Anti-apoptotic Effect Of Andrographolide Analogue On RIN-mβ Cell Apoptosis Induced By H₂O₂-A Proteomic Analysis

Objective:To screen the proteomic alteration and investigate the molecular mechanism involved in the anti-apoptotic effect of andrographolide analogue on RIN-m beta cell apoptosis induced by H2O2.Methods:The apoptosis cell model was established by H2O2 treatment. The effect of AL-1 on the cell viability was measured by MTT. Flow cytometry (FCM) with propidium iodide (PI) staining was used to detect the cell apoptosis, and fluorescent microscope to observe the morphology of the cell apoptosis. The total proteins from the drug-treated cells and control were extracted respectively, and separated by two-dimensional gel electrophoresis (2-DE). The gels were stained with sliver staining. Proteins with alterations in expression were analyzed with ImageMaster 2D-Platinum software, and identified by mass spectrometry. The obtained data were classified by bioinformatics. With Western blotting method, the expression differences of RHO GDP-dissociation 1, EF-Tu, and 14-3-3ζwere verified. The signal transduction pathway of AL-1 on Rin-m beta cell was analyzed. The differential apoptotic proteins of control and treated cells were analyzed and characterized.Results:1) The survival levels of H2O2-treated cells were increased by AL-1.2) The 2D-gel patterns of Rin-m beta cells with and without AL-1 treatment were established, and 72 proteins with different expression including thioredoxin, peroxiredoxin-1, peroxiredoxin-5, glutaredoxin-3, DJ-1,14-3-3ζ, Hsp90, HnrnpA2/B1, EF-Tu were identified. These proteins are involved in glucose metabolism, nucleic acid metabolism, protein metabolism and modification, signal transduction, cell apoptosis, cell structure and movement, immune and defense-related physiological processes.3) In the AL-1-treated cells, the pro-apoptotic proteins including isoform 3 of programmed cell death 6-interacting protein, galectin-1, nuclear mitotic apparatus protein 1 were found to be down-regulated, and the anti-apoptotic proteins including prohibitin, heat shock protein 9, Hspa5, isoform 1 of 60kDa heat shock protein were up-regulated.4) Proteins related to Akt and ERK signalling pathways were also changed in expression in AL-1-treatment cells.Conclusion:AL-1 can activate the Akt and ERK signal transduction pathways, stimulating the expression of antioxidant proteins and anti-apoptotic proteins, and thus protecting Rin-m beta cells from damage by H2O2.