Naringin and hesperidin protect ovarian granulosa cells from cisplatin-induced mitochondrial dysfunction by enhancing bioenergetics and mitophagy

Abstract

Background

Cisplatin (CDDP) is a widely deployed chemotherapeutic medication used to treat various solid tumors, but its clinical utility is limited by dose-dependent ovarian toxicity. Naringin (NG) and hesperidin (HD) are two naturally occurring flavonoids found in citrus fruits and have protective effects on mitochondrial function.

Results

The current study examined the protective effects of NG and HD on CDDP-induced mitochondrial dysfunction and cytotoxicity in granulosa cells. The cells are treated with CDDP alone or with NG or HD. CDDP reduced cell viability, ATP synthesis, oxygen consumption rate (OCR), mitochondrial membrane potential (MMP), and mitochondrial complex functions in a dose-dependent pattern. It also altered mitochondrial dynamics and enhanced glycolytic activity, as exhibited by lactate levels. MMF increased, and fatty acid composition changed, resulting in a higher unsaturated/saturated ratio. PINK1 and PARKIN levels were reduced upon CDDP treatment, suggesting mitophagy disruption. Co-treatment of NG and HD enhanced complex activity, MMP, OCR, ATP generation, and cell viability. MMF was protected by NG and HD, which also stabilized fatty acids and enhanced ion permeability and mitochondrial swelling. Compared to NG, HD preserved numerous attributes more effectively and restored them almost completely.

Conclusions

NG and HD effectively preserve mitochondrial bioenergetics, structure, and dynamics in granulosa cells, mitigating CDDP-induced dysfunction. These findings highlight their potential as natural adjuvants to reduce ovarian toxicity and support fertility preservation in female cancer patients.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13062-026-00830-3.

Background

Cisplatin (cis-diamminedichloroplatinum II; CDDP) is a widely deployed chemotherapeutic drug that has revolutionized cancer treatment since its debut. It is largely utilized for the treatment of various solid tumors, including those of the bladder, ovaries, testes, lungs, and the neck and head [1]. The efficacy of CDDP mainly arises from its capacity to create covalent bonds with DNA, leading to DNA cross-linking, which disrupts both replication and transcription, ultimately triggering death in cancer cells [2]. Despite its therapeutic effect, the application of CDDP is sometimes limited by considerable adverse effects, such as nephrotoxicity, neurotoxicity, ototoxicity, and ovarian toxicity [3–6]. CDDP-induced ovarian toxicity poses a considerable concern, particularly for young female cancer patients, potentially leading to premature ovarian failure and infertility. The toxicity primarily stems from the drug’s capacity to induce oxidative stress and DNA damage, which results in the death of ovarian granulosa cells and primordial follicles and significantly reduces the ovarian reserve [6, 7]. Recent evidence suggests that ovarian injury resulting from chemotherapeutic agents is associated with disruptions in endocrine signaling pathways. These disruptions include altered responsiveness to follicle-stimulating hormone, steroidogenic imbalances, and impaired intra-follicular communication, all of which accelerate follicular depletion and compromise reproductive competence [7, 8].

Granulosa cells play a crucial role in regulating follicular development, steroidogenesis, and oocyte maturation. Their viability is closely linked to intracellular quality-control mechanisms, such as autophagy and mitophagy. Autophagy is a crucial mechanism for maintaining the primordial follicular pool and supporting follicular viability in response to physiological and toxicological stressors. Disruption of autophagic signaling is directly associated with follicular atresia and ovarian dysfunction [9, 10]. Ultrastructural examinations of granulosa cells during follicular atresia indicate notable morphological deterioration, marked by membrane irregularities, cytoplasmic contraction, and altered intercellular interactions, highlighting their susceptibility to toxic agents [11]. The mitochondrial toxicity caused by CDDP plays a crucial role in its overall cytotoxic effects, as it accumulates in the mitochondria, alters mitochondrial DNA, disrupts the electron transport chain, and initiates intrinsic apoptotic pathways and results in the excessive production of reactive oxygen species (ROS) and initiates intrinsic apoptotic pathways [12, 13]. Strategies to mitigate mitochondrial toxicity include the use of antioxidant therapies that preserve mitochondrial function while maintaining the CDDP’s anticancer efficacy [14, 15].

Natural flavonoids were shown to have therapeutic potential to mitigate drug- and toxicant-induced organ damage [16–19]. Naringin (NG) and hesperidin (HD) are two naturally occurring flavonoids found in citrus fruits that exhibit protective effects on mitochondria under oxidative stress and mitochondrial dysfunction. These compounds have antioxidative, anti-inflammatory, and anti-apoptotic mechanisms [17, 18]. NG boosts mitochondrial function by enhancing antioxidant enzyme activity, thereby halting the disintegration of mitochondrial membrane potential (MMP) and the release of cytochrome c, which are crucial events in apoptosis [20]. HD maintains mitochondrial integrity by modulating oxidative stress markers, preserving ATP production, and suppressing lipid peroxidation, so stabilizing mitochondrial membranes and averting cellular death [21]. Both flavonoids trigger signaling pathways, such as Nrf2/ARE, which augment endogenous antioxidant synthesis and promote mitochondrial biogenesis [22]. The aforementioned properties imply that NG and HD may function as therapeutic remedies in situations where mitochondrial malfunction is evident. This is confirmed by a growing body of literature that has highlighted the effectiveness of NG against mitochondrial dysfunction in various tissues, including neuronal tissues [20] and liver [23], as well as the effectiveness of HD in intestinal [21] and brain tissue [24].

According to a review of the literature, no research has particularly explored the potential mitigating effect of NG or HD supplementation on reducing mitochondrial dysfunction brought on by CDDP exposure. Therefore, the current investigation was conducted to ascertain the protective effects of NG and HD against the detrimental impacts of CDDP on the mitochondrial function and structure in ovarian granulosa cells using in vitro experiments. Bioenergetic parameter assays and mitochondrial membrane studies were determined. To gain a comprehensive understanding of CDDP’s toxicity, molecular docking was employed to investigate the binding affinities of mitophagy proteins and mitochondrial complexes I–V.

Methods

Molecular docking (Hypothesis generating tool)

CDDP, NG, and HD have been linked with mitochondrial complexes I–V, PARKIN, and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1). SwissDock, an automated docking program, calculated ligand binding mechanisms and affinities with target proteins. The three-dimensional structures of the mitochondrial complexes, PINK1 and PARKIN, were obtained from the Protein Data Bank after removing water molecules, adding polar hydrogens, and assigning charges. SwissDock used the CHARMM force field to predict binding modes for ligand-target interactions to optimize them. AutoDock Vina was used to independently evaluate binding affinities and interactions for validation. SwissDock docking findings were visualized and interpreted using 3D figures, including hydrogen bonds, hydrophobic contacts, and π-π stacking. To determine target protein ligand compatibility and specificity, important binding residues and interactions were identified. To compare ligands to different targets, binding affinities were assessed using the estimated free energy of binding (ΔG).

Reagents and cell culture conditions

Sigma (St. Louis, Missouri, USA) provided all preparations and reagents used. The KGN human ovarian granulosa cell line (CVCL_0375, Cellcook Biotech Co., Ltd., Guangzhou, China) was grown in DMEM/F-12 media at 37 °C with 5% CO₂, 10% (vol: vol) charcoal-stripped fetal bovine serum (VivaCell), 1% penicillin/streptomycin (Beyotime, Shanghai, China), and no phenol red. Fresh culture media were added every other day following cell adhesion.

Cell growth and viability analysis (MTS)

The MTS assay is a colorimetric method used to determine cell viability by measuring the metabolic activity of the cells. KGN cells were grown in 96-well plates to 80–90% confluence, then treated with CDDP (1, 5, 10, 15, and 20 µM) or NG or HD (10, 20, 40, 80, and 160 µM) for 48 h. These concentration ranges were selected based on prior studies [25–27]. Accordingly, inclusion of lower and higher concentrations of CDDP enables characterization of sub-toxic, moderate, and severe injury responses. However, using these concentration ranges with NG or HD was used to identify the maximally effective protective dose without inducing cellular toxicity.

In each well, add 20 µl of MTS solution and 100 µl of culture media. Incubate for 1–4 h at 37 °C in a CO₂ incubator, protected from light. Use a microplate reader to measure absorbance in the 490–500 nm range. Include wells with only medium as blanks and untreated cells as controls. Assess data by comparing absorbance levels of treated and control samples to determine cell viability.

Bioenergetics parameters evaluation

For the bioenergetic assays, cells were grown and treated with CDDP (5 or 10 µM) for 48 h in the presence or absence of NG or HD (50 µM).

Assay intracellular ATP level

A luminescence-based ATP detection test kit (Abcam, Cambridge, UK; Cat. No: ab83355) measured intracellular ATP 48 h following treatment. To liberate intracellular ATP, the medium was carefully removed, and the cells were lysed using the kit’s lysis buffer. To improve signal growth, the plate was incubated in the dark at room temperature for the duration advised by the manufacturer. A Perkin Elmer “TopCount” luminometer measured light after eliminating blank well noise. To ensure reproducibility, at least three independent experiments were run in triplicate for each condition.

Rhodamine-123 (Rh-123)-based analysis of mitochondrial membrane potential (MMP)

The effects of CDDP, NG, and HD on MMP were assessed using rhodamine 123 (Rh-123), which is found mostly in energized mitochondria. The cells were treated with Rh-123 (50 nM) for 10 min and then wrapped in aluminum foil at 37 °C. Fluorescence was measured at excitation and emission wavelengths of 480 and 530 nm, respectively. Experiments were conducted in triplicate.

DepSipher dye for flow cytometry

Cells were grown to 70–80% confluence or sufficient density for investigation before being stained with DepSipher dye for flow cytometry. To maintain dye stability, a 5 µM solution of DepSipher dye (Thermo Fisher Scientific; Cat. No: DCTM06) was prepared in serum-free growth medium and protected from light exposure. Flow cytometry employed excitation at 488 nm and emission filters for green (530 nm) and red (590 nm) fluorescence. The red/green fluorescence ratio assessed MMP, with red fluorescence suggesting polarized mitochondria and green fluorescence indicating depolarized mitochondria. This method accurately measured mitochondrial health following experimental treatments.

Assessment of mitochondrial respiratory complex activities

We employed a rigorous and well-established technique to evaluate the function of the mitochondrial respiratory chain in complexes I, II, III, and IV [28]. This approach isolates mitochondrial fractions from treated cells and spectrophotometrically analyzes enzymatic activity under ideal circumstances for each complex, employing a temperature-controlled spectrophotometer (Beckman Coulter DU 800). To confirm NADH-dependent ubiquinone reduction, rotenone was employed as a complex I activity inhibitor. Malonate inhibited succinate dehydrogenase and measured Complex II activity. Antimycin A hindered the action of Complex III on ubiquinol-cytochrome c reductase. Complex IV activity was evaluated using potassium cyanide to inhibit cytochrome c oxidase. Five biological replicates were used per treatment group to assure statistical reliability in each experiment.

Assays for cellular respiration and metabolic intermediates

Biochemical analysis of lactate accumulation

The effects of CDDP, NG, and HD on cellular lactate generation were determined using a commercial lactate assay kit (Biovision, Mountain View, California, USA; Cat. No: K607-100) according to the manufacturer’s instructions.

Assessment of pyruvate dehydrogenase (PDH) enzymatic activity

The PDH assay was carried out using the Abcam ELISA kit in accordance with the protocol. Protein content is measured to ensure consistency across samples. The protocol incorporates methods for calibrating the assay using supplied standards to quantify activity and affirm reliability.

Measurement of cellular oxygen consumption rate (OCR)

The impacts of CDDP, NG, and HD on OCR were assessed following a prior study [29]. Cells were grown in 6-well plates and treated with CDDP, NG, and HD for 48 h. After treatment, the cells were trypsinized, centrifuged, and resuspended in Hank’s solution. Cell counts were measured utilizing a hemocytometer. The change in oxygen levels (ΔO₂) was calculated over 300 s. Most changes in oxygen levels had a linear slope; however, the slope for azide was measured 60 s after its administration. Each drug concentration was tested at least 7 times to assure data consistency.

Quantification of cellular α-ketoglutarate levels

The Abcam kit was used to assay α-KG (Abcam, Cambridge, UK; Cat. No: ab83431. Cells are lysed and centrifuged, with the supernatant used for examination. Protein concentrations are standardized, and a predetermined volume of each sample is introduced to a microplate containing a reaction mix. The reaction happens at 37 °C and yields a detectable colorimetric (570 nm) or fluorometric (Ex/Em 535/587 nm) signal. A standard curve is used to measure α-KG levels. The assay is very sensitive and well-suited for metabolic research in cell lines.

Evaluation of mitophagy-related proteins (PINK1 and PARKIN)

The PARKIN and PINK1 (PTEN-induced kinase 1) were assessed utilizing the Abcam ELISA kits (Abcam, Cambridge, UK; Cat. No: ab212159 and ab279886, respectively). Proteins are extracted from cultivated cells with lysis buffer, quantified, and loaded into antibody-coated wells (antibody specific to PARKIN or PINK1). Following incubation and washing, primary and enzyme-linked secondary antibodies are added. A substrate is then used to produce a colorimetric signal determined at 450 nm. Protein levels are measured using a standard curve from PARKIN or PINK1 standards.

Mitochondrial membranes studies

Mitochondrial membranes fluidity (MMF)

Mitochondria were isolated using the technique outlined by [28] to study the effects of CDDP, NG, and HD on MMF. MMF was evaluated with a TMA-DPH fluorescent probe, following the protocol established by [30].

Gas chromatography-based profiling of mitochondrial membrane fatty acids

To evaluate the effect of CDDP, NG, and HD on MMFAC, mitochondrial lipids were isolated in accordance with Oemer et al. [31] protocol. Transesterification was performed. MMFAC was analyzed using gas chromatography with a flame ionization detector and a 30 m Omega wax column. Saturated fatty acids, particularly palmitic and stearic, as well as unsaturated fatty acids such as palmitoleic, oleic, arachidonic, linoleic, and docosahexaenoic, were measured.

Mitochondrial swelling and permeability to K⁺ and H⁺ ions

The effects of CDDP, NG, and HD on mitochondrial swelling and permeability to H⁺ and K⁺ ions were investigated according to the technique established by Yu et al. [32]. A fraction enriched in mitochondria was produced, and the protein concentration was quantified using a Bradford assay. To assess the impact of CDDP, NG, and HD on mitochondrial swelling, mitochondria were exposed to solution A (0.2 M sucrose, 1 mM Tris, 1 mM Mops, 10 µM EGTA, 1 mM Na₃PO₄, 3 g/mL oligomycin, 5 mM succinate, and 2 µM rotenone). To examine the impact on H+ permeability, mitochondria (0.5 mg/ml) were suspended in solution B, which comprised 0.135 M CH3COOK, 5 mM HEPES, 0.1 mM EGTA, 2 µM rotenone, 1 1 g/ml valinomycin, and 0.2 mM EDTA. Mitochondria were suspended in solution C (0.135 M KNO₃, 5 mM HEPES, 0.1 mM EGTA, and 0.2 mM EDTA) to assess the effect on K⁺ permeation. All solutions were augmented with CDDP (2 or 20 µM) and NG and HD (0, 5, 10, and 20 µM) and thereafter incubated for 30 min. Absorbance was recorded after 20 min at 540 nm and 25 °C utilizing a multi-mode microplate reader (FLUOstar Omega).

Evaluation of the mitochondrial gene expressions

The effects of CDDP, NG, and HD on the expression of genes that encode mitochondrial complexes were investigated employing quantitative polymerase chain reaction (qPCR). Total RNA was extracted according to Qiagen’s technique. cDNA was created by reverse transcription of 200 ng of RNA using a commercial reverse transcription kit. The current study used previously reported qPCR primer sequences from [33]. The thermocycling parameters were modified using the approach described by Huang et al. [34], and amplification was performed using a Bio-Rad Laboratories CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). The GAPDH gene was used as the endogenous internal control for normalization of gene expression. Relative mRNA expression levels were calculated using the 2^−ΔΔCt method [35] and are presented as fold changes relative to the untreated control group. All qPCR experiments were performed in triplicate to ensure reproducibility and analytical reliability.

Statistical analyses

Tukey’s post-test was applied with a one-way ANOVA to evaluate data from three or more groups. Statistical studies using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) indicate statistical significance at P < 0.05. Heat map clustering and principal component analysis (PCA) were carried out using the Science and Research online plot (SRplot; https://www.bioinformatics.com.cn/en).

Results

Molecular docking

As displayed in Table 1; Fig. 1, the molecular docking study compares CDDP, NG, and HD with mitochondrial complexes (I-V), PARKIN, and PINK1 proteins. Significant variations in binding affinity and interaction patterns between ligands and targets indicate that NG and HD may protect against CDDP-prompted toxicity. Compared to CDDP (-3.48 kcal/mol), NG (-5.29 kcal/mol), and HD (-5.00) had higher affinity for mitochondrial complex I (MC1, 5XTD). NG and HD interact with Lys137 and Asp150, indicating that they can stabilize the complex and improve mitochondrial activity. NG (-6.81 kcal/mol) and HD (-6.43 kcal/mol) bind more strongly than CDDP (-4.55 kcal/mol) in mitochondrial complex II (MCII, 6MYQ), forming many hydrogen bonds with Lys227 and Glu342. This demonstrates a strong stabilizing association with MCII, which may minimize CDDP-induced oxidative damage.

Table 1.

Molecular docking data of cisplatin (CDDP), naringin (NG), and hesperidin (HD) with mitochondrial bioenergetics related proteins

Target	Ligand	Affinity (Kcal/M)	Hydrogen bonds	Hydrophobic bonds	Residues
MC1 (5xtd)	CDDP	-3.48	2	2	Asp134, Tyr147, Ile147, Ala166, Val173, Asp357, Asn339, Asp338
	NG	-5.29	2	4	Lys137, Tyr147, ASP153, Lys 151, Asp146
	HD	-5	2	4	Asp150, Ile133, Tyr147, Lys137
MCII (6myq)	CDDP	− 4.55	6	1	Lys87, Thr86, Thr82, Cys225
	NG	-6.81	5	2	Glu342, Lys227, Glu67, Arg223, Thr222, Gly228
	HD	-6.43	9	0	Lys227, Pro226, Thr82, Asn81, Lys88, Thr86
MCIII (1bgy)	CDDP	-4.46	4	2	Met190, Met194, Phe187
	NG	-6.88	2	5	Val14, Phe18, Met190, Leu37, Leu119, Leu197, Ala193
	HD	-6.79	1	4	Gly34, Leu197, Asn15, Val18, Phe14
MCIV (5b1a)	CDDP	-4.73	2	6	Phe251, Val91, Val248, Val247, Thr95
	NG	-7.38	0	7	Thr95, Phe98, Phe251, Val247
	HD	-8.39	1	8	Phe98, Trp240, Val247, Phe251
MCV (5ARA)	CDDP	-4.73	1		Gly109
	NG	-7.03	7	0	Ala389, Ile388, Ser114, His82, Gly79, Cys78
	HD	-7.1	6	0	Glu395, Ser114, Gly79, Ser83,
PARKIN (6glc)	CDDP	-5.78	1	3	Leu301, Lys48, Phe45, Tyr59, His302
	NG	-6.02	6	2	Asp58, Leu187, His227, Lys299, Leu301, Asn60,
	HD	-6.54	3	4	Leu187,Tyr59, Phe45, Lys299,Leu301
PINK1 (5OAT)	CDDP	-5.8	4	0	Val403, cys395, Glu392
	NG	-8.25	5	1	Phe401, Cys395, Ile394, Glu392
	HD	-10.1	8	3	Tyr102, Phe32, Thr100, Asp334, Cys360, Lys193, Glu214, Asn339, Gly101, Asn60, Asp357, Lys167

Fig. 1.

Molecular docking of CDDP, NG, and HD with the main proteins involved in mitochondrial bioenergetics and mitophagy: mitochondrial complex I (MCI), mitochondrial complex II (MCII), mitochondrial complex III (MCIII), mitochondrial complex IV (MCIV), mitochondrial complex V (MCV), and mitophagy proteins (PARKIN, PINK1). Involved residues are shown in the figures. Hydrogen bonds are presented as blue dotted lines, while hydrophobic contacts are represented as gray dotted lines

NG and HD bind to mitochondrial complex III (MCIII, 1BGY) with greater efficiency than CDDP (-4.46 kcal/mol). Leu197 and Met190 are among the more hydrophobic residues that NG interacts with, thereby preserving the stability and functionality of the complex. In mitochondrial complex IV (MCIV, 5B1A), HD has the highest affinity (-8.39 kcal/mol), followed by NG (-7.38) and CDDP (-4.90). HD could mitigate the mitochondrial damage caused by CDDP by interacting with Trp240 and Phe98 in a hydrogen and hydrophobic manner. NG (-7.03 kcal/mol) and HD (-7.10 kcal/mol) function better than CDDP (-4.73 kcal/mol) for mitochondrial complex V (MCV, 5ARA), exhibiting strong interactions at residues such as Ala389 and Gly79, which may protect mitochondrial ATP generation. PARKIN (6GLC) interacts with Leu187 and Lys299 and has the highest affinity for HD (-6.54 kcal/mol). NG (-6.02 kcal/mol) binds more effectively than CDDP (-5.78), which implies that it might promote PARKIN-mediated mitophagy. PINK1 (5OAT) is best bound by HD (-10.1 kcal/mol), followed by NG (-8.25) and CDDP (-5.80). HD protects mitochondrial quality by forming a complex network of interactions, including hydrogen bonds with Asp334, Cys360, and Glu214.

Effect of CDDP, NG, and HD on granulosa cells’ viability

The results of the MTT assay revealed the inhibitory effect of CDDP on cellular viability by demonstrating a dose-dependent reduction in cell viability. Cell viability was relatively high (90–95%) at lower concentrations (1 µM) but decreased to about 60–70% at intermediate concentrations (10 µM). Cell viability dramatically dropped (below 20%) at the highest tested dosages (e.g., 50 µM or above), indicating substantial cytotoxicity (Fig. 2A). The effect of NG and HD on granulosa cell line viability at various concentrations (0, 10, 20, 40, 80, and 160 µM) demonstrated that viability increased gradually as NG and HD concentrations increased, reaching a peak at 160 µM (Figs. 2B and C).

Fig. 2.

Effects of cisplatin (CDDP), naringin (NG), and hesperidin (HD) on cell viability, ATP levels, and MMP. (A) The MTT test was used to evaluate cell viability. (B) Cell viability assessed by the MTT assay. Under the identical treatment circumstances as in panel (E), (F) MMP was assessed as a percentage of control relative fluorescence units (RFU). Presenting the median and interquartile range, data are shown as box graphs. **P < 0.01 and ***P < 0.001 imply statistical significance

The MTT assay was done to investigate the effects of CDDP both solo and in combination with NG or HD at varying concentrations on the viability of granulosa cell lines. Treatment with 5 µM CDDP alone led to a substantial reduction in viability (absorbance of 64% compared to the control group). The co-treatment of CDDP with NG or HD resulted in cell viability of 81.0% and 91.9% of control, respectively, suggesting their mitigating potentials. Cell viability dramatically dropped to 42.6%, contrary to the control at 10 µM CDDP as sole treatment, indicating enhanced cytotoxicity. Concurrent treatment with NG enhanced cell viability to 62.0%, while HD further increased it to 73.0%. Our data indicate that both NG and HD could partially alleviate the cytotoxic effects of CDDP, with HD demonstrating a slightly higher mitigating effect (Fig. 2D).

Effect of CDDP, NG, and HD on ATP production and MMP

The metabolic activity of granulosa cell lines was evaluated using the ATP assay upon treatment with CDDP either alone or in combination with NG or HD. ATP levels were significantly reduced to 64.0% following treatment with 5 µM CDDP, suggesting substantial cytotoxicity. NG and HD co-treatment increased ATP levels to 85.0% and 96.0%, respectively, exhibiting their mitigating effects. ATP levels declined to 41.0% at 10 µM CDDP, implying CDDP’s dose-dependent cytotoxicity. ATP levels were partially restored to 75% of the control when NG was added, whereas HD increased these levels to 79% (Fig. 2E).

Rh-123 was employed to evaluate the effect of CDDP both independently and in tandem with NG or HD on MMP in granulosa cells. Following treatment with 5 µM CDDP, MMP was reduced to 64.74 ± 5.39% of the control, exhibiting mitochondrial malfunction. At 10 µM CDDP, MMP levels significantly decreased to 43.87 ± 5.52%, revealing a dose-dependent escalation in mitochondrial damage. The administration of NG or HD alongside CDDP resulted in a partial restoration of MMP levels, with HD displaying a more pronounced protective effect (Fig. 2F).

Granulosa MMP was evaluated by the flow cytometry assay following CDDP treatment and its combination with NG or HD. Treatment with 5 µM CDDP decreased the ULQ to 66.19 ± 8.28%, indicating a notable alteration in cell populations. The ULQ was further decreased to 52.81 ± 6.62% upon exposure to 10 µM CDDP, demonstrating the dose-dependent effects of CDDP. Co-treatment with NG or HD partially recovered ULQ values, with HD demonstrating a greater protective effect, reflecting a substantial recovery in cellular MMP (Fig. 3).

Fig. 3.

Effects of cisplatin (CDDP), naringin (NG), and hesperidin (HD) on MMP (δψm). (A) Control (Cont) and treated cells’ mitochondrial membrane potential (ΔΨm) flow cytometry dot plots are displayed here. JC-1 dye was used to label cells; red fluorescence indicates high ΔΨm, or healthy mitochondria, while green fluorescence shows mitochondrial depolarization. Low-dose (CDDPL) and high-dose (CDDPH) co-treated groups with NG or HD treatments. In the bottom, the box plots displaying median and interquartile range, (B) quantification of decipher dye orange fluorescence (indicative of intact ΔΨm) among treatment groups. (C) Quantitative data of JC-1 green fluorescence (indicative of mitochondrial depolarization) under varying treatment settings. Significant variations among treated groups shown by statistical analysis demonstrate the protective properties of NG and HD against CDDP-induced mitochondrial dysfunction. Presenting the median and interquartile range, data are shown as box graphs. *P < 0.05, **P < 0.01, and ***P < 0.001 imply statistical significance

Effect of CDDP, NG, and HD on mitophagy biomarkers

The effects of CDDP either alone or in combination with NG or HD on mitophagy proteins in granulosa cell lines were assessed using the PINK1 and PARKIN ELISA evaluation. A significant reduction in PINK1 expression was seen following treatment with 5 µM CDDP. PINK1 expression further decreased when the concentration of CDDP was increased to 10 µM, indicating a dose-dependent interaction. This decrease was mitigated by co-treatment with NG or HD, demonstrated by the partial restoration of PINK1 levels. HD displayed a nearly complete recovery to control levels (Figs. 4A and B).

Fig. 4.

Effects of cisplatin (CDDP), naringin (NG), and hesperidin (HD) on mitochondrial respiration and mitophagy markers. The assays investigated the effect of treatments on PINK1 protein levels (pg/ml) (A), PINK1 gene expression (relative expression) (B), Parkin protein levels (pg/ml) (C), and PARKIN gene expression (relative expression) (D). Lactate levels (µM/mg protein) (E), PDH fold change (F). α-KG fold change (G), OCR rate (O₂ consumption, nmol O₂/min) (H). Presenting the median and interquartile range, data are shown as box graphs. *P < 0.05, **P < 0.01, and ***P < 0.001 imply statistical significance

The PARKIN level revealed that treatment with 5 µM CDDP resulted in a decrease of PARKIN levels to 88.8%, suggesting that PARKIN expression was suppressed. Co-treatment with NG raised levels to 101.3%, whereas co-administration of HD enhanced them to 108.1%, indicating a more profound mitigating effect. PARKIN levels dramatically dropped to 68.6% at 10 µM CDDP, suggesting a dose-dependent reduction. Concurrent treatment with NG partially alleviated the suppression, resulting in levels of 82.7%. In contrast, co-treatment with HD led to a more pronounced improvement than NG, achieving levels of 101.0% (Figs. 4C and D).

Effect of CDDP, NG, and HD on lactate production

As depicted in Fig. 4E, lactate levels significantly increased after treatment with 5 µM CDDP, indicating increased glycolytic activity to compensate for the notable mitochondrial dysfunction. Levels were partially restored to 90.6% upon co-treatment with NG. However, lactate levels were further reduced to 77.2% by co-treatment with HD, suggesting a protective effect against metabolic changes brought on by CDDP. At 10 µM CDDP, lactate levels increased dramatically to 162.4%, suggesting that higher CDDP levels are associated with enhanced glycolytic activity. Co-treatment with NG produced levels of 131.5%, whereas co-treatment with HD produced levels of 112.1%, which were closer to baseline.

Effect of CDDP, NG, and HD on Krebs cycle kinetics

The PDH activity, as the main entry of the Krebs cycle, in granulosa cell lines, was examined under various treatment conditions. Treatment with 5 µM and 10 µM CDDP decreased PDH activity to 88.3% and 67.5%, respectively, suggesting an adverse impact on mitochondrial function. Co-treatment with NG resulted in a partial restoration of activity, whereas HD caused a more noticeable increase in PDH activity (normalizing function) and indicated a potential protective effect (Fig. 4F).

The levels of α-ketoglutarate (α-KG) in granulosa cell lines were assessed as a main intermediate in the Krebs cycle. Treatment with 5 µM CDDP resulted in a reduction of α-KG levels, while 10 µM CDDP significantly caused a pronounced reduction of α-KG levels, indicating substantial mitochondrial dysfunction. Co-treatment with NG partially restored α-KG levels. In contrast, co-treatment with HD elevated α-KG levels significantly, indicating protective effects of both compounds, with HD demonstrating a more significant effect (Fig. 4G).

Effect of CDDP, NG, and HD on OCR

Additionally, the OCR in granulosa cell lines was assessed under various treatment conditions. Treatment with 5 µM and 10 µM CDDP resulted in a significant reduction of OCR to 83.3% and 58.5% of the control, respectively, indicating a notable decline in mitochondrial efficiency. Co-treatment with NG resulted in a slight improvement in OCR, while co-treatment with HD yielded better improvement in recovery relative to NG, demonstrating the consistent alleviating effects of HD (Fig. 4H). As depicted in Fig. 5, CDDP dramatically reduced mitochondrial complex activities in a concentration-dependent manner. NG and HD significantly decrease the effect of CDDP on the mitochondrial complexes of human granulosa cells to varying degrees.

Fig. 5.

Effects of cisplatin (CDDP), naringin (NG), and hesperidin (HD) on mitochondrial respiratory chain complexes over time. Complex I (CI) activity in cells treated with 5 µM and 10 µM CDDP, respectively. Rotenone (RO) was used as a CI inhibitor (A, B). Complex II (CII) activity under CDDP (5 µM and 10 µM) treatment. TTFA was used as a CII inhibitor (C, D). Complex III (CIII) activity in response to CDDP (5 µM and 10 µM) treatment. Antimycin A (AA) was used as a CIII inhibitor (E, F). Complex IV (CIV) activity following treatment with CDDP (5 µM and 10 µM). Potassium cyanide (KCN) was used as a CIV inhibitor (G, H). Complex V (CV) activity after treatment with CDDP (5 µM and 10 µM). Rotenone (RO) was used as a reference control (I, J). Cells were treated with cisplatin (CDDP) at 5 µM or 10 µM, with or without neuroglobin (NG, 80 µM) and HDAC inhibitor (HD, 80 µM). Data are presented as mean ± SE

Effect of CDDP, NG, and HD on mitochondrial complexes

The effect of CDDP medications, given solo or in conjunction with NG or HD, on the expression of mitochondrial genes ND1, ND5, Cyb, Co-1, and ATP 6/8 in the granulosa cell line was investigated. Gene expression varies between treatment groups and is modulated by both dosage and combination factors. Combining CDDP with NG or HD reduces its effects to varying degrees, particularly at lower CDDP doses (Fig. 6).

Fig. 6.

Box plots present the effects of cisplatin (CDDP), naringin (NG), and hesperidin (HD) on mitochondrial gene expressions. ND1 (A), ND5 (B), Cyt b (C), CO1 (D), and ATP6/8 (E). Statistical significance was assessed using appropriate post-hoc tests, with significance levels indicated as *P < 0.05, **P < 0.01, and ***P < 0.001

Effect of CDDP, NG, and HD on mitochondrial membranes

The effect of various micromolar doses of CDDP on mitochondrial membrane swelling, permeability to H⁺ and K⁺ ions, and MMF was investigated (Fig. 7). The recorded drop in absorbance revealed a significant dose-dependent increase in mitochondrial membrane swelling, indicating mitochondrial injury (Fig. 6A). This resulted in increased penetration of H⁺ and K⁺ ions (Figs. 7B and C) and disturbances in the electrochemical gradient across mitochondrial membranes. The effect of various doses of CDDP on MMF of granulosa cells, both alone and in combination with NG or HD. CDDP5 does not significantly increase MMF (121.4%±16.2%), but when combined with NG, it slightly decreases (101.4%±13.0%). CDDP (5 µM) combined with HD exhibited comparable levels (94.8% ± 10.8%). CDDP doses of 10 µM showed a significant impact (171.7% ± 21.0%). Combining CDDP at 10 µM with NG resulted in a slight decrease in the effect (142.3%±14.5%); however, cotreatment with HD displays a potential protective effect (121.5%±12.7%) (Fig. 7D).

Fig. 7.

Effects of cisplatin (CDDP), naringin (NG), and hesperidin (HD) on various mitochondrial membranes integrity parameters. Mitochondrial swelling assay in response to CDDP treatment alone and in combination with NG or HD. (A) Potassium ion permeation (KP) assay; (B) hydrogen ion permeation (HP) assay; (C) absorbance changes at 540 nm indicating key physiological responses under the indicated treatment conditions. (D) MMF: Fluorescence polarization changes, reflecting alterations in membrane dynamics or structural integrity across treatment groups. Statistical significance is indicated as **P < 0.01 and ***P < 0.001. MMFAC was evaluated by gas chromatography. (E) Bar graph displaying fold changes in MMFAC across different experimental conditions. Various fatty acids, including saturated palmitic (PLI) and stearic (STC) fatty acids and unsaturated palmitoleic (PLO), oleic (OLC), linoleic (LIC), arachidonic (ARC), and docosahexaenoic (DOC) fatty acids, were measured. In addition, the saturated/unsaturated (S/US) fatty acid ratio was evaluated. Statistical annotations indicate significant differences between groups, with labels a, b, and c denoting P < 0.05, P < 0.01, and P < 0.001, respectively

The effect of different doses of CDDP, both alone and in tandem with NG80 or HD80, on granulosa cell MMFAC was investigated (Fig. 7E). The data revealed that CDDP dramatically reduced the amounts of saturated fatty acids (palmitic and stearic acids) while substantially increasing the levels of other examined unsaturated fatty acids, resulting in an enhanced unsaturated/saturated fatty acid ratio in membranes. The effects were significantly mitigated by cotreatment with NG and HD to varying degrees.

Multivariate analyses

Clustering heatmap revealed that CDDP and its combinations with HD and NG modulate mitochondrial function and corresponding parameters. CDDP substantially decreased mitochondrial metrics such as MMP, ATP, OCR, and expression of key mitochondrial genes and proteins. However, co-treatment with HD (CDDP5 + HD80) and NG (CDDP5 + NG80) increased MMP, ATP, and gene expression, thereby partially restoring the mitochondrial function. ND5 and CO-1 were somewhat better protected by HD than NG. These findings suggest that HD and NG may attenuate CDDP-induced mitochondrial dysfunction by enhancing bioenergetics and mitophagy (Fig. 8A). The PCA plots show how the first two principal components (PC1 and PC2) capture variation in data. Figure 8B demonstrates that PC1 accounts for 75.7% of the total variance, showing significant differences between treatment groups, whereas PC2 explains 6.3%. CDDP had a considerable impact on mitochondrial properties, with the control group clustering differently than CDDP (10 µM) and CDDP (5 µM). Co-treatment with HD or NG results in clustering closer to the control, particularly in CDDP5 + HD80 and CDDP5 + NG80, indicating that they might decrease CDDP-induced dysfunction. With a substantial distinction between the control, CDDP5, and CDDP10 treatments, PC1 explains 81.5% of the overall variation (Fig. 8C). However, Figs. 8D and E show co-treatment findings, with PC1 (61%-71.6%) and PC2 (7.4%-9.2%) indicating different co-treatments. The co-treatment groups (CDDP5 + HD80, CDDP5 + NG80, CDDP10 + HD80, and CDDP10 + NG80) cluster independently of high-dose CDDP10, indicating partial mitochondrial repair.

Fig. 8.

Clustering heatmap and PCA of the effects of cisplatin (CDDP), naringin (NG), and hesperidin (HD) on mitochondrial and metabolic parameters. (A) The heatmap displays the expression levels of various mitochondrial and metabolic markers across different experimental groups. The color scale represents normalized expression values, with red indicating upregulation and blue indicating downregulation, and hierarchical clustering reveals patterns of similarity among treatments. (B–E) PCA plots depict the variance in mitochondrial and metabolic profiles among experimental conditions. (B) shows PCA including all groups, highlighting the primary clustering along PC1 and PC2. (C) Compares CDDP5, CDDP10, and control, demonstrating a dose-dependent metabolic shift. (D) focuses on CDDP5 and its co-treatments (CDDP5 + HD80 and CDDP5 + NG80), revealing distinct metabolic effects. (E) Examination of CDDP10 with HD80 and NG80, shows a clear separation between treatment groups

Overall, the results indicate that CDDP induces dose-dependent cytotoxicity in granulosa cells. Evidence is indicated by reduced cell viability, mitochondrial dysfunction, decreased ATP production, alterations in mitochondrial membrane potential, diminished mitophagy, variations in the Krebs cycle activity, and decreased expression of key mitochondrial genes and complexes. Co-treatment with NG and HD resulted in a partial or complete reduction of these adverse effects. HD consistently demonstrated a more robust protective effect across various parameters. Multivariate analyses, including hierarchical clustering and PCA, confirmed that NG and HD restore mitochondrial function and metabolic homeostasis to levels near control, highlighting their potential as modulators of CDDP-induced mitochondrial and bioenergetic dysfunction.

Discussion

CDDP is one of the most extensively used chemotherapeutic medications worldwide [36]. Despite its therapeutic benefits, it exerts dose-dependent toxic effects, including nephrotoxicity, neurotoxicity, and reproductive toxicity [14]. CDDP toxicity engenders mitochondrial dysfunction, oxidative stress, and activation of inflammatory pathways. This cascade results in DNA injury, endoplasmic reticulum stress, and intrinsic apoptosis [37, 38]. The current study examined CDDP ovarian toxicity and the protective effect of NG and HD in human granulosa cells that exemplify an outstanding in vitro model for reproductive toxicology, owing to their crucial roles in follicular development, oocyte maturation, and hormone synthesis [39]. These cells also provide insight into cellular processes, including signaling pathways and toxin-induced changes in gene expression [40].

The molecular docking analysis was applied as a tool for generating hypotheses to inform subsequent in vitro studies. High-resolution PDB structures of mitochondrial complexes I–V, PARKIN, and PINK1 were selected based on functional relevance, and key binding residues were identified from prior literature. Docking simulations using SwissDock, validated with AutoDock Vina, evaluated ligand-target interactions, including hydrogen bonds, hydrophobic contacts, and π-π stacking, with estimated free energies of binding (ΔG) used to compare affinities. The docking results indicated that NG and HD exhibited stronger binding affinities and more favorable interaction patterns with mitochondrial complexes (I–V), PARKIN, and PINK1 compared to CDDP. This indicates their potential role in stabilizing mitochondrial function and enhancing mitophagy. NG and HD exhibited interactions with critical residues in complexes I–V, which may enhance electron transport efficiency and ATP production. Additionally, they formed strong interactions with PARKIN and PINK1, suggesting a potential role in promoting mitophagy and mitochondrial quality control. In addition, docking simulations suggest potential interactions between NG, HD, and CDDP with mitochondrial complexes and mitophagy proteins, but these results do not confirm that the ligands occupy identical binding sites. Differences in predicted binding affinities may arise from ligand size or conformational flexibility rather than specific site preference. Future studies employing site-directed mutagenesis or advanced biophysical assays are necessary to validate these interactions and clarify their functional significance. However, these computational results provide a hypothesis for the toxic effect of CDDOP and the protective effects of NG and HD, which guided the subsequent in vitro assays.

According to the current study, CDDP considerably altered the mitochondrial bioenergetics of the granulosa cells and diminished the treated cells’ ATP levels and MMP. Previous research suggests that the administration of CDDP leads to considerable depolarization of the MMP, signifying compromised mitochondrial integrity and function [41]. CDDP treatment increased ROS production and inhibited MMP in human ovarian cancer cells, hence enhancing oxidative stress and causing mitochondrial dysfunction and apoptosis [42, 43].

Current research revealed that CDDP markedly reduced the mitochondrial complex activities of granulosa cells and their encoding gene expressions according to its concentrations. This is congruent with the prior research, which found that CDDP significantly interacts with mitochondrial DNA, compromises respiratory chain complexes’ integrity, causes mitochondrial malfunction, and ultimately triggers apoptosis [13, 41]. Complex I (NADH: ubiquinone oxidoreductase) and complex II (succinate dehydrogenase) are inhibited by CDDP, resulting in decreased ATP synthesis, enhanced ROS formation, and impaired electron transport [44]. It also harms complex IV (cytochrome C oxidase), worsening oxidative phosphorylation deficits [45]. In addition to being crucial for CDDP’s anticancer efficiency, these consequences also play a role in its dose-limiting toxicities [5, 46].

Additionally, the current study demonstrated that CDDP altered the levels of expression of PINK1 and PARKIN autophagic proteins and their coding gene. Normally, PINK1 enters mitochondria and is degraded at the inner membrane. Interestingly, the import of PINK1 is dependent on mitochondrial membrane potential. When mitochondria become depolarized due to cell stress, PINK1 accumulates on the mitochondrial outer membrane, recruiting and phosphorylating PARKIN (E3 ubiquitin ligase) [47]. Upon phosphorylation, parkin is activated, causing the ubiquitination of different mitochondrial outer membrane proteins, which are then enclosed by autophagosomes and eventually degraded by autolysosomes [48]. According to previous research, CDDP frequently disrupts mitophagy, the process by which damaged mitochondria are selectively eliminated, leading to a buildup of dysfunctional mitochondria [49]. Additionally, mitophagy facilitates the recycling of damaged mitochondrial components, including proteins and lipids. Consequently, its inhibition hinders mitochondrial renewal and diminishes cellular energy metabolism [48]. CDDP compromises mitophagy in different cell lines, including auditory cell lines [50] and colorectal cancer cells [51], respectively. CDDP-induced disruption of mitophagy is indicated by diminished expression of mitophagy markers, including PINK1 and PARKIN [52]. CDDP-prompted mitochondrial malfunction is evidenced by oxidative stress and perturbations in mitochondrial dynamics [53]. These aberrations underscore the crucial role of mitochondrial dysfunction in the cytotoxic consequences of [51].

CDDP intoxication substantially promotes MMF in human granulosa cells, primarily by raising the ratio of unsaturated to saturated fatty acids in the mitochondrial membrane. The shift in lipid content raises the fluidity of the membrane, which may jeopardize the structural integrity and function of the mitochondria. Enhanced MMF may impair the efficacy of the electron transport chain, leading to decreased ATP synthesis and enhanced generation of ROS [54].

The current results revealed that NG and HD notably mitigated the effect of CDDP on the mitochondrial bioenergetics of granulosa cells in the various parameters evaluated. According to published research, the two essential citrus flavonoids have garnered considerable interest for their potential to regulate mitochondrial bioenergetics, notably in relation to oxidative stress and metabolic dysfunction [20, 21]. Several investigations have demonstrated that NG can boost mitochondrial function by increasing ATP generation while decreasing MMP dissipation. NG has been shown to protect against mitochondrial dysfunction caused by toxins like rotenone by stabilizing the mitochondrial membrane and lowering oxidative stress [55, 56]. In a similar vein, another citrus flavonoid, HD, has been demonstrated to boost mitochondrial bioenergetics by modifying mitochondrial respiration and enhancing ATP generation, particularly in cells exposed to oxidative stress [21, 57]. In tandem, these flavonoids promote mitochondrial homeostasis, minimize oxidative harm, and boost cellular energy generation. Furthermore, both NG and HD have been investigated for their synergistic effects when administered in combination with other antioxidants, implying their significance in maintaining mitochondrial function [58].

Interestingly, PCA demonstrates that PC1 primarily reflects treatment-induced mitochondrial function alterations, whereas PC2 captures additional variability. HD80 and NG80 protect, particularly at CDDP5 (lower CDDP doses). These findings imply that NG and HD can boost mitochondrial activity and ameliorate mitochondrial dysfunction. The suggested pathways underlying the protective potential of NG and/or HD against CDDP-induced mitochondrial dysfunction in ovarian granulosa cells are compiled in Fig. 9.

Fig. 9.

Proposed mechanisms behind the protective potential of naringin (NG) and/or hesperidin (HD) against cisplatin (CDDP)-induced mitochondrial dysfunction in ovarian granulosa cell

The current findings are consistent with previous research demonstrating the vulnerability of granulosa cells to chemical disturbances. A previous study highlighted that chemotherapeutic agents, including CDDP, induce ovarian toxicity through mechanisms such as oxidative stress, mitochondrial dysfunction, and apoptosis, indicating the need for protective strategies [59]. Prior studies employing caprine models have shown that granulosa cells are notably susceptible to cytotoxic agents. Sharma and Bhardwaj [60] reported apoptosis-related morphological changes in granulosa cells, whereas Bhardwaj and Saraf [61] observed mitochondrial and ultrastructural alterations following malathion exposure. These studies highlight the critical importance of mitochondrial integrity and cell viability in maintaining ovarian function, thereby supporting the investigation of natural flavonoids, such as NG and HD, as protective agents against CDDP-induced ovarian damage.

The present study demonstrates the protective effects of NG and HD against CDDP-induced mitochondrial dysfunction in granulosa cells; however, some limitations should be acknowledged. The study is limited to an in vitro model, and the cellular responses observed may not accurately reflect in vivo ovarian physiology or systemic pharmacokinetics. Secondly, while various mitochondrial parameters and mitophagy markers were examined, alternative pathways potentially contributing to CDDP toxicity, such as inflammatory signaling or DNA damage responses, were not investigated. Thirdly, the concentrations and exposure durations utilized may not align with clinically relevant doses. Fourthly, although NG and HD were tested separately in docking and in vitro assays, the potential for synergistic interactions was not explicitly assessed. Docking predictions cannot determine if these flavonoids compete for the same binding sites or exhibit synergistic effects. Future research should investigate combination therapies to assess whether NG and HD operate synergistically or additively in protecting against CDDP-induced mitochondrial dysfunction. Finally, molecular docking provided insights into the mechanisms involved; however, these computational predictions require validation in more complex biological systems. Further investigations using in vivo models and detailed mechanistic analyses are necessary to confirm the translational efficacy of NG and HD for ovarian protection. In addition, while molecular docking provided useful mechanistic hypotheses, it is inherently limited and cannot confirm functional effects of ligand-residue interactions. Advanced computational methods, such as free energy perturbation, and experimental validation are needed to substantiate the predicted interactions.

Conclusions

This study demonstrates that CDDP impairs mitochondrial bioenergetics, disrupts membrane integrity, and suppresses mitophagy, resulting in pronounced mitochondrial dysfunction in ovarian granulosa cells. These detrimental effects are efficiently counteracted by NG and HD, which restore respiratory complex activities, mitochondrial membrane potential (MMP), and ATP production. HD exhibited superior protective effects, substantially normalizing key mitochondrial parameters. The cytoprotective action of both flavonoids is further supported by molecular docking analyses. Multivariate analysis indicates that NG and HD partially attenuate CDDP-induced mitochondrial damage, particularly at lower concentrations. Collectively, these findings suggest that NG and HD are promising adjuvants for mitigating ovarian toxicity induced by CDDP, thereby potentially preserving fertility in female cancer patients. By targeting mitochondrial dysfunction, these natural antioxidants may enhance the safety profile of CDDP-based chemotherapy without compromising its anticancer efficacy.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

α-KG

Alpha-Ketoglutarate

AA

Antimycin A

ARC

Arachidonic Acid

ATP

Adenosine Triphosphate

CDDP

Cisplatin

CO1

Cytochrome C Oxidase Subunit I

Cyt b

Cytochrome b

DAB

3,3′-Diaminobenzidine

DAMP

Damage-Associated Molecular Pattern

DOC

Docosahexaenoic Acid

ELISA

Enzyme-Linked Immunosorbent Assay

GAPDH

Glyceraldehyde-3-Phosphate Dehydrogenase

HD

Hesperidin

HP

Hydrogen Ion Permeation

IHC

Immunohistochemistry

KCN

Potassium Cyanide

KP

Potassium Ion Permeation

LC3

Microtubule-Associated Protein 1 A/1B-Light Chain 3

LIC

Linoleic Acid

MC I-V

Mitochondrial Complex I-V

MMF

Mitochondrial Membrane Fluidity

MMFAC

Mitochondrial Membrane Fatty Acid Composition

MMP

Mitochondrial Membrane Potential

MTS

3-(4,5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2 H-Tetrazolium

NADH

Nicotinamide Adenine Dinucleotide (Reduced)

ND1

NADH Dehydrogenase Subunit 1

ND5

NADH Dehydrogenase Subunit 5

NG

Naringin

OCR

Oxygen Consumption Rate

OLC

Oleic Acid

PAMP

Pathogen-Associated Molecular Pattern

PARKIN

Parkin RBR E3 Ubiquitin Protein Ligase

PCA

Principal Component Analysis

PDH

Pyruvate Dehydrogenase

PINK1

PTEN-Induced Kinase 1

PLI

Palmitic Acid

PLO

Palmitoleic Acid

PTEN

Phosphatase and Tensin Homolog

Rh-123

Rhodamine-123

ROI

Region of Interest

ROS

Reactive Oxygen Species

S/US

Saturated/Unsaturated Fatty Acid Ratio

STC

Stearic Acid

TLR

Toll-Like Receptor

TNF-α

Tumor Necrosis Factor-Alpha

TTFA

Thenoyltrifluoroacetone

ULQ

Upper Left Quadrant (Flow Cytometry)

Author contributions

JL, EME, AAA, MS, SFI, MG, QG: Conceptualization. JL, EME, AAA, NA, MS, SFI, MG, ASA, QG: Software, Validation, Visualization, Data curation, and Formal analysis. EME, AAA, FSE, HMA, HEN, SKE, MAS, NE, HSW, SA: Investigation, Methodology, Formal analysis, Validation, and Visualization. All authors: Writing-original draft, Writing-review & editing. EME, QG: Supervision. All authors read and approved the final manuscript.

Funding

The authors extend their appreciation to Northern Border University, Saudi Arabia, for supporting this work through project number (NBU-CRP-2026-2510). This research was also funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R127), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Research, King Faisal University, Saudi Arabia (Grant No. KFU261460).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Institutional review board statement

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.