Daphnetin Induces Apoptosis In Fibroblast-like Synoviocytes From Collagen-induced Arthritic Rats And Its Mechanism

Objectives:To observe the effect on apoptosis in fibroblast-like synoviocytes from collagen-induced arthritic rats(CIA-FLSs)induced by daphnetin(DAP),and investigate the possible mechanism of this effect.Methods:1.To optimize the roles of caspase inhibitors.CIA-FLSs were pre-treated with four caspase inhibitors(Z-DEVD-FMK,Z-IETD-FMK,Z-LEHD-FMK,Z-VAD-FMK)at various concentrations(1.25 μM,2.5 μM,5 μM,10 μM,20 μM,40 μM)for various times(1 h,2 h,4 h),then treated with DAP(40 μg/ml)for another 48 h,cell viability was analyzed by CCK-8 assay.It provided a primary screening for the concentration and pre-treatment time of inhibitors for following experiments.2.To investigate the mechanism of apoptosis induecd by DAP in CIA-FLSs.Experimental groups:(1)Control(vehicle);(2)TP;(3)DAP;(4)DAP+Z-DEVDFMK;(5)DAP+Z-IETD-FMK;(6)DAP+Z-LEHD-FMK;(7)DAP+Z-VAD-FMK.(1)Nuclear morphology of CIA-FLSs was observed by Hoechst 33258 staining;(2)Ultrastructure of CIA-FLSs was observed by transmission electron microscopy;(3)Apoptosis of CIA-FLSs was examined by flow cytometry after Annexin V/PI double staining;(4)Relative mRNA levels of FasL,TNF,Bax,Bcl-2,Bid,Cyt-c,Caspase-3,Caspase-8,Caspase-9 were examined by Real-time PCR;(5)Relative protein levels of Bax,Bcl-2,Caspase-3,Caspase-8,Caspase-9 were examined by Cell-based ELISA;(6)The Cyt-c levels in cytosol and mitochondria were examined by Western blot respectively.Results:1.DAP(40 μg/ml)significantly inhibited the viability of CIA-FLSs by treatment for 48 h.Pre-treated with Z-DEVD-FMK(5 μM),Z-IETD-FMK(5 μM),Z-LEHDFMK(5 μM),or Z-VAD-FMK(20 μM)for 2 h,the viability of CIA-FLSs was significantly improved,compared to treatment with DAP alone.2.The effect of DAP on nuclear morphology of CIA-FLSs: after Hoechst 33258 staining,cell nuclears showed uniform blue fluorescence in Control group;DAP group appeared more hyperchromatic,dense fluorescence of apoptosis than Control group;the number of cells in Z-DEVD-FMK group,Z-LEHD-FMK group,or Z-VAD-FMK group all significantly increased compared with DAP group;there was no obvious difference between Z-IETD-FMK group and DAP group.3.The effect of DAP on ultrastructure of CIA-FLSs: CIA-FLSs in Control group were elliptical and encapsulated,with clear nucleolus;CIA-FLSs in DAP group appeared many vacuolar structures in cytoplasm,with narrow nucleus,concentrated chromatin;morphology of CIA-FLSs in Z-VAD-FMK group were not obviously differ from those in Control group;morphology of CIA-FLSs in Z-DEVD-FMK group,Z-LEHD-FMK group were more normal than those in DAP group,but still with concentrated chromatin,large number of vacuolar structures in cytoplasm;apoptosis in Z-IETD-FMK group was decreased compared with DAP group,but nuclears were obvious deformed or shrinked and there were a large number vacuolar structures in cytoplasm.4.The effect of DAP on apoptosis rate in CIA-FLSs: apoptosis rate of DAP group was significantly higher than Control group or each inhibitor group(P < 0.05);apoptosis rate of Z-VAD-FMK group was the lowest in all inhibitor groups while Z-IETD-FMK group was the highest.5.The effect of DAP on expressions of mRNA related in apoptotic pathways:(1)FasL and TNF mRNA level in DAP group were significantly higher than those in Control group,Z-DEVD-FMK group,Z-IETD-FMK group,or Z-VAD-FMK group(P < 0.05).(2)Cyt-c and Bid mRNA levels in DAP group were significantly higher than those in Control group or each inhibitor group(P < 0.05).(3)Bax mRNA level and Bax/Bcl-2 mRNA ratio in DAP group were significantly higher than those in Control group or each inhibitor group(P < 0.05),while Bcl-2 mRNA level in DAP group was significantly lower than that in Control group or each inhibitor group(P < 0.05).(4)Caspase-3 and Caspase-8 mRNA levels in DAP group were significantly higher than those in Control group or each inhibitor group(P < 0.05);Caspase-9 mRNA level in DAP group was significantly higher than that in Control group,Z-LEHD-FMK group,or Z-VAD-FMK group(P < 0.05).6.The effect of DAP on expressions of protein related in apoptotic pathways:(1)Bax protein level in DAP group was significantly higher than that in Control group,Z-DEVD-FMK group,Z-LEHD-FMK group,or Z-VAD-FMK group(P < 0.05),while Bcl-2 protein level in DAP group was significantly lower than that in Control group or each inhibitor group(P < 0.05);Bax/Bcl-2 protein ratio in DAP group was higher than that in Control group or each inhibitor group(P < 0.05).(2)Caspase-3 protein level in DAP group was significantly higher than that in Control group or each inhibitor group(P < 0.05);Caspase-8 protein level in DAP group was significantly higher than that in Control group,Z-DEVD-FMK group,Z-IETD-FMK group,or Z-VAD-FMK group(P < 0.05);Caspase-9 protein level in DAP group was significantly higher than that in Control group,Z-DEVD-FMK group,Z-LEHD-FMK group,or Z-VAD-FMK group(P < 0.05).7.The effect of DAP on the relase of Cyt-c from mitochondria: cytosol Cyt-c content in DAP group was significantly higher than that in Control group or each inhibitor group(P < 0.05),while mitochondria Cyt-c content in DAP group was significantly lower than that in Control group or each inhibitor group(P < 0.05).Conclusion:1.The viability of CIA-FLSs treated with DAP(40 μg/ml)for 48 h was significantly inhibited.Pre-treated with Z-DEVD-FMK(5 μM),Z-IETD-FMK(5 μM),Z-LEHD-FMK(5 μM),or Z-VAD-FMK(20 μM)for 2 h,the cell viability was obviously improved,compared to treatment with DAP alone.2.DAP induced apoptosis in CIA-FLSs,along with typical morphological and ultrastructural changes.Different caspase inhibitor suppressed the apoptosis in CIA-FLSs induced by DAP in varying degrees.3.The mRNA expressions of FasL,TNF,Cyt-c,Bid,Bax,Caspase-3,Caspase-8,Caspase-9 and the protein expressions of Bax,Caspase-3,Caspase-8,Caspase-9,were increased by DAP;while the mRNA expression and the protein expression of Bcl-2 were decreased by DAP.Different caspase inhibitor suppressd the effect of DAP in varying degrees.4.DAP promoted the relase of Cyt-c from mitochondria into cytosol.Different caspase inhibitor suppressd the relase of Cyt-c caused by DAP in varying degrees.In summary,DAP induced apoptosis in CIA-FLSs,mainly by caspase-dependent pathway;among the two classical apoptotic pathways,death receptor-mediated pathway and mitochondria-mediated pathway,DAP mainly dependented mitochondria-mediated pathway to induce apoptosis in CIA-FLSs.