| Osteoarthritis(OA)is a common degenerative osteoarticular lesion,which mainly involves major weight-bearing joints.OA has a slow course,and often leads to limited activity and even loss of self-care ability.At present,it is generally believed that OA is formed due to the imbalance between the synthesis and degradation of cartilage matrix,chondrocytes,and subchondral bone resulting from mechanical and biological factors.It is involved in joint mechanics,cytokines,oxidative stress,and genetic factors.A large number of studies have shown that oxidative stress plays an important role in the development of OA disease by participating in intracellular signal transduction,senescence and apoptosis of chondrocytes,synthesis and degradation of extracellular matrix,and synovial inflammation.Therefore,improving the body's antioxidant capacity is considered to be a viable option for the treatment of OA.Superoxide dismutase(SOD)is one of the important components of the body's antioxidant enzyme system.It can quickly and efficiently scavenge reactive oxygen species(ROS),which makes it a potential enzyme drug for the treatment of ROS-related diseases.In the previous studies of this research group,the O-HTCC was successfully conjugated with SOD to obtaine O-HTCC-SOD conjugate with high enzyme activity retention rate,prolonged half-life,and good cell-entry capacity.Based on this,the present study first measured the protective effect of O-HTCC-SOD on ROS-induced chondrocyte injury in vitro,and then preliminarily investigated the pharmacokinetics of O-HTCC-SOD after single intra-articular injection in rats,finally systematically evaluated the therapeutic effects of O-HTCC-SOD on established animal models of OA.The main research results obtained in this dissertation are as follows:1.Preparation and Characterization of O-HTCC-SODThe water-soluble O-HTCC was obtained through a simple three-step chemical reaction with a yield of 83.3%.The structure was confirmed by infrared and 1H NMR spectra.The content of free amino group was 81.52%as measured by the ninhydrin colorimetric method.SOD was covalently linked with O-HTCC through carbodiimide-mediated condensation reaction,and the resulting reaction retained 54.7%of enzyme activity.The reaction mixture was purified by one step weak anion exchange column chromatography to give O-HTCC-SOD conjugate.The enzyme specific activity of O-HTCC-SOD was 1.08 times that of unmodified SOD.Electrophoresis results showed that the molecular weight of the conjugate was a continuous distribution,and potential analysis showed that the conjugate was positively charged.2.Evaluation of protective effect of O-HTCC-SOD on rat chondrocytesThe rat primary chondrocytes were extracted from rat knee cartilage degraded by collagenase,and identified using the alicin blue and toluidine blue staining.The cells of the second and third generation were used for the whole cell experimental.The toxicity of O-HTCC-SOD on chondrocytes was evaluated by MTT assay.The results showed that O-HTCC-SOD and SOD had no significant effect on rat chondrocyte growth in vitro when the enzyme activity was less than 200 U/mL.MIA could induce damage of rat chondrocytes.Low-,medium-,and high-doses of O-HTCC-SOD or SOD significantly inhibited MIA-induced chondrocyte damage at 24 and 48 h(P<0.01).However,the protective effect of O-HTCC-SOD on chondrocytes was significantly higher than that of SOD after 48 h incubation(P<0.01),indicating a more lasting protective effect of O-HTCC-SOD on chondrocytes.After treated with O-HTCC-SOD(or SOD)and MIA in turn,the relative cell viability of three O-HTCC-SOD groups remained at about 90%,which was significantly better than that of the model cells(P<0.01).However,the relative cell viability of SOD groups decreased to about 60%,indicating SOD had no obvious protective effect on MIA-mediated chondrocyte damage(P>0.05).Therefore,O-HTCC-SOD significantly inhibited MIA-mediated chondrocyte damage by entry into the cells,but SOD did not.After treated with O-HTCC-SOD(or SOD)and MIA in turn,intracellular ROS levels were measured using the DCFH-DA kit.The results showed that intracellular ROS levels in the three O-HTCC-SOD groups were significantly lower than those in the model group(P<0.01),and even decreased to levels lower than those in the control group(P<0.01),while there was no significant difference in ROS levels between the three SOD groups and the model group(P>0.05).Therefore,O-HTCC-SOD played a role in scavenging ROS by entering into chondrocytes,but SOD did not.3.Preliminary study on pharmacokinetics of O-HTCC-SOD in ratsElimination of O-HTCC-SOD and SOD in rat joint cavity was studied by determining the SOD enzyme activity after a single intra-articular injection of O-HTCC-SOD and SOD in rats.The results showed that the area under the curve(AUC)from 0-168h(AUC0-168th)of SOD and O-HTCC-SOD were 18043.5(U-h),91853.478(U-h)and their AUC0-? were 20061.932(U·h),91910.091(U-h),respectively.The AUC of O-HTCC-SOD was approximately 5 times that of SOD.Therefore,the bioavailability of O-HTCC-SOD was higher than that of SOD in the joint cavity.The mean residence time(MRT)for SOD and O-HTCC-SOD was 14.915h and 27.918h,respectively.The MRT of O-HTCC-SOD was about 2 times that of SOD,indicating that O-HTCC-SOD had a longer residence time in the joint cavity.4.Efficacy evaluation of O-HTCC-SOD on osteoarthritis in ratsRat OA models were establish by intra-articular injection of MIA.The rats were treated by weekly intra-articular injection of O-HTCC-SOD,SOD,O-HTCC,and mixture of O-HTCC and SOD for four weeks.The results showed that the color of the cartilage of the femur and tibial plateau in the model group was dark and there was a cartilage defect,indicating successful modeling.The cartilage color of the femur and tibial plateau of the low-,medium-,and high-dose O-HTCC-SOD groups was the same as that of the blank group,with occasional small crevices and small holes.,while SOD group was similar to model group and the degree of articular cartilage in the O-HTCC group and the mixture group was improved to some extent compared with the model group.The results of pathological section and the general observation of the knee joint were basically consistent.It showed that O-HTCC-SOD treatment effectively protected chondrocytes and maintained the normal form of cartilage tissue.Injection of MIA in the knee joint could induce changes in the mechanical paw withdrawal threshold(PWT)of the hindlimb.After treatment of O-HTCC-SOD by intra-articular injection,the PWT gradually increased to normal level.However,SOD treatment did not improve the PWT.The levels of IL-1?,IL-6 and TNF-a in rat peripheral blood were determined by ELISA kit.The results showed that different doses of O-HTCC-SOD significantly reduced IL-1?,IL-6 and TNF-a levels(P<0.01),SOD,O-HTCC,and the mixture of SOD and O-HTCC had no obvious effect.This showed that intra-articular injection of O-HTCC-SOD effectively reduced the levels of IL-1(3,IL-6 and TNF-a in the peripheral blood and reduced the inflammatory response.The content of MDA and GSH in rat synovial tissue was measured by chemiluminescence.The results showed that O-HTCC-SOD significantly reduced the content of MDA in synovial tissue(P<0.01)and increased the content of GSH(P<0.01).The effect of SOD,O-HTCC and mixtures was not obvious.This showed that intra-articular injection of O-HTCC-SOD improved the antioxidant level in the joints.In short,O-HTCC-SOD effectively protected chondrocytes,maintained the normal morphology of articular cartilage,reduced the level of inflammatory factors and increased the body's antioxidant levels,and the bioavailability and residence time in the joint cavity were significantly improved compared with unmodified SOD.So,it might be a potential therapeutics for OA and worthy of further study and development. |
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