| Objective:To investigate the effect of ICA on IL-1β induced chondrocytes and whethe r it is related to autophagy,Then knock down the ULK1 level in SW1353 cells to in-vestigate whether the protective effect of ICA on SW1353 cells stimulated by IL-1β is related to Phosphatidylinositol 3-kinase(PI3K)/protein kinase B(A kt)/Mammaliantarget of rapamycin(mTOR)/ULK1 pathway.Methods:1.Culture SW1353 cells,use microscopy to confirm the specific morphology,and use the CCK-8 method to test the cell viability of multiple concentrations and time nodes to determine the optimal concentration and optimal time;2.Demonstration of IL-1β:using the actual ratio of 20ng/mL of IL-1β in vitro induction SW1353 cells,Western blot was selected to determine the protein expression of the extracellular matrix MMP3、MMP13、and type Ⅱ collagen(Collagen Ⅱ),and qRT PCR detected the mRNA expression levels of MMP3、Collagen Ⅱ,and proteoglycan(Aggrecan)in the extracellular matrix;3.ICA can reduce the inflammation of chondrocytes and increase autophagy:SW1353 is pretreated with 40 μM ICA for a duration of 2h,and then 20ng/mL of IL-1β stimulation is used for 48h.Western blot determined the expression status of MMP3,Collagen Ⅱ,LC3Ⅱ/I,Beclinl,p62,qRT PCR to determine the mRNA expression levels of MMP3 and Beclin1,and the synthesis of Collagen Ⅱ.Collagen Ⅱ and Aggrecan was detected by immunofluorescence method.4.After knocking down the ULK1 gene,the effect of ICA on the inflammatory,autophagy,PI3K/Akt/mTOR/ULK1 signaling pathway of chondrocytes:lentiviral transfection knockdown of ULK1 gene in SW1353 cells,40μM ICA pretreatment shRNA-ULK1 virus-infected SW1353 cells for 2h,and then 20ng/mL of IL-1βstimulation for 48h,detection of relevant protein levels and autophagy-related protein levels in the extracellular matrix of each group:Western blot detected the protein expression levels of MMP3,Collagen Ⅱ,LC3Ⅱ/Ⅰ,Beclin1,p62,ULK1,p-PI3K,PI3K,p-AKT,AKT,p-mTOR,and mTOR.Results:1.The morphology of SW1353 cells in subcultured culture is approximately fusiform,and the cytoplasm is abundant and grows in clusters.The optimal concentration and time of IL-1β detected by CCK8 method were 20 ng/mL and 48h,respectively,and the optimal concentration and time of ICA were 40 μM and 48h,respectively.2.Successful establishment of cellular OA models:Western Bolt results showed that compared with the Control group,the expression of Collagen Ⅱ in the model group showed a significant decrease(P<0.05),and the expression index of MMP3 and MMP13 was significantly improved(P<0.05);the RT-PCR assay was obtained,and compared with the Control group,the Collagen Ⅱ and Aggrecan mRNAs in the model group were significantly reduced(P<0.05),and the MMP3 mRNA index was significantly increased(P<0.05).3.ICA reduces OA inflammation and increases autophagy:Western Bolt results show that compared with the Control group,In IL-1β group,MMP3 and p62 proteins were significantly increased(P<0.05),and the expression of Collagen Ⅱ,LC3Ⅱ/Ⅰ,and Beclinl proteins were significantly reduced(P<0.05),The opposite results were shown after ICA pretreatment.Using RT-PCR detection,Compared with the Control group,the expression of Beclinl and Collagen Ⅱ mRNA in the IL-1β group SW1353 was significantly reduced(P<0.05),MMP3 mRNA was significantly increased(P<0.05),corresponding to ICA pretreatment will increase beclinl,Collagen Ⅱ mRNA expression(P<0.05),reduce MMP3 mRNA expression(P<0.05).By immunofluorescence analysis,Aggrecan and Collagen Ⅱ proteins in the IL-1β group was significantly reduced(P<0.05)compared with the Control group,and the fluorescence intensity of intracellular Collagen Ⅱ and Aggrecan proteins was significantly increased by ICA pretreatment(P<0.05).4.Knock down ULK1 gene of SW1353 cells,Western Bolt results showed that the expression of LC3Ⅱ/I,Beclinl,Collagen Ⅱ,and ULK1 proteins in the ICA+IL-1β group increased significantly compared with the IL-1β group(/P<0.05),the MMP3,p62,p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR indicators were significantly reduced(P<0.05);Compared with ICA+IL-1β+shRNA-NC,the expression of LC3Ⅱ/I,Beclinl,Collagen Ⅱ,and ULK1 proteins in the ICA+IL-1β+shRNA-ULK1 group was significantly reduced(P<0.05),and the expression of MMP3,p62,p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR was significantly increased(P<0.05).The results of RT-PCR test showed that the Aggrecan mRNA expression in the ICA+IL-1β group was significantly increased(P<0.05)and the expression of MMP3 mRNA was significantly reduced compared with the IL-1β group(P<0.05);compared with the ICA+IL-1β+shRNA-NC,Aggrecan mRNA expression in the ICA+IL-1β+shRNA-ULK1 group decreased significantly(P<0.05)and MMP3 mRNA expression increased significantly(P<0.05).Conclusion:ICA reverses IL-1β induced SW1353 inflammation,related to inhibition of P I3-K/Akt/mTOR pathway... |
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