动物实验,骨关节炎 z-bo 2020-09-03 442

　　Background: Osteoarthritis (OA) is a heterogeneous chronic disease that affects all tissues of synovial joints. At present, it is believed that the cause of disease is multifaceted, involving a variety of mechanical, biochemical and genetic factors, as well as molecular and enzymatic feedback loops. The changes of osteoarthritis are mainly in the process of remodeling, from the failure of cells to maintain a steady-state balance, resulting in tissue disorders. As the disease progresses, the catabolic process will exceed the synthesis of metabolites, leading to progressive tissue disease. The morphological changes observed in OA include varying degrees of synovial inflammation, mainly in the clinical stage of the disease, which in turn produces inflammatory mediators (inflammatory cytokines), which play a key role in the pathophysiological mechanism of OA effect. The pharmacological treatment of OA includes different types of drugs classified according to their mode of action. Current treatment programs are mainly focused on reducing pain and improving joint function, including painkillers and non-steroidal anti-inflammatory drugs; unfortunately, their use in chronic treatment is limited by the harmful side effects of cartilage and gastrointestinal tract. These drugs are classified as slow-acting drugs for OA. These drugs are expected to delay, stabilize or reverse the pathological changes of arthritis, thereby limiting the progression of the disease. Such drugs include diacerein, and anthraquinone derivatives are oral anti-inflammatory, analgesic and antipyretic agents specifically used to treat OA. The contribution of pro-inflammatory cytokines to the degradation of OA cartilage includes interleukin-1 (IL-1), which stimulates the degradation process of cartilage and inhibits the synthesis of cartilage matrix. The active metabolite of diacerein has been shown to inhibit IL-1β in vitro and in vivo, while reducing the production of collagenase by articular cartilage cells, reducing the activity of fibrinolysis in joint fluid and stimulating the production of cartilage growth factors. The purpose of this study was to test the effectiveness of oral diacerein in the treatment of early rabbit OA. Use histological techniques to compare with micro-CT (Micro-CT) and non-contrast techniques to determine cartilage and subchondral bone structure.

　　Method: Pre- and post-operative care for induced osteoarthritis: 16 adult female (6-7 months old, average weight 5 kg) healthy New Zealand white rabbits were used. All animals underwent surgery, and the veterinarian monitored the rabbits daily throughout the experiment. An anterior cruciate ligamentectomy (ACLT) and a partial meniscus resection of the knee were used to induce OA (selected at random). The contralateral knee was used as a control. The animals were preoperatively treated with medetomidine (50μg/kg, IM) and ketamine (25mg/kg, IM) to induce anesthesia, and then isoflurane (2.5-4%) was used to maintain anesthesia. Each animal received buprenorphine (1 mg/kg, IM) during perioperative and postoperative periods; enrofloxacin was used for one week of antibiotic prophylaxis (15 mg/kg, SC, once a day) and 3 days after surgery. Loxicam (20ug/kg, SC). Three weeks after surgery, 8 animals were randomly assigned to each group. The control group was given 5ml of normal saline (sodium chloride 0.9%), and the treatment group was given the same amount of diacerein (1.5mg/kg). The two treatment methods are administered directly orally using a 5 ml syringe daily.

　　Preparation of autopsy and histological specimens: After sedation of rabbits with ketamine (25 mg/kg, IM), the rabbits were killed by overdose of sodium pentobarbital (100 mg/kg, IV). An autopsy was performed on all animals to assess whether there were any changes other than the knee joint. After dissecting the joints, in the same anatomical position of each animal’s knee, the medial femoral condyle obtained two cylinders with a diameter of 2.9 mm and a length of 8 mm. Use the same procedure to obtain a part of the joint capsule close to the patellar ligament.

　　For evaluation, the samples are divided into four groups. Ctrl: healthy non-surgical treatment receiving placebo group (n = 8); OA: receiving placebo group after surgery (n = 8); Ctrl+DC: healthy non-surgical treatment receiving diacebo group (n = 8); OA+DC: animals with knee osteoarthritis plus diacerein treatment group (n = 8). After EDTA decalcification, together with the joint capsule, routine paraffin embedding, sectioning, HE and Safranin O-fast green staining, and HE staining of synovial smear

　　Microscopic evaluation: The biopsy of the synovial membrane, demineralized bone and cartilage tissue core was evaluated by two independent observers according to published guidelines. The classification of structural damage is as follows: the pathology of cartilage, chondrocytes and proteoglycan ranges from 0 (normal) to 4 (completely affected), and the synovial membrane ranges from 0 (normal) to 2 (osteoarthritis). The evaluation parameters of cartilage samples are: cartilage pathology (cartilage surface characteristics), chondrocyte pathology (cell density and distribution), proteoglycan (degree of pathological staining), and integrity of the tide line. Synovial slide: the characteristics of the inner cell (the number of superficial cell layers), hyperplasia (presence or absence), with or without cell infiltration. Quantitative histological evaluation using non-decalcified slices: evaluation parameters: subchondral bone cortical thickness (SB.TH), total cartilage thickness (Cg.Th), non-calcified (nCg.Th), calcified cartilage thickness (cCg.Th) ), cartilage surface undulation (FI), trabecular subchondral bone area (Tb.A) and trabecular bone separation (Tb.Sp). Tb A is defined as the percentage of trabecular bone in this area. Tb.Sp is the average distance between trabeculae.

　　Micro-computed tomography: Non-decalcified slices are evaluated by high-resolution micro-CT. The following direct indicators were measured: bone volume fraction (BV/TV), bone trabecular thickness (Tb.Th) and separation (Tb.Sp), and bone trabecular number (Tb.N).

　　Result: No changes in body weight or general conditions were observed in the experiment. The outstanding fact is: Diacerein treats animal urine discoloration; however, no pathological changes in the kidneys or other organs were found on autopsy. All 32 joints were collected, except for those excluded due to infection, the remaining 31 were subjected to histological analysis.

　　Quantitative histological results: The subchondral bone measurement parameters in the calcified slices obtained from the Ctrl and OA groups were similar, and there was no change in the two groups (Ctrl+DC and OA+DC). Cartilage related parameters, non-calcified cartilage thickness ((nCg.Th) ) Showed that the OA group was significantly higher than the Ctrl and Ctrl+DC groups. The OA?+?DC group (diacerein treatment group after knee surgery) showed a decrease in nCg. The total cartilage thickness between the Ctrl and OA groups (Cg.Th) ) The difference is not significant. The cartilage thickness of the OA group is significantly higher than that of the other groups. The cartilage thickness of the OA+DC group is between the control group and the OA group. There are many muscle fibrosis on the surface of the OA group. The two variables in the synovium are internal The characteristics of the layered cells and the presence of hyperplasia were statistically significant between the OA and Ctrl groups, but there was no inflammatory infiltration. The only difference between the two diacerein groups was the presence of hyperplasia.

　　Conclusion: Oral diacerein can reduce cartilage swelling, surface changes and synovial inflammation as a treatment for early OA in a rabbit surgical model. Regarding subchondral bone, Diacerein has the ability to increase bone mass and bone density for healthy non-arthritis, but its results have not been announced so far. This study also proved the validity of the animal model. MicroCT can detect the morphological changes of articular cartilage. Further research is needed to determine the efficacy of diacerein in long-term OA animal models.