Study On Osteoarthritis-like Lesions And Chondrocyte Senescence Induced By STZ Combined With High-Energy Diet In Rats

Osteoarthritis is the most common joint disease,often occurring in middle-aged and elderly animals,and is also the main cause of disability in middle-aged and elderly animals.Osteoarthritis seriously affects the quality of life of the diseased body,which also has a great impact on the development of the livestock industry and the pet industry.Some scholars have observed that clinical type II diabetes and osteoarthritis are usually occur together,and the symptoms are usually more obvious than patients with non-diabetes complicated osteoarthritis.To this end,this experiment will explore changes in the degradation,damage,and aging of cartilage tissue and cells after osteoarthritis and diabetes occur together.At the same time,chondrocytes are cultured at different glucose concentrations in vitro to explore the effect of high-concentration glucose environment on chondrocytes.In order to provide an experimental reference for the in-depth study of the aging mechanism in the future.In vivo experiments,through the use of injection streptozotocin(STZ)combined with highenergy diet modeling method,to establish a type II diabetes elderly rat model.By observing the posture and behavior of each group of rats,detecting the content of IL-6 and IL-8 in the serum of each group of rats,observation and Mankin score of the distal femoral articular surface of the rats,HE staining and Saffron O fast green staining of knee joint tissue to observe the pathological changes of the cartilaginous sclera,using Alizarin red S staining to observe the calcification of the cartilaginous sclera,using immunohistochemical staining to detect type II collagen,MMP-13 and p21 expression in cartilage tissue,evaluate the changes in the cartilage tissue metabolism and agingrelated indicators of the experimental group.In vitro experiments,culture and passage of 17-dayold SD rat knee chondrocytes.Using the first and fifth generation chondrocytes,morphological observation,toluidine blue staining and immunofluorescence detection of each group to identify the cultured chondrocytes.The first generation of chondrocytes are divided into 3 groups and the fifth generation into 3 groups: 1st generation low concentration glucose group,1st generation medium concentration glucose group,1st generation high concentration glucose group,5th generation low concentration glucose group,5th generation medium concentration glucose group,5th generation high concentration glucose group,culture respectively with three cell culture media containing different concentrations of glucose from the primary generation.Immunofluorescence detect the expression of type II collagen in each group of cells,Alizarin red S staining compare the calcification of each group of cells,and Western Blotting detect the type II collagen,MMP-13,p21,p53,p16 protein expression level of each group of cells,detect the level of ROS produced by each group of cells,and then evaluate the effect of different concentrations of glucose culture environment on chondrocytes.The results showed that the diabetes model of the experimental group was successfully established.Compared with the control group,the articular cartilage of the experimental group was thinner and the color was dull,the HE and safranine O green staining results showed,the experimental group’s cartilaginous sclera is significantly thinner,the surface layer crack increase,fibrosis is obvious.the number of chondrocytes decreases and the arrangement is disordered,and the Mankin score is increased.IL-6 and IL-8 content increase significantly,immunohistochemistry results showed that the content of type II collagen in the cartilaginous sclera of the experimental group was greatly reduced,and the content of MMP-13 and p21 increased significantly.The results of in vitro culture of chondrocytes showed that with the increase of the number of passages and the treatment of high concentration glucose,the Alizarin red S of the 5th generation high concentration glucose group cells have the strongest staining intensity and the most ROS production.The results of immunofluorescence show that the staining intensity of type II collagen is the weakest,and the protein expression level of type II collagen is also the lowest in Western Blotting test results,the expression of MMP-13 is the highest,and the proteins expression of p21,p53,p16 in 6 groups are gradually increasing.The experimental results show that the cartilage degradation and cartilage damage of articular cartilage tissue of elderly rats with diabetes are more serious,the cartilaginous sclera is thinner,and the aging degree of cartilage tissue is also increased;by using high concentration glucose to culture chondrocytes,the cartilage will be aggravated,the level of oxidative stress of cells accelerates,the degradation of their matrix,and also increases the aging degree of chondrocytes.In general,the changes of 5th generation cells are more obvious than 1st generation cells.