Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Salidroside pretreatment alleviates ferroptosis induced by myocardial ischemia/reperfusion through mitochondrial superoxide-dependent AMPKα2 activation.

doi: 10.1016/j.phymed.2024.155365

Figure Lengend Snippet: Fig. 2. Sal pretreatment alleviates A/R damage-induced ferroptosis in H9c2 cells. H9c2 cells underwent pre-treatment with 10 μM Sal for 3 h, 2 μM Fer-1 for 24 h, or 10 μM MitoTEMPO for 24 h, resulting in the following observations. After A/R injury, PGTS2 mRNA levels (A), Fe2+ contents (B), MDA and 4-HNE levels (C), intracellular/mitoROS levels (D-E) were assayed in H9c2 cells. F Western blot detected the changes of phosphorylated AMPKα, AMPKα2, and GPX4 in H9c2 cell lysates. G Relative intensities of p-AMPKα and AMPKα2 in H9c2 cell lysates. H Relative intensity of GPX4 in H9c2 cell lysates. I AMP/ATP ratio in H9C2 cells. All data represent mean ± SD (n = 3 or 4). NS, no significance. **p < 0.01, compared with the indicated groups.

Article Snippet: Antibodies against AMPKα2 (2757), p-AMPKα (8208S), GAPDH (2118), and β-actin (3700) were purchased from Cell Signaling Technology.

Techniques: Western Blot

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Salidroside pretreatment alleviates ferroptosis induced by myocardial ischemia/reperfusion through mitochondrial superoxide-dependent AMPKα2 activation.

doi: 10.1016/j.phymed.2024.155365

Figure Lengend Snippet: Fig. 3. Sal pretreatment alleviates A/R damage-induced ferroptosis in H9c2 cells via the AMPKα2 pathway. H9c2 cells were transfected with pAD/AMPKα2- shRNA for 24 h and then treated with or without 10 μM Sal for 3 h prior to A/R damage; the below findings were also obtained using this method. After A/R damage, PGTS2 mRNA levels (A), Fe2+ contents (B), MDA and 4-HNE levels (C), intracellular/ mitoROS levels (D-E) were assayed in H9c2 cells. F Western blot detected the changes of phosphorylated AMPKα, AMPKα2, PGC-1α, and GPX4 in H9c2 cell lysates. G Relative intensity of PGC-1α in H9c2 cell lysates. H Relative intensities of p- AMPKα and AMPKα2 in H9c2 cell lysates. I Relative intensity of GPX4 in H9c2 cell lysates. All data represent mean ± SD (n = 3 or 5). NS, no significance. *p < 0.05, compared with the indicated groups. **p < 0.01, compared with the indicated groups.

Article Snippet: Antibodies against AMPKα2 (2757), p-AMPKα (8208S), GAPDH (2118), and β-actin (3700) were purchased from Cell Signaling Technology.

Techniques: Transfection, shRNA, Western Blot

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Salidroside pretreatment alleviates ferroptosis induced by myocardial ischemia/reperfusion through mitochondrial superoxide-dependent AMPKα2 activation.

doi: 10.1016/j.phymed.2024.155365

Figure Lengend Snippet: Fig. 5. Under mild mitoROS generation Sal activited AMPKα2 enhancing mitoETC complex I activity. H9c2 cells were pretreated with 10 μM Sal for various durations (0–12 h). A Western blot detected the changes of phosphorylated AMPKα and AMPKα2 and their relative intensities in H9c2 cells. B The AMP/ATP ratio of 10 μM Sal for different treating times (0–12 h) in H9c2 cells. C AMP/ATP ratio of Sal different treating concentrations (0.1–10 μM) for 3 h in H9c2 cells. D OCR curves and histograms of indices related to mitochondrial vitality in H9c2 cells. (E) Western blot detected the changes of phosphorylated AMPKα and AMPKα2 and their relative intensities in H9C2 cells. F MitoETC complex I and III activity in H9C2 cells. G MMP levels were visualized using flow cytometry and expressed as the fluorescence ratio in the upper and lower right quadrants. All data represent mean ± SD (n = 3–5). NS, no significance. *p < 0.05, compared with the indicated groups. **p < 0.01, compared with the indicated groups.

Article Snippet: Antibodies against AMPKα2 (2757), p-AMPKα (8208S), GAPDH (2118), and β-actin (3700) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Western Blot, Flow Cytometry, Fluorescence

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Salidroside pretreatment alleviates ferroptosis induced by myocardial ischemia/reperfusion through mitochondrial superoxide-dependent AMPKα2 activation.

doi: 10.1016/j.phymed.2024.155365

Figure Lengend Snippet: Fig. 7. Sal pretreatment alleviated I/R injury-induced ferroptosis in the rat myocardium by upregulating AMPKα2 and inhibiting oxidative and energy stress. A Western blot detected the changes of phosphorylated AMPKα, AMPKα2, PGC-1α, NDUFB8, UQCRC2, and GPX4 in rat myocardial lysates. B Relative in tensity of PGC-1α expression. C Relative intensities of p-AMPKα and AMPKα2 expression. D Relative intensity of GPX4 expression. E Relative intensities of NDUFB8 and UQCRC2 expression. F PGTS2 mRNA and Fe2+ levels in rat myocardial lysates. G MDA and 4-HNE levels in rat myocardial lysates. H Representative images of DHE/MitoSOX staining and relative fluorescence intensities of DHE/MitoSOX in rat myocardial sections. The scale bars represent 20 μm. I Suc and α-KG levels in rat myocardial lysates. J ATP content of rat myocardial lysates. K SOD, CAT, and GSH-Px activity in rat myocardial lysates. lThe GSH/GSSG ratio in rat myocardial lysates. All data represent mean ± SD (n = 3, 5 or 6). NS, no significance. **p < 0.01, compared with the indicated groups.

Article Snippet: Antibodies against AMPKα2 (2757), p-AMPKα (8208S), GAPDH (2118), and β-actin (3700) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Staining, Fluorescence, Activity Assay

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Salidroside pretreatment alleviates ferroptosis induced by myocardial ischemia/reperfusion through mitochondrial superoxide-dependent AMPKα2 activation.

doi: 10.1016/j.phymed.2024.155365

Figure Lengend Snippet: Fig. 8. Diagram presenting how Sal pretreatment protects the myocardium in response to I/R damage. Sal pretreatment upregulates and phosphorylates AMPKα2, which enhances mitoETC complex I activity under mild mitoROS generation, thereby alleviating iron accumulation, inhibiting oxidative stress and aberrant lipid metabolism, improving energy metabolism, preventing mitochondrial dysfunction, and protecting the myocardium against I/R-induced ferroptosis.

Article Snippet: Antibodies against AMPKα2 (2757), p-AMPKα (8208S), GAPDH (2118), and β-actin (3700) were purchased from Cell Signaling Technology.

Techniques: Activity Assay

Journal: Chinese medicine

Article Title: Salidroside protects against myocardial infarction via activating MIF-mediated mitochondrial quality control.

doi: 10.1186/s13020-025-01076-3

Figure Lengend Snippet: Fig. 2 Salidroside activated MIF signaling and decreased mitochondrial damage in myocardial infarction model mice. A Salidroside was administered at various concentrations following MI surgery, and heart tissue samples were collected. The mitochondrial ultrastructure was observed via transmission electron microscopy, and damaged mitochondria were quantified. The yellow or blue regions indicate damaged mitochondria or intact mitochondria, respectively, scale bar = 2 or 1 μm. B The ratio of mtDNA to nuclear DNA was determined via quantitative real-time PCR. C ATP content was measured. D Western blot analysis of the expression of MIF. E Representative images of MIF immunofluorescence staining and data analysis of heart sections. The cell nuclei were stained blue with DAPI, and MIF was stained red, scale bar = 100 μm. F Western blot analysis of the expression of p-AMPK, LC3I, LC3II, PINK1, BNIP3, OPA1, and MFN1 were performed. Statistical analysis was performed via one-way ANOVA followed by Bonferroni’s multiple comparisons test. n = 3. *P < 0.05 vs. the MI-Ctrl group; **P < 0.01 vs. the MI-Ctrl group; #P < 0.05 vs. the MI-Sal-L group; ##P < 0.01 vs. the MI-Sal-L group

Article Snippet: After sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE), electrophoretic transfer, and nonspecific binding blocking, the transferred proteins were incubated overnight with primary antibodies against GAPDH (1:5000, 10494-1-AP, Proteintech), Bcl-2 (1:2000, ab182858, Abcam), Bax (1:2000, ab32503, Abcam), cleaved caspase-3 (1:1000, #9661, CST), MIF (1:1000, ab187064, Abcam), AMPK (1:1500, 66536-1-Ig, Proteintech), p-AMPK (1:1000, ab23875, Abcam), microtubule-associated protein 1 light chain 3 II (LC3II, 1:1000, 14600-1-AP, Proteintech), PTEN-induced putative kinase 1 (PINK1, 1:1000, 23274-1-AP, Proteintech), BCL2interacting protein 3 (BNIP3, 1:1000, ab109362, Abcam), Optic Atrophy 1 (OPA1, 1:2000, 27733-1-AP, Proteintech), and Mitofusin 1 (MFN1, 1:1000, 2398-1-AP, Proteintech), followed by incubation with the corresponding Band intensity was analyzed using a gel documentation system (Bio-Rad, Hercules, CA, USA) and normalized to that of GAPDH.

Techniques: Transmission Assay, Electron Microscopy, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Chinese medicine

Article Title: Salidroside protects against myocardial infarction via activating MIF-mediated mitochondrial quality control.

doi: 10.1186/s13020-025-01076-3

Figure Lengend Snippet: Fig. 5 Effects of MIF on oxygen‒glucose deprivation (OGD)-induced apoptosis and mitochondrial dysfunction in cardiomyocytes. A Cardiomyocytes were subjected to oxygen‒glucose deprivation (OGD) or control conditions and transfected with siMif or incubated with recombinant MIF (rMIF) for 24 h. Then, western blot analysis of the expression of MIF was performed. B Western blot analysis of the expression of Bax, Bcl-2, and caspase-3 was performed. C Cardiomyocyte apoptosis was detected and quantified by flow cytometry. D Mitochondria were quantified by mitochondrial labeling, and the data were analyzed. Scale bar = 100 μm. E ATP content was measured. F Western blot analysis of p-AMPK, LC3-I, LC3-II, PINK1, BNIP3, OPA1, and MFN1 was performed. The data are presented as the means ± SDs. Statistical analysis was performed via one-way ANOVA followed by Bonferroni’s multiple comparisons test. n = 3. *P < 0.05 vs. the Ctrl group, **P < 0.01 vs. the Ctrl group, #P < 0.05 vs. the OGD group, ##P < 0.01 vs. the OGD group

Article Snippet: After sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE), electrophoretic transfer, and nonspecific binding blocking, the transferred proteins were incubated overnight with primary antibodies against GAPDH (1:5000, 10494-1-AP, Proteintech), Bcl-2 (1:2000, ab182858, Abcam), Bax (1:2000, ab32503, Abcam), cleaved caspase-3 (1:1000, #9661, CST), MIF (1:1000, ab187064, Abcam), AMPK (1:1500, 66536-1-Ig, Proteintech), p-AMPK (1:1000, ab23875, Abcam), microtubule-associated protein 1 light chain 3 II (LC3II, 1:1000, 14600-1-AP, Proteintech), PTEN-induced putative kinase 1 (PINK1, 1:1000, 23274-1-AP, Proteintech), BCL2interacting protein 3 (BNIP3, 1:1000, ab109362, Abcam), Optic Atrophy 1 (OPA1, 1:2000, 27733-1-AP, Proteintech), and Mitofusin 1 (MFN1, 1:1000, 2398-1-AP, Proteintech), followed by incubation with the corresponding Band intensity was analyzed using a gel documentation system (Bio-Rad, Hercules, CA, USA) and normalized to that of GAPDH.

Techniques: Control, Transfection, Incubation, Recombinant, Western Blot, Expressing, Flow Cytometry, Labeling

Journal: Chinese medicine

Article Title: Salidroside protects against myocardial infarction via activating MIF-mediated mitochondrial quality control.

doi: 10.1186/s13020-025-01076-3

Figure Lengend Snippet: Fig. 6 Salidroside alleviates mitochondrial dysfunction and apoptosis in oxygen‒glucose deprivation (OGD)-induced cardiomyocytes via MIF signaling. A Cardiomyocytes were transfected with siMif or treated with recombinant MIF and then treated with salidroside or PBS with or without oxygen‒glucose deprivation (OGD) for 24 h. Proteins were subsequently harvested for western blotting to determine the level of MIF. B Western blotting was performed to determine the expression levels of Bax, Bcl-2, and cleaved caspase 3. C Cardiomyocyte apoptosis was detected and quantified by flow cytometry. D ATP content was measured. E Mitochondria were quantified by mitochondrial labeling. Scale bar = 100 μm. F, G Western blot analysis of the levels of p-AMPK, LC3I, LC3II, PINK1, BNIP3, OPA1, and MFN1 was performed. The data are presented as the means ± SDs. Statistical analysis was performed via one-way ANOVA followed by Bonferroni’s multiple comparisons test. n = 3. *P < 0.05 vs. the Ctrl group, **P < 0.01 vs. the Ctrl group, #P < 0.05 vs. the OGD group, ##P < 0.01 vs. the OGD group, @P < 0.05 vs. the Sal group, @@P < 0.01 vs. the Sal group

Article Snippet: After sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE), electrophoretic transfer, and nonspecific binding blocking, the transferred proteins were incubated overnight with primary antibodies against GAPDH (1:5000, 10494-1-AP, Proteintech), Bcl-2 (1:2000, ab182858, Abcam), Bax (1:2000, ab32503, Abcam), cleaved caspase-3 (1:1000, #9661, CST), MIF (1:1000, ab187064, Abcam), AMPK (1:1500, 66536-1-Ig, Proteintech), p-AMPK (1:1000, ab23875, Abcam), microtubule-associated protein 1 light chain 3 II (LC3II, 1:1000, 14600-1-AP, Proteintech), PTEN-induced putative kinase 1 (PINK1, 1:1000, 23274-1-AP, Proteintech), BCL2interacting protein 3 (BNIP3, 1:1000, ab109362, Abcam), Optic Atrophy 1 (OPA1, 1:2000, 27733-1-AP, Proteintech), and Mitofusin 1 (MFN1, 1:1000, 2398-1-AP, Proteintech), followed by incubation with the corresponding Band intensity was analyzed using a gel documentation system (Bio-Rad, Hercules, CA, USA) and normalized to that of GAPDH.

Techniques: Transfection, Recombinant, Western Blot, Expressing, Flow Cytometry, Labeling

Journal: Heliyon

Article Title: LOX-1 attenuates high glucose-induced autophagy via AMPK/HNF4α signaling in HLSECs

doi: 10.1016/j.heliyon.2022.e12385

Figure Lengend Snippet: LOX-1 silencing increased autophagy of HLSECs through AMPK/HNF4α signaling pathway. All groups were treated with 25 mM of glucose for 72 h, except for the normal control group. The cells in the transfection group HG + LV-LOX-1 or HG + LV-CON cells were blocked with AMPK (10 μM), HNF4α (0.6 μM), or autophagy (6 mM) inhibitors. The untreated intact samples were run as a control in each experiment. (A–C) HLSECs were cultured under the above conditions for 72 h, and the mRNA expression levels of LOX-1, Beclin-1, and LC3II in each group were determined by qRT-PCR. (D) The protein expression levels of each group were determined using western blotting, and β-actin was used as a loading control (see original blots in Supplemental Figure 6D). (E–K) Western blotting analyzed the relative protein expression of LOX-1, Beclin-1, LC3II, AMPK, p-AMPK, HNF4α, and p-HNF4α in each group (n = 3 independent experiments). All data are expressed as mean ± SD. from three independent experiments. ∗ P < 0.05, ∗∗∗ P < 0.01 versus the NC group; # P < 0.05, ### P < 0.001 versus HG + LV-CON group; x P < 0.05, xx P ​< 0.05, xxx P < 0.01 versus HG + LV-LOX-1 group.

Article Snippet: Antibodies against AMPK (bs-1115R), p-AMPK (bs-14318R), HNF4α (bs-3828R), p-HNF4α (bs-4001R), LC3 (bs-8878R), beclin-1 (bs-1353R), HRP-conjugated secondary antibodies were purchased from Bioss Biotechnology Co., Ltd. (Beijing, China).

Techniques: Transfection, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot