthritis have not been related to systematic study, and O3 treatment of the rheumatoid arthritis-related mechanisms have not been reported. This led to controversy O3 for the treatment of RA, and limited the application of O3 in the treatment of RA. In this study, we used different concentrations O3 (10μg/mL, 20μg/mL, 30μg/mL,40μg/mL,50μg/mL) to treat RA rats, and observed the general situation and joint swelling degrees before and after treatment with different concentrations of O3 in RA rats. Observe the effect of O3 on the RA rats and the impact of synovial tissue by histological section and evaluate the effect of different concentrations of O3 for the treatment of RA, to guide clinical selection of appropriate O3 concentration.Current consensus is that dysfunction, cartilage damage, bone erosion and deformity caused by RA are closely related with apoptosis disorders of synovial cells. Apoptosis is a genetically controlled programmed cell death pattern, and the apoptosis barrier is considered an important cause of the most cancer and autoimmune diseases. Therefore, to achieve good efficacy, control the apoptosis of synovial cells is critical. Apoptosis of RA synovial cells are related with a variety of signal transduction pathways. In these signal transduction pathways, cysteine-containing aspartate-specific proteases (Caspase) family plays a key role.There are three Caspase-3 mediated apoptotic pathway, including Intracellular pathway, extracellular pathways and ER Netcom Way, and the first two pathway research more than the third. In these three pathways, TNF-αparticipate in the outside cell-mediated apoptosis pathway. TNF-αand tumour necrosis factor receptor-Ⅰ(TNFRⅠ) combined, trimmers of the TNFRⅠthrough the intracellular death domain, and tumor necrosis factor receptor-associated death domain and the cytoplasm associated death domain combined, downstream apoptotic signaling, activation of upstream caspase-8 protein, and activation of downstream caspase-3 protein, leading to apoptosis. Current consensus is that dysfunction, cartilage damage, bone erosion and deformity caused by RA are closely related with apoptosis disorders of synovial cells. B cell lymphoma/lewkmia-2 (Bcl-2) family proteins primarily in the mitochondrial pathway of apoptosis are the intracellular pathway of apoptosis play a role. B cell lymphoma/lewkmia-2 associated protein x (Bax) and Bcl-2 was the important representative antiapoptotic and proapoptotic molecule in Bcl-2 family. Under normal physiological conditions, intracellular Bcl-2 with the same family of pro-apoptotic molecules forms heterodimers by BH3 domain, in order to maintain pro-apoptotic proteins in the cell localization. In the process of apoptosis, activated by the apoptosis-related factors, Bcl-2 translocation to the mitochondria membrane within the cell, destroy the integrity of mitochondria, and release the pro-apoptotic factors of mitochondria. After signal transduction activation of upstream Caspase protein, and produce cascade activation of downstream Caspase proteins, and then gradually enlarge the signal transduction pathway of apoptosis, eventually leading to apoptosis. Bax is a main regulatory factorss of Bcl-2 activity, and it is also the most widely studied pro-apoptotic protein in Bcl-2 family. Bax is mainly through Bcl-2 heterodimer to regulate the apoptosis process. When the Bcl-2 in cells increased, Bcl-2 and Bax heterodimer formation also increased, decreased apoptosis trends.When the Bax content in cells is higher than Bcl-2 content, homodimer formation of Bax itself play a major role, then prone to apoptosis. Apoptotic signal can activate Bax, and make Bax translocation from the cytoplasm to the mitochondrial membrane. After bax translocation to the mitochondrial membrane, quickly formed a homodimer, and promote apoptosis, while Bcl-2 limited Bax's capabilities during the progress of induced apoptosis. Therefore, the content of Bcl-2 and Bax and the ratio of Bcl-2/Bax are decided whether the cells into apoptosis.Course treatment with different concentrations of O3 RA collagen-induced arthritis model rats treated with different concentrations of O3 in rats on RA synovitis and synovial hyperplasia, and by different concentrations of O3 on RA synovial apoptosis and Caspase-3 content and expression, from the O3 of RA synovial cells apoptosis, the mechanism of interpretation of O3 treatment of RA. O3 concentrations were also observed on RA synovial cells extracellular and intracellular pathways of the apoptosis, and further elaborated mechanisms of O3 treatment of RA.Objective1. Assess the O3 effect of synovitis and synovial hyperplasia, in order to guide the selection of appropriate concentration to treat RA in clinical.2. Research the effect of synovial apoptosis and Caspase-3 content and expression by using different concentrations of O3, in order to probe the mechanism of using O3 to treat RA.3. Research the effect that content of the RA rats serum and synovial TNF-α, TNFRⅠ, TNFRⅡby using different concentrations of O3, in order to elaborate the O3 mechanism of treating RA by extracellular apoptotic pathway.4. Research the effect that the expression of RA synovial Bcl-2, Bax by using different concentrations of O3, in order to elaborate the O3 mechanism of treating RA by apoptosis apoptotic pathway.Methods1. Rat animal model of RAChoose 48 healthy male Wistar rats, weighing 160-180 g.Adaptive feeding the rats a week, and then randomly divided into control (CON group, n= 6), RA group (RA group, n= 6), pure oxygen injection group (O2 group, n= 6) and O3 treatment group (O3 group n= 30). According to intra-articular injection of ozone concentrations (μg/mL) O3 groups were divided into 5 subgroups:①O3-10 group, ②O3-20 group,③O3-30 group,④O3-40 group,⑤O3-50 group, each sub-group n= 6. Specific methods as follows:first, the certain amount of the bovine collagen typeⅡwas dissolved in theO.1M acetic acid solution, and preparation of a concentration of 2 mg/mL solution, and then placed in 4℃refrigerator overnight. Conditions in an ice bath to take an equal volume of the above solution and complete Freund's adjuvant and its run-in with a mortar into a liquid form, Conditions in an ice bath with the solution, mixing an equal volume of complete Freund's adjuvant. And then repeatedly drawn and push in emulsifier until fully emulsified, made of collagen concentration of 1 mg/mL. Second, the rats in RA group, O2 and O3 groups were anesthetized by intraperitoneal injection of sodium pentobarbital. After disinfection with 75% alcohol, using a Hamilton syringe to take collagen solution 1mL, which prepared in the first step on both sides of the dorsal spine and tail subcutaneously. The third, one week after injection of 0.5mL to booster, induced CIA model. CON group received no special treatment.2. O3 treatmentAfter the modeling, rats in O2 group injected with pure oxygen 1mL, Injected once 1 week, continuous injection three weeks. Five subgroups in O3 group were injected with a concentration of 10μg/mL,20μg/mL,30μg/mL,40μg/mL,50μg/mL of O31mL, Injected once 1 week, continuous injection 3 weeks. CON group and the RA group did not give any treatment.3. MaterialsOne week after treatment, intraperitoneal injection of sodium pentobarbital anesthesia, with 75% alcohol disinfection of rat hind limbs and tail, along the median longitudinal skin incision knee, exposing the knee, complete stripping of synovial tissue. All animals in the O2 group and the O3 group in the last intra-articular injection were killed after one week, and then Synovium obtained, fixed overnight by using 4% paraformaldehyde. Meanwhile, used the tail blood, and centrifuged in the centrifuge, extracted supernatant in the end.4. Effect of joint swelling in rats by using different concentrations of O3From the modeling to the animals were killed, one time weekly record.5. Effect of the thickness of rat paw by using different concentrations of O3After the modeling, measuring the thickness of mouse feet hind paws by electronic vernier caliper, measured once a week until death. Weekly measurements were measured three times.6. Effect of RA rat synovial sections by using different concentrations of O3The rat synovial specimens, the conventional method of dehydration, dipping into the paraffin wax embedded blocks, paraffin slicing machine, made of 4μm paraffin sections. And then observed the situation of synovial tissue proliferation, inflammatory cell infiltration, connective tissue hyperplasia, pannu by using HE staining.7. Effect of RA rat Synovial apoptosis index by using different concentrations of O3Obtained paraffin sections of the knee joint synovial tissue, and used transferase-mediated deoxyuridine triphosphate-biotin nick end labeling in situ end labeling-Tunel staining, then calculated apoptotic number along the synovial tissue with a×400 field of vision, count five horizons per field record number of apoptotic, used the mean value. Normal synovial cells showed blue-purple, apoptosis of synovial cells showed brown. Method of apoptosis index (AI)= (the number of apoptotic cells/the total number of synovial cells)×100%.8. Caspase-3 spectrophotometerSynovial supernatant obtained by spectrophotometer atλ= 405nm measured absorbance. Determine the activities content of Caspase-3 by calculating the OD. 9. Caspase-3 in the West-blotting detectionSynovial drawn, the extraction of synovial proteins, 10μg of sample to 20μl2×second hydroxyl sulfate gel loading buffer in boiling water, boil 8 minutes to denature the proteins, 10000rpm centrifuge for 5 minutes. The sample voltage of 100 volts after the transfer film one hour,10% skimmed milk powder in a closed fluid closed for two hours. Add primary antibody (1:500), secondary antibody (1:1000) for one hour each, follow-up instructions by ECL luminescence kit until to complete. Glyceraldehyde-3--phosphate dehydrogenase (37 KD) was served as the internal control.10. Serum and synovial TNF-α, TNFRⅠ, TNFRⅡcontent detectionTake synovial supernatant and plasma, use serum TNF-α, TNFRⅠ, TNFRⅡto the enzyme-linked immunosorbent assay (ELISA) test.11. Effect of Bcl-2, Bax expression in RA rat by using different concentrations of O3The synovial tissue sections at 60℃for 60min bake baking sheet box. Observed two sections each joint, the positive cell count to take the mean of two sections. Under the observation of each slice 5×400 field of vision.12. Statistical analysisAll statistical analyses were performed by SPSS version 13.0 statistical software. Repeated measures analysis of variance and One-way analysis of variance were used to analyze data. Measurement data were expressed as (x±s). P<0.05 was considered significantly different.Results1. Effect of joint swelling in rats by using different concentrations of O37d modeling, the rats showed symptoms of synovitis, each joint and foot padfeet appeared red swelling, joint red swelling significantly 14d modeling.21d after modeling the most severe joint swelling appeared in rats. RA group, O2 group, O3-10 group, O3-20 group gradually reduced joint swelling after 28d, joint swelling were still existed before killed.The first and the second injection of O3 one week after, O3-30 group, O3-40 group are significantly reduced redness or swelling. One week after the third injection of O3, O3-30 group, O3-40 group is no longer swelling. One week after the first injection of O3, redness and swelling of O3-50 group rats are more significant than before the injection. O3 week after the second injection and were killed before, O3-50 rats, the phenomenon of swelling was still evident, but more for the first time been reduced. One week after the second O3 injection and were killed before, rats, the phenomenon of swelling in the O3-50 group was still evident. However, compare the first time, the swelling had been improved.2. Effect of the thickness of rat paw by using different concentrations of O3Before the experiment, the paw thickness between each group rat was no significant difference (p> 0.05). In addition to CON group rats, the paw thickness of rats began to increase after 7 d,21d after the model the maximum joint thickness. Compared with the rats after the modeling 21d, O3-30 group, the paw thickness of O3-40 group which after modeling 28d was significantly reduced (p<0.01), difference between the two groups was not significant.After modeling 35d, RA group, compared with CON group, paw thickness of O2 group O3-10 group, O3-20 group, O3-40 group, O3-50 group increased, the difference was statistically significant (p<0.01). O3-30 group and the CON group compared with the RA group, the difference was statistically significant (p<0.05).After modeling 42d, the paw thickness of O3-40 group decreased more than it after modeling 35d, the difference was statistically significant (p<0.05). Compared with the CON group, RA group, O2 group, O3-10 group, O3-20 group, O3-30 group,the paw thickness increases, the difference was statistically significant (p-<0.05). Compared with the CON group, the paw thickness of O3-50 group significantly increased, the difference was statistically significant (p<0.01).3. Effect of RA rat synovial sections by using different concentrations of O3In CON group, normal joint structure, have no congestion and edema of synovial tissue, synovial cells without proliferation, no pannus formation. In RA group and O2 group, showed obvious proliferation of synovial cells and connective tissue, disorganized, inflammatory cell infiltration, pannus formation within the synovial tissue. In O3-10 group and O3-20 group, slightly reduce the proliferation of synovial cells, congestion and edema of synovial tissue reduced, angiogenesis and the number of infiltrating inflammatory cells reduced, pannus formation reduced. In O3-30 group and O3-40 group, synovial cell proliferation was not obvious, synovial tissue significantly reduced congestion and edema, angiogenesis, and the number of infiltrating inflammatory cells reduced, pannus formation was significantly reduced. Compared with the CON group, the synovial tissue of the O3-50 group had decreased and disorder, connective tissue hyperplasia, inflammatory cell infiltration obviously.4. Effect of RA rat Synovial apoptosis index by using different concentrations of O3There were scattered apoptosis in the synovial of the CON group rats. Compared with the CON group, RA group, O2 group of normal and apoptosis of synovial cells increased, the apoptosis rate decreased,the difference was statistically significant (p<0.05). Compared with the model group, the apoptosis rate increased in each O3 treatment group. Compared with the RA group, the rate of synovial cell apoptosis in the O3-10 group, O3-20 group was no statistically significant (p>0.05). Compared with the RA group, the rate of synovial cell apoptosis in O3-30, O3-40 group and O3-50 group was statistically significant (p<0.05).5. Effect of RA rat Caspase-3 content of synovial by using different concentrations of O3Compared with the CON group, RA group,02 group O3-10 group Caspase-3 content of synovial decreased, the difference was statistically significant (p<0.05). Compared with the three groups, the difference was not statistically significant (p> 0.05). Compared with the CON group, O3-30 group, O3-40 group of Caspase-3 levels increased, the difference was statistically significant (p<0.05). Caspase-3 in O3-40 group was higher than the O3-30 group, the difference was statistically significant (p<0.05). Compared with the CON group, O3-20 group, O3-50 group Caspase-3 levels in the synovial membrane, the difference was not statistically significant (p> 0.05).6. Effect of Caspase-3 protein expression in RA rat's synovial by using different concentrations of O3Compared with CON group, Caspase-3 protein expression of synovial in RA group, O2group, O3-10 group and O3-50 group decreased, the difference was statistically significant (p<0.01). Compared with CON group, Caspase-3 protein expression of synovial in RA group decreased, the difference was statistically significant (p<0.05).Compared with CON group, Caspase-3 protein expression of synovial in the O3-30 group increased, the difference was not statistically significant (p>0.05). Compared with CON group, Caspase-3 protein expression of synovial in the O3-40 group increased, the difference was statistically significant (p<0.01).7. Effect of Bcl-2, Bax expression in RA rat's synovial by using different concentrations of O3Synovial tissue of rats were seen positive cells of Bcl-2 and Bax, which CON group expressed the weakest, RA group expressed the strongest. Compared with CON group, the number of Bcl-2 positive cells in RA group, O2 group, O3-10 group increased, while Bax-positive cells were decreased, the difference was statistically significant (p<0.01). Compared with the three groups, the difference was not statistically significant (p> 0.05). Compared with RA group, the number of Bcl-2 positive cells in the O3-20 group, O3-50 group decreased, while Bax-positive cells were increased, the difference was statistically significant (p<0.05). Compared the two groups, thedifference was not statistically significant (p> 0.05). Compared with RA group, the number of Bcl-2 positive cells in the O3-30 group, O3-40 group decreased, while Bax-positive cells were increased, the difference was statistically significant (p<0.05). Compared the two groups, the difference was statistically significant (p< 0.05).Conclusion1. Intra-articular injection of O3 concentration 40μg/mL can achieve best results. It can significantly reduce the synovial infiltration of inflammatory cells, reduce the formation of connective tissue and pannus, also can reduce the joint swelling.2. The mechanism of O3 treatment in RA is related to increasing apoptosis of RA synovial cells and increased expression of Caspase-3 protein.3. O3 can reduced the activity of synovial TNF-a, TNFRⅡand increase the expression of TNFR I, particularly use Intra-articular injection of O3 concentration 40μg/mL.4. The main mechanism of O3 treatment RA rats are that the expression of Synovial Bcl-2 decreased and the expression of Bax increased.