| Osteoarthritis(OA)is a very common chronic degenerative bone and joint disease.The lesion mainly involves the weight-bearing large joints.The course of the disease progresses slowly and progressively worsens.The daily activities of patients with advanced OA are obviously limited,even may lose the ability to daily life.It is generally believed in the academic community that OA is caused by the imbalance of metabolism of articular cartilage matrix,articular chondrocytes and subchondral bone due to mechanical load and biochemical factors.The pathological process involves joint mechanics,inflammatory factors,oxidative stress.(Free radicals)and congenital genetic factors and other factors.A large number of reports have shown that Oxidative Stress(OS)can regulate the conduction of intracellular signaling pathways,synthesis and degradation of extracellular matrix,biofilm permeability,cellular senescence and apoptosis,and transcription and translation of genes.The process of promoting the development of multiple stages of OA disease.Reactive oxygen species(ROS)are the direct cause of OS damage,senescence and apoptosis in OA chondrocytes.Therefore,maintaining a balance between oxidation and anti-oxidation in the body and eliminating excess ROS is considered a feasible solution for the treatment of OA.Dopamine is a neurotransmitter that helps cells transmit pulsed chemicals.This kind of brain secretion is related to human lust and feeling,and it conveys excitement and happy information.Recent studies have shown that dopamine has significant antioxidant capacity,while dopamine has the ability to activate autophagy to remove damaged organelles and harmful substances,and that dopamine has good biocompatibility and microcytotoxicity,so dopamine is a good treatment for OA.Potential drugs.However,the rapid degradation and the in vivo environment have a great influence on the physicochemical properties of dopamine.It is difficult to achieve ideal therapeutic effect by simply applying dopamine.In view of these characteristics of dopamine,dopamine melanin nanoparticles(DM)were successfully prepared by melanin-modified melatonin.DM well compensated for the short half-life of dopamine in vivo,and preserved dopamine autophagy.The activation effect,the excellent properties of antioxidant scavenging ROS.Based on this,the subject firstly measured the therapeutic effect of DM on interleukin-1Bata(IL-1?)-mediated OA chondrocytes;secondly,the effect of DM on IL-1?-mediated autophagy of OA chondrocytes;To detect the effect of DM on IL-1?-mediated ROS in OA chondrocytes in vitro;further study the intrinsic relationship between DM and OA to activate autophagy and clear ROS;finally establish OA animal model and systematically evaluate the therapeutic effect of DM on OA.The main research results of this dissertation are as follows:1.Preparation and characterization of DMDopamine hydrochloride was dissolved in an ethanol/water mixed solution,and added to an ammonium hydroxide solution as a catalyst.After reacting for 24 hours in the air,the product was collected by centrifugation.The synthesized DM can be stably dispersed in water to form a transparent colloidal solution.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)showed that the product nanoparticles were uniform spheres with an average diameter of 112.5 nm.Dynamic light scattering(DLS)experiments found that the nanoparticle has a hydrodynamic size of 230 nm.Zeta potential analysis showed that the surface of the nanoparticles was negatively charged(13.5 m V).This was attributed to the multiple catechol groups on the surface of the nanoparticles and was confirmed by Fourier-transform infrared spectroscopy(Fr-IR)and nuclear magnetic resonance spectroscopy(1H NMR)analysis.2.Evaluation of the therapeutic effect of DM on OA chondrocytesThe rat knee articular cartilage was degraded with type II collagenase and primary chondrocytes were extracted and identified by toluidine blue and alexin blue staining.The whole in vitro cell experiments were partially treated with second generation chondrocytes.The toxicity of DM to chondrocytes was evaluated by CCK-8 method.The results showed that the DM dose did not exceed 100 ?g/m L,and had no significant effect on the cell viability of SD rat chondrocytes.Induction of classical in vitro OA cell model using IL-1?,low,medium and high doses of DM solution for incubated with OA cells for 24 h significantly up-regulated the expression of autophagy-related indicators,decreased ROS levels and inhibited IL-1? in OA cells.Mediated inflammatory factor secretion(p < 0.01).The rat knee articular cartilage was degraded with type II collagenase and primary chondrocytes were extracted and identified by toluidine blue and alexin blue staining.The whole in vitro cell experiments were partially treated with second generation chondrocytes.The toxicity of DM to chondrocytes was evaluated by CCK-8 method.The results showed that the DM dose did not exceed 100 ?g/m L,and had no significant effect on the cell viability of SD rat chondrocytes.Induction of classical in vitro OA cell model using IL-1?,low,medium and high doses of DM solution for incubated with OA cells for 24 h significantly up-regulated the expression of autophagy-related indicators,decreased ROS levels and inhibited IL-1? in OA cells.Mediated inflammatory factor secretion(p < 0.01).3.Evaluation of the efficacy of DM on OA ratsThrough minimally invasive surgery,the rat anterior cruciate ligament transection and medial meniscus resection were performed to establish a classic animal model of OA,and the successful model was verified by the front drawer test.At 4 weeks after surgery,low,medium and high concentrations of DM solution were injected into the joint cavity,and injected twice a week,0.2 ml each time,for four weeks.The results of the general observation showed that the femoral condyle and tibial plateau cartilage tissue of the osteoarthritis model group were gray and dark,and obvious cartilage defects appeared.This result further indicated the successful modeling and the damage of the joints of osteoarthritis;low and medium The femoral and tibial plateau cartilage color of the high-concentration DM nanoparticle treatment group was significantly different from that of the osteoarthritis model group,and the joint damage caused by the surgery was significantly alleviated,which was consistent with the results of tissue staining.In addition,the threshold of mechanical contraction of hind limbs in the OA group was significantly reduced,and the threshold of mechanical contraction was gradually increased to normal level after three treatments in the DM group.The levels of IL-6,MMP-13 and TNF-a in rat joint fluid were determined by ELISA kit.The results showed that inflammatory factor levels(IL-6,MMP-13 and TNF-a)were observed in different doses of DM treatment group.Significantly decreased(P<0.01),indicating that injection of DM into the joint cavity can effectively reduce the levels of IL-6,MMP-13 and TNF-a in the joint fluid of rats,and reduce the inflammatory response;the level of ROS is detected by extracting the joint fluid of each group.The results showed that the ROS level in the OA group was significantly higher than that in the normal group,and the DM group significantly reduced the level of ROS in the synovial fluid.The MDA and GSH kits were used to detect the content of MDA and GSH in the synovial fluid of different groups of rats.The results showed that the DM nanoparticle treatment group can significantly reduce the content of MDA in synovial fluid and increase the content of GSH in synovial fluid compared with the OA model group..It is indicated that the oxidized level of joint tissue is improved by intra-articular injection of DM nanoparticles.In conclusion,DM can protect chondrocytes more effectively,maintain the normal morphology of articular cartilage,activate autophagy,increase the body's antioxidant level and reduce the level of inflammatory factors,and it has higher biological activity and longer half-life in the joint cavity.Time is a potentially clinically significant OA drug. |
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