| Objective:To investigate whether RES could exert anti-osteoarthritic effect on SW1353 cell line via TLR4 and PI3K/Akt signaling pathway.The study further clarify the pathogenesis and provides experimental basis for nutrition treatment of OA.Methods : SW1353 cell line was purchased from Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.The cell proliferation viability was determined by CCK-8 assay in presence or absence of IL-1β at various concentrations of RES(0 ~100 μmol/L),and the effect of different concentrations of CLI-095 or LY294002.The level of MMP-13 in cell culture supernatant was measured by ELISA.The protein expression of TLR4、p-Akt and Akt were examined by Western blot.Results:1、The effect of RES and/or IL-1β on SW1353 cell proliferation viabilityCompare with blank control group,DMSO has no inhibition effects on cell viability(P>0.05);compared with DMSO group,RES(3.125 ~ 25 μmol/L)could significantly increase the cell proliferation activity(P<0.01),however RES(100 μmol/L)could decrease the cell proliferation activity obviously(P<0.05);IL-1β(10 ng/ml)has no inhibition effects on cell viability of SW1353 cell(P>0.05);while under the combined of IL-1β and RES(6.25 ~ 50 μmol/L),cell proliferation activity could significantly increase(P<0.01),but under the combined of IL-1β and RES(100 μmol/L),cell proliferation activity was decreased significantly(P<0.05).2、The effect of RES on levels of MMP-13 in SW1353 cell lineThe levels of MMP-13 were significantly upregulated in the presence of IL-1b(10ng/ml)(P<0.01),while RES(50 μmol/L)treatment could dramatically inhibite the effect of IL-1β,the level of MMP-13 production were significantly reduced(P<0.01).3、The effect of RES on TLR4 signaling pathway in SW1353 cell lineIL-1 b(10 ng/ml)could activate the TLR4 signaling pathway,the expression of TLR4 protein were significantly upregulated in the presence of IL-1β(P<0.01),while RES(50 μmol/L)treatment could dramatically downregulate the TLR4 production in SW1353 cell line(P<0.01).4、The effect of IL-1b on PI3K/Akt signaling pathway in SW1353 cell lineNo obvious difference was found in the expression of Akt protein compared with the control group(P>0.05).While the PI3K/Akt signaling pathway could be activate by IL-1b(10 ng/ml)at different time points,the expression of p-Akt protein were significantly upregulated(P<0.05).5、The effect of RES on PI3K/Akt signaling pathway in SW1353 cell lineNo obvious difference was found in the expression of Akt protein compared with the control group(P>0.05).While the PI3K/Akt signaling pathway could be activate by RES(50 μmol/L)at different time points,the expression of p-Akt protein were significantly upregulated(P<0.05).6、Effects of RES on PI3K/Akt Signaling Pathway in IL-1β-treated SW1353 cells after inhibition of TLR4 signaling pathway6.1 The effect of different concentrations of CLI-095 on SW1353 cell viabilityCompared with the blank control group,DMSO had no significant effect on cell proliferation activity(P>0.05).Compared with DMSO group,CLI-095(0.25 μg/mL)could significantly increased cell proliferation activity(P<0.05),while CLI-095(4、16μg/mL)could significantly inhibite cell proliferation activity(P<0.05).6.2 Effect of RES on TLR4 expression induced by IL-1β after inhibit the TLR4 signaling pathwayCompared with IL-1β group or IL-1β + RES group,CLI-095 pretreatment could significantly inhibit the TLR4 signaling pathway,the expression of TLR4 protein was significantly decreased(P<0.05).6.3 Effect of RES on PI3K/Akt signaling pathway after inhibit the TLR4 signaling pathwayCompared with IL-1β group or IL-1β + RES group,CLI-095 pretreatment could significantly inhibit the PI3K/Akt signaling pathway,the expression of p-Akt protein has significantly decreased(P<0.05);while no obvious difference was found in the expression of Akt protein.7 、 Effects of RES on TLR4 Signaling Pathway in IL-1β-treated SW1353 cells after inhibition of PI3K/Akt signaling pathway7.1 The effect of different concentrations of LY294002 on SW1353 cell proliferation viabilityCompared with the blank control group,DMSO had no significant effect on cell proliferation activity(P>0.05).Compared with DMSO group,each concentrations of LY294002 could inhibit cell proliferation viability,however LY294002(15 ~ 45 μmol /L)could inhibit cell proliferation more significantly(P<0.01).7.2 Effect of LY294002 pretreatment on RES activation of PI3K/Akt signaling pathwayCompared with IL-1β group or IL-1β + RES group,LY294002 pretreatment could inhibit the effect of RES which has activate the PI3K/Akt signaling pathway,and the expression of p-Akt protein was significantly decreased(P<0.05);while there was no obvious effect on the expression of Akt protein(P>0.05).7.3 After inhibited the PI3K/Akt signaling pathway,the effect of RES on TLR4 protein expressionCompared with IL-1β group,LY294002 pretreatment could significantly enhance the effect of IL-1β on TLR4 signaling pathway and further increase the expression of TLR4 protein(P<0.01).Compared with IL-1β + RES group,LY294002 was inhibited the effect of RES,the expression of TLR4 protein was significantly increased(P<0.01).Conclusion:1.RES exert anti-OA effect on SW1353 cells which induced by IL-1β.2.IL-1β may inducing OA by activate TLR4 signaling pathway,while RES may exert its inhibitory effect on OA by inhibit the TLR4 signaling pathway and activate the PI3K/Akt signaling pathway.3.Some cross-talk may exist between TLR4 signaling pathway and PI3K/Akt signaling pathway when RES exerts its anti-OA effect. |
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