Study On The Expression Of Complement Complex In Rat Osteoarthritis Model

Background & Objective: Osteoarthritis(OA)is well known in the world.It mainly involves the knee,and is the main risk factor of the elderly lower limb disability.Many domestic and foreign scholars have studied a lot of years persistently,but the exact etiology and pathogenesis are still unclear.OA treatments are mainly in three aspects: to reduce pain,improve function,delay or prevent the progression of the disease.However,there are still lack of early effective treatments,treatment methods include non drug conservative treatment,steroidal anti-inflammatory drugs to alleviate pain,aminoglycosides and joint cartilage nutrition medicine lubricant sodium hyaluronate,but these treatments are not according to the pathological pathogenesis,the contro of progress of the diseasel is not good.Eventually the patient had to undergo joint replacemen,the operation is not only a burden to the patients,but also to the national health system and causes a huge economic burden.Therefore,it is very important to investigate the pathological damage mechanism of OA in early stage.More and more studies show that OA in synovium is in low level,synovitis is not the main reason that causes cartilage defect.Early in the last century,it has been found that complements in synovium and cartilage are activated,but the terminal complement complex MAC in synovium and cartilage could eventually form in cartilage and synovium and who plays the pathological roles remains unclear.In this study,we established a rat osteoarthritis model to investigate whether complement complex MAC plays a pathological role in OA cartilage and synovium,and whether it is one of the main pathological factors in the progress of OA.Methods:Part One Animal Model Establishment and Detection of Gross Specimen 1.Animal model establishment and pathological score: S30 SD rats were randomly divided into control group,4 week group,8 week group and 12 week group.There were10 rats in each opertation group.Anterior cruciate ligament transection plus partial medial meniscectomy was used to manufacture rat osteoarthritis model in rats hind knee join in operation group,the other side suffered only skin incision and exposed articular cavity as control group.4 weeks after surgery,8 weeks and 12 weeks on Both sides of knee articular cartilage were adopted for gross observation after 4 weeks,8 weeks and12 weeks,HE staining,safranin O-fast green staining and toluidine blue staining were used to estimate the degree of cartilage degeneration and pathological Mankin score,HE staining was only used to estimate synovial pathological score.2.Immunohistochemistry,Western blot and Elisa were used to detect the deposition of MAC in cartilage and synovium.Part Two Chondrocytes Culture and Complement Attack Experiment in Vitro1.primary chondrocytes culture and identification: Use toluidine blue staining and type II collagen immunofluorescence to identify the primary cultured rat chondrocytes.2.Complement attack assay and immunofluorescence detection of MAC formation:Cartilage cells were attacked different serum concentration,and immunofluorescence staining was used to detect the deposition of MAC on the surface of chondrocytes.Results:Part One Animal Model Establishment and Detection of Gross Specimen1.animal model and pathological score: compared with the control group,the knee joint cartilage surgery group rats of 4 weeks,8 weeks and 12 weeks showed cartilage erosion,rough,crack formation,defect and osteophyte formation in OA early,middle and late stage.Mankin score: the control group(0.11 ± 0.17),4 weeks group(3.89 ± 1.31),8weeks group(8.17 ± 1.67),12 weeks group(12.89 ± 1.05),there was no significant difference among all groups(p<0.01),the damage gradually increased.Synovial score:the control group:(0 ± 0)points,1 week group:(3.47 ± 0.25)points,2 weeks group:(4.58±0.51)points,4 weeks group:(5.17 ± 0.75)points,8 weeks group:(4.17 ±0.75)points,12 weeks group:(2.83 ± 0.75)points,there was statistical significance between the groups(p<0.01).2.Immunohistochemical staining showed that MAC appeared around the chondrocytes,but there was no MAC in synovium.Western blot showed that the experimental group had MAC bands,while the control group did not.After the application of Elisa method,the amount of MAC increased gradually with the prolongation of incubation time,while the synovial membrane and PBS tended to be similar with the prolonging of incubation time of OA cartilage.Part Two Chondrocytes Culture and Complement Attack Experiment in Vitro1.the primary culture and identification of chondrocytes: the chondrocytes grew well,which were triangular,polygonal or spindle shaped.Toluidine blue stained cells were polygonal,triangular,fusiform or similar to the round.The cartilage matrix was stained with light blue nucleus mechanism,while nuclei dyed dark blue.Type II collagen Immunofluorescence staining showed that the cytoplasm was stained red.Cells were polygonal or circular.2.complement attack experiment and immunofluorescence detection of MAC formation:After complement attack of cartilage cells,immunofluorescence showed that chondrocytes lost normal long fusiform,polygonal or triangular shape,became rounded cells,cytoplasm reduced,MAC deposited on the surface of the cell membrane and distributed in a circle.Conclusions: Using knee joint anterior cruciate ligament and partial meniscectomy can successfully produce the model of osteoarthritis in early,middle and late stage.The complement complex affects mainly cartilage,not the synovium,and synovitis is in low level in OA.The complement system plays an important pathological role in the progression of OA.