Diosmetin ameliorates inflammation and apoptosis in the pathomechanism of PCOS through the NRF2/AKT/PPARγ signalling pathway

Abstract

Ethnopharmacological relevance

Diosmetin (DIO) is a flavonoid extracted from the traditional Chinese medicine Schizonepeta tenuifolia Briq. The anti-inflammatory and neuroprotective properties of DIO have shown promise. However, the underlying mechanisms need further elucidation.

Study aim

This research aimed to explore how DIO reduces oxidative stress and inflammation in the ovaries and slows the pathological development of polycystic ovary syndrome by influencing the AKT/PPARγ signalling pathway.

Materials and methods

DIO targets were screened via network pharmacology tools. The protective effect of DIO on polycystic ovary syndrome was assessed via haematoxylin‒eosin (H&E) staining. Immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence were used to determine the effects of DIO on ovarian granulosa cell inflammation. In addition, we performed Western blotting to determine the expression of TNF-α, IL-6 and AKT/PPARγ pathway proteins.

Results

This research demonstrated an increase in TNF-α and IL-6 expression in a rat model of polycystic ovary syndrome (PCOS) induced by letrozole. Histological analysis indicated that the ovaries of rats in the PCOS group showed significant follicular loss and vacuolation changes compared with those in the normal control (NC) group. Treatment with DIO improved the cystic changes in the ovaries. Metabolic assessments revealed that the PCOS group presented significantly altered levels of FSH (4.2 ± 0.3 IU/L), TG (0.65 ± 0.2 mmol/L), E2 (106 ± 14 pg/L), TC (3.9 ± 0.7 mmol/L), LH (7.8 ± 0.2 IU/L), and TEST (11 ± 3 ng/mL) compared with those in the NC group (FSH: 6.3 ± 1.7 IU/L; TG: 1.2 ± 0.2 mmol/L; E2: 147 ± 21 pg/mL; TC: 2.2 ± 0.4 mmol/L; LH: 5.8 ± 1.2 IU/L; and TEST: 5.5 ± 2 ng/mL), indicating hyperandrogenaemia. Additionally, at the conclusion of the study, the PCOS group (310 ± 7 g) presented a significant increase in body weight compared with the NC group (310 ± 7 g), whereas treatment with 50 mg/kg DIO (351 ± 6 g) or 100 mg/kg DIO (342 ± 8 g) mitigated this weight gain. Immunohistochemistry, Western blot, and immunofluorescence results revealed that DIO reduced inflammation and alleviated the pathological changes associated with PCOS. Furthermore, DIO improved the inflammatory condition of the ovaries in the PCOS group by inhibiting the AKT/PPARγ signalling pathway. The suppression of AKT and PPARγ diminished the anti-inflammatory effects of DIO. Additionally, DIO countered inflammation and apoptosis in testosterone-induced ovarian granulosa cells by enhancing the expression of AKT/PPARγ signalling.

Conclusion

The present study confirms that DIO has important therapeutic potential for treating polycystic ovary syndrome by inhibiting ovarian inflammation and oxidative stress through the modulation of AKT/PPARγ signalling.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13048-025-01788-y.

Introduction

Polycystic ovary syndrome (PCOS) is an endocrine disorder prevalent in women of childbearing age and is usually characterized by altered ovarian morphology, hyperandrogenaemia, and irregular menstruation, which have significant negative physical and psychological effects on women [1–3]. PCOS affects approximately 15% of women of childbearing age globally (Rotterdam 2003) and causes 75% of anovulatory infertility [4, 5]. An epidemiological study of 8993 women revealed that the prevalence of PCOS was 19.6% among Indian women between the ages of 18 and 40 years [6]. Patients with PCOS exhibit a markedly elevated risk of long-term complications, including metabolic syndrome, endometrial cancer, and cardiovascular disease, relative to women without this condition. There is no effective treatment for PCOS, potentially due to the lack of understanding of the molecular mechanisms underlying its pathogenesis.

Several studies have reported that chronic low-grade inflammation and apoptosis are prominent features of PCOS, and the Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway is strongly associated with this inflammatory state [7–9]. Studies have shown that patients with PCOS have increased levels or gene expression of inflammatory markers, including C-reactive protein (CRP), interleukin-18 (IL-18), tumour necrosis factor (TNF-α), and IL-6. One study described the interplay among hyperinsulinaemia, metabolic syndrome, and the inflammatory state in patients with PCOS [10]. In addition, therapies targeting inflammatory and apoptosis have shown great potential for treating PCOS [11, 12].

Diosmetin (DIO) is a naturally occurring flavonoid found in large quantities in Schizonepeta tenuifolia Briq with anti-inflammatory, anticancer, antimicrobial, phytoestrogen, and neuroprotective properties [13, 14]. Studies have shown that DIO exerts cytoprotective effects against lipopolysaccharide-induced acute lung injury through inhibition of the nucleotide-binding structural domain-like receptor protein 3 (NLRP3) inflammasome and the activation of nuclear factor erythroid 2-related factor 2 (NRF2) [15]. DIO has been reported to exert antioxidative stress effects by altering the conformation of polyphenol oxidase [16]. In another study, DIO was found to alleviate the neuronal damage induced by advanced glycosylation end products by targeting peroxisome proliferator-activated receptor γ [17]. Furthermore, it has been demonstrated that isomers of DIO exert antiproliferative and anti-inflammatory effects on TNF-α-stimulated fibroblast-like synoviocytes by modulating the Akt and NF-κB signalling pathways [18]. However, whether DIO can alleviate pathological changes in PCOS by inhibiting inflammation and apoptosis remains unclear. In this study, the effect of DIO intervention on the expression of NRF2/AKT1/PPARγ signalling pathway components in PCOS OGCs was investigated.

Materials and methods

General

This project (JN No. 20241030s0240315[575]) was formally approved by the Animal Ethics Committee of Jiangnan University and was conducted in accordance with the principles of the Declaration of Helsinki. Eight-week-old female Sprague–Dawley (SD) rats were purchased from Jiangnan University Laboratory Animal Center. All rats were housed in a sterile pathogen-free (SPF) environment (temperature, 23 ± 1 °C; humidity, 40–55%; light/dark cycle, 12 h). All rats were allowed to drink and eat freely.

Network pharmacology and molecular docking

The TCMSP (https://old.tcmsp-e.com/tcmsp.php) and SWISS (http://www.swisstargetprediction.ch/) databases were searched for 100 targets related to the action of the compound diosmetin, which were grouped around the regulation of isoenzymes. Polycystic ovary syndrome was entered into the GeneCards (https://www.genecards.org) database, and information on 6445 targets was downloaded. A total of 61 targets at the intersection of drugs and diseases were obtained to produce Venn diagrams. The intersecting target information obtained in the previous step was entered into the STRING database (https://cn.string-db.org/), multiple proteins were selected, the intersecting target information was entered, and Homo sapiens was selected as the organism to obtain the PPI network. The intersecting targets AKT1, SRC, ESR1, EGFR, PPARG, MMP2, and IGF1R were all found to be closely related to the regulation of inflammation, oxidative stress and apoptosis. Thus, the structural files for diosmetin and the proteins AKT1, SRC, ESR1, EGFR, PPARG, IGF1R, and NRF2 were individually downloaded, and molecular docking was performed using Discovery Studio. Autodock Tools 1.5.6 software was utilized to enhance the preparation of the ligand small molecules by incorporating hydrogen atoms and assigning charges. The optimized structure was obtained and stored as a pdbqt structure file for subsequent molecular docking. The combined stable conformations were used for visual pattern analysis and imported into PyMOL 2.4 mapping software to construct 3D molecular structure maps.

Animal models

Forty-eight female SD rats were randomly divided into four groups (12 rats per group): (1) the sham group (NC); (2) the PCOS group; (3) the PCOS + 50 mg/kg DIO group; and (4) the PCOS + 100 mg/kg DIO group. To establish a PCOS model, rats were administered letrozole [19–21] (1 mg/kg; Jiangsu Hengrui Pharmaceutical Co., Ltd.) via gavage daily for 30 days [22]. DIO (50 mg/kg or 100 mg/kg) was also administered [23] by gavage to the rats in the treatment group once a day for 30 days. The dosages for DIO are based on previously published literature [23, 24]. DIO was dissolved in ultrapure water and given to the rats through gavage for treatment purposes. Rats in the sham group received the same amount of ultrapure water via gavage. Each rat was administered a daily gavage dose in a volume of 0.5 ml. Throughout the experiment, there were no fatalities among the rats, and no notable negative drug reactions were detected. After one month, the rats were euthanized, and their intact ovaries were harvested. These isolated ovaries were then utilized for immunohistochemical (IHC) and histological analyses.

Histological and IHC examinations

The collected ovarian samples were subjected to IHC staining. Sections were dewaxed and hydrated after the inactivation of endogenous enzymes using hydrogen peroxide, antigen repair, and serum closure. The sections were incubated with p-AKT, p-PPARγ, cleaved caspase-3, TNF-α, and IL-6 antibodies (1:200) overnight at 4 °C. Diaminobenzidine staining was performed after incubation with the secondary antibody, and the staining results were observed using a microscope.

Cell culture

The Cell Bank of the Chinese Academy of Sciences provided the human KGN OGCs. The cells were cultured in Dulbecco's modified Eagle’s medium (Gibco) supplemented with 10% foetal bovine serum (Gibco) and 100 U penicillin/streptomycin (Gibco). Cells in the NC group were only exposed to medium. The PCOS cell model was constructed by adding 10 μM testosterone according to the literature. Cells in the treatment group were treated with 5 or 20 μM DIO with or without AKT1-IN-1 (100 nM) or ML385 (1 μM) for a total of 2 days before being collected for further experiments.

Cell Counting Kit-8 (CCK-8) assays

KGN cells were cultured in vitro, and different concentrations of DIO (0, 2.5, 5, 10, 20, 40, 80, and 160 μM) were added to 96-well plates according to the results of a pharmacokinetic study of DIO. The plates were incubated in a CO₂ incubator for 24 h before being treated with Cell Counting Kit-8 (CCK-8) solution. A microplate reader was used to detect the absorbance of each well at 450 nm.

Western blotting

Total protein was extracted from KGN cells using lysis buffer, with a sample volume of 20 µL per well for Western blotting (WB). The proteins in the gel were transferred to a PVDF membrane after sample electrophoresis. Following blocking with skim milk powder, the PVDF membranes were incubated with the following membranes for 10 h at 4 °C: antibodies against NRF2 (1:1000; Affinity), AKT (1:1000; Affinity), p-AKT (1:1000; Affinity), PPARγ (1:1000; Affinity), p-PPARγ (1:1000; Affinity), IL-6 (1:1000; Affinity), and TNF-α (1:1000; Affinity). The membranes were washed with TBST and then incubated with secondary antibodies containing horseradish peroxidase, followed by protein band analysis by chemiluminescence.

Immunofluorescence assays

After intervention, ovarian granulosa cells in Petri dishes were used for immunocytofluorescence assays. The cells were fixed in paraformaldehyde, the cell membranes were ruptured with Triton X-100, and the samples were blocked with goat serum. The cells were sequentially incubated with TNF-α and IL-6 antibodies for 12 h at 4 °C (1:100). Subsequently, the cells were incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 594) or goat anti-rabbit IgG H&L (Alexa Fluor® FITC) secondary antibodies at 37 °C for 1 h. After washing with phosphate-buffered saline, the cells were stained with DAPI. Immunofluorescence images were immediately obtained using laser confocal microscopy. TUNEL staining and detection of ROS levels were performed according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay

Rat venous blood was centrifuged at 2800 rpm to collect the serum, and the levels of reactive oxygen species (ROS), total cholesterol (TC), triglycerides (TG), testosterone (TEST), oestradiol (E2), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured using the corresponding kits. Ovarian tissue homogenates were prepared using cold saline and then centrifuged at 3000 rpm at 4 °C to collect the supernatant. The levels of TNF-α, IL-6, and ROS were assessed using corresponding kits.

Statistical analysis

Statistical analysis and visualization of the results were conducted using GraphPad Prism version 8.0.2, and the data are presented as the means ± standard deviations (means ± SDs). Group comparisons were made using one-way ANOVA, with a P value of less than 0.05 indicating a statistically significant difference (*P < 0.05, **P < 0.01.). Each experiment was conducted three times.

Results

Network pharmacology results for Schizonepeta tenuifolia Briq and PCOS

To investigate the potential pharmacological mechanism of action of Schizonepeta tenuifolia Briq in PCOS, we searched for the targets of action of Schizonepeta tenuifolia Briq and PCOS separately. Among them, the total number of targets of Schizonepeta tenuifolia Briq was 302 (Fig. 1A), and the total number of targets of PCOS was 937 (Fig. 1A). The total number of intersecting targets of Schizonepeta tenuifolia Briq and PCOS was 49 (Fig. 1A-E), including AKT1, SRC, ESR1, EGFR, PPARG, MMP2, and IGF1R. Interestingly, these intersecting targets are closely related to inflammatory and apoptotic signalling pathways. In addition, we screened (Fig. 1B) the active ingredients schizonepetoside B, schkuhrin I, diosmetin, sitosterol, 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one, luteolin, quercetin, beta-sitosterol, stigmasterol, supraene, and campest-5-en-3beta-ol from Schizonepeta tenuifolia Briq by pharmacological screening (OB ≥ 30% and DL ≥ 0.18). Therefore, as the broad pharmacological effects of diosmetin coincide with the inflammation and apoptosis revealed by the intersecting targets and PPI profiles (Fig. 1D-H), we next focused on validating its potential pharmacological effects on PCOS.

Fig. 1.

Network pharmacology data on DIO and polycystic ovary syndrome. A Venn diagram of Schizonepeta tenuifolia Briq. and PCOS. B Intersecting targets of PCOS and Schizonepeta tenuifolia Briq. C-E PPI network map of intersecting PCOS and Schizonepeta tenuifolia Briq targets. F-G Heatmap of the molecular targets of Schizonepeta tenuifolia Briq. H Pie chart of DIO molecular targets

DIO ameliorates the pathologic changes of PCOS in rats

To investigate the effects of DIO on PCOS progression, we constructed a rat model of PCOS. After 1 month of PCOS model replication and pharmacological intervention, ovarian tissues were collected for histological studies. The results of the body weight analysis (Fig. 2C, D) revealed that the rats with PCOS gained significant amounts of weight, and the DIO intervention ameliorated this metabolic change. Gross observation (Fig. 2A) and HE staining revealed that (Fig. 2B) the white membrane of the ovary was thickened and that the cortical surface was more fibrotic in the PCOS model group than in the NC group. In addition, an increased number of immature cystically dilated follicles could be observed. Moreover, FSH, TG, and E2 expression was decreased in the model group (Fig. 2F, I, L), whereas TC, LH, TEST, and HOMA-IR expression (Fig. 2E, G, H, K) was elevated. Administering low doses of DIO resulted in a slight improvement in the ovarian cystic alterations caused by letrozole in rats. Additionally, compared with the low-dose group, the high-dose group presented a more significant reduction in follicular cystic lesions and hyperandrogenaemia in rats with PCOS. In summary, DIO may exert protective effects against PCOS by ameliorating pathological changes in follicular atresia and cystic dilatation; reducing TC, LH, TEST, and ROS levels; increasing HOMA-IR expression (Fig. 2E, G, H, K, M, N); and increasing FSH, TG, and E2 expression (Fig. 2F, I, L).

Fig. 2.

DIO relieves pathologic changes in polycystic ovary syndrome. A Pictures of the ovaries of rats in each group. B HE staining of rat ovaries. DIO (50 mg/kg and 100 mg/kg) treatment was administered to rats in the PCOS group. C-D Body weight analysis of the rats in each group. E-N Endocrine metabolic changes in various groups of rats

DIO protects against PCOS by suppressing inflammation

To investigate the potential mechanisms by which DIO protects the ovaries in PCOS, the expression of inflammatory factors in OGCs was studied in vitro and in vivo. As shown in Fig. 3, ovarian tissue inflammation was enhanced in the PCOS model, with increased TNF-α and IL-6 expression. However, DIO treatment inhibited TEST-induced inflammation, including decreasing TNF-α and IL-6 expression (Fig. 3A–D). The effect of DIO on OGC viability was further assessed in vitro by CCK-8 assay (Fig. 3E). DIO at doses less than 20 μM did not affect cell viability. The low- and high-dose options for drug treatment were 5 μM and 20 μM, respectively. TEST (10 μM) was used to stimulate KGN cells to establish a PCOS model. Consistently, the WB (Fig. 3F–H) and IF (Fig. 3I–L) results showed that TEST could upregulate TNF-α and IL-6 expression. DIO reversed the TEST-induced damage and inhibited the inflammatory response of KGN cells in the PCOS model. Importantly, the impact of DIO on the inflammation of KGN cells in PCOS was dependent on the dosage, as 20 μM DIO exerted a somewhat greater anti-inflammatory effect than 5 μM DIO.

Fig. 3.

DIO relieves the inflammatory response in polycystic ovary syndrome. A-D Immunohistochemical results showing that DIO downregulated the expression of TNF-α and IL-6. E A CCK-8 kit was used to determine the cytotoxicity of DIO (0, 2.5, 5, 10, 20, 40, 80 and 160 μM) on OGCs. F-H Western blot analysis revealed that DIO alleviated TNF-α and IL-6 expression. I-L Immunofluorescence detection of DIO-induced decreases in the expression of TNF-α and IL-6. *P < 0.05, **P < 0.01

DIO protects against PCOS by suppressing apoptosis and ROS production

Next, we investigated whether DIO could alleviate apoptosis in the context of pathological changes in PCOS. As shown by the results of immunohistochemistry and TUNEL staining (Fig. 4A, B, G, H), the apoptosis of ovarian granulosa cells was increased in the PCOS model group, whereas apoptosis was significantly reduced after DIO intervention. The WB results (Fig. 4C-F) revealed that the expression of Bax and cleaved caspase-3 was significantly downregulated, whereas the expression of Bcl-2 was significantly increased after DIO treatment compared with that in the PCOS model group. In addition, the TEST-induced PCOS cell model presented obvious signs of ROS production; however, the immunofluorescence results (Fig. 4I–J) revealed that DIO significantly reduced ROS production. Moreover, compared with 5 μM DIO, 20 μM DIO demonstrated considerably greater antiapoptotic effects and a greater capacity to reduce ROS production.

Fig. 4.

DIO protects against PCOS by suppressing apoptosis and ROS production. A-B Immunohistochemical results of cleaved caspase-3 protein expression in ovarian tissues. C-F Western blotting was used to assess the expression of Bax (C, D), Bcl-2 (C, E), and cleaved caspase-3 (C, F). H-I Immunocytofluorescence evaluation of TUNEL staining and ROS production in each group. *P < 0.05, **P < 0.01

Molecular docking and WB reveal potential molecular targets of DIO for PCOS relief, such as AKT1 and PPARγ

The protein and ligand structures underwent pretreatment processes, including the removal of water molecules, the addition of hydrogen atoms, and charge optimization, to facilitate the molecular docking of DIO with the six key targets mentioned earlier. The binding energies for these targets were found to be SRC: −3.49, NRF2: −4.29, PPARG: −4.92, AKT1: −4.33, EGFR: −2.97, ESR1: −3.92, and IGF1R: −3.26, with PPARG showing the strongest binding affinity to DIO. The results were then visualized and analysed using PyMOL software, which provided the docking patterns of DIO with the key target molecules. The results revealed that (Fig. 5A-F) all 6 targets could stably bind to DIO, especially AKT1 and PPARγ. Next, we explored the effects of DIO on the phosphorylation of the 6 target proteins in the cells. The Western blot results revealed that (Fig. 5G-M) the levels of the phosphorylated forms of the target proteins, with the exception of SRC, were altered in the PCOS model group compared with those in the control group. Interestingly, DIO reversed the alterations in p-AKT and p-PPARγ in PCOS(Fig. 5G, H, L). These findings suggest that AKT and PPARγ may be potential molecular targets of DIO.

Fig. 5.

Molecular docking and WB revealed potential molecular targets of DIO for PCOS relief, such as AKT1 and PPARγ. A-F Molecular docking results of DIO with AKT1, SRC, ESR1, EGFR, PPARG, and IGF1R. Western blotting was performed to detect the expression of p-AKT (G, H), p-SRC (G, I), p-ESR1 (G, J), p-EGFR (G, K), p-PPARγ (G, L), and p-IGF1R (G, M). *P < 0.05, **P < 0.01

DIO alleviates PCOS-related OGC inflammation, oxidative stress and apoptosis by targeting AKT

AKT1-IN-1, a specific inhibitor of AKT, was used to further validate whether DIO alleviates inflammation, oxidative stress, and apoptosis in PCOS by targeting the AKT1/PPARγ signalling pathway. The IHC results (Fig. 6A-D) revealed that DIO intervention significantly upregulated p-AKT and p-PPARγ expression in the ovarian tissues of PCOS rats. The Western blot results revealed that DIO promoted AKT phosphorylation, but AKT1-AN-1 eliminated the phosphorylation of AKT. In addition, AKT1-IN-1 treatment resulted in decreased expression of p-AKT (Fig. 6E, F), p-PPARγ (Fig. 6F, G), and Bcl-2 (Fig. 6E, I) and increased expression of Bax (Fig. 6E, H), cleaved caspase-3 (Fig. 6E, J), IL-6 (Fig. 6E, K), and TNF-α (Fig. 6E, L), impairing the protective effect of DIO on TEST-induced KGN cells. The immunofluorescence results (Fig. 6M‒P) of IL-6 and TNF-α were consistent with the WB results, indicating that AKT1-IN-1 reversed the inhibitory effect of DIO on TEST-induced inflammation in KGN cells. In addition, the results of the ROS assay confirmed that AKT1-IN-1 eliminated the inhibitory effect of DIO on oxidative stress. The TUNEL staining results (Fig. 6Q, R) were consistent with the Western blot results, and AKT1-IN-1 similarly reversed the mitigating effect of DIO on TEST-induced apoptosis in KGN cells. In conclusion, DIO alleviated TEST-induced inflammation, oxidative stress and apoptosis in KGN cells by activating the AKT/PPARγ signalling pathway.

Fig. 6.

DIO alleviates PCOS-related OGC inflammation, oxidative stress and apoptosis by targeting AKT. A-D Immunohistochemical results of p-AKT and p-PPARγ protein expression in ovarian tissues. Western blotting assays were performed to detect the expression of p-AKT (E, F), p-PPARγ (E, G), Bax (E, H), Bcl-2 (E, I), cleaved caspase-3 (E, J), IL-6 (E, K), and TNF-α (E, L). M-T Immunocytofluorescence evaluation of TUNEL staining, ROS production, and TNF-α and IL-6 expression in each group. *P < 0.05, **P < 0.01

DIO inhibits PCOS-related OGC inflammation, oxidative stress and apoptosis by modulating the NRF2/AKT1/PPARγ signalling pathway

DIO significantly upregulates NRF2 activity. To explore the mechanism by which DIO inhibits inflammatory expression in PCOS, we investigated whether DIO modulates the AKT/PPARγ signalling pathway by activating NRF2 expression. First, we performed molecular docking of DIO and NRF2, and the results revealed that DIO and NRF2 binding was more stable (Fig. 7A). The IHC results (Fig. 7B, C) demonstrated that NRF2 expression was downregulated in the ovarian tissues of the PCOS model rats. DIO dose-dependently upregulated NRF2 expression (Fig. 7D, E), and in KGN cells, DIO ameliorated the TEST-induced inhibition of NRF2 expression. This effect was reversed by the NRF2 inhibitor ML385. In addition, ML385 inhibited the expression of NRF2 and downregulated the expression of p-AKT (Fig. 7D, F), p-PPARγ (Fig. 7D, G), and Bcl-2 (Fig. 7D, I) and upregulated the expression of Bax (Fig. 7D, H), cleaved caspase-3 (Fig. 7D, J), TNF-α (Fig. 7D, L) and IL-6 (Fig. 7D, K). TNF-α and IL-6 showed similar results in terms of their expression via immunofluorescence (Fig. 7J-L). TUNEL staining and ROS assays (Fig. 7Q-T) revealed that ML385 reversed the protective effect of DIO against oxidative stress and apoptosis in KGN cells. Overall, DIO mediated the inflammatory response in PCOS ovarian granulosa cells through activation of the AKT/PPARγ signalling pathway, which is regulated by NRF2 expression.

Fig. 7.

DIO inhibits PCOS-related OGC inflammation, oxidative stress and apoptosis by modulating the NRF2/AKT1/PPARγ signalling pathway. A Molecular docking results of DIO with NRF2. B, C Immunohistochemical results of NRF2 protein expression in ovarian tissues. Western blotting wa performed to detect the expression of NRF2 (D, E), p-AKT (D, F), p-PPARγ (D, G), Bax (D, H), Bcl-2 (D, I), cleaved caspase-3 (D, J), IL-6 (D, K), and TNF-α (D, L). M-R Immunofluorescence was used to evaluate the expression of TUNEL, TNF-α and IL-6. S-T Immunofluorescence was used to evaluate the level of ROS. *P < 0.05, **P < 0.01

Discussion

PCOS is a prevalent endocrine disorder in women of childbearing age that usually leads to an increased risk of cardiovascular disease and metabolic syndrome [2]. There is no effective therapeutic regimen to cure PCOS, so it is clinically important to investigate its pathogenesis and develop new therapeutic targets. Inflammation, apoptosis, and oxidative stress, as core pathological features, connect NRF2/AKT/PPARγ signalling and PCOS disease progression. We demonstrated that DIO, a natural compound from Schizonepeta tenuifolia Briq, may inhibit inflammation and apoptosis in OGCs and ameliorate pathological changes in PCOS by modulating the oxidative stress-dependent AKT/PPARγ signalling pathway in vitro and in vivo.

Numerous studies have confirmed that most people with PCOS have chronic inflammation and excess subcutaneous fat [25]. Recent studies have established that elevated levels of numerous pro-Inflammatory cytokines, including TNF-α, CRP, IL-6, IL-8, and IL-18, are present in patients with PCOS [26] [27]. Studies have indicated that the levels of inflammatory mediators, such as IL-6 and TNF-α, in the ovarian tissue of rats with PCOS are notably elevated[28]. Furthermore, the levels of inflammation in PCOS patients are consistent with a high body mass index and elevated TEST levels, suggesting that high levels of activated inflammation may be related to metabolic regulation of TEST [29]. This aligns with earlier findings regarding the clinical and pathological alterations observed in individuals with PCOS[30]. Consistently, our study revealed increased TNF-α, IL-6, and TEST levels in the serum of PCOS model rats. This increase was accompanied by increased body weight and increased ovarian cystic follicular enlargement in the rats. Moreover, we found that intervention with DIO not only reduced the body weight of PCOS rats but also effectively alleviated the alterations in inflammatory and hormone metabolic indices, such as TNF-α, IL-6, FSH, LH, and TEST. These findings suggest that DIO may ameliorate the pathological progression of PCOS by suppressing the inflammatory response. Nevertheless, certain studies have indicated that rats with PCOS do not consistently display increased testosterone levels or decreased oestradiol levels, which contrasts with our results. This discrepancy could be attributed to differences in modelling techniques, the specific strains of the experimental animals, variations in their ages, and the absence of synchronization in the oestrous cycles of the animals used in the experiments.

Furthermore, in vitro experiments revealed that DIO alleviated TEST-induced inflammation, apoptosis and ROS production in KGN cells. To further explore the molecular mechanisms, we molecularly docked DIO to six high-potential interaction targets based on the results of network pharmacology. The molecular docking and WB results showed that DIO has good binding stability with AKT and PPARγ. As a serine/threonine kinase, AKT plays a key role in ovarian metabolism, cell survival and the cell cycle. Growth hormone has been reported to inhibit ROS accumulation and apoptosis in ovarian granulosa cells in polycystic ovary syndrome by regulating PI3K/Akt signalling [31]. In addition, the level of AKT phosphorylation is closely related to the degree of embryonic trophoblast apoptosis in PCOS mice [32]. Many studies have also demonstrated that AKT signalling also affects the core pathological features of PCOS by regulating inflammation and oxidative stress [33, 34]. PPARγ, a key member of the intranuclear receptor transcription factor superfamily, is mainly responsible for regulating the expression of target genes. Its activation state plays a key role in cell growth, apoptosis, an inflammatory processes and is closely associated with cardiovascular disease, diabetes, and PCOS [35, 36]. Hypericin attenuates PCOS-associated adipogenesis and insulin resistance by modulating PPARγ ubiquitination and degradation [37]. In this study, AKT and PPARγ were involved in inflammation, apoptosis, and ROS production in KGN cells. DIO also upregulated AKT expression and PPARγ phosphorylation, which corresponded with the results of binding and interaction modelling. Research indicates that PPARγ may be a promising target for treating type 2 diabetes and nonalcoholic fatty liver disease [38, 39]. Specifically, thiazolidinedione medications effectively reduce blood sugar levels and alleviate liver inflammation and fibrosis by functioning as PPARγ agonists. Given the regulatory influence of DIO on PPARγ, future studies should investigate the potential of DIO as a treatment for diabetes and nonalcoholic liver disease.

NRF2 is a potential target for regulating inflammation and oxidative stress in PCOS. As a transcription factor, NRF2 consists of seven Neh domains (NRF2 ECH homologous structural domains). It exerts antioxidative-stress effects by binding to the promotor region of antioxidant response elements (AREs). In addition, NRF2 has been associated with TEST-induced inflammation in PCOS. Many studies have confirmed that NRF2 activation elicits various anti-inflammatory effects, including downregulating NF-κB and decreasing the activity of a range of inflammatory mediators [40, 41]. The buildup and clustering of misfolded proteins lead to excessive generation of reactive oxygen species (ROS) from the mitochondria, the ER, and other sources, triggering the activation of NRF2 [42, 43]. NRF2 is a hub that compiles emergency signals from the accumulation of misfolded proteins to facilitate coordinated transcriptional responses. AKT has the ability to form a dimer with NRF2 at antioxidant response elements (AREs), leading to HO-1 expression (He et al.2001). This process helps safeguard cells from proteotoxic stress. CREB-binding protein and its cofactor p300 can be corecruited by NRF2 to AREs for transcriptional activation through the Neh4/5 structural domain of NRF2 [44]. NRF2 reduces the oxidative stress-induced activation of NF-κB by lowering the intracellular levels of reactive oxygen species (ROS). Furthermore, NRF2 stops the proteasomal breakdown of IκB-α and prevents the nuclear translocation of NF-κB, thereby decreasing inflammation. Under ER stress conditions, ER membrane-associated E-ubiquitin ligases, such as HRD1, bind to the Neh4/5 structural domain of NRF2 and mediate its ubiquitination and degradation [45]. In this study, we discovered that NRF2 levels were lower in PCOS rats than in control rats, which aligns with earlier studies [46]. Additionally, activating Nrf2 can help inhibit cell enlargement and inflammation in the adipose tissue of mice with PCOS [47].

Hyperandrogenaemia and insulin resistance reportedly contribute to mitochondrial damage in the reproductive system by mediating inflammation and oxidative stress imbalance [48]. Furthermore, by targeting NRF2/NF-κB signalling, serpentine ameliorated symptoms accompanied by reduced intracellular ROS accumulation and attenuated oxidative stress in PCOS mice [49]. Additionally, the combination of Guizhi Fuling Wan and rosiglitazone improved the oestrous cycle, ovarian histological changes, and follicular development in PCOS rats, possibly through activation of the NRF2/HO-1 pathway. ML385 also eliminated the ameliorative effect of DIO on ovarian dysfunction in PCOS by inhibiting NRF2 activity. Overall, these results suggest that the alleviation of the pathological progression of PCOS by DIO is closely related to the suppression of inflammation and oxidative stress.

DIO is a natural flavonoid compound with anti-inflammatory, antioxidant, anti-infective, and neuroprotective pharmacological properties [50]. DIO reportedly alleviates lipopolysaccharide-induced acute lung injury by targeting inflammatory effects [51]. Furthermore, DIO in glial cells has been shown to activate Keap1/Nrf2 signalling and relieve neuropathic pain [52]. In the present study, we found that DIO mitigated the symptoms of PCOS and related hormonal and metabolic disturbances and that DIO suppressed AKT/PPARγ signalling-induced inflammation and oxidative stress in PCOS by enhancing NRF2 expression. Unfortunately, studies related to the alleviation of PCOS by DIO are currently lacking, and further research is still needed. This study has certain limitations, including the absence of vaginal smears to identify the oestrous cycle in rats while developing the PCOS rat model, which was done to eliminate the effects of hormone metabolism on the findings. Future research will aim to resolve these limitations. Importantly, in comparison with low-dose DIO, high-dose DIO notably enhanced the apoptosis of KGN cells, although it did not significantly affect inflammation. Additionally, higher doses could increase the likelihood of negative drug reactions. Future careful determination of the DIO dosage still needs to be confirmed through in vivo pharmacokinetic studies.

Conclusion

In this study, we found that DIO protected against TEST-induced inflammation, apoptosis, and oxidative stress in KGN cells by inhibiting NRF2/AKT1/PPARγ signalling in vitro and in vivo. The potential molecular mechanisms by which DIO protects against PCOS may be related to the activation of NRF2 expression and inhibition of the AKT1/PPARγ pathway in OGCs.

Abbreviations

PCOS

Polycystic ovary syndrome

ERS

Endoplasmic reticulum stress

DIO

Diosmetin

AKT

Protein kinase B

PPARγ

Peroxisome proliferator-activated receptor γ

BAX

B-cell lymphoma-2-associated x

NRF2

Nuclear factor erythroid 2-related factor 2

TNF-α

Tumour necrosis factor-α

IL-6

Interleukin 6

SRC

Sarcoma

ESR1

Oestrogen receptor 1

EGFR

Epidermal growth factor receptor

IGF1R

Insulin-like growth factor 1

MMP2

Matrix metalloproteinase 2

NLRP3

NOD-like receptor protein 3

BCL-2

B-cell lymphoma-2

TLR4

Toll-like receptor 4

NF-κB

Nuclear factor-Κb

CRP

C-reactive protein

IL-18

Interleukin 18

SD

Sprague–Dawley

SPF

Sterile pathogen-free

IHC

Immunohistochemical

CCK-8

Cell counting kit-8

ROS

Reactive oxygen species

TC

Total cholesterol

TG

Total glyceride

TEST

Testosterone

E₂

Oestradiol

LH

Luteinizing hormone

FSH

Follicle-stimulating hormone

Authors’ contributions

Mengting Chen and Jingwen Meng: Writing–original draft, Visualization, Software, Methodology, Investigation, Formal analysis, and Data curation. Yuan Liu: Visualization, Formal analysis, and Data curation. Yafang Jin: Software, Methodology, and Formal analysis. Xiong Yuan, Zhiquan Qin and Xiaohui Cao: Writing–review & editing, Validation, Supervision, Project administration, Funding acquisition, and Conceptualization.All authors reviewed the manuscript.

Funding

This study was financially supported by the Wuxi Maternal and Child Health Hospital Elite Talent Program (JY2023012) and the Scientific Research Program of Wuxi Municipal Health Commission (Q202305).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study was approved by the Ethics Committee of JNU (JN. No. 20241030s0240315[575]), and all participants provided informed consent.

Consent for publication

All authors of this paper agreed to publish the study.

Competing interests

The authors declare no competing interests.