Metabolic switch induced by Cimicifuga racemosa extract prevents mitochondrial damage and oxidative cell death
Abstract Background
Cimicifuga racemosa extract is a well-established therapy for menopausal symptoms. The mechanisms underlying the multiple therapeutic effects of Cimicifuga extract, e.g. reducing hot flushes and profuse sweating are not well defined. Recent studies revealed pronounced effects of Ze 450, a Cimicifuga racemosa extract that was produced by a standardized procedure, on energy metabolism through activation of AMP-activated protein kinase in vitro and beneficial anti-diabetic effects in vivo.
Purpose
The aim of the study was to investigate the effects of Ze 450 on energy metabolism. Since mitochondria are the key regulators of cellular energy homeostasis, we wanted to elucidate whether Ze 450 affects mitochondrial resilience and can provide protection against oxidative damage in neuronal and liver cells.
Methods/study design
In this study, we investigated the effects of Ze 450 (1-200 µg/ml) on mitochondrial integrity and function, and cell viability in models of oxidative stress induced by erastin and RSL-3 in neuronal and liver cells. The effects of Ze 450 in control conditions and after induction of oxidative stress were analyzed using FACS for detecting lipid peroxidation (BODIPY), mitochondrial ROS formation (MitoSOX), mitochondrial membrane potential (TMRE) and cell death (AnnexinV/PI staining). Furthermore, we determined metabolic activity (MTT assay), ATP levels and mitochondrial respiration and glycolysis (oxygen consumption rates; Seahorse).
Results
Ze 450 preserved mitochondrial integrity and ATP levels, and prevented mitochondrial ROS formation, loss of mitochondrial membrane potential and cell death. Notably, Cimicifuga racemosa extract alone did not alter mitochondrial ROS levels, and subtle inhibitory effects on cell proliferation were reversed after withdrawal of the extract. In addition, Ze 450 did not exert toxic effects to liver cells, but rather protected these from the oxidative challenge. Further analysis of the mitochondrial oxygen consumption rate and the extracellular acidification rate revealed that Ze 450 mediated a switch from mitochondrial respiration to glycolysis, and this metabolic shift was a prerequisite for the protective effects against oxidative damage.
Conclusion
In conclusion, the bioenergetic shift induced by Ze 450 exerted protective effects in different cell types, and offers promising therapeutic potential in age related diseases involving oxidative stress and mitochondrial damage.
Keywords: Black cohosh, Cimicifuga racemosa, mitochondria, neuron, liver, oxidative stress, metabolic regulation
Introduction
Cimicifuga racemosa extract is commonly used in the treatment of menopausal symptoms (Schellenberg et al., 2012). Hormonal changes, such as estrogen depletion, lead to menopausal complaints; many women undergoing menopause suffer from vasomotoric symptoms like hot flushes and sweating, mood changes and weight gain. In the treatment of these symptoms, hormone replacement therapy (HRT) is widely used, but this therapy is limited due to adverse effects such as increased risk for breast cancer or thromboembolic 2-DG events (Warren et al., 2000; Rossouw et al., 2002; Beral, 2003). Further, HRT is not approved for peri- menopausal women, and the lack of therapeutic alternatives, as well as the preference of these patients for herbal medicines, illustrates the requirement for further in-depth investigation of Cimicifuga racemosa extract. In fact, Cimicifuga racemosa extract provides beneficial therapeutic effects with a positive benefit-risk profile and is listed in a monograph by the Committee on Herbal Medicinal Products (HMPC) of the European Medicines Agency (EMA) (HMPC, 2010).
Ze 450 is a Cimicifuga racemosa extract that was produced by a standardized procedure and is approved for the treatment of peri-menopausal complaints in doses of 6.5 to 13 mg per day in many European countries. Estrogen depletion is well known to induce common menopausal complaints; however, mounting evidence suggests additional effects, such as increasing oxidative stress which may contribute to the development of metabolic disorders which do not respond to HRT (Sekhon and Agarwal, 2013) and menopausal complaints like hot flushes. In this context, research focuses on the liver as the organ being responsible for the majority of metabolic processes. In addition, altered metabolic regulations in the hypothalamus are of interest, representing the superordinate endocrine control of metabolism and thermoregulatory center. Notably, release of critical amounts of reactive oxygen species (ROS) in this brain region was linked to disrupted neuroendocrine regulation and impaired metabolic homeostasis, thereby inducing e.g. type II diabetes (Drougard et al., 2015). Recent studies revealed that Ze 450 increased AMP-activated kinase A (AMPK) phosphorylation in vitro and ameliorated metabolic disorders in ob/ob mice and in ovariectomized rats in vivo, indicating that effects of the Cimicifuga extract on the bioenergetic metabolism significantly contributed to therapeutic effects against menopausal complaints (Moser et al., 2014; Sun et al., 2016) Thus, investigating effects of Ze 450 on oxidative stress and metabolic regulation in hypothalamic neurons and liver cells shall reveal novel insights into the mechanism of action of the Cimicifuga extract.
In all cells of the body, including neuronal and liver cells, mitochondria are key organelles of energy metabolism and a major source of ROS upon electron leakage. Mitochondrial ROS are important mediators of cellular and mitochondrial damage and often elevated under certain pathological conditions and oxidative stress (Galluzzi et al., 2009). Thus, mitochondria may represent a common target of Ze 450. Increased ROS formation is considered as a driving force for mitochondrial and cellular dysfunction with significant impact on impaired metabolic regulation, inflammatory processes, and cell death. Recent studies revealed that mitochondrial resilience attenuated age-related, immunological and metabolic pathologies, such as diabetes mellitus type II (Held and Houtkooper, 2015). Hence, protecting mitochondria has been proposed a promising therapeutic strategy in the treatment of metabolic diseases related to oxidative stress. Therefore, the major aim of this study was to elucidate effects of Ze 450 on neuronal and liver cells with respect to mitochondrial parameters and oxidative cell toxicity utilizing well-established in vitro models of oxidative stress (Neitemeier et al., 2017; Jelinek et al., 2018).
Material and methods
Cell culture
HT22 cells (kindly provided by David Schubert, Cellular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA) and mHypo cells (Cedarlane®, Cellutions Biosystems Inc., Canada) were grown in Dulbecco‟s modified Eagle medium (DMEM, Capricorn Scientific GmbH, Germany) supplemented with 10 % heat-inactivated fetal calf serum (Merck KGaA, Germany), 100 U/ml penicillin, 100 mg/mL streptomycin (Capricorn Scientific GmbH, Germany) and 2 mM L-glutamine (Merck KGaA, Germany). To induce cell death, 1 µM erastin (Calbiochem®, Germany) was added to the medium for the indicated amount of time (8-16 h).
HepG2 (ATCC®™ HB-8065™) cells were grown in Eagle´s minimum essential medium (EMEM, Merck KGaA, Germany) supplemented with 10 % heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 mg/mL streptomycin. 1S, 3R-RSL-3 (4 µM) (Neitemeier et al., 2017) which was kindly provided by Prof. Diederich (University of Marburg), was added to induce oxidative cell death.
Cimicifuga racemosa extract Ze 450
The ethanolic (60% v/v) Cimicifuga racemosa dry extract Ze 450 was manufactured of dried roots and rhizomes and obtained from Max Zeller and Soehne AG (Romanshorn, Switzerland). The content of triterpeneglycosides was 6.4 %. Ze 450 was dissolved in 60% ethanol (v/v) (Carl Roth GmbH, Germany) for all experiments. Ze 450 conforms to the herbal preparation B, which was granted well-established use in the 2010 Community herbal monograph on Cimicifuga racemosa by the HMPC (HMPC, 2010). HPLC fingerprint of Ze 450 batch is presented in the supplementary material (Fig. S-1).
Cell viability
Cell proliferation was analyzed in real time by measuring electrical impedance (Diemert et al., 2012). Metabolic activity as an indicator for cell viability was quantified using the MTT assay (Neitemeier et al., 2017). Viable and metabolically active cells convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Merck KGaA, Germany), which was added at a concentration of 2.5 mg/mL for 1 hour at 37°C to the culture medium, into purple formazan. Absorbance was measured at 570 nm versus 630 nm with FluoStar (BMG Labtech, Germany) after dissolving in DMSO (Carl Roth GmbH, Germany).
Cell death of HT22, mHypo and HepG2 cells treated with Ze 450, erastin or 1S, 3R-RSL-3 was detected using the Annexin-V-FITC/PI Detection Kit (PromoCell, Germany) followed by fluorescence-activated cell sorting (FACS, guava easyCyte, Merck KGaA, Germany). Annexin-V-FITC was excited at 488 nm, emission was detected through a 525 ± 30 nm bandpass filter. Propidium iodide was excited at 488 nm, fluorescence emission was detected using a 690 ± 50 nm bandpass filter. Data were collected from at least 5‟000 cells with at least three replicates per condition. EC50 values were calculated (non-linear fit: log (Ze 450) vs. response, variable slope) of at least three independent assays using GraphPad Prism Software
6.05 (GraphPad Software, Inc., CA, USA).
Lipid peroxidation
After the indicated treatments, HT22, mHypo and HepG2 cells were stained with BODIPY 581/591 C11 (Invitrogen, USA) for 1 h (37°C, 4.5% CO2) and harvested for FACS analysis. Lipid peroxidation was analyzed by recording green (emission: 525nm/30) and red (emission: 585nm/50) fluorescence with 488 nm excitation wavelength of at least 5‟000 cells of at least three replicates per condition. Levels of lipid peroxidation were calculated by the analysis of the fluorescence shift from green to red fluorescence.
Mitochondrial ROS formation
MitoSOX red (Invitrogen, USA) is selectively targeted to the mitochondria, where it is oxidized by superoxides exhibiting red fluorescence. For detection of mitochondrial ROS formation, MitoSOX red was applied for 30 min at 37°C and cells were harvested for FACS analysis. Increasing red fluorescence correlating with the formation of mitochondrial ROS was detected by FACS analysis (excitation 488 nm, emission 690/50 nm). Data were collected from at least 5‟000 cells and three replicates per condition.
Mitochondrial morphology
MitoTracker® Deep Red FM (Invitrogen, USA, 200 nM) was used to visualize changes in mitochondrial morphology. HT22 cells were seeded in 8-well ibidi slides (Ibidi GmbH, Germany) with 14‟000 cells per well and treated with Ze 450 and erastin. After the indicated treatment time MitoTracker® Deep Red was added to cells and incubated for 30 min at 37°C. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature (RT). Mitochondrial morphology was analyzed by categorizing cells as previously described (Grohm et al., 2010). Category I mitochondria were organized in a tubular network, category II mitochondria were fragmented but equally distributed throughout the cytosol, whereas category III mitochondria were fully fragmented and surrounding the nucleus. At least 500 cells per condition were counted without knowledge of treatment history. Images were acquired using a Leica DMI6000 epi- fluorescence microscope (63x objective), using an excitation wavelength of 620 nm (bandpass filter) and detecting emissions using a 670 nm long pass filter.
ATP measurements
ATP levels were detected using the ViaLightTMplus Kit (Lonza, USA). Twenty four hours post seeding in 96-well plates (6`000 cells per well), cells were treated with Ze 450 and erastin or 1S, 3R-RSL-3. At the indicated time points after treatment (8 and 16 h), cells were transferred into a white 96 well plate and ATP levels were analyzed by luminescence detection with FluoStar OPTIMA (BMG Labtech, Germany).
Seahorse measurements
To determine oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as measures of mitochondrial respiration and glycolysis, respectively, simultaneous real-time measurement were performed using the XF Extracellular Flux Analyzer (Agilent Technologies, United States) as previously described (Neitemeier et al., 2017). Briefly, HT22 cells were plated in XF96-well microplates (6‟000 cells per well, Seahorse Bioscience) and treated with Ze 450 and erastin. At indicated treatment conditions, the growth medium was replaced by ~180 μl of assay medium (with 4.5 g/l glucose as the sugar source, 2 mM glutamine, 1 mM pyruvate, pH 7.35) and cells were incubated at 37°C for 60 min. Three baseline measurements were recorded before adding the compounds. Oligomycin (ATP synthase inhibitor) (Merck KGaA, Germany) was injected into port A (20 µl) at a final concentration of 3 µM, FCCP (uncoupling agent) (22.5 µl into port B) (Merck KGaA, Germany) at a concentration of 0.5 µM, rotenone/antimycin A (complex I/III inhibitors) (25 µl into port C) (Merck KGaA, Germany) at a concentration of 100 nM and 1 µM and 2-deoxyglucose (Carl Roth GmbH, Germany) (glycolysis inhibitor) at a concentration of 50 mM (27.5 µl into port D), respectively. Three measurements were performed after the addition of each compound (4 min mixing followed by 3 min detection).
Mitochondrial membrane potential
After treatment with Ze 450 and erastin or 1S, 3R-RSL-3, cells were stained with TMRE (0.4 nM for 30 min at 37 C, MitoPT ΔΨm Kit, Immunochemistry Technologies, USA) and harvested for TMRE fluorescence measurement via FACS analysis. Upon loss of the mitochondrial membrane integrity and, thus, membrane potential, a loss of TMRE fluorescence can be detected by FACS analysis (excitation 488 nm, emission 690/50 nm). Data were collected from at least 5‟000 cells and three wells per condition.
Statistical analysis
All data are given as mean + or ± standard deviation (SD). Statistical comparison between treatment groups was performed by analysis of variance (two-way ANOVA) followed by Scheffé‟s post hoc test and a p value < 0.05 was considered to be statistically significant. Calculations were executed with Winstat standard statistical software (R. Fitch Software, Germany) and visualized using GraphPad Prism software (GraphPad Software, United States).
Results
Ze 450 prevents neuronal cell death in model systems of oxidative stress
HT22 and mHypo cells were treated for 16 hours with Ze 450 (1 - 200 µg/ml) and oxidative stress was induced with erastin co-treatment (Fig. 1A, C, Fig. S-5 A and B). Inhibition of system Xc- by erastin, provokes cellular depletion of glutathione thereby disrupting the glutathione peroxidase 4 (GPX4) redox homeostasis leading to massive ROS formation, mitochondrial damage and cell death (Landshamer et al., 2008; Neitemeier et al., 2017).
Ze 450 did not lead to an increase in Annexin V/PI positive cells (Fig.1 A and C), but prevented erastin- mediated cell death at concentrations greater than 50 µg/ml. Metabolic activity was determined using the MTT assay. Ze 450 slightly reduced metabolic activity at concentrations higher than 100 µg/ml of the extract in HT22 cells (Fig.1 B), while metabolic activity of mHypo cells was not affected by Ze 450 (Fig.1 D). Reduced metabolic activity indicating cell death after exposure to erastin was partly preserved by Ze 450 in mHypo and HT22 cells (Fig.1 B and D). Further, real-time impedance measurements showed that proliferation of HT22 cells was reduced at Ze 450 concentrations greater than 100 µg/ml compared to controls (Fig. 1 E). These real-time measurements revealed that 10 and 50 µg/ml of Ze 450 conducted to a transient protection against erastin-toxicity while at concentrations higher than 100 µg/ml the extract mediated a permanent protective effect (Fig. 1 F). The same effects were observed with the erastin challenge in mHypo cells (Fig. S-2 A and B).
Ze 450 mediates protection against erastin-induced mitochondrial impairment
Lipid peroxidation is an early event in the model system of erastin-induced oxidative cell death. Cellular ROS formation, due to an increased activity of 12/15 lipoxygenases, was investigated using C11- BODIPY FACS analysis at 8 h and 16 h of treatment in HT22 and mHypo cells. Ze 450 did not increase cellular ROS formation, but abolished erastin-mediated increases in lipid peroxidation at concentrations higher than 50 µg/ml as detected at both time points in HT22 cells (Fig. 2 A and B, Fig. S-5 C and D). The same effect was observed in mHypo cells (Fig. S-3 A). We further analyzed the influence of Ze 450 on ATP levels to gain insights into mitochondrial involvement in the neuroprotective effects of Ze 450. In mHypo and HT22 cells, Ze 450 reduced cellular ATP levels to ~70 % upon concentrations greater than 150 µg/ml under standard culture conditions (Fig. S-3 B, Fig. 2 C). After erastin- exposure, ATP depletion was partly prevented by concentrations greater than 100 µg/ml in HT22 cells and by 50-150 µg/ml of Ze 450 in mHypo cells (Fig. 2 C, Fig. S-3 B).
Mitochondrial ROS are important mediators of cellular stress, and mitochondrial dysfunction is associated with increased mitochondrial ROS formation and associated pathways of oxidative cell death. Mitochondrial ROS formation was investigated using MitoSOX red staining and FACS analysis in HT22 and mHypo cells at 16 h after treatment. In both neuronal cell lines, Ze 450 did not affect mitochondrial ROS accumulation at basal conditions (Fig. 2 D, Fig. S-3 C, Fig. S-5 E). In contrast, erastin-induced increase in mitochondrial ROS formation was prevented by concentrations of Ze 450 higher than 50 µg/ml (Fig. 2 D, Fig. S-3 C). This effect was also observed in a post-treatment paradigm, when Ze 450 was applied up to 1 h after onset of the erastin exposure (Fig. 2 E).
Next, mitochondrial membrane potential was analyzed after 16 h of treatment in HT22 and mHypo cells using TMRE. Mitochondrial membrane potential was not affected by Ze 450 in both neuronal cell lines under standard culture conditions (Fig. 2 F, Fig. S-3 D, Fig, S-5 F), but loss of mitochondrial membrane potential due to erastin exposure was prevented by concentrations greater than 50 µg/ml of Ze 450 (Fig. 2 F, Fig. S-3 D). Overall, Ze 450 demonstrated strong protective effects at the level of mitochondria in the applied model system of erastin-induced oxidative stress.
Ze 450 induces a bioenergetic shift from mitochondrial respiration to glycolysis
Mitochondria are crucial organelles responsible for the energy metabolism and energy supply for the whole cell and play a decisive role in regulated oxidative cell death. As we have shown in Fig. 2 A and B, Ze 450 mediates protection against erastin-mediated lipid peroxidation. To gain further insights into potential metabolic effects such as alterations of mitochondrial respiration and metabolic status of cells exposed to Ze 450, OCR and ECAR were investigated using the Agilent Seahorse XF96 system for detection of oxidative phosphorylation and glycolysis, respectively. These functional metabolic parameters are essential for further elucidating the mechanism of action of Ze 450 at mitochondria. Fig. 3 A shows that Ze 450 reduced the OCR compared to control cells; especially the basal and maximal respiration were affected. After erastin exposure, OCR was not rescued by Ze 450 (Fig. 3 B). Cells performed less mitochondrial respiration, but showed an enhanced glycolysis rate compared to control cells. 100 µg/ml of Ze 450 alone slightly increased the ECAR compared to control cells (Fig. 3 C). Fig. 3 D depicts a dose dependent rescue of erastin-mediated reduction of ECAR. Fig. 3 E illustrates that the response after oligomycin injection (port A) was much steeper in control cells compared to cells treated with 100 µg/ml Ze 450 and erastin. Oligomycin inhibits the ATP-Synthase of the respiratory chain and thereby increases ECAR. Ze 450 induced a shift towards glycolysis and as a consequence the increase in glycolysis by oligomycin was not as prominent in the cells pretreated with Ze 450 compared to the corresponding controls. Overall, Ze 450 reduced OCR in a concentration-dependent manner under basal conditions and after erastin exposure, and prevented erastin- mediated reduction of the ECAR, thereby, preventing mitochondrial ROS formation, deletion of mitochondrial membrane potential and preserving ATP levels.
The question remained, whether this observed bioenergetic shift to glycolysis induced by Ze 450 was linked to the observed cytoprotection by Ze 450. To answer this question, glycolysis was blocked using 2-DG, and cell viability was analyzed by Annexin V/PI staining and FACS analysis. 100 mM of 2-DG in combination with 100 µg/ml of Ze 450 slightly increased Annexin V and PI positive cells under control conditions (Fig. 3 F). As observed previously, erastin- cytotoxicity was rescued by 100 µg/ml of Ze 450, but 2-DG abolished this protective effect of the extract (Fig. 3 F, Fig. S-6 A).
In addition to investigating mitochondrial functionality, we further analyzed mitochondrial morphology. Mitochondrial shape was evaluated by classifying mitochondria as previously described (Grohm et al., 2010). Mitochondria build a tubular network throughout the cytosol of the cell under standard culture conditions; in contrast, mitochondrial fragmentation is a common feature of oxidative induced neuronal cell death (Grohm et al., 2010). In the present study, cytotoxicity induced by erastin was associated with massive fragmentation of mitochondria and accumulation of the organelles around the nucleus (Fig. 4 B). Ze 450 did not cause mitochondrial fragmentation under standard culture conditions and the cells showed
similar mitochondrial morphology as the control cells (Fig. 4 B). Furthermore, concentrations greater than 50 µg/ml of Ze 450 prevented mitochondrial fragmentation despite erastin treatment (Fig. 4 A and B).
Ze 450 prevents cell death and mitochondrial dysfunction in HepG2 liver cells exposed to oxidative stress
The hepatoma cell line HepG2 is widely used for toxicological studies (Doostdar et al., 1988). Here, we focused on aspects of cytotoxicity and mitochondrial involvement in response to oxidative stress. Since erastin treatment only exerted minor toxicity in the hepatoma cells, the GPX4 inhibitor was applied in this liver cell line to induce oxidative stress. RSL-3 (Yang et al., 2014) acts downstream of the cysteine/glutamate antiporter and directly blocks the GPX4 independent of glutathione depletion thereby leading to lipid peroxide accumulation and to similar death signaling pathways such as erastin treatment. Fig. 5 A shows that Ze 450 did not induce cell death at concentrations up to 200 µg/ml. Moreover, at concentrations higher than 50 µg/ml Ze 450 protected against RSL-3- mediated cytotoxicity (Fig. 5 A, Fig. S-7 A). As calculated from further analyses using AnnexinV/PI staining and FACS analysis, Ze 450 was only toxic to liver cells at very high concentrations above the therapeutic range relevant for the treatment of postmenopausal complaints (EC50 of 362.8 µg/ml) (Fig. S-4 A and B).
As detected in the neuronal cell lines, Ze 450 affects mitochondrial function inducing a bioenergetic shift from mitochondrial respiration towards glycolysis that mediated the protection of mitochondrial integrity and reduced mitochondrial ROS formation. Therefore, we also investigated the effects of Ze 450 on mitochondrial parameters on HepG2 cells. ATP levels remained unaltered by Ze 450 at concentrations up to 200 µg/ml, while RSL-3- mediated ATP depletion was rescued by Ze 450 at concentrations higher than 100 µg/ml (Fig. 5 B). Further analyses of lipid ROS levels using C11-BODIPY staining and FACS analysis revealed that Ze 450 did not increase lipid-peroxidation after 8 h and 16 h (Fig. 5 C and D, Fig. S-7 B and C). In the model system of HepG2 cells, Ze 450 attenuated RSL-3- mediated mitochondrial ROS formation, while Ze 450 alone did not induce an increase in mitochondrial ROS levels (Fig. 5 E, Fig. S-7 D). Moreover, mitochondrial membrane potential was not affected by Ze 450, while the RSL-3- induced loss of mitochondrial membrane potential was prevented by concentrations of Ze 450 greater than 10 µg/ml (Fig. 5 F, Fig. S-7 E). Overall, these results showed that Ze 450 did not exert toxic effects to the liver cells, but rather protected these from the oxidative challenge.
Discussion
The aim of the present study was to investigate the effects of Ze 450 on neuronal and liver cells with a particular focus on mitochondrial parameters in model systems of oxidative stress induced by erastin or RSL-3. The results show that Ze 450 protects against erastin- mediated mitochondrial damage and cell death in different cell types, including neuronal HT22 cells, which were established as a suitable model system to study oxidative stress (Li et al., 1997; Neitemeier et al., 2017) and mHypo cells, because the hypothalamus functions as the superordinate endocrine control center of metabolism and thermoregulation, and is thought to be mainly involved in mediating menopausal symptoms such as hot flushes and associated metabolic impairments. Similar protective effects were also detected at the level of metabolic activity and cell proliferation which were rescued by Ze 450 after the induction of oxidative stress. Most intriguingly, these protective effects of Ze 450 at the level of mitochondria and on cell viability were attributed to a bioenergetic shift from oxidative phosphorylation to glycolysis; in particular, concentrations of Ze 450 higher than 100 µg/ml were found to reduce ATP levels, while cell viability was not affected. Monitoring cell proliferation in real-time showed that Ze 450 (>100 µg/ml) reduced cell growth (Fig. 1 E), but this effect was reversible after Ze 450 withdrawal (Fig. S-2 C). These data imply a major effect of Ze 450 on the bioenergetic metabolism of the cells which is likely linked to the previously established activation of AMPK as a prerequisite for the observed protective effects against damage of mitochondria and cells exposed to oxidative stress, and which may as well underlie the therapeutic effects of Ze 450 against post-menopausal complaints.
Previous studies have shown that mitochondria play a crucial role in determining cell viability in conditions of oxidative stress (Landshamer et al., 2008; Tobaben et al., 2011); once mitochondrial function and integrity are impaired, cells are unable to survive (Galluzzi et al., 2009). Mitochondrial damage was revealed as a major hallmark of neuronal oxidative death, for example, after erastin treatment (Neitemeier et al., 2017). Further, oxidative stress and associated mitochondrial impairments are involved in the pathogenesis of metabolic diseases like diabetes mellitus type II (Reagan et al., 2000) underlining the importance to identify strategies to protect mitochondria by increasing their ability to handle oxidative stress and associated metabolic dysfunction. Besides this, oxidative stress has been closely linked to menopausal complaints like hot flushes (Agarwal et al., 2013). The present study demonstrates a strong influence of Ze 450 on mitochondrial function, thereby preventing hippocampal and hypothalamic cells from oxidative damage. It has been shown that massive ROS production due to an increase in 12/15 lipoxygenases activity (Seiler et al., 2008; Tobaben et al., 2011) leads to functional mitochondrial impairments and following perturbation of cellular redox balance (Liot et al., 2009). In particular, oxidative stress-induced lipid peroxidation was completely abolished by concentrations higher than 50 µg/ml of Ze 450. Notably, this pronounced protective effect was present after 8 h, but also after 16 h of the oxidative challenge. This finding suggested that even the secondary ROS burst initiated through mitochondrial impairment was compensated by Ze 450 (Fig. 2 A and B). As previously described, mitochondrial impairment, namely mitochondrial ROS formation and loss of mitochondrial membrane potential, are crucial events marking the so called “point of no return” in neuronal cell death (Doti et al., 2014; Neitemeier et al., 2014; Reuther et al., 2014; Neitemeier et al., 2017). Ze 450 mediates protection against these processes, thereby, preserving mitochondrial integrity and cell viability (Fig. 1 A, Fig. 2 D and F, Fig. 4 A and B).
Mitochondria are the powerhouses of the cell; therefore, respiratory chain activity and oxidative phosphorylation are important in meeting cellular energy demands. Assessing the oxygen consumption rate is a helpful and established method to investigate mitochondrial respiration; surprisingly, we observed a concentration-dependent reduction of OCR by Ze 450 (Fig. 3 A), without affecting cell viability. Concomitantly, cells treated with 100 µg/ml of Ze 450 showed an increase in ECAR suggesting that the energy metabolism was switched from oxidative phosphorylation to glycolysis in cells exposed to Ze 450 (Fig. 3 C). Ze 450 may act as an inhibitor of respiratory chain complexes leading to a reduction of mitochondrial respiration, but also other mechanisms leading to an enhanced rate of glycolysis, like an activation of AMPK (Moser et al., 2014), may contribute to this observed shift in energy metabolism. In a previous study, AMPK has been exposed as a target of Ze 450 in HepaRG cells crucially involved in the regulation of cell metabolism (Moser et al., 2014). Hence, AMPK activation by Ze 450 is proposed as a potential mechanistic link between mitochondrial parameters and cellular responses to oxidative stress. In turn, however, the previously described activation of AMPK by Ze 450 could also result from the bioenergetic shift and the drop of ATP detected here after incubation of cells with Ze 450 under control conditions.
Notably, inhibition of glycolysis by 2-DG supported the proposed link between the bioenergetic shift induced by Ze 450 and the observed protective effects. In fact, 2-DG abolished the protection against oxidative stress (Fig. 3 F), and, consequently, it was concluded that mitochondrial protection and cell survival after oxidative stress was attributed to the metabolic switch from mitochondrial respiration to glycolysis.
Moreover, the protective effects of Ze 450 in neuronal cells were confirmed in liver cells. This is of particular interest, since there is an ongoing debate about Cimicifuga extract causing liver toxicity in vivo. For example, there are reports associating Cimicifuga racemosa extract to hepatic side effects in patients with possible yet unknown risk-factors (Huntley and Ernst, 2003; Low Dog et al., 2003). In rats, Ze 450 caused microvesicular steatosis of the liver at a very high dose of 1000 mg/kg body weight (Lüde et al.,2007). However, the authors concluded that these high concentrations are very unlikely to be reached in humans treated with the recommended daily doses of 6.5 to 13 mg extract (Lüde et al., 2007). Our results obtained in cultured liver cells show a pronounced protection against oxidative damage and further demonstrate that the concentrations leading to the impairment of liver cells are much greater than the concentrations required for protective effects (Fig. S-4 A and B). We found protective effects of Ze 450 (50- 200 µg/ml) against RSL-3 induced cell death, and showed that 1-200 µg/ml of Ze 450 did not affect cell viability (Fig. 5 A). In particular, in liver cells we further observed strong protective effects of Ze 450 on ATP depletion, lipid peroxidation, mitochondrial ROS formation and loss of mitochondrial membrane potential (Fig. 5 B, C, D, E and F) which were consistent with similar findings in neuronal cells. These findings rather suggest that Ze 450 shows beneficial effects on diseases related to oxidative liver damage, but will need further evaluation in vivo.
In conclusion, Ze 450 mediates protective effects on neuronal and liver cells exposed to oxidative stress. The results further suggest that the bioenergetic shift induced by Ze 450 from mitochondrial oxidative phosphorylation to glycolysis plays an important role in the observed resilience of the cells exposed to oxidative stress. Therefore, Ze 450 offers promising therapeutic potential in diseases related to oxidative stress, including age-related diseases affecting neuronal maintenance and function as well as metabolic diseases associated with mitochondrial dysfunction. The observed bioenergetic shift may be also a key to the mechanistic understanding of Ze 450-mediated symptom relief in menopausal women and, further, offers therapeutic potential in the RSL3 treatment of metabolic diseases such as diabetes mellitus type II.