Experimental Study On Effects And Machenism Of Asperosaponin In Treatment Of Osteoarthritis

Objective:To observe the pathophysiological changes of the knee joint'sarticular cartilage of osteoarthritis model of rabbit,to furtherexplore the mechanism of Chinese medicine dipsacus total saponins in the treatment of osteoarthritis.Method:1.Modeled experimental osteoarthritis model rabbit using reference Hulth method.After modeling for a week,each group of animals was treated as the a preset program for consecutive six weeks.Detected the morphology and pathophysiology relevant factors of lesions in cartilage tissue:(1)Prepared the cartilage tissue samples and sections,and observed the morphological changes of cartilage lesionsin the light microscope.(2)Extracted the joint fluid before cutting lesions of the knee.(3)Detected the IL-1 ?,IL-6,TNF-a and PGE2using ELISA assay of synovial fluid.(4)Detected the MMP-1 and MMP-3 protein content with Western blot.In vitro experiments:(1)promoting growth analysis:72h after intervention,flow cytometry was used to the cell number of GO/G1,S and of G2/M phase.Detected the cyclinDl,CDK4,p21 protein content with Western blot.(2)Inhibiting apoptosis assay:Transmission electron microscopy was used to analysis the chondrocytes morphology after 24h,flow cytometry was used to analysis the apoptosis,Bcl-2,Bax and NF-K B protein expression were detected by Western-Blot,colorimetric detection was used to evaluate the Caspase-3 and Caspase-9 protein activity.Detected the IL-1 ?,IL-6,TNF-a and PGE2using ELISA assay of synovial fluid.Results:1.In vivo experiments:observe the rabbit articular cartilage tissue sections,the tissue surface was smooth,cartilage cells were arranged in neat and evenly distributed in control group,Surface tissue was appeared to be thinner,cartilage cells arranged clutter in model group;the treatment group and the control group cartilage damage has been reduced than model group.E1ISA detect synovial fluid IL-1 ?,IL-6,TNF-a and PGE2 content assay results showed that:compared with the model group,IL-1?,IL-6,TNF-a and PGE2 expression in joint synovial fluid of the treatment group was significantly lower(P<0.05);compared with the control group,there was no significant difference in the content of IL-1 ?,TNF-? and PGE2 in the treatment group(P>0.05),butIL-6 was slightly higher than controlgroup(P<0.05).Western-blot results showed that MMP-1,MMP-3 protein in model group was significantly higher than that in the treatment group and the control group.(2)In vitro results:Dipsacus total saponins promote chondrocyte proliferation:Morphological Structure:72h after drug intervention,Middle and high-dose group were seen more nuclei splitting,rich in its cytoplasmic rough endoplasmic reticulum.Cell cycle distribution:The study found thatGO/Gl phase ratio was significantly lower in the middle and high dose group compared with control group(P<0.01),while the G2/M phase was significantly higher than the lowdose group and control group(P<0.05).CyclinDl,CDK4 and p21 protein expression:CyclinDl and CDK4 protein expression in the middle and high dose groupwas significantly higher in the low-dose group(P<0.05);while p21 protein expression in the control group,low dose group was significantly higher that in the middle and high dose group(P<0.05).dipsacus total saponins inhibiting chondrocytes apoptosis:Chondrocytes morphology observed:Compared with the control group,there was lots of necrosis and apoptosis of chondrocytes in model group;the middle and high dose groups could be seen a small amount of necrosis and apoptosis of chondrocytes.Bax,Bcl-2,NF-? B protein expression:protein Bax in middle and Bcl-2 expression in middle and high dose group was significantly higher than the low-dose group;NF-? B expression in middle and high dose group was significantly lower than the control group and low dose group.Western blot detection of Caspase-3,Caspase-9 protein expression:middle and high dose group was significantly lower than the model group.IL-1 p,IL-6,TNF-a and PGE2 content of the measurement result:compared with model group,IL-1 ?,IL-?,TNF-a and PGE2 expression in high,medium and low dose group of was significantly lower(P<0.05)?Conclusion:1,Dipsacus total saponins can delay degeneration of cartilage tissue in OA model,which can inhibit the inflammatory factors IL-1 ?,IL-6,TNF-a and PGE2 expression,and inhibit the expression of MMPs.There is a certain amount of the inhibitory effect relationship.2,Dipsacus total saponins can promote the proliferation of chondrocytes in vitro,It can promote a positive regulator of cell cycle CyclinDl,CDK4 expression and inhibit expression of negative regulators in order to promote cartilage cell proliferation.3.Dipsacus total saponins can inhibit apoptosis of cartilage cells in vitro caused by SNP.It can inhibit apoptosis factor expression such as Bax,NF-K,Caspase-3andCaspase-9,and inhibit cytokine expression,and promote anti-apoptotic factor Bcl-2 expression.