Study On The Mechanism Of Aucubin In Regulating Cellular Energy Metabolism And Delaying Articular Cartilage Degeneration

Part ? Study on the effect of Aucubin in delaying the degeneration of articular cartilageObjective To clarify the interventional effect of aucubin on the degeneration of articular chondrocytes and its potential mechanism.Methods In this part of the in vivo animal study,36 10-week-old male C57BL/6J mice were randomly divided into 3 groups,sham operation group,model group and aucubin group.Weeks),each group contains 6 mice at each time point.In this experiment,mouse medial meniscus instability was used to establish an animal model.After the operation,mice in the aucubin group were given intraperitoneal injection of aucubin solution,and mice in the sham operation group and model group were given an equal volume of 0.9%normal saline.The degeneration of articular cartilage was observed by ABH staining,OARSI score and immunohistochemical staining.The abnormal bone remodeling of subchondral bone was evaluated by Micro-CT scan and analysis.At the same time,the apoptosis of chondrocytes was observed by TUNEL staining.The in vitro study took mouse primary articular chondrocytes as the research object.First,the CCK-8 kit was used to detect the toxicity of aucubin to chondrocytes and determine the drug intervention concentration.At the same time,IL-1? was used to interfere with chondrocytes for cell modeling.The cell experiment was divided into control group,model group and different gradient concentration aucubin intervention group.RT-PCR and Alcian staining were used to evaluate the intervention effect of aucubin on chondrocyte degeneration.Results In vivo experiments found that 6w and 12w after modeling,the aucubin group had less damage to the cartilage surface of the knee joint in the model group,and there was no obvious defect in the articular cartilage,and the OARSI score was higher.Micro-CT three-dimensional reconstruction images showed that the BV/TV and Tb.Th of the subchondral bone of the proximal tibia of the aucubin group mice were significantly reduced,and the Tb.Sp was also significantly higher than that of the model group.TUNEL staining confirmed that the number of apoptotic chondrocytes in the knee joint of the aucubin group was significantly less than that of the model group.Immunohistochemical staining results showed that aucubin effectively reduced the expression levels of MMP13 and AD AMTS 5 proteins in articular cartilage after DMM modeling.In vitro cell experiments found that compared with the IL-1? group,the aucubin group at different concentrations can significantly inhibit the expression of MMP13 and COL 10 genes,and increase the expression of ACAN and COL2 genes in a dose-dependent manner.The results of alcian blue staining show that aucubin can reduce the degradation of chondrocyte matrix.Conclusion The intervention of aucubin can significantly inhibit the catabolism of chondrocytes and reduce the apoptosis of chondrocytes,thereby delaying the degeneration of articular cartilage in mice.Part ? Study on the mechanism of aucubin in regulating energy metabolism and delaying chondrocyte degenerationObjective To explore the regulatory mechanism of aucubin in inhibiting chondrocyte catabolism by affecting chondrocyte glucose transport and energy metabolism.Methods This part first analyzed the possible targets and pathways of aucubin in delaying chondrocyte degeneration through network pharmacology.Subsequently,in in vivo animal studies,immunohistochemical staining was used to observe the effect of DMM model on articular cartilage glucose transport and energy metabolism,as well as the interventional efficacy of aucubin.In the in vitro study,mouse primary articular chondrocytes were used as the research object,and IL-1? was used to intervene chondrocytes for cell modeling.The cell experiment was divided into control group,model group and different gradient concentration aucubin intervention groups,using RT-PCR And extracellular flux analyzer to evaluate the effect of aucubin on the glucose transport and energy metabolism of chondrocytes.At the same time,on the basis of IL-1? intervention,use 2DG to inhibit the glycolysis pathway of chondrocytes and observe its effect on cartilage cell catabolism.Results The results of network pharmacological analysis showed that glucose metabolism may be the main way for aucubin to protect chondrocytes.In vivo experiments confirmed that the expression of Glut1 and LDHA protein in mouse cartilage was up-regulated after DMM modeling,and aucubin could significantly reduce the expression level.In vitro studies have found that compared with IL-1? group,different concentrations of aucubin group can significantly inhibit Glut1 and LDHA gene expression.The Seahorse energy metabolism test and analysis results show that IL-1? promotes the glycolysis pathway of chondrocytes,and the aucubin group at different concentrations can effectively inhibit the level of glycolysis.At the same time,inhibiting the level of glycolysis(2DG intervention in chondrocytes)can significantly inhibit the expression of catabolism-related genes(MMP13,ADAMTS5 and COL10).In addition,in vivo and in vitro studies have found that aucubin has a regulatory effect on pSMAD2 protein expression and Tgfbr2 gene expression.Conclusion Aucubin inhibits chondrocyte glycolysis by regulating the expression levels of Glut1 and LDHA,thereby inhibiting catabolism and delaying cartilage degeneration,and the mechanism of action may be related to the activation of the TGF-? signaling pathway.Part ? Study on the mechanism of aucubin in regulating cell energy metabolism through TGF-? signaling pathwayObjective To explore the molecular mechanism of aucubin in regulating glucose transport and energy metabolism of chondrocytes through TGF-? signaling pathway.Methods We used conditional gene knockout mice Tgfbr2Col2ER(Col2-CreER;Tgfbr2fx/fx)to block the TGF-? signaling pathway.Mice in the aucubin group were given intraperitoneal injection of aucubin solution,and mice in the negative control group(Tgfbr2fx/fx mice)and Tgfbr2Col2ER group mice were treated with an equal volume of 0.9%saline.ABH staining,OARSI score and immunohistochemical staining were used to observe articular cartilage degeneration,glucose transport and energy metabolism changes.Micro-CT scan and analysis were used to evaluate abnormal bone remodeling of subchondral bone.The in vitro study used mouse primary articular chondrocytes as the research object.At the same time,based on the intervention of aucubin,SB525334 was used to inhibit the TGF-? signaling pathway of chondrocytes.RT-PCR and extracellular flux analyzer were used to evaluate its effect on cartilage.The effect of cellular glucose transport and energy metabolism.Results Tgfbr2Col2ER mice had obvious knee cartilage defects and subchondral bone collapse.The above-mentioned pathological changes of the aucubin group mice were similar to those of the Tgfbr2Col2ER group mice without any improvement.There was no statistical difference in BV/TV,Tb.Th and Tb.Sp of subchondral bone.The expression levels of Glut1 and LDHA in the knee cartilage of mice in the Tgfbr2Col2ER group were significantly higher than those in the negative control group.At the same time,in vitro studies confirmed that Glut1 and LDHA gene expression was significantly up-regulated after SB525334 induction,and aucubin could not achieve the regulation of Glut1 and LDHA expression levels after blocking the TGF-? signaling pathway.In addition,in vivo experiments have found that aucubin can up-regulate the expression level of TGF-?1 in the extracellular mechanism of chondrocytes.Conclusion The protective effect of aucubin on the articular cartilage of OA mice may promote the secretion of TGF-?1,activate the TGF-? signaling pathway,inhibit the glucose transport and glycolysis of chondrocytes,and ultimately affect the catabolism.