Fig 2: NMN administration restored SIRT3 function and alleviated oxidative stress and apoptosis in AGEs-treated human NP cells via AMPK/PGC-1a pathway. (A) Human NP cells were treated with AGEs (100–200 µg/ml) or PBS for 36 h. Representative Western blotting assay and quantitation of the levels of AMPK, p-AMPK, PGC-1a, p-PGC-1a proteins. *p < 0.05 versus PBS. (B) Western blotting assay of AMPK, p-AMPK, PGC-1a, p-PGC-1a levels in NP cells stimulated with AGEs (200 µg/ml) in the presence or absence of A-769662 (50 µM), RAGE antibody (10 µg/ml), NMN (100 µM) or Compound C (50 uM). The quantitation of the protein levels: *p < 0.05 versus AGEs (200 µg/ml). ns versus AGEs (200 µg/ml). # p < 0.05 versus AGEs +NMN. (C) Western blotting assay of SOD2, catalase, TRX2 and TRXR2 levels in NP cells stimulated with AGEs (200 µg/ml) in the presence or absence of A-769662 (50 µM) or NMN (100 µM). The quantitation of the protein levels: *p < 0.05 versus AGEs. (D) Western blotting assay of SOD2, catalase, TRX2 and TRXR2 levels in siRNA transfected NP cells stimulated with AGEs (200 µg/ml) in the presence or absence of A-769662 (50 µM) or NMN (100 µM). *p < 0.05 versus AGEs+NMN+siCON. #p < 0.05 versus AGEs+A-769662+siCON. (E) Representative fluorescence images with MitoSOX (red) and MitoTracker (green) double-staining in siRNA transfected NP cells stimulated with AGEs (200 µg/ml) in the presence or absence of A-769662 (50 µM) or NMN (100 µM). (F) Cell apoptosis was measured by Annexin V-APC/7-AAD staining under flow cytometry analysis. *p < 0.05 versus AGEs (200 µg/ml). # p < 0.05 versus AGEs+NMN+siCON. ##p < 0.05 versus AGEs+ A-769662+siCON.

Fig 3: Impairment of SIRT3 function was involved in AGEs-induced oxidative stress and apoptosis in human NP cells and SIRT3 overexpression could alleviate these processes. (A, B) Human NP cells were treated with AGEs (100–200 µg/ml) or PBS for 36 h. (A) Representative Western blotting assay and quantitation of the level of SIRT3 protein. (B) The analysis of SIRT3 deacetylase activity using mitochondrial fractions as described under Materials and Methods. *p < 0.05 versus PBS. #p < 0.05 versus AGEs (100 µg/ml at 36 h). (C) Representative Western blotting assay and quantitation of the level of mitochondrial antioxidants including SOD2, catalase, TRX2 and TRXR2. *p < 0.05 versus PBS. #p < 0.05 versus AGEs (100 µg/ml at 36 h). (D) Representative Western blotting assay and quantitation of the level of SIRT3 protein. *p < 0.05 versus PBS+siCON. #p < 0.05 versus AGEs+siCON. (E) The analysis of SIRT3 deacetylase activity using mitochondrial fractions. *p < 0.05 versus PBS+siCON. #p < 0.05 versus AGEs+siCON. (F) Representative Western blotting assay and quantitation of the level of SIRT3 protein. *p < 0.05 versus PBS+Lenti-Vector (blank vector). #p < 0.05 versus AGEs+Lenti-Vector. (G) The analysis of SIRT3 deacetylase activity using mitochondrial fractions. *p < 0.05 versus PBS+Lenti-Vector. #p < 0.05 versus AGEs+Lenti-Vector. (H) Scrambled siRNA (siCON) or SIRT3 siRNA (siSIRT3) transfection were performed before AGEs treatment (200 µg/ml, 36 h). The SOD2, catalase, TRX2 and TRXR2 protein levels were analyzed and quantitated by Western blotting. *p < 0.05 versus PBS+siCON. #p < 0.05 versus AGEs+siCON. (I) Lenti-Vector or Lenti-SIRT3 infection were performed before AGEs treatment (200 µg/ml, 36 h). The SOD2, catalase, TRX2 and TRXR2 protein levels were analyzed and quantitated by Western blotting. *p < 0.05 versus PBS+Lenti-Vector. #p < 0.05 versus AGEs+Lenti-Vector. (J) Representative fluorescence images with MitoSOX (red) and MitoTracker (green) double-staining. Cell nuclei are stained by DAPI. (K, L) Cell apoptosis was measured by Annexin V-APC/7-AAD staining under flow cytometry analysis. (K) *p < 0.05 versus PBS+siCON. #p < 0.05 versus AGEs+siCON. (L) *p < 0.05 versus PBS+Lenti-Vector. #p < 0.05 versus AGEs+Lenti-Vector.