An Experimental Study On The Regulation Of ECM In SW1353 Cells By The Specific RIP1 Inhibitor Necrostatin-1

Part I Expressions of RIP1 in AGEs-induced SW1353 cells and the effect of Nec-1 on degradation of collagen II and proteoglycan in AGEs-induced SW1353 cellsObjective1.To study the effects of different concentrations of Nec-1 on the proliferation of SW1353 cells.2.To study the expression of RIP1 in AGEs-induced SW1353 cells and the effect of Nec-1,a specific inhibitor of RIP1,on the degradation of collagen Ⅱ and proteoglycan in AGEs-induced SW1353 cells,so as to provide experimental basis for the further study of the effect of Nec-1 on the degradation of extracellular matrix.Methods1.SW1353 was cultured with four Nec-1 concentrations(0,50,100 and 150 uM)for 24 hours,and then cell proliferation activity was detected by CCK8.2.SW1353 cells were classified into four groups:(1)blank control group;(2)50μg/mL AGEs group;(3)100 μg/mL AGEs group;(4)200 μg/mL AGEs group.Different concentrations of AGEs were used to culture SW1353.Western blot and RT-PCR were used to detect the expression of RIP1.3.The SW1353 cells were classified into the following groups:(1)blank control group;(2)100μg/mL AGEs positive control group;(3)50μM Nec-1 pretreatment,100μg/mL AGEs induction group;(4)100μM Nec-1 pretreatment,100μg/mL AGEs induction group.After 24 hours,the total protein was extracted,and the levels of collagen II and proteoglycan were detected by Western blot.Results1.50 and 100 μM Nec-1 had no significant effect on the activity of SW1353(P>0.05),while 150 μM Nec-1 had significant inhibition on the activity of SW1353(P<0.05).2.50 μg/mL,100 μg/mL,200 μg/mL of AGEs could significantly induce the expression of RIP1(P<0.05).The expression of RIP1 in SW1353 cells was dose-dependent(P<0.05).3.100 μg/mL AGEs could dramatically increase the degradation of type Ⅱ collagen and proteoglycan in SW1353(P<0.05),while Nec-1 could significantly reduce the degradation of type Ⅱ collagen and proteoglycan(P<0.05),and the mitigation effect enhanced with the concentration increasing(P<0.05).ConclusionAs stated above,50 and 100 μM Nec-1 were safe;AGEs promoted the degradation of ECM of chondrocytes:RIP1 participated in the degradation of ECM of chondrocytes induced by AGEs;Nec-1 could alleviate the degradation of ECM of chondrocytes.Part Ⅱ The effects of Nec-1 on ECM(collagen Ⅱ and proteoglycan)degrading enzymes and inhibitors of AGEs-induced SW1353ObjectiveTo study the effect of Nec-1 on degrading enzymes and their inhibitors of ECM in SW1353 induced by AGEs.Clarify the theory of molecular biology about antagonizing the development of OA by Nec-1.MethodsSW1353 cells were classified into the following groups:(1)blank control group;(2)100μg/mL AGEs positive control group;(3)50μM Nec-1 pretreatment,100μg/mL AGEs induction group;(4)100μM Nec-1 pretreatment,100μg/mL AGEs induction group.After 24 hours,the total protein and RNA were extracted from the samples.The levels of MMP-3,MMP-13,TIMP-1,TIMP-2,ADAMTS-4 and ADAMTS-5 were detected by Western blot and RT-PCR.Results1.AGEs could significantly promote the expression of MMP-3 and MMP-13,ADAMTS-4 and ADAMTS-5 in SW1353 cells(P<0.05),and significantly inhibit the expression of TIMP-1 and TIMP-2(P<0.05).2.The expressions of MMP-3,MMP-13,ADAMTS-4 and ADAMTS-5 decreased significantly after Nec-1 pretreatment(P<0.05),while the expression of TIMP-1 and TIMP-2 increased significantly(P<0.05),and the effect was dose-dependent(P<0.05).ConclusionAGEs induced degradation of ECM by promoting the expression of matrix-degrading enzymes and inhibiting their inhibitor;Nec-1 ameliorated degradation of ECM by inhibiting matrix-degrading enzymes and increasing their inhibitor expression.Part Ⅲ:Mechanisms of Nec-1 in mitigating the degradation of extracelluLar matrix of chondrocytesObjectiveTo clarify the mechanism of Nec-1 in alleviating the degradation of extracellular matrix of chondrocytesMethods1.SW1353 cells were classified into the following groups:(1)blank control group;(2)100μg/mL AGEs positive control group;(3)50μM Nec-1 pretreatment,100μg/mL AGEs induction group;(4)100μM Nec-1 pretreatment,100μg/mL AGEs induction group.24 hours later,samples were collected to extract the total protein.Western blot was used to detect the expression of total JNK and phosphorylated JNK.The nucleoprotein was extracted and the expression of p65 was detected by Western blot.2.SW1353 cells were transfected with plasmid containing promoter of NF-κB and AP-1(c-Jun/c-fos),respectively.After 6 hours of transfection,SW1353 cells were cultured in the following groups:(1)blank control group;(2)100 μg/mL AGEs control group;(3)Nec-1 50μM pretreatment,100 μg/mL AGEs treatment group;(4)Nec-1 100 μM pretreatment,100 μg/mL AGEs treatment group.Luciferase activity was detected by luciferase reporter gene after intervention.Results1.AGEs could significantly promote the phosphorylation of JNK in SW1353 cells(P<0.05)without altering the total amount of JNK(P>0.05),while Nec-1 could inhibit the phosphorylation of JNK induced by AGEs in a concentration-dependent manner(P<0.05).2.AGEs could significantly promote the expression of c-Jun and c-Fos in SW1353 cells(P<0.05),while Nec-1 could inhibit the expression of c-Jun and c-Fos induced by AGEs in a concentration-dependent manner(P<0.05).3.AGEs could significantly promote the expression of p65 in SW1353 cell nucleus(P<0.05),while Nec-1 could inhibit the expression of p65 induced by AGEs in a concentration-dependent manner(P<0.05).4.AGEs significantly increased the luciferase activity of AP-1 and NF-κB promoter in SW1353 cells(P<0.05),while Nec-1 inhibited the luciferase activity of the two promoter induced by AGEs in a concentration-dependent manner(P<0.05).ConclusionNec-1 inhibits extracellular matrix degradation through JNK/AP-1 signaling pathway and NF-κB signaling pathway.