Ginsenoside Rg5 alleviates hypoxia-induced myocardial apoptosis by targeting STAT3 to promote Tyr705 phosphorylation

Abstract

Background: The heart, as the body's blood-pumping organ, is extremely sensitive to changes in oxygen levels. Myocardial injury caused by hypoxia is a challenging issue, and there are currently no definitive specific drugs available for its treatment. Ginsenoside Rg5, one of the main rare saponins in ginseng, has shown significant efficacy in treating myocardial injury. This study aims to investigate the role and mechanisms of Rg5 in the treatment of hypoxic myocardial injury.

Methods: The cardioprotective effect against acute hypoxia of Rg5 was studied by assessing heart function, myocardial injury markers, inflammation, and oxidative stress in C57 mice, as well as apoptosis and reactive oxygen species (ROS) levels in H9c2 cardiomyocytes. Thermal proteome and target validation techniques were used to confirm the target protein of Rg5. The further protective mechanisms against hypoxia-induced damage were explored using immunocoprecipitation, immunofluorescence and rescue experiments in vivo and in vitro.

Results: The experimental results demonstrated that Rg5 effectively improved cardiac function in mice, reduced inflammation, oxidative stress, and the release of myocardial injury markers, decreased cardiomyocyte apoptosis, and lowered ROS levels. Further, using target protein screening and validation techniques, Signal transducer and activator of transcription 3 (STAT3) was verified as a direct target for Rg5's cardioprotective effect. It was observed that Rg5 specifically promoted the phosphorylation of Tyr705 in STAT3 via the JAK2/STAT3 pathway, leading to the translocation of phosphorylated STAT3 into the nucleus where they induce the expression of anti-apoptotic protein and protect cells from hypoxic damage.

Conclusion: Rg5 could be a potential therapeutic agent for preventing and treating myocardial hypoxic injury, providing scientific evidence for its application in anti-hypoxic therapy.

Keywords: Ginsenoside Rg5; Hypoxia; Myocardial apoptosis; Stat3; Tyr705 phosphorylation.

Fig. 1

Rg5 alleviated myocardial damage caused by acute hypoxia in vivo A. Schematic diagram of animal experiment procedure. A total of 18 mice were divided into three groups: Ctrl group, Mod group and Rg5 group. The Mod and Rg5 groups were exposed at 6500 m altitude for three days. B. Content changes of IL-1β, IL-6, and TNF-α in peripheral blood (n = 6). C. Changes of MDA, GSH and SOD activity in peripheral blood (n = 6) D. Representative echocardiographic image of mice in three groups. E. Rg5 improved EF, ejection fraction and FS, left ventricular fractional shortening of mice exposed to hypoxia (n = 6). F. Content changes of myocardial injury markers in the peripheral blood. CK, Creatine kinase; cTnI, Cardiac troponin I; CK-MB, Creatine kinase-MB; Myo, Myoglobin (n = 6). G. Representative HE staining image of heart tissues in three group of mice (n = 3).. Scale bar = 50 μm. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated

Fig. 2

Rg5 improved H9c2 cardiomyocyte injury induced by acute hypoxic exposure A. Rg5 increased the cell viability of H9c2 cardiomyocytes after hypoxic exposure (n = 6). B. The content ratio of CKMB/CK in H9c2 culture medium supernatant was significantly reduced after Rg5 treatment (n = 6). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated

Fig. 3

Rg5 mitigated oxidative stress and mitochondrial damage of H9c2 induced by acute hypoxia A. The ROS content in H9c2 cells was determined by flow cytometry (n = 3). B, C. MitoSOX dye was used to determine the content of ROS in mitochondria after treatment with Rg5 in H9c2 cells. Scale bar = 10 μm. D. The aerobic respiration capacity of H9c2 cells after hypoxia exposure was measured by a Seahorse XF Cell Mitochondrial Stress Test Kit. E–F. ATP production and basal respiration were quantified from Fig. 3D. G. Calcein AM dye was used to determine the MPTP after treatment with Rg5 in H9c2 cells. Scale bar = 10 μm. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated

Fig. 4

Rg5 mitigated the apoptosis of H9c2 cardiomyocytes induced by acute hypoxia. A Representative flow cytometry images using Annexin V/PI label to detect the hypoxia-induced apoptotic rate of H9c2 cells. B Statistical results of apoptotic rate detected by flow cytometry (n = 5). C. JC-1 mitochondrial membrane potential assay was performed using flow cytometry. D Quantitative comparison of mitochondrial membrane potential detected in Fig. 4c (n = 3). E Effect of Rg5 on apoptosis-related protein content in hypoxic H9c2 cardiomyocytes. F-J. WB quantitative results of apoptosis-related proteins: Bax (F), Cytochrome-c (G), Cytochrome-c (H), Cleaved caspase3 (I), Bcl2 (J). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated

Fig. 5

Evidence of Rg5 targeting STAT3 A. Thermal proteome screening data indicated that STAT3 was a potential target of Rg5. B. Schematic diagram of peak intensity of a representative peptide of STAT3 in the thermal proteome data. C. STAT3 peptide AILSTKPPGTFLLR was identified with good confidence. D. Molecular docking results showed that Rg5 and STAT3 had good binding ability. E Schematic diagram of binding sites for molecular docking of Rg5 & STAT3. F Schematic diagram of binding energy between Rg5 and STAT3

Fig. 6

STAT3 is the key target of Rg5 in alleviating acute hypoxic myocardial injury. A Cellular thermal shift assay (CETSA) showed that the degradation of STAT3 slowed down after Rg5 treatment. B WB quantitative results of CESTA. C The degradation of STAT3 was hindered by Rg5 in Drug affinity responsive target stability (DARTS) assay. D WB quantitative results of DARTS. E–F. MitoSOX dye was used to determine the content of ROS in mitochondria after treatment with Rg5, Rg5 + stattic (sta) and colibelin (col) in H9c2 cells. Scale bar = 10 μm. G. The ROS content in H9c2 cells was determined by flow cytometrya. H. The statistical quantification of ROS content based on Fig. 6E (n = 3). I. Representative flow cytometry images using Annexin V/PI label to detect the effects of STAT3 inhibitors and agonists on apoptotic rate. J. Statistical results of apoptotic rate detected by flow cytometry (n = 3). Data are shown as mean ± SD. ***P < 0.001 as indicated

Fig. 7

Rg5 enhanced phosphorylation of STAT3 Tyr705 and promoted nuclear transfer of STAT3 A. Rg5 specifically promoted the phosphorylation of Stat3 Tyr705 in whole cell lysate. B The statistical quantification of Try705 phosphorylation (p705) based on Fig. 7a (n = 3). C. WB results of p705 in the nucleus after Rg5 treatment. D. The content changes of p705 in nucleus calculated based on Fig. 7c (n = 3). E. Immunofluorescence results showed that Rg5 promoted the aggregation of p705 in the nucleus. Scale bar = 10 μm. F-G. mRNA levels of the target proteins STAT3 transcriptionally regulated (n = 3). Bcl2 (F), Mcl-1 (G). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated

Fig. 8

Stattic, a STAT3 p705-specific inhibitor, exacerbated hypoxic-induced apoptosis of H9c2 cardiomyocytes A-B. MitoSOX staining results showed that stattic (sta) further increased the accumulation of ROS caused by hypoxia. Scale bar = 10 μm. C, D. The ROS content in H9c2 cells was determined by flow cytometry after sta treatment (n = 3). E–F. Annexin V/PI results detected by flow cytometry showed that sta increased the apoptotic rate of H9c2 cardiomyocytes compared with Mod group (n = 3). G-H. Sta caused more drastic changes in mitochondrial membrane potential compared with Mod group (n = 3). I. Sta inhibited the accumulation of STAT3 p705 in the nucleus. Scale bar = 10 μm. J. Sta inhibited the content of STAT3 p705 compared with the Mod group. Data are shown as mean ± SD. **P < 0.01, ***P < 0.001 as indicated

Fig. 9

Stattic reversed the improvement of Rg5 on the apoptosis of H9c2 cardiomyocytes induced by acute hypoxia A. Immunofluorescence results showed that the accumulation of STAT3 p705 in the nucleus was significantly reduced after Stattic intervention compared to the Rg5 group. Scale bar = 10 μm. B. WB results of STAT3 p705 in nucleus of H9c2 cardiomyocytes. C. Sta significantly reduced the amount of STAT3 p705 in the nucleus compared to Rg5 group, as calculated in Fig. 3b (n = 3). D. WB results of Bcl2 in H9c2 cardiomyocytes treated by sta. E. Sta significantly reduced the amount of Bcl2 compared to Rg5 group, as calculated in Fig. 4j (n = 3). F. Immunoprecipitation results showed that Rg5 enhanced the interaction between JAK2 and STAT3. G. Molecular docking results showed that Rg5, STAT3, JAK2 had good binding ability. H Schematic diagram of binding energies of Rg5, STAT3, JAK2. I. WB results of p705 and pJAK2 in H9c2 cardiomyocytes treated by IL-6. WB quantitative results of proteins: Bax (J), Cytochrome-c (K). Data are shown as mean ± SD. *P < 0.05, **P < 0.01 as indicated

Fig. 10

Rg5 improved acute hypoxic heart injury by targeting STAT3 in vivo A. Representative echocardiographic image of mice in three groups. B Sta reversed Rg5’s improvement in cardiac function of hypoxic mice. EF, ejection fraction and FS, left ventricular fractional shortening (n = 6). C Content changes of myocardial injury markers in the peripheral blood affect by sta. CK, Creatine kinase; cTnI, Cardiac troponin I; CK-MB, Creatine kinase-MB; Myo, Myoglobin (n = 6). D Content changes of IL-1β, IL-6, and TNF-α in peripheral blood affected by sta (n = 6). E Representative HE staining image of heart tissues in three group of mice. Scale bar = 50 μm. F WB results of STAT3 p705 and apoptosis-related proteins of mouse hearts. G Content changes of STAT3 p705 and apoptosis-related proteins based on Fig. 10g (n = 3). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated