Effect Of Tetramethylpyrazine On Proliferation And Apoptosis Of Rabbit Articular Chondrocytes Induced By IL-1β

ObjectiveThis research through the IL-1beta(IL-1β) induced rabbit primarychondrocyte,and simulated the pathology environment of osteoarthritis(OA), adding tetramethylpyrazine(TMP) coculture, viability, iNOS and NOexpression differences of rabbit primary chondrocyte were observated. OnIL-1β induced cartilage cell proliferation and apoptosis, as applied toclinical treatment of TMP osteoarthritis cartilage degeneration ofexperimental and theoretical basis. To investigate the effects of TMP onIL-1β induced chondrocyte proliferation and apoptosis, and provideexperimental and theoretical basis of TMP on clinical application for theprevention and treatment of osteoarthritis cartilage progressivedegeneration.Methods1. Experimental groups: control group, IL-1β10ng/ml group, IL-1β10ng/ml+TMP5ug/ml group, IL-1β10ng/ml+TMP10ug/ml group, IL-1β 10ng/ml+TMP15ug/ml group and IL-1β10ng/ml+TMP20ug/ml group.2. Application of mechanical-enzyme digestion method to obtainchondrocytes, construction of primary chondrocytes system cultured invitro, and cartilage cells were identified.3.10ng/ml of IL-1β plus varied concentrations tetramethylpyrazine(5,10,15,20ug/ml) were added into culture medium for48hours, flowcytometry was used for the detection of each cartilage cell cycle andapoptosis and MTT assay was applied to monitor chondrocytesproliferation.4. Experimental groups were treated after48h, the method of reversetranscription polymerase chain reaction (RT-PCR) andimmunohistochemistry were used to detecte the gene and proteinexpression of iNOS, nitrate reduction assay was used to detecte the levelof NO.Results1. Microscopically visible cartilage cell is a triangle or a polygon.Toluidine blue staining showed purple blue metachromatic granules in thecartilage cell cytoplasm and the collagen type Ⅱprotein in cartilage cellcytoplasm, even in nucleus.2.10ng/ml of IL-1β plus varied concentrations tetramethylpyrazine(5,10,15,20ug/ml) were added into culture medium for48hours, comparedwith the control group, IL-1β induced cartilage cell block in G1,and chondrocyte proliferation decreased significantly (P<0.01);adding differentconcentrations of TMP can significantly decrease the IL-1β blocked effecton cartilage cell G1,TMP increased the percentage of S phase and G2phase,proliferation index(PI) and chondrocyte proliferation significantly (P<0.05or P<0.01).3.10ng/ml of IL-1β plus varied concentrations TMP (5,10,15,20ug/ml)were added into culture medium for48h, compared with the control group,IL-1β induced cartilage cell apoptosis rate, iNOS and NO expression ofcartilage cells were significantly increased, with statistical significance(P<0.01), adding different concentrations of TMP can inhibit IL-1β inducediNOS and NO expression in cartilage cell, with statistical significance（P<0.05or P<0.01）.Conclusions1. Effects of TMP on IL-1β induced rabbit primary chondrocytes caneffectively increase cartilage cell proliferation due to increase cellproliferation index (S+G2%) and reduct the cell G1percentage, so it canpromote cell growth and protect cartilage cells.2. TMP can effectively inhibit IL-1β induced rabbit cartilage cellapoptosis, iNOS and NO expression.