Fig 2: PGC-1α was required for songorine induction of mitochondrial biogenesis. (A) PGC-1α gene and protein expression in primary neonatal rat ventricular myocytes (NRVMs) stimulated with LPS for 24 h; (B) the immunoprecipitation assay of the binding of PGC-1α to Nrf2 in NRVMs stimulated with LPS for 24 h; (C, D) luciferase report activities of Nrf1 and Tfam in H9C2 cells stimulated with LPS for 24 h; (E) gene expression of Nrf1 and Tfam in NRVMs exposed to LPS for 24 h; (F) mitochondrial number labeled with nonyl acridine orange (NAO) was viewed using flow cytometry in NRVMs stimulated with LPS for 24 h; (G, H) MTCO1 and VDAC1 protein expression in NRVMs treated with LPS for 24 h. All data are presented as mean ± SEM (n = 5); *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS or LPS + pEX3-NC treatment; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. indicated treatment. p values are determined by one-way ANOVA followed by Tukey's test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Cardiac Nrf2 deficiency attenuated protective effects of songorine. Male C57BL/6 mice were injected with AAV9-shNfe2l2 or AAV9-shNC through the caudal vein. Mice were administrated with songorine (i.p.) 1 h before and 12 h after LPS challenge (10 mg/kg), and the heart was examined 24 h later: (A) ejection fraction (EF), shortening fractional (FS), left ventricular end-systolic volumes (LVVs), left ventricular end-diastolic volumes (LVVd) and left ventricular mass (LV Mass) (n = 7); (B) immunofluorescence staining of cardiac SIRT1 protein (scale bar: 100 μm), one presentation of five mice; (C) immunoprecipitation examination of the binding of PGC-1α to Nrf2 (n = 5); (D-F) cardiac genes expression of Nrf1 and Tfam,ND1 and COX1, Cycs, Ndufv1 and Ndufs1were detected; (G) MTCO1 and VDAC1 protein expression in heart tissue. All data are presented as mean ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS + AAV9-shNC treatment; #p < 0.05,##p < 0.01, ###p < 0.001 vs. indicated treatment. p values are determined by one-way ANOVA followed by Tukey's test.

Fig 4: Rg3 suppresses the production of mitochondrial ROS induced by DEX. C2C12 myotubes were treated with DEX with vehicle or Rg3 for additional one day (D4). (A) Mitochondrial ROS production analysis. Fluorescence intensity is given as arbitrary units and values presented as means ± SD (n=5). Rg3/DEX-treated group was compared with DEX-treated group, **P<0.002; one-way ANOVA with post Bonferroni's multiple comparison test. (B) Mitochondrial membrane potential analysis. Fluorescence ratio (590 to 530 nm) was used for quantitative analysis. Values presented are as means ± SD (n=4). Rg3/DEX-treated group was compared with DEX-treated group, **P<0.002; one-way ANOVA with post Bonferroni's multiple comparison test. (C) The determination of ATP content. The total ATP content was measured using ATP determination kit. Values presented are as means ± SD (n=4). Rg3/DEX-treated group was compared with DEX-treated group, *P<0.033; one-way ANOVA with post Bonferroni's multiple comparison test. (D) Model of the effect and molecular mechanisms of ginsenoside Rg3 on DEX-induced myotube atrophy. Rg3 protects DEX-induced myotube atrophy through the activation of Akt/mTOR signaling and suppresses muscle-specific ubiquitin ligases. In addition, Rg3 efficiently prevents DEX-triggered mitochondrial dysfunction of myotubes through PGC1-α activities and its mitochondrial biogenetic transcription factors, NRF1 and Tfam. Rg3, ginsenoside Rg3; ROS, reactive oxygen species; DEX, dexamethasone; PGC1-α, peroxisome proliferator-activated receptor γ coactivator 1-α; NRF1, nuclear respiratory factor 1; Tfam, mitochondrial transcription factor A.

Fig 5: Effects of MORE on the expressions of biogenesis-regulating factors in C2C12 myoblasts. The C2C12 myoblasts were differentiated into myotubes in DMEM with 2% horse serum medium and then treated with 0.5, 1, and 2 mg/mL MORE or 2.5 mM/mL Met for 24 h. (A) Expression of TFAM, NRF1, SIRT1, and TFAM was determined by Western blot. (B–E) Relative expression of each protein was normalized to β-actin. All data are presented as the mean ± SD of three independent experiments (n = 3). Nor, nontreated normal cells; MORE0.5, cells treated with 0.5 mg/mL MORE; MORE1, cells treated with 1 mg/mL MORE; MORE2, cells treated with 2 mg/mL MORE; and Met, cells treated with 2.5 mM/mL metformin. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs Nor.