Study On The Protective Effect Of Astragaloside IV On Inflammatory Damaged Chondrocytes By Regulating Autophag

PART Ⅰ Screening out the best intervention concentration and time of astragaloside Ⅳ and Interleukin-1βObjective:To screen the appropriate doses and time of action of AS-I V on IL-1β-induced inflammatory injury chondrocyte cell.Methods:Chondrocytes were isolated and cultured from the knee joints of SD neonatal mice,then type Ⅱ collagen immunofluorescencetest were performed.The blank control group,the model group and the AS-IV concentrationgradient group(50,100,200 μmol/L)were set up to intervene chondrocytes.The cell viability was detected by MTT Assay at different cultured period,respectively,and theappropriate concentration and time were screened.Results:The collagen Ⅱ immunofluorescence suggested that the isolated cells were chondrocytes.After exposure to AS-IV,the cellsexhibited the characteristic of cell rise.It was much more prominment while the treating time prolong or the concentration increased within a certain range of concentration and treating time.Conclusions:In conclusion,we demonstrated that AS-IV could enhanced the proliferation of the chondrocytes in present study.The appropriate doses of AS-IV was 200μmol/L and the appropriate reaction time was 48h.PART Ⅱ Protective effect of AS-IV on inflammatory injury of chondrocytes through autophagy pathwayObjective:To investigate the protective effect of AS-IV on rat chondrocytes treated with IL-1β,and to explore its mechanism based on autophagy pathway.Methods:Chondrocytes were separated by three groups.Model group was used to replicate IL-1β-induced inflammatory chondrocyteinjury.ASIV group was used to add AS-IV and IL-1β into chondrocytes for coculture.A control group was also set up.The morphology and cytoskeleton of cells were tested by Ghost pen cyclic peptide dyeing.Flow cytometry was used to detect the apoptosis rate.TNF-α and GAG concentrations in cell supernatant were tested by ELISA.The expression of LC3,p62 mRNA was detected through RT-qPCR.The expression of p62 protein and LC3 Ⅱ/Ⅰ ration was detected by Western Blot.Results:Ghost pen cyclic peptide dyeing showed that,compared with blank control group,cytoskeleton of model group showed fusiform changes.Compared with the model group,the cytoskeleton ofAS-IV group was more polygonal.The flow cytometry analysis showed that the apoptosis in the model group was higher than that of control group,but the apoptosis in the AS-IV group was decreased compared with the control group.The ELISA showed that the levelof TNF-α in the model group was higher than that of control group,but the level of TNF-α in the AS-IV group was decreased compared with the control group.At the same time,the level of GAG in the model group was lower than that of control group,but the level of GAG in the AS-IV group was increased compared with the control group.The RT-qPCR showed that the expression of p62 mRNA in the model group was higher than that of control group,but the expression of p62 mRNA in the AS-IV group was decreased compared with the control group.At thesame time,the expression of LC3 mRNA in the model group was lower than that of control group,but the expression of LC3 mRNA in the AS-IV group was increased compared with the control group.The Western Blot showed that the expression of p62 protein in the model group was higher than that of control group,but the expression of p62 protein in the ASIV group was decreased compared with the control group.At the samet ime,the expression of LC3Ⅱ/Ⅰ ration in the model group was lower than that of control group,but the expression of LC3Ⅱ/Ⅰ ration in the AS-IV group was also decreased compared with the control group.Conclusions:AS-IV can reduced the inflammatory injury of chondrocytes induced by IL-1β through enhancing autophagy of chondrocytes probably.