Effects Of HongHua Injection On The Proliferation And Metabolism Of Rabit Articular Chondrocytes Induced By TNF-a In Vitro

Objective:To study the method of isolation and culture of rabbit articular chondrocytes for biological characteristic studies;To observe the effect of HongHua injection on proliferation of rabbit articular chondrocyte and the protective effect of on chondrocyte lesion induced by TNF-a,and discuss the mechanism of HongHua injection for treating osteoarthritis.Methods:Articular chondrocytes were isolated from the cartilages of 3 week-old New Zealand rabbits were cultured in vitro.The morphological changes and growth feature of primary culture and subculture of chondrocytes were observed under the inverted microscope each day.Toluidine blue staining and immunocytochemistry were used to indentify the chondrocytes.The proliferation rates of chondrocytes which were normal and induced by TNF-a were detected by MTT chromometry,after Cultured in different concentrations of HongHua injection(10%,5%,2.5%,1.25%, 0.625%,0.312%).The content of SOD and NOS in supernate are tested;The production of proteoglycan were estimated respectively by determination of Alician Blue method.Results:The primarily cultured chondrocytes was in polygonal shape,and became dedifferentiation after 3 passage.The chondrocytes maintained the morphology and immunochemical staining pattern within the first 3 passages.Compared with the control,2.5%,1.25%,0.625%,0.312%of HongHua injection could promote the proliferation of chondrocytes with significant difference.1.25% HongHua injection had the greatest function in the six doses with a significant difference from other groups.10%,5%,HongHua injection had no significant promotion effect difference;After induced by TNF-a,2.5%,1.25%,0.625% HongHua injection could promote the proliferation of chondrocytes which were induced by TNF-a and compare with control group have remarkable difference.TNF-a could promote the NOS activity and depress SOD activity in chondrocytes.HongHua injection could altered the above mentioned results in a concentration dependent manner.1.25%HongHua injection had the greatest function.Compare with the blank control group,The 1.25%HongHua injection could promoted the anabolism of proteoglycan obviously.While 2.5%,1.25%of HongHua injection group also promoted the anabolism of proteoglycan compare with model group.Conclusions:The method used in this work for isolation and culture ofchondrocytes is simple and feasible.The chondrocytes cultured in vitro maintained the specific chondrocytes phenotype in the first 3 passages.The chondrocytes induced by TNF-a,the SOD activity could be reduced,the NOS activity could be enhanced.The following experiment suggested that HongHua injection can effectively promote the proliferation of chondrocytes and prevent and protect against chondrocytes lesion induced by TNF-a,and regulate anabolism of chondrocytes.thus it can defer the degeneration of osteoarthritis cartilage.It demonstrated that HongHua injection was effective to prevent and cure osteoarthritis.