Effect Of Advanced Oxidation Protein Products On Articular Cartilage And Synovium In A Rabbit Model Of Knee Osteoarthritis

Backgrouds:Osteoarthritis (OA) is a chronic degenerative joint disease that characterized by degradation and loss of articular cartilage, subchondral bone remodeling, and inflammation of the synovial membrane at the clinical stage of the disease. The final common pathway of cartilage destruction results from a failure of chondrocytes to maintain a homeostatic balance between matrix synthesis and degradation.OA is thought to be the most prevalent chronic joint disease. The incidence of OA is rising because of the ageing of population and the epidemic of obesity.OA counts among the most common joint disease which are influencing human health. The occurrence of OA has no varies in regions and races. It is one of the most common types of disabling disease for the people whose age more than 50 years, behind cardiovascular disease. Based on this survey, the disability rate of OA is 2%-6% in human disease. According to the statistics, in USA, the cost of OA is about 150 billion dollar, tripled rheumatoid arthritis. Half of the cost result from work disability. Because of OA can be more easily affect the function of lower limbs than other disease, it becomes one of the most serious disabling disease that caused great economic losses and hinder the social development. Knee osteoarthritis(KOA) is a chronic progressive joint disease that characterized by degradation of articular cartilage and the loss of chondrocyte. The morbidity of KOA is 40% in the eldrly aged 55-64. Although there are multiple pathogenic factors, a similar biological characteristics and morphological characteristics has been observed in the impairment of KOA. In the course of KOA, not only influence the articular cartilage but also subchondral bone, ligament, joint capsule, synovial membrane and muscle around the joint. Finally, arthralgia and dysfunction of joint lead to a lower quality of patient’s life. Therefore, revealing the pathogenesis of OA is of great significance to the prevention and control of the OA.Imbalance between oxidant and anti-oxidant system results in the accumulation of reactive oxygen species (ROS), which then case oxidative damage to macromolecules such as proteins, lipid, DNA, RNA. This is the pathological process which we called oxidative stress. Proteins tend to be damaged more easily by ROS than others. AOPPs are the dityrosine-containing and cross-linking protein products formed during oxidative stress, which were firstly found in the plasma of dialysis patients. AOPPs can be incubated by by mixing serum albumin with hypochlorous acid (HOC1), and the level of AOPPs depends on the mount ofHOCI. In vivo, plasma concentration of AOPPs closely correlate with levels of dityrosine, a hallmark of oxidized protein, and pentosidine, a marker of protein glycoxidation tightly related to oxidative stress. AOPPs are now widely regarded as novel markers of oxidative stress. There is elevated plasma AOPPs level in many diseases. Piwowar et al found that patients of type 2 diabetes mellitus had higher AOPPs than healthy subjects, and those with subclinical complications had higher AOPPs than those without. AOPPs increase in the early phase of CKD, and and there is a positive correlation between AOPPs level and clearance of creatinine rate (Ccr). AOPPs may be involved in the pathological process of atherosclerosis too. Clinical evidence has indicated that AOPPs are highly correlated to carotid intima media thickness and may even be related to atherosclerotic cardiovascular events-Barsotti et al pointed out that AOPP/Thiol ratio elevated in patients with acute coronary syndromes and may represent a reliable marker of oxidative unbalance in this setting of patients. Other diseases with increasing AOPPs include IgA nephropathy, Henoch-Schonlein purpura, active ulcerative colitis, allergic rhinitis, inflammatory bowel disease, obesity, nonalcoholic steatohepatitis. In vivo study found that AOPPs increased renal macrophage infiltration and upregulated the expression of monocyte chemoattractant protein-1(MCP-1) and transforming growth factor-0 5 and accelerated the deterioration of renal structure and function. Similar changes happened in a AOPPs treated remnant kidney model. Moreover, AOPPs promoted inflammation and atherosclerotic plaque area oxidized low-density lipoprotein (oxLDL) deposition in artery of rabbit. In above animal models, elevated AOPPs levels were found in both plasma and tissues. Moreover, Witko-Sarsat et al demonstrated that AOPPs could activate monocytes and trigger respiratory burst. Guo et al found that AOPPs isolated from CKD or incubated in vitro could both activate vascular endothelial cells via a RAGE-mediated, ROS sensitive pathway. AOPPs also increase the generation of superoxide in mesangial cell, resulting in upregulation of fibronectin and collagen IV genes and proteins and overexpression of TGF-β. Zhou et al pointed out that AOPPs induced inflammatory response and insulin resistance in cultured adipocytes via induction of endoplasmic reticulum stress, and inhibited differentiation and activate inflammation in 3T3-L1 preadipocytes. More over, AOPPs induce cardiomyocyte death and podocyte apoptosis. In a word, AOPPs are not only the result of oxidative stress, they trigger ROS generation too. AOPPs are very important mediators of inflammation and may be involved in the pathological base of many important diseases.Our research group has proved that exposure of firoblast-like synoviocytes(FLSs) to AOPPs upregulated the mRNA and protein expression of TNF-α, IL-1β, MMP-3, MMP-13 and VEGF in a concentration dependent manner. At present the etiology and pathogenesis of OA has not yet fully understood, but numerous studies have found that the expression and activity of matrix metalloproteinases(MMPs) and Interleukin(IL) play a key role in the degradation of cartilage matrix. Matrix metalloproteinases is a proteolytic enzyme that rely on zinc ion and calcium ion. Extra cenularmatrix (ECM), part of the protein and signaling protein can be degraded by MMPs specifically. MMP-3 belongs to a kind of stromelysin, substrates include Collagen Ⅱ, Ⅳ, Ⅸ, Ⅹ, Ⅺ, gelatin. MMP-13 also named Collagenase 3, Substrates include Collagen Ⅰ,Ⅱ, Ⅲ, Ⅳ, Ⅸ, Ⅹ, ⅪⅤ, gelatin. MMP-3 and MMP-13 both are the common test index in the study of cartilage destruction.It is still unknow that whether AOPPs has an action on articular cartilage and synovial membrane. So we suppose that AOPPs has a negative influence on articular cartilage and synovial membrane, it may be an important pathogenic factors of OA. In order to confirm our hypothesis, we choose an vivo study to explore the effect of AOPPs on articular cartilage and synovium in a rabbit model of knee osteoarthritis.Objectives:1. To investigate the effect of AOPPs on the gross morphology of condyles of femur in rabbit OA model.2. To investigate the effect of AOPPs on the expression quantity of MMP-3 and MMP-13 of synovium in a rabbit OA model.Material and Methods:1. AOPPs-RS A preparationAOPPs-RSA was prepared in vitro as described previously. In brief, RSA (Sigma, USA) solution (20 mg/ml) was incubated with 40 mM HCLO in phosphate buffered saline (PBS, pH 7.4) for 30 min at 37℃. The reaction was then stopped via overnight dialysis against PBS to remove free HCLO. To remove contaminated endotoxin, all samples were passed through a Detoxi-Gel column. Endotoxin levels in AOPPs-RSA and un-modified RSA were then measured using a Limulus Amoebocyte Lysate kit andwere found to be below 0.05 ng/mg protein. Prepared AOPPs-RSA was stored in -20℃2. Animals and treatments and samples harvestForty-eight 5-month-old male New Zealand rabbits (body weight 2.0-2.5Kg) were randomly divided into 3 groups:AOPPs group (n=16), PBS group (n=16) and sham-operated group (n=16).OA model were created in AOPPs group and PBS group by anterior cruciate ligament transection and medial meniscus resection, then intra-articular injection of lml AOPPs or PBS were performed for 4 weeks and 8 weeks (every other day) in AOPPs group and PBS group, respectively. In sham-operated group, the anterior cruciate ligament was just exposed without transection, and then the incision was sutured.All rabbits were sacrificed after 4 weeks or 8 weeks of intervention, respectively.Both sides of the knee were obtained. The tibias and the femurs were excised, covered with normal saline (NS) gauze and stored at -20℃.3. Endpoints measurements(1) The gross morphology of condyles of femur. India ink scoring system for assessment of macroscopic cartilage changes in rabbits OA model.(2) Safranine O and fast green staining of condyles of femur section. Mankin scoring system for assessment of microscopic cartilage changes in rabbits OA model.(3) Extract the protein of synovium in rabbits OA model and detect the protein expression level of MMP-3 and MMP-13.Results:1. Effect of AOPPs administration on gross morphology of articular cartilageThe India ink score of two time points were 4.19 ± 0.60、5.75 ± 0.60 in AOPPs group, and 1.06±0.18、1.38±0.60 in sham-operated group,2.50±0.46、3.06±0.62 in PBS group, respectively. The India ink score of PBS group at two time points are both higher than sham-operated group, the differences at two time points were both statistically significant between the two groups. (P<0.01). In addition, The India ink score of AOPPs group at two time points the differences were both higher than the other two groups, and differences were both statistically significant among the three groups(P<0.01).2. Effect of AOPPs administration on microscopic changes of articular cartilageThe Mankin score of two time points were 8.19±0.70.11.94±0.90 in AOPPs group, and 0.75±0.53、1.06±0.73 in sham-operated group,4.25±1.46、4.50±0.89 in sham-operated group,respectively. The Mankin score of PBS group at two time points are both higher than sham-operated group, the differences at two time points were both statistically significant between the two groups. (P<0.01). In addition, The Mankin score of AOPPs group at two time points the differences were both higher than the other two groups, and differences were both statistically significant statistically significant among the three groups(P<0.01).3. Effect of AOPPs administration on MMP3 and MMP13 in synoviumThe protein expression level of MMP3 and MMP13 on synovial at two time points were 1.006±0.080、1.098±0.088 in AOPPs group, and 0.066±0.006、0.053 ±0.011 in sham-operated group,0.552±0.024、0.839±0.084 in sham-operated group,respectively. The MMP-3 and MMP-13 expression level of PBS group at two time points are both higher than sham-operated group, the differences at two time points were both statistically significant between the two groups. (P<0.01).In addition, The MMP-3 and MMP-13 expression level of AOPPs group at two time points the differences were both higher than the other two groups, and differences were both statistically significant statistically significant among the three groups(P<0.01).Conclusions:In summary, the present study determined that AOPPs significantly accelerate the regression of articular cartilage in a rabbits OA model. Meanwhile, AOPPs also upregulated the the protein expression of MMP3 and MMP13 in synovium.These date may provide new information toward understanding the pathogenic basis of OA and may provide targets for intervention.