Flavonoids from Potentilla anserina protect H9C2 cells from hypoxia/reoxygenation injury by inhibiting mitochondrial apoptosis

Abstract

Potentilla anserina L., a valued traditional Chinese medicinal herb and edible plant, has a long history of use for its antioxidant and immunomodulatory properties. Flavonoids extracted from the tuberous roots of P. anserina (silverweed cinquefoil root flavonoids, SCR-F) exhibit protective effects against hypoxia/reoxygenation (H/R) injury in H9C2 cells. In our study, we observed that H/R injury reduces cell viability, increases lactate dehydrogenase release, and elevates levels of reactive oxygen species (ROS) and malondialdehyde (MDA). The protective action of SCR-F on H/R-injured H9C2 cells was concentration-dependent, with efficacy increasing alongside dosage. Notably, the strongest protective effect occurred at a concentration of 208.32 μg/ml of SCR-F. Treatment with SCR-F significantly improved cell viability decreased ROS and MDA levels, and enhanced the activity of antioxidant enzymes, including catalase, superoxide dismutase, and glutathione peroxidase (GSH-px). Furthermore, SCR-F upregulated the expression of the anti-apoptotic protein Bcl-2, downregulated the pro-apoptotic protein Bax, and decreased caspase-3 and -9 activity. A reduction in voltage-dependent anion channel 1protein coding gene (VDAC)1 expression was also observed, suggesting inhibition of the mitochondrial apoptotic pathway. Our findings indicate that SCR-F protects H9C2 cells from H/R injury by reducing oxidative stress and mitochondrial-mediated apoptosis, underscoring its potential as a therapeutic agent for hypoxia-induced myocardial damage.

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Introduction

Hypoxic conditions in high-altitude regions lead to reduced oxygen availability, affecting human and livestock survival. Hypoxia decreases total antioxidant capacity (T-AOC), depletes ATP, and increases reactive oxygen species (ROS) accumulation, triggering oxidative stress and inflammation (Ajith, 2010; Dubouchaud et al., 2018). Excessive ROS oxidises unsaturated fatty acids, producing malondialdehyde (MDA), which exacerbates oxidative stress (Turton et al., 1997). Hypoxia also increases vascular permeability and stimulates the production of inflammatory mediators, thereby intensifying inflammatory responses (Zhou et al., 2022). Inflammatory tissues can further aggravate hypoxia due to increased cellular metabolic demands coupled with substrate shortages, with these factors often coexisting and interacting (Hsu et al., 2019).

The NF-κB/TNF-α pathway is pivotal in regulating inflammatory responses and is significantly influenced by endogenous ROS, as demonstrated in studies by Lee et al. (2016) and Subhan et al. (2017). Oxidative stress induces upregulation of TNF-α expression, which activates NF-κB, a downstream target. This activation triggers the expression of related genes and receptors, leading to NF-κB translocation into the nucleus, where it binds to κB sequences, promoting transcription of inflammation-related genes. Studies have shown that reducing inflammatory responses can significantly protect against and improve recovery from hypoxia-induced damage (Hoesel & Schmid, 2013; Lee et al., 2016). Xu and Lu (2019) found that immunosuppressive therapy can alleviate pulmonary inflammation, reduce ROS production, and thereby mitigate lung damage.

Hypoxia also disrupts energy metabolism by inhibiting the respiratory chain, reducing ATP production, and increasing glycolysis. Lactate dehydrogenase (LDH) activity rises, shifting metabolism towards lactate production, impairing the tricarboxylic acid (TCA) cycle, and causing mitochondrial damage (Ebert et al., 2019; Long et al., 2020; Umemoto et al., 2018). Mitochondrial dysfunction promotes apoptosis by reducing membrane potential, releasing cytochrome c (Cyt c), and activating caspase cascades (Brentnall et al., 2013; Gupta et al., 2016; He et al., 2017; Park et al., 2019; Zhang et al., 2013).

Currently, anti-hypoxia medications available on the market fall into two categories: chemical pharmaceuticals and traditional Chinese medicine extracts. Chemical drugs include a variety of compounds such as acetazolamide, dexamethasone, aminophylline, nifedipine, and nimodipine. Studies have shown that these drugs enhance ventilation and oxygenation by relaxing vascular smooth muscle and bronchial tissue, increasing pulmonary arterial blood flow, and activating the Nrf-2 pathway, ultimately increasing oxygen saturation (Lisk et al., 2013; Lu et al., 2020). Additionally, these drugs can help reduce pulmonary hypertension and hypoxia-induced inflammatory responses in the lungs (Crea et al., 1994). However, while chemical drugs have some antihypoxic effects, long-term use can lead to side effects, such as kidney damage. This has led to a growing interest in highly effective antihypoxic drugs of natural origin that have minimal side effects.

Potentilla anserina L., a perennial herb from the Rosaceae family, is characterised by its bulbous, large root, commonly referred to as “ginseng fruit.” Known for its medicinal and edible qualities, P. anserina is traditionally used to relieve cough and phlegm, ease tissue contractions, reduce glandular secretions, treat haemorrhaging, and aid in digestion (Li et al., 2024). Reports indicate that P. anserina exhibits various beneficial properties, including antihypoxia, antioxidant, anti-inflammatory, antidiabetic, and weight loss effects (Tang et al., 2022). As a natural plant, it exhibits a favourable safety profile with no toxic side effects. Numerous researchers have demonstrated its safety through murine experiments and cellular studies. Notably, even when mice were administered the maximum dose of 345.6 g/kg extract within 12 hr, no abnormalities were observed in renal function, hepatic function, or other pathological examinations, with no signs of toxicity detected (Dram et al., 2020; Kurskaya et al., 2022). Current research on P. anserina mainly focuses on the extraction and efficacy validation of its polysaccharides (Ghasemi et al., 2024; Olennikov et al., 2015), polyphenols (Olennikov et al., 2015), and saponins (Zhao et al., 2008), with relatively fewer studies examining the plant’s flavonoid content.

Flavonoids are known for their broad range of biological activities; research has shown that plant-derived flavonoids, such as those from Iberis amara, can protect myocardial tissue from low-pressure and hypoxic damage by alleviating oxidative stress, enhancing energy metabolism, and modulating apoptosis-related proteins (Liu et al., 2021). Additionally, total flavonoids from Rosa laevigata have been shown to reduce myocardial cell damage caused by hypoxia/reoxygenation (H/R) by upregulating miR-1247-3p expression, inhibiting oxidative stress, and suppressing apoptosis (Liu et al., 2023). Studies have also shown that flavonoids are the most abundant compounds in P. anserina L., accounting for approximately 37.66% of the total (Li et al., 2024). Many of these flavonoid compounds can enhance antioxidant capacity by inhibiting enzyme activity (Yang et al., 2021a).

In our previous study, we identified 42 distinct flavonoid compounds in the total flavonoids extracted from Xizang P. anserina, which demonstrated significant antioxidant activity in vitro (Yan et al., 2023). Based on these findings, we hypothesise that the flavonoids in Xizang P. anserina may offer a protective effect against hypoxia-induced myocardial tissue damage. This study, therefore, focuses on Xizang P. anserina, using a low-pressure, hypoxic myocardial injury model to investigate the protective effects of its flavonoids on myocardial tissue under hypoxic conditions. Additionally, this research aims to explore the potential mechanisms underlying these protective effects.

Materials and methods

Extraction and preparation of total flavonoids from P. anserina root

P. anserina L., sourced from Xizang, China, was finely ground and passed through a 60-mesh sieve. A measured quantity of this powder was mixed with 71% ethanol and subjected to sonication at 75 °C for 70 min. The ethanol was then removed using a rotary evaporator, and the extract was dried to yield crude flavonoids from P. anserina root with an extraction yield of 1.3%. After purification with AB-8 macroporous resin, total flavonoids from the root were obtained, achieving a purity of 73%.

Construction of the cell culture model

H9C2 myoblast cells were revived from a frozen state using DMEM medium containing 10% foetal bovine serum and cultured in an incubator at 37 °C with 5% CO₂. The medium was refreshed every 1–2 days, and the cells were passaged and seeded into 96-well plates for experimentation. To establish a H/R injury model, H9C2 cells were first washed twice with phosphate-buffered saline and then cultured in a sugar-free, serum-free medium at 2% oxygen concentration for 8 hr. This was followed by culturing in a normal medium at 21% oxygen concentration for 3 hr. The blank control group (Con group) was maintained under normal conditions without hypoxia treatment. In addition to the Con and H/R groups, other groups of myoblast cells were pretreated with different concentrations of silverweed cinquefoil root flavonoids (SCR-F) before H/R. The culture medium was supplemented with SCR-F at concentrations of 52.08, 104.16, and 208.32 μg/ml (selected based on preliminary experiments showing dose-dependent bioactivity within 50–200 μg/ml) for 24 hr. Cells were then divided into five groups: Con group, H/R group, H/R + 52.08 (low concentration) group, H/R + 104.16 (medium concentration) group, and H/R + 208.32 (high concentration) group.

Cell viability assay

Cells were seeded into a 96-well plate, and 10 μl of Cell Counting Kit-8 (CCK-8) reagent was added to each well. After incubating for 2 hr in a CO₂ incubator in the dark, the optical density (OD) at 450 nm was measured using a microplate reader. Cell viability (%) was calculated as follows:

where OD₁ is the OD value of the experimental group, and OD₂ is the OD value of the control group.

LDH assay

The supernatant from each group of H9C2 cells (100 μl per well) was transferred to a new 96-well plate. According to the LDH assay instructions, the reaction mixture was added, and the plate was incubated at room temperature for 30 min. Absorbance of LDH was measured at 490 nm using a microplate reader.

Membrane potential assay

The culture medium in each group’s 96-well plate was replaced with 500 μl of 2 μmol/L JC-1 dye, and the cells were incubated in the dark at room temperature for 30 min. Fluorescent images were captured using a 40× fluorescence microscope, and fluorescence intensity was quantified using ImageJ software.

ROS assay

Cells were seeded into a 6-well culture plate at a density of 2 × 10⁵ cells/ml and incubated for 24 hr. The fluorescent dye 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was diluted to a concentration of 10 μM in serum-free DMEM, and 1 ml of this solution was added to each well for the treatment groups. After a 30-min incubation at 37 °C, the cells were rinsed three times with serum-free DMEM and imaged using an inverted fluorescence microscope.

Cardiac injury marker assay

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was performed. Microwells precoated with antibodies specific to cardiac myoglobin (MYO/MB), cardiac troponin (cTn), and creatine kinase isoenzyme MB (CK-MB) were used. Samples, standards, and horseradish peroxidase (HRP)-labelled detection antibodies were added sequentially, followed by incubation and thorough washing. Absorbance (OD value) was measured at 450 nm with a microplate reader, and sample concentrations were calculated based on standard curves.

MDA content and antioxidant enzyme activity assay

Following stimulation, the cell culture medium was removed, and the cells were collected to prepare a cell homogenate. After centrifugation, the supernatant was used for analysis. Assays for superoxide dismutase (SOD), MDA, glutathione peroxidase (GSH-px), and catalase (CAT) were conducted according to standardised protocols, with absorbance readings taken at specific wavelengths: SOD at 560 nm; GSH-px at 412 nm; CAT at 550 nm; and MDA at 450, 532, and 600 nm. The absorbance data were applied to the standard curve to determine the concentrations of the respective components.

Western blot analysis of antioxidant stress proteins and apoptosis-related protein expression levels

Total protein from each group of myoblast cells was extracted using protein lysis buffer, and protein concentrations were quantified via Bicinchoninic Acid Assay (BCA) method. Proteins were denatured at high temperatures and separated by 10% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Following separation, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane at a constant voltage of 90 V. The membrane was blocked for 2 hr in a solution containing 5% skim milk powder. It was then incubated overnight at 4 °C with primary antibodies specific to Bax, Bcl-2, caspase-3, caspase-9, survivin, and VDAC1. After three washes with TBST, the membrane was incubated with secondary antibodies. Chemiluminescence was developed using an enhanced chemiluminescence (ECL) reagent, and band intensity was quantified using Image J software.

Data analysis

Data were analysed using SPSS 21.0 statistical software. Quantitative data that conformed to a normal distribution were represented by the mean ± SD of n = 5 replicates. The independent-samples t-test was used for comparison between two groups, and one-way ANOVA was used for comparison among multiple groups. A difference was considered statistically significant at p < .05.

Results and discussion

Morphology and viability of myocardial cells in each group

As shown in Figure 1, cells in the control group and the H/R + 52.08 μg/ml SCR-F group displayed a prismatic shape with good adhesion and a compact structure. Figure 1F reveals that, compared to the control group, cell viability significantly decreased in the H/R group and in the H/R groups treated with 52.08, 104.16, or 208.32 μg/ml SCR-F (p < .05). However, compared to the H/R group, SCR-F improved myocardial cell proliferation in a concentration-dependent manner across the 52.08, 104.16, and 208.32 μg/ml concentrations. Cell viability in the H/R + 52.08 μg/ml, H/R + 104.16 μg/ml, and H/R + 208.32 μg/ml SCR-F groups was significantly higher than in the H/R group (p < .01). These findings indicate that H/R injury inhibits myocardial cell proliferation, while medium to high concentrations of SCR-F can effectively counteract this inhibition.

Comparison of myocardial cell injury levels in each group

The heart is highly sensitive to hypoxia due to its substantial oxygen demands. Research indicates that exposure to low-pressure hypoxia can damage myocardial structure and function, primarily through disruptions in the electron transport chain during mitochondrial oxidative phosphorylation, leading to substantial ROS production (Singh et al., 2014). Reactive oxygen species levels were measured using the DCFH-DA probe, which emits green fluorescence under a fluorescence microscope. The results showed a significant increase in ROS levels in the H/R group compared to the control (Con) group. Conversely, the SCR-F group displayed a marked reduction in ROS release relative to the H/R group, as illustrated in Figure 2A.

Myocardial injury leads to the release of membrane-bound enzymes, resulting in elevated serum markers. Lactate dehydrogenase and CK-MB are highly sensitive and specific indicators, making their detection a reliable measure of myocardial cell damage. Studies have demonstrated that rats exposed to low-pressure hypoxia experience myocardial tissue damage, reflected in increased levels of CK-MB and LDH in the bloodstream (Figure 2D and E). Furthermore, the concentration of inflammatory cytokines in the blood is significantly higher in the hypoxic group compared to the normoxic group (Nehra et al., 2017).

In this study, both cellular viability and the concentrations of LDH and CK-MB were notably higher in the H/R group than in the Con group, confirming the successful establishment of the H/R injury model. Silverweed cinquefoil root flavonoid treatment significantly mitigated these effects, reducing LDH and CK-MB secretion (p < .01) and helping maintain myocardial function markers at normal levels. These results suggest that SCR-F enhances cellular activity, reduces damage, and alleviates hypoxia-induced myocardial injury. Notably, the 208.32 μg/ml SCR-F group exhibited the most pronounced reduction in these biomarkers, further supporting the hypothesis that higher concentrations provide greater protective effects.

Oxidative stress can stem from both enzymatic and nonenzymatic sources. Although nonenzymatic sources contribute minimally to oxidative stress, MYO-MB serves as a nonenzymatic antioxidant, assisting in cellular defence against ROS. Cardiac troponin (cTN), a regulatory protein specific to myocardial tissue, is one of the most sensitive serum markers of myocardial injury. Results from this study indicated that, compared to the Con group, the H/R group showed decreased MYO-MB levels and a significant increase in cTN concentrations, further supporting the presence of myocardial cell injury under hypoxic conditions.

Effects of SCR-F on antioxidant enzyme activity in H/R myocardial cells

The precise mechanism by which low-pressure hypoxia damages myocardial tissue remains partially understood; however, a widely accepted theory suggests that hypoxia-induced oxidative stress plays a significant role in this damage (Dong et al., 2023). Low-pressure hypoxia disrupts the electron transport chain in mitochondrial oxidative phosphorylation, leading to the excessive production of ROS (Figure 2A). Elevated ROS levels can damage biological membranes by inducing lipid peroxidation, which substantially increases MDA levels within myocardial tissue. In vitro studies indicate that the total flavonoids extracted from P. anserina possess strong antioxidant properties (Yan et al., 2023), demonstrating more potent scavenging activity against hydrogen peroxide, DPPH radicals, superoxide anions, and hydroxyl radicals compared to the lipophilic antioxidant vitamin E. Moreover, these flavonoids show greater in vitro antioxidant efficacy than polysaccharides derived from the same plant.

To evaluate membrane damage, the experiment measured intracellular MDA levels. In H9C2 myocardial cells exposed to H/R, MDA content rose significantly. Pretreatment with SCR-F effectively reduced this increase, resulting in a 36% decrease in MDA levels in the group treated with 208.32 μg/ml of SCR-F. This finding indicates that SCR-F mitigates lipid peroxidation induced by H/R, thereby protecting H9C2 myocardial cells from oxidative damage (Figure 3).

The body’s endogenous antioxidant defence system, which includes both enzymatic and nonenzymatic antioxidants, serves to protect cells from ROS-induced damage. Key antioxidant enzymes such as SOD, CAT, and GSH-Px form the primary defence against free radicals. Superoxide dismutase converts superoxide anions into hydrogen peroxide, which is then further broken down into water by CAT and GSH-Px (Sies, 1999). Compared to the Con group, SOD, CAT, and GSH-Px activities were significantly reduced in the H/R group. Treatment with SCR-F, however, effectively restored the activities of these enzymes. These results suggest that SCR-F enhances the body’s endogenous antioxidant defence mechanisms, reducing oxidative damage caused by free radicals. Additionally, SCR-F improves resilience under low-pressure hypoxic conditions, playing a crucial role in protecting H9C2 myocardial cells from hypoxia-induced injury. The activity of MDA and the three key antioxidant enzymes SOD, CAT, and GSH-px exhibited a similar dose-dependent trend. At a concentration of 208.32 μg/ml, the oxidative-reduction balance of the cells was best maintained.

Expression and enzyme activity of apoptosis-related proteins in myocardial cells across groups

Oxidative stress in cardiac tissue induces apoptosis and inflammation, potentially leading to heart failure (Ferrannini et al., 2016). Apoptosis is regulated by various genes, notably members of the Bcl-2 family, where Bax promotes apoptosis, and Bcl-2 inhibits it. The balance between these proteins, along with the activation of downstream proteases known as caspases, is essential for apoptosis regulation (Clemente-Moragón et al., 2020). Positioned upstream of the mitochondria, Bcl-2 and Bax play crucial roles in controlling mitochondrial permeability. Bax, typically inactive in the cytoplasm, undergoes a conformational shift upon receiving apoptotic signals. This change allows Bax to form homodimers or multimers with Bcl-2 that integrate into the mitochondrial outer membrane, triggering the release of Cyt c and advancing the apoptotic cascade (Heiser et al., 2004). Caspase-3 and Caspase-9 are key proteases in the terminal stages of apoptosis and are considered principal executors in this process, particularly in damaged cells or tissues (Yang et al., 2021b).

Survivin, an antiapoptotic protein, directly interacts with the caspase family, specifically inhibiting Caspase-3 and modulating Caspase-9 activity. The p53 protein initiates apoptosis in response to certain stress signals, with numerous triggers capable of rapidly increasing p53 levels. Emerging evidence suggests that hypoxia also contributes to p53 accumulation (Wei et al., 2021).

Western blot analysis results revealed significant upregulation of Bax (Figure 4A and B), caspase-3 (Figure 5C and D), caspase-9 (Figure 5A and B), and p53 (Figure 6C and D) in the H/R group compared to the control group. Conversely, Bcl-2 (Figure 4C and D) and survivin (Figure 6A and B) levels were markedly reduced. Compared to the H/R group, the SCR-F treatment group demonstrated a dose-dependent decrease in Bax, caspase-3, caspase-9, and p53 expression, alongside a significant increase in Bcl-2 and survivin expression. Among them, the 208.32 μg/ml SCR-F group exhibited the lowest Bax expression and the highest Bcl-2 expression, indicating that high-dose SCR-F had the most significant effect on inhibiting cell apoptosis. In addition, the activities of caspase-3 and caspase-9 gradually decreased with increasing SCR-F concentration, suggesting that SCR-F may reduce the release of Cyt c, thereby inhibiting the amplification of apoptotic signalling cascades and ultimately reducing myocardial cell damage.

These findings suggest that SCR-F treatment effectively suppresses the apoptotic pathway, with flavonoids substantially alleviating myocardial ischemia–reperfusion injury. This therapeutic effect likely results from the inhibition of apoptosis via the mitochondrial pathway.

Comparison of expression levels of mitochondrial apoptosis pathway proteins Cyt c and VDAC1 in myocardial tissue across groups

VDAC plays a critical role in balancing energy metabolism between mitochondria and the cell, acting as a conduit for metabolic product exchange. Located on the outer membrane of the mitochondrial permeability transition pore (mPTP), VDAC is a multifunctional protein that forms hydrophilic voltage-gated channels, enabling the transport of ions, energy, and metabolites between the cytoplasm and mitochondria. Cytochrome c functions as an activator of apoptosis with a molecular weight of 11–15 kDa, primarily residing within the mitochondria of healthy cells. When mitochondrial membrane potential (ΔΨm) decreases and membrane permeability increases, Cyt c is released into the cytoplasm. Following H/R injury, substantial amounts of Cyt c are released from mitochondria, indicating apoptotic pathway activation.

Previous research has shown that ischemia–reperfusion injury elevates VDAC1 expression on the outer mitochondrial membrane in H9C2 myocardial cells. This upregulation triggers the opening of mPTP, reducing mitochondrial membrane potential and facilitating the release of Cyt c, which subsequently initiates the cell’s apoptotic programme (Olsen et al., 2006). VDAC1 is implicated in apoptosis regulation, where reducing its expression and activity can diminish cellular apoptosis. Our findings showed that, after H/R treatment in H9C2 myocardial cells, VDAC1 protein expression was significantly elevated (p < .01) compared to the control group, with a notable increase in Cyt c release (p < .01), suggesting activation of the mitochondrial apoptosis pathway (Figure 7). In contrast, in the SCR-F treatment group, VDAC1 protein expression was markedly downregulated (p < .01) compared to the H/R group, and Cyt c release was also significantly reduced (p < .01). High-dose SCR-F treatment led to a considerable decrease in VDAC1 protein expression, impacting mPTP opening and reducing mitochondrial membrane potential (Figure 2C). This reduction limited the release of Cyt c into the cytoplasm, thereby inhibiting mitochondrial apoptosis pathway activation. Additionally, evidence indicates that VDAC can interact with antiapoptotic proteins Bcl-2 and Bcl-xL (Huang et al., 2013). VDAC may also form channels either independently or in conjunction with Bax, promoting Cyt c release from mitochondria and contributing to apoptosis.

Potential protective response of SCR-F to H/R injury

Hypoxia, arising from low oxygen levels in the blood, impaired oxygen transport, or insufficient cellular oxygen uptake, can initially stimulate gene expression aimed at tissue repair. However, prolonged hypoxic exposure leads to functional impairments, oxidative stress, and diminished ATP production. Under these conditions, cells switch to anaerobic glycolysis, generating lactic acid and disrupting the cellular redox balance. Reoxygenation further exacerbates damage through the production of ROS, causing lipid peroxidation, protein oxidation, and DNA damage, which can lead to apoptosis. Thus, the regulation of ROS accumulation by the antioxidant enzyme system is crucial to prevent cell death. Silverweed cinquefoil root flavonoids significantly enhanced the activity of antioxidant enzymes, including SOD, CAT, and GSH-px, in cells subjected to hypoxia/reoxygenation injury. Following treatment with higher SCR-F concentrations (>100 μg/ml), antioxidant enzyme activity in damaged cells was restored to levels comparable to those in undamaged cells (Figure 3). Flavonoids serve as secondary antioxidants, not only supporting other ROS scavenging systems but also enhancing antioxidant enzyme activity when it declines (Shen et al., 2022). In vitro antioxidant studies on P. anserina flavonoids indicated that their antioxidant activity surpasses that of vitamin E, a well-known lipophilic antioxidant (Yan et al., 2023). At lower doses, SCRF may act as direct free radical scavengers, neutralising reactive oxygen species (ROS) generated during H/R. Higher doses might upregulate endogenous antioxidant systems (e.g., SOD, CAT, GSH-px) via activation of the Nrf 2/ARE pathway, enhancing cellular resilience to oxidative stress.

Multiple apoptotic pathways, including the mitochondrial, endoplasmic reticulum stress, and death receptor pathways, contribute to programmed cell death. This study demonstrates that hypoxia/reoxygenation injury influences the expression of mitochondrial pathway proteins. Pro-apoptotic proteins (Bad, Bid, Bax) and anti-apoptotic proteins (Bcl-2, Bcl-xL) respond to death signals by translocating to mitochondria, where they form channels that regulate Cyt c release through alterations in mitochondrial membrane permeability (Figure 8). The Bcl-2 family proteins modulate this permeability, with VDAC1 playing a key role in pore formation, facilitating the release of pro-apoptotic proteins (Shoshan-Barmatz et al., 2017). VDAC1 interacts with Bcl-2 family proteins, such as Bax and Bak, which promote its opening, while Bcl-2 and Bcl-xL binding induces closure, thereby regulating mitochondrial membrane permeability.

After SCR-F treatment, the expression levels of Bax and VDAC1 decreased, while Bcl-2 expression increased. This shift in expression was positively correlated with the SCR-F dose, suggesting that SCR-F modulates mitochondrial membrane permeability and consequently influences Cyt c release. The translocation of Cyt c from mitochondria to the cytoplasm is a critical event in the apoptotic signalling pathway (Ott et al., 2002). Upon release, Cyt c binds to apoptotic protease-activating factor-1, forming an apoptosome complex with procaspase-9, which activates caspase-9. This activation triggers a caspase cascade, ultimately leading to apoptosis. Low-to-moderate doses could inhibit mitochondrial apoptosis by stabilising mitochondrial membrane potential and suppressing Bax/Bcl-2 imbalance. Higher doses may further modulate downstream caspases (e.g., caspase-3/9) or inhibit endoplasmic reticulum stress-related apoptosis pathways.

In the apoptotic signalling pathway, initiator caspases such as caspase-9 are closely linked with pro-apoptotic signals, and their activation cleaves and activates downstream effector caspases, such as caspase-3, to execute apoptosis. Survivin is a key protein that regulates cellular signalling pathways involving cytokines, transcriptional networks, and gene modifications, affecting cell proliferation and apoptosis by stabilising the cellular environment. In the mitochondrial apoptosis pathway, survivin inhibits caspase-3 and caspase-9, preventing apoptosis. The P53 protein binds to survivin’s promoter region; however, its activity can be suppressed by certain drugs. Survivin and P53 maintain a balance within the mitochondrial apoptosis pathway. P53 can also translocate to mitochondria, bind with Bcl-2 or Bcl-xL, activate Bax, or disrupt Bak/Mcl-1 or Bak/Bcl-2 interactions to induce apoptosis (Wei et al., 2021).

The study findings indicate that SCR-F modulates mitochondrial-mediated apoptosis by decreasing p53 expression and increasing survivin expression. Overall, SCR-F reduces oxidative damage and promotes cardioprotection by attenuating oxidative stress and preventing apoptosis through the mitochondrial pathway.

Conclusion

This study examined the mechanisms by which SCR-F inhibit oxidative stress and apoptosis in hypoxic cardiomyocytes through in vitro experiments. Following hypoxia–reoxygenation injury, a decrease in cellular activity and an increase in LDH release were observed in cardiomyocytes. Treatment with SCR-F, particularly at a dose of 208.32 μg/ml, demonstrated substantial cardioprotective effects on hypoxia/reoxygenation-injured H9C2 cells. This protection likely results from the scavenging of excessive ROS, reduction of oxidative stress, and inhibition of the mitochondrial-mediated apoptotic pathway, providing a protective effect on hypoxic cardiomyocytes. The mitochondrial pathway plays a central role in cellular apoptosis, characterised by the release of Cyt c from mitochondria into the cytoplasm through the permeabilised outer membrane. This process is regulated by the balance between pro-apoptotic and anti-apoptotic proteins in the Bcl-2 family, as well as the actions of initiator caspase-9 and effector caspase-3, with involvement from proteins such as VDAC1. SCR-F treatment reduced apoptosis in H9C2 cells under hypoxic conditions, as indicated by a significant increase in anti-apoptotic Bcl-2 expression, a marked decrease in pro-apoptotic Bax expression, and reduced expression of caspase-9 and caspase-3. Concurrently, the reduced expression of VDAC1 inhibited its binding with apoptotic proteins, preserving mitochondrial metabolic and energetic functions and suppressing Cyt c release and cellular apoptosis. Xizang P. anserina contains 42 flavonoid compounds, with 23 isolated and identified for the first time. Silverweed cinquefoil root flavonoids have shown strong in vitro antioxidant activity. Further research is needed to identify the specific flavonoids with the most pronounced anti-hypoxia effects and to explore whether SCR-F can influence other endogenous or exogenous pathways involved in cellular apoptosis.

Data availability

Data will be made available on request.

Author contributions

Yingying Yan: Writing—original draft, Visualisation, Software, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation. Yuhong Zhang: Methodology, Investigation, Data curation. Wenhui Zhang: Methodology, Investigation, Formal analysis, Data curation. Jifeng Zhang: Writing—review & editing, Supervision. Yingying Yan (Conceptualisation, Data curation, Formal analysis, Methodology, Software, Writing—original draft [equal]), Yuhong Zhang (Conceptualisation, Methodology, Supervision, Writing—review & editing [equal]), Wenhui Zhang (Conceptualisation, Funding acquisition, Project administration, Resources [equal]), and Jifeng Zhang (Formal analysis, Methodology, Supervision [equal])

Funding

This work is supported by Major science and technology projects in the Xizang Autonomous Region (XZ202201ZD0001N).

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors thank the Major science and technology projects in the Xizang Autonomous Region for the funding.

References