Journal: The Journal of Headache and Pain

Article Title: SS-31 alleviated nociceptive responses and restored mitochondrial function in a headache mouse model via Sirt3/Pgc-1α positive feedback loop

doi: 10.1186/s10194-023-01600-6

Figure Lengend Snippet: Mitochondria were injured and mitochondrial homeostasis was altered after repeated IS infusion in a headache male mouse model. Male C57BL/6 mice were sham treated or dural-infused of inflammatory soup (IS) for 7 consecutive days, and then sacrificed for mitochondrial assessment. A - C Representative immunoblots and quantification of the protein levels of Pgc-1α ( **** p < 0.0001), Tfam ( *** p = 0.0009), Drp1 ( ** p = 0.0050), Mfn2, Fis1 ( * p = 0.0108), Beclin1, P62 ( ** p = 0.0021), Pink1 ( **** p < 0.0001), and Parkin in the TNC. n = 6 per group; Student’s t -test. D The levels of ATP ( ** p = 0.0022), MMP ( ** p = 0.0043), ROS ( *** p = 0.0002) and MDA ( * p = 0.0136) were detected and normalized by total protein concentrations in the TNC. n = 6 per group; Student’s t -test. E Mitochondrial ultrastructure in the TNC by TEM analysis. The abnormal mitochondria count percent ( *** p = 0.0002) and area percent ( **** p < 0.0001) were calculated. Red arrowhead, damaged mitochondria (swollen mitochondria and reduction of mitochondrial cristae). Black arrowhead, normal mitochondria. Scale bar, 1 μm. n = 4 per group; Student’s t -test. F Immunofluorescence staining of VDAC1 and nucleus (DAPI) in the TNC. The mean mitochondria length ( *** p = 0.0006) and area ( * p = 0.0285) were calculated. Scale bar, 5 μm. n = 6 per group; Student’s t -test. Data are represented as Mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as compared to sham group

Article Snippet: The target protein information of the specific primary antibody was used as follows: Pgc-1α (1:1000, Novus Biologicals), mitochondrial transcription factor A (Tfam, 1:1000, Abcam), mitofusin 2 (Mfn2, 1:5000, Proteintech), fission 1 (Fis1, 1:2000, Wuhan Fine bio), dynamin related protein 1 (Drp1, 1:2000, Wuhan Fine bio), Beclin1 (1:1000, Wanleibio), PTEN-induced putative kinase 1 (Pink1, 1:1000, Wanleibio), Parkin (1:1000, Wanleibio), P62 (1:1000, Cell Signaling Technology), c-fos (1:5000, Proteintech), Sirt3 (1:1000, Novus Biologicals/ Wanleibio), proliferating cell nuclear antigen (Pcna, 1:500, Wanleibio), cytochrome oxidase subunit IV (CoxIV, 1:5000, Proteintech) and β-actin (1:3000, Servicebio).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: The Journal of Headache and Pain

Article Title: SS-31 alleviated nociceptive responses and restored mitochondrial function in a headache mouse model via Sirt3/Pgc-1α positive feedback loop

doi: 10.1186/s10194-023-01600-6

Figure Lengend Snippet: SS-31 restored mitochondrial function and mitochondrial homeostasis in an IS-induced headache mouse model. C57BL/6 mice received sham, IS or SS-31 treatment for 7 consecutive days, and were sacrificed to evaluate the effects of SS-31 on mitochondrial function and mitochondrial homeostasis in TNC. A - C Western blot analysis was used to asses expression levels of Pgc-1α (F(2, 15) = 5.434, * p = 0.0168; * p = 0.0384, + p = 0.0249), Tfam (F(2, 15) = 6.457, ** p = 0.0095; * p = 0.0443, ++ p = 0.0100), Mfn2, Drp1, Fis1 (F(2, 15) = 15.74, *** p = 0.0002; ** p = 0.0015, +++ p = 0.0003), P62 and Pink1 (F(2, 15) = 5.143, * p = 0.0199; * p = 0.0153) in different groups. n = 6 per group; One-way ANOVA. D Western blot analysis and quantification of Sirt3 (F(2, 15) = 10.66, ** p = 0.0013; ** p = 0.0011, + p = 0.0208) protein level normalized to β-actin. n = 6 per group; One-way ANOVA. E The levels of ROS (F(2, 15) = 25.33, **** p < 0.0001; **** p < 0.0001, +++ p = 0.0003), ATP (F(2, 15) = 7.704, ** p = 0.0050; * p = 0.0413, ++ p = 0.0045), MMP (F(2, 15) = 25.89, **** p < 0.0001; **** p < 0.0001, +++ p = 0.0009) and MDA (F(2, 15) = 3.854, * p = 0.0446; + p = 0.0362) were detected and normalized by total protein concentrations in different groups. n = 6 per group; One-way ANOVA. (F) Mitochondrial ultrastructure in TNC by TEM analysis. The abnormal mitochondria count percent (F(2, 15) = 14.39, *** p = 0.0003; *** p = 0.0004, ++ p = 0.0034) and area percent (F(2, 15) = 13.70, *** p = 0.0004; *** p = 0.0003, + p = 0.0120) were calculated. Red arrowhead, damaged mitochondria (swollen mitochondria and reduction of mitochondrial cristae). Black arrowhead, normal mitochondria. Scale bar, 500 nm. n = 6 per group; One-way ANOVA. Data are represented as Mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as compared to sham group. + p < 0.05, ++ p < 0.01 and +++ p < 0.001 as compared to IS group

Article Snippet: The target protein information of the specific primary antibody was used as follows: Pgc-1α (1:1000, Novus Biologicals), mitochondrial transcription factor A (Tfam, 1:1000, Abcam), mitofusin 2 (Mfn2, 1:5000, Proteintech), fission 1 (Fis1, 1:2000, Wuhan Fine bio), dynamin related protein 1 (Drp1, 1:2000, Wuhan Fine bio), Beclin1 (1:1000, Wanleibio), PTEN-induced putative kinase 1 (Pink1, 1:1000, Wanleibio), Parkin (1:1000, Wanleibio), P62 (1:1000, Cell Signaling Technology), c-fos (1:5000, Proteintech), Sirt3 (1:1000, Novus Biologicals/ Wanleibio), proliferating cell nuclear antigen (Pcna, 1:500, Wanleibio), cytochrome oxidase subunit IV (CoxIV, 1:5000, Proteintech) and β-actin (1:3000, Servicebio).

Techniques: Western Blot, Expressing

Journal: The Journal of Headache and Pain

Article Title: SS-31 alleviated nociceptive responses and restored mitochondrial function in a headache mouse model via Sirt3/Pgc-1α positive feedback loop

doi: 10.1186/s10194-023-01600-6

Figure Lengend Snippet: SS-31 restored mitochondrial function and mitochondrial homeostasis in H 2 O 2 -induced PC12 cells. A PC12 cells were treated with SS-31 (100 nM) for 2 h, followed by additional H 2 O 2 (300 nM) for 12 h. B Western blot analysis of Sirt3 and Pgc-1α (F(2, 6) = 7.438, * p = 0.0237; * p = 0.0452, + p = 0.0292). Protein levels of Sirt3 and Pgc-1α were quantified normalized to β-actin. n = 3 per group; One-way ANOVA. (C) Western blot analysis of Tfam (F(2, 6) = 6.981, * p = 0.0272; + p = 0.0254), Drp1, Fis1 (F(2, 6) = 8.292, * p = 0.0188; * p (H2O2) = 0.0200, * p (SS-31+H2O2) = 0.0485) and Pink1 (F(2, 6) = 20.93, ** p = 0.0020; ** p (H2O2) = 0.0037, ** p (SS-31+H2O2) = 0.0030). Protein levels were quantified normalized to β-actin. n = 3 per group; One-way ANOVA. D and E Flow cytometric analysis was used to examine MMP (F(2, 6) = 16.90, ** p = 0.0034; ++ p = 0.0045) and ROS (F(2, 6) = 7.564, * p = 0.0229; * p (H2O2) = 0.0314, * p (SS-31+H2O2) = 0.0388) levels in different groups. n = 3 per group; One-way ANOVA. Data are represented as Mean ± SD; * p < 0.05 and ** p < 0.01 as compared to con group. + p < 0.05 and ++ p < 0.01 as compared to H 2 O 2 group

Article Snippet: The target protein information of the specific primary antibody was used as follows: Pgc-1α (1:1000, Novus Biologicals), mitochondrial transcription factor A (Tfam, 1:1000, Abcam), mitofusin 2 (Mfn2, 1:5000, Proteintech), fission 1 (Fis1, 1:2000, Wuhan Fine bio), dynamin related protein 1 (Drp1, 1:2000, Wuhan Fine bio), Beclin1 (1:1000, Wanleibio), PTEN-induced putative kinase 1 (Pink1, 1:1000, Wanleibio), Parkin (1:1000, Wanleibio), P62 (1:1000, Cell Signaling Technology), c-fos (1:5000, Proteintech), Sirt3 (1:1000, Novus Biologicals/ Wanleibio), proliferating cell nuclear antigen (Pcna, 1:500, Wanleibio), cytochrome oxidase subunit IV (CoxIV, 1:5000, Proteintech) and β-actin (1:3000, Servicebio).

Techniques: Western Blot

Journal: The Journal of Headache and Pain

Article Title: SS-31 alleviated nociceptive responses and restored mitochondrial function in a headache mouse model via Sirt3/Pgc-1α positive feedback loop

doi: 10.1186/s10194-023-01600-6

Figure Lengend Snippet: The effects of SS-31 on mitochondrial function and homeostasis were partially counteracted by inhibiting Sirt3/Pgc-1α. C57BL/6 mice received PBS + sham + DMSO, PBS + IS + DMSO, SS-31 + IS + DMSO, SS-31 + IS + 3-TYP or SS-31 + IS + SR-18292 treatment for 7 consecutive days, followed by sacrifice to evaluate mitochondrial function and mitochondrial homeostasis in TNC. A ROS levels were examined in different groups and normalized by total protein concentrations. n = 6 per group; One-way ANOVA. F(4, 25) = 7.078, *** p = 0.0006; * p (PBS+IS+DMSO) = 0.0222, ** p (SS-31+IS+SR-18292) = 0.0029, + p (SS-31+IS+DMSO) = 0.0259, ## p (SS-31+IS+SR-18292) = 0.0034. B ATP levels were examined in different groups and normalized by total protein concentrations. n = 9 per group; One-way ANOVA. F(4, 40) = 4.753, ** p = 0.0031; ** p (PBS+IS+DMSO) = 0.0096, * p (SS-31+IS+3-TYP) = 0.0301, + p (SS-31+IS+DMSO) = 0.0393. C Western blot analysis showed that expression of Sirt3 (F(4, 40) = 13.71, **** p < 0.0001; **** p (PBS+IS+DMSO) < 0.0001, **** p (SS-31+IS+3-TYP) < 0.0001, **** p (SS-31+IS+SR-18292) < 0.0001, + p (SS-31+IS+DMSO) = 0.0153, ## p (SS-31+IS+3-TYP) = 0.0487, ## p (SS-31+IS+SR-18292) = 0.0216) and Pgc-1α (F(4, 40) = 15.15, **** p < 0.0001; *** p (PBS+IS+DMSO) = 0.0002, **** p (SS-31+IS+3-TYP) < 0.0001, **** p (SS-31+IS+SR-18292) < 0.0001, ## p (SS-31+IS+3-TYP) = 0.0044, ## p (SS-31+IS+SR-18292) = 0.0039) in different groups. n = 9 per group; One-way ANOVA. (D) Representative immunoblots and quantification illustrated the levels of Tfam (F(4, 40) = 9.868, **** p < 0.0001; *** p (PBS+IS+DMSO) = 0.0004, *** p (SS-31+IS+3-TYP) = 0.0001, *** p (SS-31+IS+SR-18292) = 0.0003, + p (SS-31+IS+DMSO) = 0.0417, # p (SS-31+IS+3-TYP) = 0.0177, # p (SS-31+IS+SR-18292) = 0.0363), Drp1 (F(4, 40) = 5.655, ** p = 0.0011; *** p (PBS+IS+DMSO) = 0.0004, * p (SS-31+IS+SR-18292) = 0.0492, + p (SS-31+IS+DMSO) = 0.0429), Fis1 and Pink1 in different groups. n = 9 per group; One-way ANOVA. E Immunofluorescence staining of VDAC1 and nucleus (DAPI) in TNC. The mean mitochondria length (F(4, 25) = 5.225, ** p = 0.0034; * p (PBS+IS+DMSO) = 0.0232, * p (SS-31+IS+3-TYP) = 0.0410, + p (SS-31+IS+DMSO) = 0.0312) and area (F(4, 25) = 6.964, *** p = 0.0007; * p (PBS+IS+DMSO) = 0.0396, ++ p (SS-31+IS+DMSO) = 0.0033, # p (SS-31+IS+3-TYP) = 0.0150, ## p (SS-31+IS+SR-18292) = 0.0047) were calculated. Scale bar, 5 μm. n = 6 per group; One-way ANOVA. Data are represented as Mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as compared to PBS + Sham + DMSO group. + p < 0.05 and ++ p < 0.01 as compared to PBS + IS + DMSO group. # p < 0.05 and ## p < 0.01 as compared to SS-31 + IS + DMSO group

Article Snippet: The target protein information of the specific primary antibody was used as follows: Pgc-1α (1:1000, Novus Biologicals), mitochondrial transcription factor A (Tfam, 1:1000, Abcam), mitofusin 2 (Mfn2, 1:5000, Proteintech), fission 1 (Fis1, 1:2000, Wuhan Fine bio), dynamin related protein 1 (Drp1, 1:2000, Wuhan Fine bio), Beclin1 (1:1000, Wanleibio), PTEN-induced putative kinase 1 (Pink1, 1:1000, Wanleibio), Parkin (1:1000, Wanleibio), P62 (1:1000, Cell Signaling Technology), c-fos (1:5000, Proteintech), Sirt3 (1:1000, Novus Biologicals/ Wanleibio), proliferating cell nuclear antigen (Pcna, 1:500, Wanleibio), cytochrome oxidase subunit IV (CoxIV, 1:5000, Proteintech) and β-actin (1:3000, Servicebio).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining