Objective: To study the effects and potential mechanisms of the aquaporin 1(AQP1) inhibitor acetazolamide(AZ) on the proliferation, apoptosis, and inflammatory response of rheumatoid arthritis(RA) fibroblast-like synoviocytes(FLS). Methods: TNF-α-induced RA-FLS was served as in vitro RA model. MTT assay, IF staining, and EdU incorporation assay were applied to study AZ′s effects on RA-FLS proliferation. Hoechst staining, flow cytometry analysis of Annexin V-FITC/PI-stained cells, and mitochondrial membrane potential detection experiments were used to detect cell apoptosis. ELISA and quantitative real-time PCR methods were used to measure pro-inflammatory cytokine production. Cell autophagy was evaluated using IF staining and mCherry-GFP-LC3B puncta assay. Western blot was performed to detect the levels of autophagy, apoptosis, and proliferation-related proteins. Moreover, the role of autophagy inhibition in AZ′s effects on RA-FLS was examined by co-treating with the autophagy activator rapamycin(RAPA) or the autophagy inhibitor 3-methyladenine(3-MA). Results: AZ dose⁃dependently inhibited cell proliferation , promoted apoptosis , and reduced the production of pro⁃inflammatory cytokines in RA⁃FLS. Furthermore , AZ suppressed cytoprotective autophagy in these cells , as evidenced by decreased LC3B levels ( P < 0. 05 ) , increased p62 expression ( P < 0. 05 ) , and reduced autophagic flux ( P <0. 01) . Particularly , AZ ′s beneficial effects were reversed by RAPA⁃induced autophagy activation and enhanced by 3 ⁃MA⁃induced autophagy inhibition. Conclusion This study provides the first evidence that AZ hinders cytoprotective autophagy , thereby alleviating the hyperproliferation , apoptosis resistance , and aberrant inflammatory response of RA⁃FLS , revealing the core role of autophagy inhibition in AZ ′s anti⁃RA effects.

Objective The present study was designed to investigate whether pulsing DCs with tumor-derived extracts is an ef- fective way to induce CTL. and antitumor immunity, Methods DCs were propagated from bone marrow (BM)of C57BL/6J(H-2Kb. I- Ab)mice in vitro with GM-CSF + IL-4tumor associated antigen (TAA) extracted from actively growing Hepa 1-6 cells was used to activate DCs. The phenotypes of DCs were detected by FACS, the cytotoxicity of CTL was as- sayed by 3H-TdR labbel assay. Result and Conclusion The TAA extract pulsed DCs exhibited much more and longer cell processes and increased expression of MHC- Ⅰ, MHC-Ⅱ, CD80 (B7-1 ) 、 CD86 (B7-2 ). This experiment has shown that DCs pulsed with TAA extracts of C57B/6J cells could stimulate effectively the responsiveness of syngenic splenic T cells to induce specific CTL against C57BL/6J cells.