| Objective:The aim of this study was to investigate the existence of macroautophagy and chaperone-mediated autophagy(CMA)in primary cultured rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS),the pathogenesis of these phenomena in rheumatoid arthritis(RA),and the effects of isochalactone(IATL)on RA-FLS proliferation,apoptosis and autophagy.Methods:The knee synovial tissues from RA patients were digested with II type collagenase to obtain primary RA-FLS.Osteoarthritis fibroblast-like synoviocytes(OA-FLS)from knee joint synovial tissue of patients with osteoarthritis(Osteoarthritis,OA)were used as a control group.Observed the cell morphology,performed subculture,and choose cells at good state from 3-7 generations for subsequent experiments.1.3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide(MTT)was used to detect the proliferation of RA-FLS and OA-FLS;Real-time quantity polymerase chain reaction(RT-qPCR)and Western Blot were used to detect the mRNA and protein expression of macroautophagic markers(LC3 and P62)in RA-FLS and OA-FLS,respectively.2.RT-qPCR and Western Blot were used to detect the mRNA and protein expression of CMA(Hsc70 and LAMP2A)in RA-FLS and OA-FLS,respectively.Co-localization of Hsc70 and LAMP2A in RA-FLS and OA-FLS were detected by immunofluorescence,which further indicated the level of CMA.3.RA-FLS cells were treated with different concentrations of IATL(0,6,12,24μmol/L)for 24 hours.MTT was used to detect the proliferation of RA-FLS;flow cytometry was used to detect the apoptosis of RA-FLS cells;RT-qPCR and Western blot were used to detect the mRNA and protein expression of LC3 and p62 in RA-FLS.In addition,RA-FLS was transfected with mRFP-GFP-LC3 double-labeled adenovirus,LC3 protein labeled by RFP and GFP were observed under the confocal microscope.The yellow spots(superposition of red-green fluorescence)indicated the autophagosomes,while red spots represent autophagylysosomes.Chloroquine,an inhibitor of advanced stage of macroautophagy,was used as a positive control for different concentrations of IATL.From this,the autophagy progress in treated RA-FLS cells was observed.Besides,RT-qPCR and Western blot were used to detect the mRNA and protein expression of Hsc70 and LAMP2A in RA-FLS,and immunofluorescence was used to detect the Co-localization of Hsc70 and LAMP2A.Results:1.RA-FLS and OA-FLS were successfully isolated from the knee synovial tissues of patients.The MTT results showed that the cell proliferation of RA-FLS was significantly increased compared with that of OA-FLS.RT-qPCR and Western Blot results showed that the level of LC3 in RA-FLS was significantly higher than that in OA-FLS,while the level of P62 was significantly lower.The differences were statistically significant(P<0.05).2.The RT-qPCR and Western Blot results of showed that the levels of Hsc70 and LAMP2A in RA-FLS were both increased compared with OA-FLS,the difference between the two groups was statistically significant(P<0.05).Hsc70 and LAMP2A proteins were labeled with TRITC-red fluorescence and FITC-green fluorescent antibodies,respectively.The red and green fluorescence in RA-FLS was significantly enhanced compared with OA-FLS;and the yellow fluorescence(red-green fluorescence overlap),which represented the co-expression of Hsc70 and LAMP2A,were also significantly increased.3.After the treatment with IATL at different concentrations(0,6,12,24μmol/L)for 24 hours,the cell proliferation of RA-FLS cells was decreased in a dose-dependent manner.Flow cytometry showed that the apoptosis of RA-FLS was increased after IATL treatment:except the IATL 6μmol/L group,the apoptosis rates of the IATL 12 μmol/L and 24μmol/L group were statistically higher than the control group(5.61 ±0.11)%(P<0.05).Macroautophagy:Except LC3 protein in IATL 6 μmol/L group,the levels of LC3 and P62 in RA-FLS were increased in comparison with non-medicated group,the differences between the groups were statistically significant(P<0.05).In the RA-FLS after mRFP-GFP-LC3 adenovirus transfection,it can be observed that the number of yellow spots significantly increased,while that of red spots decreased,which is paralleling with the increase of IATL concentration.The result was consistent with the positive control group-chloroquine,indicating that the late macroautophagy process was inhibited in RA-FLS.CMA:RT-qPCR and Western Blot showed that the levels of Hsc70 and LAMP2A in IATL(24μmol/L)induced RA-FLS were statistically increased compared with the non-medicated group(P<0.05).Immunofluorescence results showed that the number of yellow spots increased significantly,and the protein co-localized expression of Hsc70 and LAMP2A significantly enhanced.Conclusions:1.Compared with OA-FLS,the cell proliferation of RA-FLS was significantly enhanced,and the levels of macroautophagy and CMA were also significantly increased.2.IATL inhibits the proliferation and promotes apoptosis of RA-FLS cells in a concentration-dependent manner.In RA-FLS,it reduces the level of macroautophagy via blocking the late autophagy process,and inhibits the activation of the CMA pathway.IATL may have therapeutic effects on RA through autophagy pathway. |
