Ginsenosides mixture from Panax ginseng C.A.Meyer improves CoCl2-induced oxidative stress and mitochondrial dysfunction through NAD+-dependent SIRT1 activation in cardiomyocytes

doi:10.21203/rs.3.rs-4882234/v1

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Ginsenosides mixture from Panax ginseng C.A.Meyer improves CoCl2-induced oxidative stress and mitochondrial dysfunction through NAD+-dependent SIRT1 activation in cardiomyocytes

https://doi.org/10.21203/rs.3.rs-4882234/v1

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Introduction: There is growing recognition that medical therapies aimed at mediating mitochondrial ROS production may be important strategies to ameliorate cardiac disorders. Total ginsenosides (GS), a primary constituent extracted from Panax ginseng C.A.Meyer (ginseng) roots shows a strong therapeutic activity in heart disease and health benefits for hypoxia-related diseases. However, it is unclear whether GS protects hypoxic cardiomyocytes based on ROS production and mitochondrial function, as well as its molecular mechanism. Methods: In this study, mitochondrial respiratory function, ATP production, mitochondrial biosynthesis, glucose uptake, and NAD⁺-dependent SIRT1 activation in hypoxic and GS-pretreated H9c2 cells were investigated.

Results: We found that GS protected cells from oxidative damage and also maintained normal mitochondrial function in CoCl₂-stimulated cardiomyocytes. GS significantly reduced the glucometabolism disorder and mitochondrial respiration dysfunction as well. Further studies confirmed GS increased mitochondrial contents through regulating the NAD⁺ dependent SIRT1 activation, which was completely abrogated by nicotinamide. Importantly, we found that the ginsenoside Rg1, Re, Rf, Rb1, Rc and Rb2 are the key substances in GS associated with the anti-hypoxic action.

Conclusion: This study may provide new insights into the protection of ginseng against cardiac hypoxia damage.

ginsenoside

hypoxia

oxidative stress

mitochondrial damage

NAD+-SIRT1/PGC1α signaling

Oxygen (O₂) is essential for regular energy metabolism in mammals (24). Hypoxia is an important factor, which induces cardiomyocytes oxidative stress and mitochondrial damage, leading to cardiomyocytes loss, cardiac insufficiency, and even heart failure (45, 40). Park et al demonstrated that the dysfunction of mitochondrial played a crucial role in the reprogramming of glucose uptake, and stimulated the excessive production of reactive oxygen species (ROS) (29). Furthermore, glucose uptake affects energy metabolism, including oxidative phosphorylation (OXPHOS) and glycolysis, and adenosine triphosphate (ATP) production (3). During this process, the completeness of the mitochondrial membrane is involved in ATP production (7). Thus, the inhibition of oxidative stress and mitochondrial damage might be a potential and effective programme for the treatment of hypoxia.

Oxidized nicotinamide adenine dinucleotide (NAD⁺) is cosubstrate in signaling pathways and coenzyme in metabolic reactions of cells (26). Increased levels of NAD⁺ promote sirtuin 1 (SIRT1), the silent information regulator 2 homologue 1, which can regulate many physiological and pathological processes (14). One of the functions is to regulate peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC1α) that can affect mitochondrial biogenesis (39). Additionally, PGC-1α also buffers oxidative stress occurring during differentiation by promoting the expression of antioxidant enzymes (9). Recent research has shown that NAD⁺ improves cognitive function and reduces neuroinflammation by ameliorating mitochondrial damage and decreasing ROS production in chronic cerebral hypoperfusion models through SIRT1/PGC-1α pathway (48).

It has been believed from ancient days that ginseng is a reliable drug for longevity, fighting fatigue and stress. It has a variety of heart beneficial activities, can protect heart cells from cardiotoxicity (2), and is extensively used in patients with myocardial infarction, heart failure and cardiogenic shock all over the world (41, 49, 43). Ginsenosides, as the main active substances of Panax ginseng C.A.Meyer (ginseng), are widely responsible for the protective effects against oxidative stress, inflammation, visceral diseases, aging and other diseases (15, 1). Among the ginsenosides against hypoxia, Rb1, Rg3 and Rg1 are the most abundant studied ginsenosides at present (17, 28, 34), and Rg5, Rd, Rh2 and Rg2 have also been researched (46, 33, 19, 36). Mostly they amplify the response by stimulating hypoxia inducible factor 1 alpha (HIF1α) pathway (16), mitochondrial-mediated apoptotic pathway (20), phosphatidylinositol-3-OH kinase (PI3K)/protein kinase B (Akt)-mediated autophagy (47) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) signaling (42). Furthermore, these ginsenosides can block calcium over-influx into cells and eliminate the free radicals (30). However, the anti-hypoxic effect and molecular mechanism of total ginsenosides (GS), a mixture of active components from ginseng root in cardiomyocytes remain largely unclear up to now. Similarly, the molecular mechanisms of GS regulate anti-oxidative defense and mito-biogenesis for protecting heart from hypoxic stress still maintains elusive.

In the present study, we first investigated the effects of GS on hypoxia-induced mitochondrial membrane potential injure and reactive oxygen species accumulation in H9c2 cardiomyocytes for determining the protective effects of GS against hypoxia. Secondly, we compared hypoxia with hypoxia-GS treated H9c2 cells to examine the mitochondrial function related indexes, including the ability of glucose uptake, oxygen uptake and glycolysis, as well as explored the molecular mechanism of GS in anti-hypoxic pharmacological effect. In addition, NAM, an NAD⁺ precursor inhibitor, is known to prevent SIRT1 trigger (38) was used to verify the signaling pathway. Finally, HPLC was used to identify the ginsenoside monomers in GS, we preliminarily explored the anti-hypoxic ginsenoside monomers which playing a critical role in GS. This is a study to reveal the molecular mechanism of GS upregulate NAD⁺ is essential for the SIRT1/PGC1α mediated inhibition of hypoxia induced oxidative stress and mitochondrial dysfunction.

2.1. Materials and cell culture

H9c2 cardiomyocytes were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Culture related reagents, including fetal bovine serum (FBS), antibiotics, L-glutamine, and trypsin-EDTA were purchased from Invitrogen/Gibco (Carlsbad, CA, USA). CoCl₂ was purchased from Sigma-Aldrich (60818; St. Louis, MO, USA). Nicotinamide (NAM) was obtained from Med Chem Express (HYB0150; NJ, USA), which was stocked at 5 M in DMSO, and stored at -20 ^oC. Briefly, the H9c2 cardiomyocytes were cultured in plates with 5% CO₂ and 95% air at 37 ^oC in high-glucose Dulbecco's modified Eagle's medium with the coexistence of 10% FBS. Once H9c2 cells reached 80% confluence, they were incubated with 0.05% trypsin containing 1 mM EDTA for 2 min, washed with PBS for twice, centrifuged at 1,000 rpm for 5 min, and replated under the same culture technique.

2.2. Standard constituents and GS samples

The ginsenoside standards used in this study (Rg1, Rc, Rf, Rb1, Rc, Rh1, Rb2, Rb3, F1, Rd, F2, Rk3, Rg3, R-Rg3, PPT, Rk1, Rg5, Rh2) were purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). The GS (purity ≥ 80%, UV method, S25997; YUANYE, Shanghai, China) fine powder was weighed (20mg), dissolved in 1.0 mL of 100% methanol, The solution was traversed through a 0.22 µm syringe filter and injected into the HPLC system directly. The voucher specimen (No. 20170020ltt) was deposited in the Research Center of Traditional Chinese Medicine.

2.3. High-performance liquid chromatography (HPLC) analysis

The HPLC equipment for the experiment was Shimadzu 2010 HPLC system (Shimadzu, Japan), and the column used was Elite Kromasil C18 column (4.6 mm×250 mm; 5 µm). The mobile phases were a mixture of acetonitrile (HPLC grade; Sigma-Aldrich, St. Louis, MO, USA) and ultrapure water (HPLC grade, JT Baker). The proportion of acetonitrile was gradually increased from 18–21% (40 min), 21–26% (42 min), 26–32% (46 min), 32–33.8% (66 min), 33.8–38% (71 min), 38–49.1% (78 min), 49.1% (82 min), 49.1–50.6% (83 min), 50.6–59.6% (88 min), 59.6–65% (89.8 min), 65% (97 min), 65–85% (102 min), 85% (119 min), and 85–18% (140 min). The temperature of column was set at 35 ^oC, and the flow rate of mobile phases was 1.0 mL/min. The chromatogram elution curves were obtained at 203 nm using a UV detector.

2.4. Pretreatment with test drug and hypoxia induction

As the preliminary experiment, chemical hypoxia was achieved by adding different concentrations of CoCl₂ 800 µM to H9c2 cells for (3 h, 6 h and 12 h) to determine the appropriate hypoxia modeling conditions. Flow cytometry (FCM) was used to assist in determining the appropriate conditions. To explore the influences of GS in CoCl₂-induced cell damage, (0.2, 2 or 20 µg/mL) GS-treated H9c2 cells were maintained with CoCl₂ in complete medium. The control cells were incubated without GS or CoCl₂. In order to determine the molecular pathway, H9c2 cells were randomly divided into control group (Ctrl), CoCl₂ group, GS treatment group and NAM group (CoCl₂ + NAM 12 h after GS 48 h). Next, we sought to identify ginsenoside monomers in GS responsible for the inhibitory effect on CoCl₂-stimulated hypoxic injury in H9c2 cells. To clarify this speculation, H9c2 cells were randomly divided into control, CoCl₂, GS treatment and ginsenoside monomer groups (CoCl₂ for 12 h afte Rg1, Re, Rf, Rb1, Rc, Rb2, Rd, F2, Rk3, Rg3 and Rg5 48h).

2.5. Flow cytometry

To detect intracellular superoxide levels, cells were incubated with 1 µM DCFH-DA reactive oxygen species probe (S0033; Beyotime, Shanghai, China) in the dark for 20 min at 37 ^oC atmosphere and directly analyzed by FCM without fixing. Measurement of mitochondrial membrane potential was carried out essentially according to the manufacturer’s instruction. In brief, cells were washed and resuspended in staining buffer and loaded with JC-1 indicator (C2006; Beyotime, Shanghai, China) in the dark for 20 min at 37 ^oC atmosphere. Intracellular fluorescent products were measured immediately by FCM. For mitochondrial biogenesis measurements, cells were harvested and stained in suspension with Mito-Traker Green (C1048; Beyotime, Shanghai, China) in the dark for 20 min at 37 ^oC atmosphere and analyzed by FCM. Measurement of glucose uptake in H9c2 cells, as an index of cell vitality, using 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose (2-NBDG, N13195; Invitrogen, Carlsbad, CA, USA) in the dark circumstances for 20 min at 37 ^oC and performed by FCM following the directions (44).

2.6. Assessment of OCR and ECAR using a fluorescence-based assay

We used MitoXpress Xtra Oxygen Consumption Assay (MX-200; Agilent, Palo Alto, CA, USA) and pH-Xtra Glycolysis Assay (PH-200; Agilent, Palo Alto, CA, USA) to detect mitochondrial respiration and glycolysis, respectively. The operation was according to the manufacturer's instructions, then read the plate in a Cytation 5 multi-mode plate reader (BioTek, Winooski, VT, USA) and measured every 2 min over 2.5 h. When measurement was completed, the OCR and ECAR of each well were determined by linear regression (13).

2.7. Quantification of ATP levels and measurement of NAD⁺/NADH ratio

Following the manufacture's instruction, Cellular ATP and NAD⁺/NADH content were measured using ATP content kit (FF2000; Promega, Madison, WI, USA) and NAD/NADH assay kit (G9071; Promega, Madison, WI, USA). ATP and NAD⁺/NADH contents were measured with a luminometer (Cytation 5; BioTek, Winooski, VT, USA).

2.8. Western blotting

Whole cell extracts of cultured cardiomyocytes were lysed in RIPA buffer (R0010; Solarbio, Beijing, China) supplemented with protease inhibitor and phosphatase inhibitor cocktail (04693116001, 04906837001; Roche, Basel, Switzerland), which were determined to detect protein concentrations using a BCA protein assay kit (P0011; Beyotime, Shanghai, China). The sample proteins (50 µg per well) were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and eletrotransfer onto polyvinylidene fluoride (PVDF) membranes (Amersham, Life Technologies). After blocking in 5% non-fat milk for 1h, and then immunoblotted with the following antibodies: SIRT1 (ab189494; Abcam, Cambridge, MA, USA), PGC1 alpha (ab54481; Abcam, Cambridge, MA, USA) and GAPDH (60004-1-lg; Proteintech, Chicago, USA) overnight at 4°C. Band densitometric quantifications were determined and normalized using a chemiluminescent imaging system (FluorChem, ProteinSimple, San Jose, CA, USA) after incubating with the corresponding secondary antibodies for 1 h at room temperature.

2.9. Statistical analysis

Data are presented as mean ± standard derivation (SD) from three independent experiments. One-way analysis of variance (ANOVA) and Tukey’s test were used to analyze statistical significance using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). For all of the statistical analyses, a significant difference was accepted at P < 0.05.

3.1 GS inhibits cobalt chloride-stimulated oxidative stress and mitochondrial damage in H9c2 cells

To establish hypoxia cell models, H9c2 cells were incubated with 800 µM CoCl₂ medium for different times, we detected ROS and MMP by FCM. The results of analysis showed that CoCl₂ incubation for 6h and longer times remarkably elevated ROS levels and JC-1 monomers-positive cells (Fig. 1A, B). Taken together, these results mean that CoCl₂ caused oxidative stress and mitochondrial damage in hypoxia models. Based on the above results, treatment with 800 µM CoCl₂ medium for 12 h in H9c2 cells (model group) was selected as the optimal condition for the subsequent experiments. To evaluate the anti-hypoxic pharmacological effect of GS, H9c2 cells were incubated with different concentration of GS, and exposed to CoCl₂. As shown in Fig. 1C, GS pretreatment for 24 h significantly decreased ROS levels induced by CoCl₂. As the results showed in Fig. 1D, CoCl₂ induced a 2-fold increment of JC-1 monomers-positive cells compare to control group. GS significantly reduced JC-1 monomers-positive cells 0.5-0.8-fold over the CoCl₂-induced group. These results indicated that GS exerts its inhibitory effect on oxidative stress and mitochondrial damage in CoCl₂-stimulated cardiomyocytes.

3.2 GS regulates glucometabolism to exert anti-hypoxic pharmacological effects

Now, the new study has shown that the inhibition of glucose uptake was related to the ROS overproduction (22). We investigated the effect of the GS on glucometabolism, the amount of glucose uptake (2-NBDG) influenced by GS was determined as well as the mitochondrial respiratory function (OCR/ECAR), and ATP production (by chemiluminescence-based assays). FCM analysis revealed that GS visibly excited the rates of glucose uptake suppressed by CoCl₂ in a concentration-dependent manner (Fig. 2A). Further, OCR and ECAR analysis showed that GS pretreatment for 24h significantly reversed the decrease of OXPHOS and the increase of glycolysis induced by CoCl₂ (Figs. 2B, C). In addition, cellular ATP content was measured and revealed that CoCl₂ decreased the levels of ATP in H9c2 cells, which were recovered by GS pretreatment (Figs. 2D). These results suggest that GS can be able to protect cardiomyocytes from CoCl₂-induced glucometabolism disorder and mitochondrial respiration dysfunction.

3.3 The NAD⁺-SIRT1/PGC1α pathway involves in cardiomyocytes protection of GS on hypoxic stress

It has been reported that ROS level and mitochondrial function are mediated and critically controlled by NAD⁺-SIRT1/PGC1α pathway (23). Based on these studies, we analyzed the effect of GS on the ratio of NAD⁺/NADH and the expression of SIRT1 and PGC1α. As shown in Fig. 2E, NAD⁺/NADH content results revealed that CoCl₂ significantly decreased the ratio of NAD⁺/NADH, which was obviously increased with GS treatment, in the dose-dependent manner. Western blot results showed that CoCl₂ obviously reduced the expression of SIRT1 and PGC1α, which were both visibly increased by GS treatment (Fig. 2F). PGC-1α, as an integrator of the transcriptional network, which could regulate mitochondrial biogenesis and function (12). CoCl₂ induction caused a decrease of the content of mitochondrial in H9c2 cells, which was obviously antagonized by GS treatment in a concentration-dependent manner (Fig. 2G). These results suggest that GS might play a role in mitochondrial mass and function improvement to anti-hypoxic injury by targeting NAD⁺-SIRT1/PGC1α signaling.

3.4 GS reverses loss of mitochondrial content and regulate glucometabolism in cardiomyocytes via NAD ⁺ dependent deacetylase Sirtuin 1 activation

To confirm the cardiomyocytes protective effect of GS on the inhibition of hypoxic stress, we used NAM to further investigate whether NAD⁺ signaling regulates SIRT1/PGC1α mediates the regulation of mito-biogenesis and glycometabolism by GS treatment in the hypoxia-induced cardiomyocytes. We first investigated the cellular NAD⁺/NADH content, SIRT1 and PGC1α expression effect of NAM on H9c2 cells to identify molecular mechanism. As shown in Fig. 3A, GS significantly increased the NAD⁺/NADH ratio in CoCl₂ conditions, but this effect was blocked by NAM. Consistently, the level of SIRT1 and PGC1α increased by GS was significantly reversed by NAM treatment as shown in Fig. 3B. Moreover, FCM analysis showed that GS with NAM has a worse effect on mitochondrial content than GS (Fig. 3C). As expected, GS-mediated increase of glucose uptake were reduced by NAM (Fig. 3D). For mitochondrial energy metabolism, the increase of OXPHOS level and ATP content and the decrease of glycolysis level induced by GS were inhibited by the combination of GS and NAM (Fig. 3E-G). These results indicated that GS protects cardiomyocytes from hypoxia-induced mito-biogenesis and glucose metabolism disorder by activating the NAD⁺-SIRT1/PGC1α signaling pathway.

3.5 GS downregulates ROS and reverses mitochondrial function damage in H9c2 cells with NAD⁺-SIRT1/PGC1α activation

We further tested whether mitochondrial function was affected by NAM combined with GS. We found that NAM prevented the effects of GS treatment, reduced the anti-oxidative defense and increased the level of MMP damage over those of the GS treated group (Fig. 4A, B). Recent evidence advocated that PGC-1α coordinates a cellular antioxidant program which prevents accumulation of ROS (23). Which might explain GS elevated PGC-1α expression induces ROS degradation to orchestrate mitochondrial biogenesis. Correspondingly, during NAM incubation, depletion of PGC-1α increases ROS levels and elevates mitochondrial oxidative processes. Taken together, these results direct indicated that the NAD⁺-SIRT1/PGC1α pathway is crucial in cardiomyocytes protection of GS on hypoxic stress.

3.6 Effects of ginsenoside monomers in GS on SIRT1-mediated mitochondrial function in hypoxia-treated H9c2 cardiomyocytes

Next, we sought to determine whether ginsenoside monomers in GS were in charge of the inhibitory effect on CoCl₂-stimulated mitochondrial damage in H9c2 cells. To test this assumption, the main ginsenoside monomers namely Rg1, Re, Rf, Rb1, Rc, Rb2, Rd, F2, Rk3, Rg3 and Rg5 were added to H9c2 cells based on the compositions and content of GS from HPLC analysis (Fig. 5). Results indicate that ginsenoside Rg1, Re, Rf, Rb1, Rc, and Rb2, significantly increased CoCl₂-induced decrease of MMP, while ginsenosides Rd, F2, Rk3, Rg3 and Rg5 showed no statistically significant changes (Fig. 6A). These results authenticate that these ginsenoside monomers can promote mitochondrial function. For SIRT1 expression, compared to CoCl₂ stimulation, the results indicate that ginsenoside Rg1, Re, Rf, Rb1, Rc, and Rb2, especially Rg1, significantly increased the reduction of SIRT1 expression induced by CoCl₂ in H9c2 cells (Fig. 6B). Taken together, these results demonstrated the ginsenoside monomers in GS, including Rg1, Re, Rf, Rb1, Rc, and Rb2, which promote mitochondrial physiology and activate SIRT1 to protect cardiomyocytes from hypoxia-induced injury.

Here, we revealed the mechanism by which GS improves CoCl₂-induced oxidative stress and mitochondrial dysfunction in cardiomyocytes. In this study, GS pretreatment increased glucose uptake, aerobic cellular respiration and ATP production. Moreover, GS activated the NAD⁺-SIRT1/PGC1α pathway to promote mitochondrial biosynthesis which was inhibited by NAM. Rg1, Re, Rf, Rb1, Rc and Rb2 may be the major ginsenoside monomers for activating SITR1 expression and inhibiting mitochondrial dysfunction, which should be further investigated and compared to explore their functions and molecular mechanisms. The present research provides evidence for the antioxidant effects and mitochondrial protective properties of GS on hypoxic damage, which supported the clinical application of ginseng for the benefit of human health and hypoxic diseases.

When O₂ supply to the body is insufficient in the systemic or the local, such as in myocardial infarction or cellular hypoxia, it is enough to result in disorder of metabolic function and and damage of vitality (8). Previous investigations demonstrated that there were numerous compensatory mechanisms at the whole and regional levels to permit cells to function in a hypoxic environment. A large amount of evidence indicates that oxidative stress is related to myocardial ischemia and that antioxidant treatment may be beneficial in cardiac injury during ischemia (35, 10, 18). Moreover, it has been shown that mitochondrial dysfunction plays an essential role in ischemic-hypoxic myocardial damage (40); when respiration-competent mitochondria transplanted to ischemic heart tissue, the results included reduced cell death, increased energy production, and improved contractile function (5). In the current study, we demonstrated that preconditioning with GS abolished the decrease in MMP levels after CoCl₂ incubation, preventing H9c2 cells from hypoxic stress via reduction of ROS. Both aerobic respiration (mitochondrial dynamics, ROS generation) and glycolysis (glucose metabolism) affect cardiomyocytes function and viability (32, 6). In this study, we also demonstrated that GS abolished the reduction in glucose update. Moreover, the entry of glucose and subsequent aerobic respiration/glycolysis, and ATP synthesis promote the glucometabolism.

NAD⁺ can regulate mitochondrial respiratory function and activate sirtuins to maintain energy homeostasis (37). We demonstrated that, the ratio of NAD⁺/NADH in the hypoxic cardiomyocytes was increased by GS pretreatment, which can activate SIRT1 to match cellular energy requirements. SIRT1-mediated deacetylation leads to an increase in PGC1α levels, thereby inducing mitochondrial biogenesis and function (31). Our data suggest that SIRT1 and its downstream pathway were significantly activated by GS, which may be the molecular mechanism of GS on the improvement of mitochondrial content and function to anti-hypoxic injury. Based on the cross-talk between NAD⁺-SIRT1/PGC1α signaling pathways and mitochondria mass (4), we used NAM, a NAD⁺ precursor inhibitor to verify the above results. As showed, GS-mediated mitochondrial respiration, ATP production, reduction of ROS and increase in MMP levels were abolished by NAM, our findings indicate that restoring NAD by GS is essential for the SIRT1/PGC1α-mediated protective effects in hypoxic stress induced damage. Based on our results and those previously reported, we proposed a mechanism for the anti-hypoxic activity of GS in Fig. 7. GS activates NAD⁺-SIRT1/PGC1α pathway to reduce ROS level and inhibit mitochondrial function damage, and finally leading to the increase in mitochondrial number.

In addition, our studies for GS were not limited to the holistic mixture, as well as the bioactive compositions that exert the protective functions have also been studied. Our data indicate that ginsenoside Rg1, Re, Rf, Rb1, Rc and Rb2 were the person liable for activating SITR1 expression in GS, and might be the key substances responsible in GS with the anti-hypoxia action. We all know that the separation characteristics of reverse column packed with C₁₈ bonded silica gel are as follows: the components with large polarity pass through the column preferentially and are captured by the detector (25). Based on our results, we speculate that the ginsenoside monomers with large polarity in GS isolated from ginseng roots may be the most active components mitigate CoCl₂-induced hypoxia injury-mediated oxidative stress and mitochondrial damage by activating the NAD⁺ dependent deacetylase Sirtuin 1 in H9c2 cardiomyocytes. More importantly, recent study found ginsenoside Rb1 reversibly inhibits the activity of mitochondrial complex I to protect neurons against ischemic stroke (27). Using molecular docking and SIRT1 activity detection, Huang et al (11) found that ginsenoside Rc strongly binds and promotes SIRT1 activity, which promotes energy metabolism. In hypoxia/reoxygenation (H/R)-induced H9c2 cell models of ischemia/reperfusion injury, ginsenoside Rg1 suppressed H/R-mediated cell apoptosis, cytotoxicity, cellular ROS levels and mitochondrial damage (21). These results highlight the regulatory effects of ginsenoside monomers on mitochondrial function and SIRT1 activity. Based on these findings, we will further explore the molecular mechanism of Rg1, Re, Rf, Rb1, Rc and Rb2 against hypoxia-induced cardiomyocyte injury in subsequent experiments, which will be our next research direction. Furthermore, animal experiments are necessary for a deeper and complete research.

In conclusion, our study shows that GS activates the SIRT1/PGC1α pathway to promote mitochondrial biosynthesis in hypoxia induced cardiomyocytes injury. GS pretreatment increased glucose uptake, improved energy metabolism remodeling and ATP production, also maintained intracellular ROS and mitochondrial membrane stability. NAM inhibited this process, confirming that GS promotes mitochondrial biogenesis and function in a NAD⁺ dependent manner. More importantly, ginsenoside monomers Rg1, Re, Rf, Rb1, Rc and Rb2 are the main components involved in activating SIRT1 and improving mitochondrial function in GS. This study provides a potentially promising therapeutic option into the extensive application of ginseng for cardic protection in patients with hypoxic diseases.

2-NBDG 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose

Akt protein kinase B

ATP adenosine triphosphate

CoCl₂ cobalt chloride

ECA/ECAR extracellular acidification rates

FBS fetal bovine serum

FCM flow cytometry

ginseng Panax ginseng C.A.Meyer

GS ginseng saponins

HIF1α hypoxia inducible factor 1 alpha

HPLC high-performance liquid chromatography

H/R hypoxia/reoxygenation

MMP mitochondrial membrane potential

NAD⁺/NADH nicotinamide adenine dinucleotide

NAM nicotinamide

NF-κB nuclear factor kappa-light-chain-enhancer of activated B

O₂ oxygen

OCR oxygen consumption rates

OXPHOS oxidative phosphorylation

PGC1α peroxisome proliferator-activated receptor γ coactivator-1 alpha

PI3K phosphatidylinositol-3-OH kinase

ROS reactive oxygen species

SIRT1 sirtuin 1, silent information regulator 2 homologue 1

Ethics approval

Not applicable.

Competing interests

The authors have no conflicts of interest to declare.

Authors' contributions

(applicable for submissions with multiple authors) Tingting Lou: Conceptualization, Writing – original draft. Qingxia Huang: Supervision, Writing – review & editing. Xiangyan Li: Project administration. Daqing Zhao: Funding acquisition.

Funding

This work was supported by the Science and Technology Development Plan Project of Jilin Province (No. YDZJ202201ZYTS266, 202002053JC and 20200201419JC), and the National Natural Science Foundation of China (No. 82104432 and U19A2013).

Availability of data and material

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Ahuja, A., Kim, J. H., Kim, J. H., Yi, Y. S. and Cho, J. Y. (2018) Functional role of ginseng-derived compounds in cancer. J Ginseng Res, 42, 248-254.
Al-Kuraishy, H., Al-Hussaniy, H., Al-Gareeb, A., Negm, W., El-Kadem, A., Batiha, G., N Welson, N., Mostafa-Hedeab, G., Qasem, A. and Conte-Junior, C. (2022) Panax ginsengCombination of C. A. Mey and Febuxostat Boasted Cardioprotective Effects Against Doxorubicin-Induced Acute Cardiotoxicity in Rats. Frontiers in pharmacology, 13, 905828.
Chen, Y., Liu, L., Xia, L., Wu, N., Wang, Y., Li, H., Chen, X., Zhang, X., Liu, Z., Zhu, M., Liao, Q. and Wang, J. (2022) TRPM7 silencing modulates glucose metabolic reprogramming to inhibit the growth of ovarian cancer by enhancing AMPK activation to promote HIF-1α degradation. Journal of experimental & clinical cancer research : CR, 41, 44.
Chitra, L. and Boopathy, R. (2013) Adaptability to hypobaric hypoxia is facilitated through mitochondrial bioenergetics: an in vivo study. Br J Pharmacol, 169, 1035-1047.
DB, C., R, Y., JK, T., D, Z., PJ, D. N. and JD, M. (2017) Transit and integration of extracellular mitochondria in human heart cells. Scientific reports, 7, 17450.
Fuhrmann, D. C. and Brune, B. (2017) Mitochondrial composition and function under the control of hypoxia. Redox Biol, 12, 208-215.
Gasanov, S., Kim, A., Yaguzhinsky, L. and Dagda, R. (2018) Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity. Biochimica et biophysica acta. Biomembranes, 1860, 586-599.
Giordano, F. J. (2005) Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest, 115, 500-508.
Han, B., Jiang, W., Liu, H., Wang, J., Zheng, K., Cui, P., Feng, Y., Dang, C., Bu, Y., Wang, Q., Ju, Z. and Hao, J. J. T. (2020) Upregulation of neuronal PGC-1α ameliorates cognitive impairment induced by chronic cerebral hypoperfusion. 10, 2832-2848.
Hanson, E. S. and Leibold, E. A. (1999) Regulation of the iron regulatory proteins by reactive nitrogen and oxygen species. Gene Expr, 7, 367-376.
Huang, Q., Su, H., Qi, B., Wang, Y., Yan, K., Wang, X., Li, X. and Zhao, D. (2021) A SIRT1 Activator, Ginsenoside Rc, Promotes Energy Metabolism in Cardiomyocytes and Neurons. Journal of the American Chemical Society, 143, 1416-1427.
Huss, J. M. and Kelly, D. P. (2005) Mitochondrial energy metabolism in heart failure: a question of balance. J Clin Invest, 115, 547-555.
Hynes, J., Nadanaciva, S., Swiss, R., Carey, C., Kirwan, S. and Will, Y. (2013) A high-throughput dual parameter assay for assessing drug-induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays. Toxicology in vitro : an international journal published in association with BIBRA, 27, 560-569.
Katsyuba, E., Mottis, A., Zietak, M., De Franco, F., van der Velpen, V., Gariani, K., Ryu, D., Cialabrini, L., Matilainen, O., Liscio, P., Giacchè, N., Stokar-Regenscheit, N., Legouis, D., de Seigneux, S., Ivanisevic, J., Raffaelli, N., Schoonjans, K., Pellicciari, R. and Auwerx, J. (2018) De novo NAD synthesis enhances mitochondrial function and improves health. Nature, 563, 354-359.
Kim, J. H., Yi, Y. S., Kim, M. Y. and Cho, J. Y. (2017) Role of ginsenosides, the main active components of Panax ginseng, in inflammatory responses and diseases. J Ginseng Res, 41, 435-443.
Kong, H. L., Li, Z. Q., Zhao, S. M., Yuan, L., Miao, Z. L., Liu, Y. and Guan, R. M. (2015) Apelin-APJ effects of ginsenoside-Rb1 depending on hypoxia-induced factor 1alpha in hypoxia neonatal cardiomyocytes. Chin J Integr Med, 21, 139-146.
Li, F., Fan, X. X., Chu, C., Zhang, Y., Kou, J. P. and Yu, B. Y. (2018) A Strategy for Optimizing the Combination of Active Components Based on Chinese Medicinal Formula Sheng-Mai-San for Myocardial Ischemia. Cell Physiol Biochem, 45, 1455-1471.
Li, Z., Xu, X., Leng, X., He, M., Wang, J., Cheng, S. and Wu, H. (2017) Roles of reactive oxygen species in cell signaling pathways and immune responses to viral infections. Arch Virol, 162, 603-610.
Lian, L. H., Jin, Q., Song, S. Z., Wu, Y. L., Bai, T., Jiang, S., Li, Q., Yang, N. and Nan, J. X. (2013) Ginsenoside Rh2 Downregulates LPS-Induced NF- kappa B Activation through Inhibition of TAK1 Phosphorylation in RAW 264.7 Murine Macrophage. Evid Based Complement Alternat Med, 2013, 646728.
Liu, Z., Chen, J., Huang, W., Zeng, Z., Yang, Y. and Zhu, B. (2013) Ginsenoside Rb1 protects rat retinal ganglion cells against hypoxia and oxidative stress. Mol Med Rep, 8, 1397-1403.
Liu, Z., Pan, H., Zhang, Y., Zheng, Z., Xiao, W., Hong, X., Chen, F., Peng, X., Pei, Y., Rong, J., He, J., Zou, L., Wang, J., Zhong, J., Han, X. and Cao, Y. (2022) Ginsenoside-Rg1 attenuates sepsis-induced cardiac dysfunction by modulating mitochondrial damage via the P2X7 receptor-mediated Akt/GSK-3β signaling pathway. Journal of biochemical and molecular toxicology, 36, e22885.
Llanos, P. and Palomero, J. J. C. (2022) Reactive Oxygen and Nitrogen Species (RONS) and Cytokines-Myokines Involved in Glucose Uptake and Insulin Resistance in Skeletal Muscle. 11.
Luo, C., Widlund, H. R. and Puigserver, P. (2016) PGC-1 Coactivators: Shepherding the Mitochondrial Biogenesis of Tumors. Trends Cancer, 2, 619-631.
MacIntyre, N. R. (2014) Tissue hypoxia: implications for the respiratory clinician. Respir Care, 59, 1590-1596.
Merkyte, V., Longo, E., Jourdes, M., Jouin, A., Teissedre, P. L. and Boselli, E. (2020) High-Performance Liquid Chromatography-Hydrogen/Deuterium Exchange-High-Resolution Mass Spectrometry Partial Identification of a Series of Tetra- and Pentameric Cyclic Procyanidins and Prodelphinidins in Wine Extracts. J Agric Food Chem, 68, 3312-3321.
Neves, D., Goodfellow, B., Vieira, S. and Silva, R. (2022) The role of NAD metabolism in neuronal differentiation. Neurochemistry international, 159, 105402.
Ni, X., Wang, H., Cai, Y., Yang, D., Alolga, R., Liu, B., Li, J. and Huang, F. (2022) Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke. Redox biology, 54, 102363.
Oh, J., Yoon, H. J., Jang, J. H., Kim, D. H. and Surh, Y. J. (2019) The standardized Korean Red Ginseng extract and its ingredient ginsenoside Rg3 inhibit manifestation of breast cancer stem cell-like properties through modulation of self-renewal signaling. J Ginseng Res, 43, 421-430.
Park, C. M., Kim, K. T. and Rhyu, D. Y. (2021) Exposure to a low concentration of mixed organochlorine pesticides impairs glucose metabolism and mitochondrial function in L6 myotubes and zebrafish. J Hazard Mater, 414, 125437.
Park, J. K., Namgung, U., Lee, C. J., Park, J. O., Jin, S. H., Kwon, O. B., Ko, S. R., Kim, S. W., Kang, E. J., Ko, J. H., Lee, S. M., Kim, D. H. and Won, M. H. (2005) Calcium-independent CaMKII activity is involved in ginsenoside Rb1-mediated neuronal recovery after hypoxic damage. Life Sci, 76, 1013-1025.
Peng, J., Yang, Z., Li, H., Hao, B., Cui, D., Shang, R., Lv, Y., Liu, Y., Pu, W., Zhang, H., He, J., Wang, X. and Wang, S. J. I. j. o. m. s. (2023) Quercetin Reprograms Immunometabolism of Macrophages via the SIRT1/PGC-1α Signaling Pathway to Ameliorate Lipopolysaccharide-Induced Oxidative Damage. 24.
Plecita-Hlavata, L. and Jezek, P. (2016) Integration of superoxide formation and cristae morphology for mitochondrial redox signaling. Int J Biochem Cell Biol, 80, 31-50.
Pokharel, Y. R., Kim, N. D., Han, H. K., Oh, W. K. and Kang, K. W. (2010) Increased ubiquitination of multidrug resistance 1 by ginsenoside Rd. Nutr Cancer, 62, 252-259.
Qin, L., Fan, S., Jia, R. and Liu, Y. (2018) Ginsenoside Rg1 protects cardiomyocytes from hypoxia-induced injury through the PI3K/AKT/mTOR pathway. Pharmazie, 73, 349-355.
Redza-Dutordoir, M. and Averill-Bates, D. A. (2016) Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta, 1863, 2977-2992.
Shuangyan, W., Ruowu, S., Hongli, N., Bei, Z. and Yong, S. (2012) Protective effects of Rg2 on hypoxia-induced neuronal damage in hippocampal neurons. Artif Cells Blood Substit Immobil Biotechnol, 40, 142-145.
Sun, C., Bai, S., Liang, Y., Liu, D., Liao, J., Chen, Y., Zhao, X., Wu, B., Huang, D., Chen, M., Wu, D. J. B., Biomedecine, p. and pharmacotherapie. (2023) The role of Sirtuin 1 and its activators in age-related lung disease. 162, 114573.
Suzuki, E., Okuda, H., Nishida, K., Fujimoto, S. and Nagasawa, K. (2010) Protective effect of nicotinamide against poly(ADP-ribose) polymerase-1-mediated astrocyte death depends on its transporter-mediated uptake. Life Sci, 86, 676-682.
Tian, F., Li, Q., Shi, L., Li, J., Shi, M., Zhu, Y., Li, H. and Ge, R. (2022) In utero bisphenol AF exposure causes fetal Leydig cell dysfunction and induces multinucleated gonocytes by generating oxidative stress and reducing the SIRT1/PGC1α signals. Toxicology and applied pharmacology, 447, 116069.
Umbrasas, D., Jokubka, R., Kaupinis, A., Valius, M., Arandarcikaite, O. and Borutaite, V. (2019) Nitric Oxide Donor NOC-18-Induced Changes of Mitochondrial Phosphoproteome in Rat Cardiac Ischemia Model. Medicina (Kaunas), 55.
Wang, J., Chen, P., Cao, Q., Wang, W. and Chang, X. (2022) Traditional Chinese Medicine Ginseng Dingzhi Decoction Ameliorates Myocardial Fibrosis and High Glucose-Induced Cardiomyocyte Injury by Regulating Intestinal Flora and Mitochondrial Dysfunction. Oxidative medicine and cellular longevity, 2022, 9205908.
Wang, J., Tian, L., Khan, M. N., Zhang, L., Chen, Q., Zhao, Y., Yan, Q., Fu, L. and Liu, J. (2018) Ginsenoside Rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of NF-kappaB mediated epithelial-mesenchymal transition and stemness. Cancer Lett, 415, 73-85.
Xu, P., Zhang, W., Xie, J., Wen, Y., Zhang, G. and Lu, S. (2020) Shenfu injection prevents sepsis-induced myocardial injury by inhibiting mitochondrial apoptosis. Journal of ethnopharmacology, 261, 113068.
Xu, W., Lai, Y., Pan, Y., Tan, M., Ma, Y., Sheng, H. and Wang, J. (2022) m6A RNA methylation-mediated NDUFA4 promotes cell proliferation and metabolism in gastric cancer. Cell death & disease, 13, 715.
Yang, X., He, T., Han, S., Zhang, X., Sun, Y., Xing, Y. and Shang, H. (2019) The Role of Traditional Chinese Medicine in the Regulation of Oxidative Stress in Treating Coronary Heart Disease. Oxid Med Cell Longev, 2019, 3231424.
Ying, A., Yu, Q. T., Guo, L., Zhang, W. S., Liu, J. F., Li, Y., Song, H., Li, P., Qi, L. W., Ge, Y. Z., Liu, E. H. and Liu, Q. (2018) Structural-Activity Relationship of Ginsenosides from Steamed Ginseng in the Treatment of Erectile Dysfunction. Am J Chin Med, 46, 137-155.
Zeng, D., Wang, J., Kong, P., Chang, C., Li, J. and Li, J. (2014) Ginsenoside Rg3 inhibits HIF-1alpha and VEGF expression in patient with acute leukemia via inhibiting the activation of PI3K/Akt and ERK1/2 pathways. Int J Clin Exp Pathol, 7, 2172-2178.
Zhao, Y., Zhang, J., Zheng, Y., Zhang, Y., Zhang, X., Wang, H., Du, Y., Guan, J., Wang, X. and Fu, J. J. J. o. n. (2021) NAD improves cognitive function and reduces neuroinflammation by ameliorating mitochondrial damage and decreasing ROS production in chronic cerebral hypoperfusion models through Sirt1/PGC-1α pathway. 18, 207.
Zhou, P., Gao, G., Zhao, C., Li, J., Peng, J., Wang, S., Song, R., Shi, H. and Wang, L. (2022) In vivo and protective effects of shengmai injection against doxorubicin-induced cardiotoxicity. Pharmaceutical biology, 60, 638-651.

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You are reading this latest preprint version

Ginsenosides mixture from Panax ginseng C.A.Meyer improves CoCl2-induced oxidative stress and mitochondrial dysfunction through NAD+-dependent SIRT1 activation in cardiomyocytes

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Materials and cell culture

2.2. Standard constituents and GS samples

2.3. High-performance liquid chromatography (HPLC) analysis

2.4. Pretreatment with test drug and hypoxia induction

2.5. Flow cytometry

2.6. Assessment of OCR and ECAR using a fluorescence-based assay

2.7. Quantification of ATP levels and measurement of NAD⁺/NADH ratio

2.8. Western blotting

2.9. Statistical analysis

3. Results

3.1 GS inhibits cobalt chloride-stimulated oxidative stress and mitochondrial damage in H9c2 cells

3.2 GS regulates glucometabolism to exert anti-hypoxic pharmacological effects

3.3 The NAD⁺-SIRT1/PGC1α pathway involves in cardiomyocytes protection of GS on hypoxic stress

3.5 GS downregulates ROS and reverses mitochondrial function damage in H9c2 cells with NAD⁺-SIRT1/PGC1α activation

3.6 Effects of ginsenoside monomers in GS on SIRT1-mediated mitochondrial function in hypoxia-treated H9c2 cardiomyocytes

4. Discussion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

Ginsenosides mixture from Panax ginseng C.A.Meyer improves CoCl2-induced oxidative stress and mitochondrial dysfunction through NAD+-dependent SIRT1 activation in cardiomyocytes

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Materials and cell culture

2.2. Standard constituents and GS samples

2.3. High-performance liquid chromatography (HPLC) analysis

2.4. Pretreatment with test drug and hypoxia induction

2.5. Flow cytometry

2.6. Assessment of OCR and ECAR using a fluorescence-based assay

2.7. Quantification of ATP levels and measurement of NAD+/NADH ratio

2.8. Western blotting

2.9. Statistical analysis

3. Results

3.1 GS inhibits cobalt chloride-stimulated oxidative stress and mitochondrial damage in H9c2 cells

3.2 GS regulates glucometabolism to exert anti-hypoxic pharmacological effects

3.3 The NAD+-SIRT1/PGC1α pathway involves in cardiomyocytes protection of GS on hypoxic stress

3.5 GS downregulates ROS and reverses mitochondrial function damage in H9c2 cells with NAD+-SIRT1/PGC1α activation

3.6 Effects of ginsenoside monomers in GS on SIRT1-mediated mitochondrial function in hypoxia-treated H9c2 cardiomyocytes

4. Discussion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

2.7. Quantification of ATP levels and measurement of NAD⁺/NADH ratio

3.3 The NAD⁺-SIRT1/PGC1α pathway involves in cardiomyocytes protection of GS on hypoxic stress

3.5 GS downregulates ROS and reverses mitochondrial function damage in H9c2 cells with NAD⁺-SIRT1/PGC1α activation