| ObjectiveBased on "Wnt/β-catenin" signal pathway to explore the mechanism of drug-containing serum of Zhuanggujianxi Formula which intervened the recessionary chondrocyte, expound the microscopic mechanism of Zhuanggujianxi Formula protecting cartilage from the level of molecular biology, the goal was to provid the evidence of Zhuanggujianxi Formula for clinical practice.Methods1.40clean level SD rats, randomly divided into the blank control group (0.9%saline irrigated stomach) and medicine group (Zhuanggujianxi Formula irrigated stomach), continuous irrigated stomach for5days, took blood from abdominal aortic which used for normal serum and contains Zhuanggujianxi Formula medicine serum at the end time of irrigating stomach after3h.2.Cartilage was isolated from the knee joint of two-week old SD ratsand primary chondrocytes were cultured. Through the morphological observation, toluidine blue staining and the Ⅱ collagen type dyeing to appraisal; MTT method was used to detect generation of1～5chondrocyte proliferation and drew chondrocyte proliferation curve.3.Used10ng/ml IL-1β to establish induced degeneration chondrocyte model, the mRNA expression of β-catenin, GSK-3β, Dkk-1, sFRP-3in normal and degeneration chondrocyte were detected by RT-PCR. The protein expression of the normal and induced degeneration chondrocyte’s Dkk-1, sFRP-3were detected by Western Blot method.4.Used normal serum and Zhuanggujianxi Formula drug-containing serum intervened chondrocyte which induced by IL-1β. The mRNA expression of β-catenin, GSK-3β, Dkk-1, sFRP-3in Zhuanggujianxi Formula drug-containing serum acted on degeneration chondrocyte were detected after different period (24h,36h,48h,72h) by RT-PCR. The Dkk-1, sFRP-3protei expression of Zhuanggujianxi Formula drug-containing serum acted on degeneration chondrocy were detected after different period (24h,36h,48h,72h) by Western Blot. The Dkk-1, sFRP-protein expression in culture solution of Zhuanggujianxi Formula drug-containing serum acted c degeneration chondrocyte were detected after different period (24h,36h,48h,72h) by ELISA.Results1.The culture chondrocyte was conformed to the biological characteristies by the inverte microscope morphological observation, toluidine blue staining and the Ⅱ collagen type dyein; Chondrocyte within three generations could maintain phenotype stable.2.The mRNA expression of GSK-3β, β-catenin:normal chondrocyte were lower than IL-1induced degeneration chondrocyte; and normal chondrocyte higher than degeneratic chondrocyte in the Dkk-1, sFRP-3mRNA expression, while the expression of Dkk-1, sFRP-proteinin normal chondrocyte were higher than degeneration chondrocyte.3.Different serums acted on degeneration chondrocyte after different period, including tl mRNA expression of β-catenin in medicine group were gradually weaken while enhanced in tl blank serum group over time. Including the mRNA expression of GSK-3β, Dkk-1and sFRP-and the protein expression of Dkk-1and sFRP-3in medicine group to intensified with time whi the blank group decreased. The expression of Dkk-1, sFRP-3protein in culture solution we strengthened gradually in medicine group while gradually weakened in the blank group were wi time.ConclusionThe chondrocyte degeneration process induced by IL-1β was accompanied1"Wnt/β-catenin" signaling pathways leading to the activation. Zhuanggujianxi Formu drug-containting serum made the protective effect to the induced degeneration chondrocyte. Tl mechanism may be lower the β-catenin level, promote GSK-3β, Dkk-1, sFRP-3mRNA ai protein’s expression in degeneration chondrocyte by Zhuanggujianxi Formula drug-containi(?) serum, which made the nuclear β-catenin reducedand inhibiting "Wnt/β-catenin" signal pathwa activated relevant. |
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