Fig 2: Construction of the SDHD mutant. The gene sequence of SDHD with CRISPR targeting sites is shown a. The sgRNA and PAM sequences predicted by the online idtdna program are depicted in blue and red colors, respectively; the specific site for mutation (at 118-bp) is shown with a green arrow. Expression of SDHD protein is shown b. The protein extracts reacted with rabbit polyclonal antibodies to SDHD and rabbit polyclonal antibodies to ß-Actin are shown. The DNA level recombination events in the genome of the mutant are illustrated c. The bases c and a (highlighted and in purplr color) of the parent have been replaced with the base g (highlighted and in red color) in the mutant. Expression of representative subunits from all five ETC complexes are shown d-i. The expression of NDUFB8 (complex i; d and e), SDHB (comlex II; d and f), UQCRC2 (complex III; d and g), MTCO1 (complex IV; d and h) and ATP5A (complex V; d and i) were normalized to the expression of ß-Actin

Fig 3: Knockdown of Aifm1 or Chchd4 significantly reversed the protective effect of Sirt5 overexpression on NP cells under compression.Rat NP cells were cultured in a compression culture chamber and subjected to 1 MPa static compression for 0 h or 24 h. Lenti-Sirt5 and siRNA of Aifm1 or Chchd4 were used to verify the protective effect of SIRT5 in rat NP cells under compression. Mitochondrial proteins were extracted from rat NP cells in each group. a–f Western blotting analysis and quantification of ATP5F1A, UQCRC2, SDHB, NDUFB8, and COX IV protein expression extracted from mitochondria (normalized to TOM20 expression, n = 3). g Quantification of ATP levels in NP cells in each group (n = 5). TOM20 IF staining was performed to visualize mitochondrial morphology. h Representative immunohistochemical staining of the mitochondrial structure in NP cells (n = 3). White scale bar = 10 μm; green scale bar = 2 μm. i Representative dot plot of cell apoptosis by flow cytometry analysis after Annexin V/PI dual staining (n = 3). j Quantification of the percentage of TUNEL-positive cells (n = 5). k–p Western blotting analysis and quantification of Aggrecan, MMP13, SIRT5, AIFM1, and CHCHD4 protein expression (normalized to β-actin expression, n = 3). Differences among multiple groups were analyzed by one-way ANOVA. The data in the figures represent the mean ± S.D. The P value is shown, *P < 0.05, **P < 0.01.

Fig 4: Protective effects of MK801 against LPS-induced mitochondrial dysfunction in HUVECs.A Flow cytometry graphs and B analysis of mitochondrial transmembrane potential in HUVECs treated with LPS in the absence or presence of indicated doses of MK801. Representative of traces (C) and quantification at 10 min (D) of mitochondria Ca2+ influx labeled with Rhod-2 AM in HUVECs treated with LPS in the presence or absence of Ru360 (mitochondrial calcium uptake inhibitor), DS16570511 (MCU inhibitor), or MK801 for 2 h. E–H O2 consumption rates (D), basal (E) and maximal respiration rate (F), and ATP production (G) in HUVECs treated with LPS in the presence or absence of MK801. Representative of western blotting images (I) and quantification (J) of NDUFB8, SDHB2, UQCRC2, MTCO1, and ATP5a in HUVECs treated with LPS in the presence or absence of MK-801 at indicated doses. (Data are presented as mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05 vs. Control; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. LPS).

Fig 5: Knockdown of Sirt5 impairs mitochondrial homeostasis and leads to a decrease in ETC complex subunits in NP cells.a Representative TEM images of mitochondria in NP cells from the si-NC and si-Sirt5 groups (n = 3). Yellow arrow: normal mitochondria. Red arrow: unhealthy mitochondria. Black scale bar = 2 µm; white scale bar = 1 µm. b Representative immunohistochemical staining of the structure of mitochondria in NP cells from the si-NC and si-Sirt5 groups (n = 3). White scale bar = 50 µm; green scale bar = 10 µm. c Seahorse analysis of the oxygen consumption rate (OCR) in the si-NC and si-Sirt5 NP cells. The OCR was measured continuously throughout the experimental period at baseline and in the presence of the indicated drugs: 1 µM oligomycin, 2 µM FCCP, and 1 µM rotenone with 1 µM antimycin A (R + A). d Quantification of maximal respiration, ATP production, and basal respiration of the data from mitochondrial stress tests (n = 3). e Quantification of ATP levels in NP cells from the si-NC and si-Sirt5 groups (n = 6). f, g Western blotting analysis and quantification of ATP5F1A, UQCRC2, SDHB, NDUFB8, and COX IV protein expression (normalized to ß-actin expression, n = 3). Differences between two groups were analyzed by Student’s t test. The data in the figures represent the mean ± S.D. The P value is shown, *P < 0.05, **P < 0.01.