Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder characterized primarily by motor impairments, including bradykinesia, tremor, and postural instability1,2 Pathologically, it involves the abnormal aggregation of alpha-synuclein (α-syn) and the formation of Lewy bodies3. Epidemiological studies indicate that the incidence of PD in postmenopausal women is twice that of men, with a faster disease progression and higher mortality rate4, which correlates with fluctuations in endogenous estrogen levels5,6. The mechanisms underlying PD onset in postmenopausal women remain unclear but are associated with gender, hormonal changes, and reproductive history4,7. By 2030, there will be an estimated 1.2 billion postmenopausal women globally, imposing significant healthcare economic burdens6. Therefore, understanding the pathogenesis of PD in postmenopausal women and identifying therapeutic targets is crucial.

Estrogen is produced by the placenta and ovaries in female animals. It can cross the blood-brain barrier to accumulate in various parts of brain tissue, where it acts as an important central nervous system neurotransmitter, exerting neuroprotective and cognitive-enhancing effects8. Research indicates a significant correlation between estrogen deficiency and neurodegenerative diseases9, highlighting its protective role in the central nervous system. However, clinical estrogen replacement therapy may increase the risk of uterine cancer and cardiovascular diseases10. Therefore, finding new estrogen-based therapeutic drugs is crucial for the prevention and treatment of postmenopausal PD11.

The estrogen receptor (ER) is a nuclear steroid receptor involved in estrogen signaling, influencing mood and neurodevelopment12,13. ER serves as a key target for estrogen’s actions. At the same time, phosphatidylinositol 3-kinase (PI3K)/Protein kinase B (Akt) constitutes an intracellular signaling pathway for estrogen, exerting anti-apoptotic effects and promoting cell proliferation and differentiation14,15. The anti-apoptotic mechanism of PI3K/Akt primarily involves phosphorylation, where activated Akt inhibits apoptotic protein activity and enhances B-cell lymphoma-2 (Bcl-2) expression16,17, phosphorylated apoptotic factors containing cysteine-aspartic acid protease-3 (Caspase-3) further suppress subsequent apoptotic responses18. Moreover, the interaction between ER and PI3K/Akt is a critical mechanism for neuroprotection19.

Epimedium brevican Maxim, a plant belonging to the Berberidaceae family, is one of the traditional Chinese medicines. It is used in Chinese medical practice to treat conditions such as osteoporosis. It contains a rich array of flavonoid chemical compounds, among which the flavonoid compound Icaritin (ICT) the main active ingredient extracted from Epimedium. ICT possesses anti-inflammatory and neuroprotective properties, it enhances the effection of estrogen20. Additionally, preliminary cell experiments conducted by the research group have found that ICT has potential anti-PD capabilities21,22,23. Therefore, we hypothesize that ICT exerts estrogen-like effects by activating ER and the PI3K/Akt signaling pathway, regulating apoptotic proteins, and promoting estrogen’s protective effects on the central nervous system, thereby delaying the pathological changes of postmenopausal PD.

Epimedium brevican Maxim is a plant belonging to the Berberidaceae family, widely used in traditional Chinese medicine (TCM) as a tonic herb known for its benefits in kidney reinforcement, enhancing sexual function, and strengthening bones and muscles. The main active constituents of Epimedium include ICT, icariin (ICA), and epimedin B. ICT is recognized as a significant flavonoid compound with notable biological activities. ICT can be generated in vivo through the metabolic hydrolysis of ICA, which serves as one of the direct precursors of ICT. Furthermore, ICT effectively crosses the blood-brain barrier, indicating its potential physiological functions within the central nervous system24. These characteristics highlight the importance of Epimedium in TCM, particularly regarding its roles in promoting skeletal health and providing neuroprotective effects. Research shows that ICT can alleviate the discomfort caused by reduced hormones in postmenopausal women, reduce the drug-like side effects produced by hormones20, and simulate the benefits of endogenous estrogen action, making it a new candidate drug for preventing neurodegenerative diseases25. Preliminary studies have proven that ICT has a neuroprotective effect in PD models and has made substantial progress26,27. Therefore, based on the preliminary studies on the anti-PD effection of ICT, this study further explores the estrogenic mechanism of ICT in postmenopausal PD models. It is committed to providing a basis for the treatment and prevention of PD after menopause.

Materials and methods

Drugs, antibodies, and reagents

ICT (purity: 99.0%, catalog: Y-049), from Chengdu Ruifensi Biotechnology Co., Ltd. (China). Fulvestrant (ICI182780), from Med Chem Express (USA). MPTP, from Med Chem Express (USA). Antibodies: Bcl-2 (cat. 26593-1-AP), Bax (cat. 50599-2-lg), pro-Caspase-3 (cat. 19677-1-AP), β-actin (cat. 20536-1-AP), ER (cat. 21244-1-AP), goat anti-rabbit and mouse IgG-HRP (cat. SA00001-2), PI3K (cat. 60225), Akt (cat. 66444-1-1 g), from Wuhan Sanying (China). p- PI3K (p85) (cat. 17366s), p-Akt (473) (cat. 4058s), from Cell Signaling Technology (USA). ELISA kits: Mouse E2, FSH, LH, from Shanghai Jianglai Biotechnology Co., Ltd. (China). Hematoxylin and Eosin staining, from Seville (China). SDS-PAGE Gel Preparation Kit, BCA Protein Concentration Determination Kit, from Jiangsu Biyun Tian Biotechnology Research Institute (China). Aniline Blue (JT8570) (China), Sodium Dihydrogen Phosphate (1010580101700) (Tianjin), Formaldehyde (1340040101602) (AR Grade) (China), Hydrochloric Acid (7647-01-0) (AR Grade) (China), Neutral Gum (1004160), Anhydrous Ethanol (100092680) (AR Grade) (China).

Animals and methods

Animals

Seventy-five female 8-week-old C57BL/6J mice (18–22 g) were obtained from Tianqin Biotechnology Co., Ltd., Changsha, Hunan Province, China, and housed under SPF conditions at the Center Laboratory Animal Facility of the Third Affiliated Hospital of Zunyi Medical University, with a temperature of 23 °C (± 2 °C) and a 12-hour light/dark cycle, with free access to food and water. Animal care followed NIH guidelines (Publication No. 80 − 23, revised 1978), and experimental procedures were approved by the Ethics Committee of the Third Affiliated Hospital of Zunyi Medical University.

Model preparation

After one week of acclimatization, mice were intraperitoneally injected with 1% pentobarbital sodium at a dose of 0.03 ml/kg for anesthesia. A longitudinal incision approximately 1–2 cm long was made bilaterally on the midline of the back, about 1 cm from each side, to access the abdominal cavity and locate the ovaries (attached to the white adipose tissue). Bilateral OVX was performed by ligating and removing the ovaries using a 4 − 0 suture, followed by a layered closure incision28,29. In the Sham group, ovaries weren’t removed; equivalent adipose tissue was excised. Mice received 1% pentobarbital sodium injections for abdominal anesthesia for three days postoperatively, with one day of fasting but access to water. Vaginal cytology was observed in both the Sham and OVX groups. A ≥ 50% reduction in keratinized cells in vaginal smears compared to the Sham group indicated successful OVX.

Following successful OVX, mice were randomly assigned into five groups (n = 12 per group): Sham, OVX, MPTP model, ICT, ICI182780. Three days after surgery, MPTP (20 mg/kg) was intraperitoneally injected once daily for 7 consecutive days to induce the PD model30. The Sham and OVX groups received intraperitoneal DMSO (1.5%Safe concentration) injections31. The ICI182780 and treatment groups were orally administered ICT (20 mg/kg) via gavage, while the Sham, OVX, and MPTP model groups received DMSO vehicle solution orally once daily, the concentration of DMSO was 1.5%. After 14 days of consecutive gavage, mice were anesthetized abdominally, blood was collected from the eyeball, and they were euthanized by cervical dislocation for brain, uterus, and left femur extraction, as outlined in (Fig. 1a).

Behavioral testing

Behavioral experiments were conducted daily from 8:30 a.m. to 5:30 p.m. Mice were acclimated to the laboratory environment for at least one hour before testing. The test area was cleaned with 75% ethanol before each session to eliminate olfactory interference.

Rotarod test

Before the experiment, mice acclimated on the rotarod for 30 min at 10 rpm. During the test, mice are placed on a rotating rod (3 cm diameter, 8 cm lane width) with the speed increasing from 4 to 40 rpm over 300 s. Each mouse underwent three trials at 15-minute intervals. The average time spent on the rod across trials was recorded for limb coordination and the duration each mouse maintained its position assessed synchronous movement ability. Test results represent the average duration from the three trials.

Pole climbing test

A 23 mm diameter wooden ball is fixed at the top of a 53 cm long, 0.8 cm diameter wooden pole, wrapped with gauze. Mice are placed on the wooden ball, and the time taken to climb from the top to the bottom of the pole is recorded. The experiment was conducted three times consecutively, with each trial separated by a 10–15-minute interval.

Hanging wire test

A 0.1 cm diameter, 30 cm long wire was horizontally fixed 30 cm above the ground. The mouse’s two front paws suspended on the wire. Hanging scores were recorded: both hind paws grasping within 10 s scored 3; one hind paw 2; neither hind paw 1, falling 0. at 5-minute intervals, and the average score was calculated.

Enzyme-linked immunosorbent assay (ELISA)

Whole blood samples were stored at room temperature for 1 h or overnight at 4 °C, then centrifuged at 3000 × g for 15 min at 2–8 °C. The supernatant was collected and stored at -80 °C. ELISA was used to detect hormones, with separate wells for standard, sample, and blank. Competitive assays were employed for E2 and LH, while an assay was used for FSH.

Western blot analysis of midbrain protein expression levels

Midbrain tissues were collected post-ICT intervention for protein analysis via Western blotting. proteins studied included α-syn, Bcl-2, Bax, pro-caspase-3, cleaved caspase-3, ER, PI3K, Akt, phosphorylated p-PI3K (p85), and p-Akt (Ser473). Midbrain tissue was placed in a 1.5 ml EP tube with protease inhibitor PMSF phosphatase inhibitor mixture in RIPA homogenized on ice. Following a 30-minute lysis and centrifugation at 12,000 × g for 15 min at 4 °C, the supernatant was collected protein concentration was determined using the BCA method. After washing, the membrane was incubated with HRP-conjugated secondary antibodies, and proteins were visualized using ECL detection. Band intensities were quantified using Quantity One software (Bio-Rad).

Aniline blue staining of the left femur and hematoxylin-eosin (HE) staining of the uterus, along with dual-energy X-ray absorptiometry (DEXA) scan for whole body bone density in mice

Aniline Blue staining of the left femur: Tissue fixation and embedding procedures, the fixed tissues were dehydrated using an automatic dehydration machine with the following schedule: 75% ethanol for 2 h, 85% ethanol for 1 h, 95% ethanol for 1 h, 100% ethanol I for 20 min, 100% ethanol II for 20 min, 100% ethanol I for another 20 min, and 100% ethanol IV for 20 min. Subsequently, the tissues were cleared using clearing agent I for 25 min and clearing agent II for 30 min, followed by infiltration with paraffin I for 30 min, paraffin II for 1 h, and paraffin I again for 1 h; Sectioning and staining procedures: Deparaffinization and hydration, The sections were deparaffinized in deparaffinization solution for 130 min, followed by deparaffinization solution II for 30 min. The sections were then dehydrated in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, and 75% ethanol for 5 min, followed by rinsing in running tap water for 5 min; Staining, The sections were air-dried and then immersed in aniline blue staining solution for 2–3 min; Washing, The sections were washed with distilled water.; Dehydration and mounting: Gradient ethanol dehydration and clearing were performed, followed by embedding in neutral gum.

The uterus was also fixed in 4% PFA for > 24 h. Both underwent gradual ethanol dehydration (70%, 80%, 90%, 95%, 100%, 100%) for 40 min each, paraffin embedding, sectioning, and slide mounting. Deparaffinization: xylene I (10 min), xylene II (10 min), ethanol I (5 min), ethanol II (5 min), 95% alcohol (5 min), 85% alcohol (5 min), 75% alcohol (5 min), and 55% alcohol (5 min). Slides were then stained with hematoxylin (0.5–1 min), rinsed, differentiated in 1% HCl alcohol, rinsed, counterstained with 1% ammonia solution, and rinsed. Those coverslips use a neutral mounting medium. For the DAX whole-body bone density scan, mice were anesthetized, placed on the DAX imaging table, scanned, and data recorded.

Immunofluorescence staining protocol and immunohistochemistry staining protocol (refined version)

Immunofluorescence Staining Protocol; Sample Preparation: Heat paraffin-embedded sections at 60 °C for 1 h to remove excess paraffin, followed by deparaffinization in xylene (3 × 5 min) and rehydration through a graded ethanol series (100%, 95%, 75%, and 50%, each for 3 min) ending in distilled water. Antigen Retrieval: Perform antigen retrieval by incubating the sections in EDTA buffer (pH 8.0) at 95 °C for 15–20 min using a water bath or microwave. Cool to room temperature, then wash the slides in PBS (3 × 5 min). Blocking: Block nonspecific binding sites by incubating the sections with 5% BSA in PBS at room temperature for 1 h. Primary Antibody Incubation: Incubate the sections with the specific primary antibody diluted in antibody diluent (typically 1–5% BSA in PBS) overnight at 4 °C in a humidified chamber. Washing: Wash the sections with PBS (3 × 5 min) to remove unbound primary antibody. Secondary Antibody Incubation: Apply the fluorophore-conjugated secondary antibody diluted in PBS to the sections and incubate for 1 h at room temperature in the dark. Counterstaining: Add DAPI (1 µg/mL in PBS) to the sections for 5 min at room temperature to stain nuclei. Final Wash and Mounting: Wash the slides with PBS (3 × 5 min), mount them with an anti-fade mounting medium, and seal with coverslips. Imaging: Visualize using a fluorescence microscope equipped with appropriate filter sets.

Immunohistochemistry Staining Protocol; Sample Preparation: Follow the same heat, deparaffinization, and rehydration steps as in the immunofluorescence protocol. Antigen Retrieval: Perform antigen retrieval as described above. Blocking: Block nonspecific binding by incubating the sections with 5% BSA or 10% normal serum (matching the secondary antibody species) in PBS for 1 h at room temperature. Primary Antibody Incubation: Incubate the sections with the primary antibody diluted in antibody diluent overnight at 4 °C in a humidified chamber. Washing: Wash the sections in PBS (3 × 5 min) to remove unbound primary antibody. Secondary Antibody Incubation: Apply an HRP-conjugated secondary antibody and incubate for 1 h at room temperature. Color Development: Prepare the chromogenic substrate (e.g., DAB) according to the manufacturer’s instructions. Apply the substrate to the sections and incubate until a visible color develops (typically 5–10 min under visual monitoring). Counterstaining: Counterstain the sections with hematoxylin for 1–2 min, then wash thoroughly in tap water to remove excess stain. Dehydration and Clearing: Dehydrate the sections through a graded ethanol series (50%, 75%, 95%, and 100%, each for 3 min), followed by clearing in xylene (2 × 5 min). Mounting: Mount the slides with neutral resin and cover with coverslips. Imaging: Examine under a brightfield microscope to visualize the staining pattern.

Data analysis

Statistical analysis of data was performed using SPSS 22.0 software, and results were reported as mean ± standard deviation. Multiple-factor analysis of variance (ANOVA) was used to compare data across multiple groups. Post-hoc tests included the LSD test for homogeneity of variances and Dunnett’s T3 test for heterogeneity of variances. A p-value < 0.05 was considered statistically significant for all analyses.

Results

The effect of ICT on behavioral changes in PD disease model mice after OVX

Healthy adult female C57BL/6J mice were acclimatized for 1–7 days, underwent bilateral ovariectomy on day 832, received subcutaneous penicillin injections from days 9–11, intraperitoneal MPTP injections from days 12-1830, oral administration of ICT from days 19–32, underwent behavioral testing from days 33–35 and were euthanized on day 36 for tissue sampling (Fig. 1a).

In the rotarod test, the duration was reduced for the MPTP and ICI182780 groups, indicating a decline in motor function and confirming the successful establishment of the PD) model. After ICT treatment, the ICT group’s mice showed prolonged stay times on the rod (Fig. 1b), demonstrating improved motor coordination and balance. In the pole climbing test (Fig. 1c), climbing times were prolonged for both the MPTP and ICI182780 groups. Administration of 20 mg/kg ICT improved motor function and coordination in the PD model mice. Reduced hanging scores in the MPTP and ICI182780 inhibitor groups indicated severe emotional distress induced by MPTP in the PD model mice. However, the ICT and surgery group showed improved hanging scores compared to the MPTP group (Fig. 1d), suggesting that 20 mg/kg ICT can facilitate the gradual damage associated with estrogen deficiency. The results from the rotarod, pole climbing, and hanging tests show a decline in duration, climbing times, and hanging scores in the MPTP group, indicating a successful PD model establishment and that ICT intervention can improve motor functions in PD mice.

Fig. 1
figure 1

The impact of ICT on the behavioral aspects of PD model mice after OVX (n = 7–10). (a) Experiment timeline diagram. (b) Rotarod Test (F = 9.275, P < 0.000). (c) Pole Climbing Test (P < 0.000). (d) Hanging Wire Test (P < 0.000), n = 3, all bar graphs are presented as mean ± SD.

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The effect of ICT on serum E2, FSH, and LH levels in OVX female PD model mice

In this study, we investigated the effects of ICT on serum levels of E2, FSH, and LH in OVX female PD model mice. Normal female C57BL/6J mice exhibited a decrease in serum E2 levels following OVX. The E2 levels in the MPTP and ICI182780 groups decreased, while those in the ICT group increased (Fig. 2a). Serum FSH levels increased dramatically in the MPTP and ICI182780 groups, whereas they were significantly lower in the ICT group compared to the MPTP group (Fig. 2b). Serum LH levels were elevated in the MPTP and ICI182780 groups compared to the sham surgery group, while the ICT group had lower LH levels than the MPTP group (Fig. 2c). These findings indicate that hormonal changes in mice after OVX are similar to those seen in menopausal women, with significant alterations observed in the OVX PD model mice. After intervention with 20 mg/kg ICT, serum E2 levels increased, while FSH and LH levels decreased, suggesting that ICT plays a role in modulating hormonal changes and exerting estrogenic neuroprotective effects.

Fig. 2
figure 2

The Effect of ICT on Serum Hormone Levels in OVX Female PD Model Mice. (a) Serum E2 levels in each group(P < 0.018). (b) Serum FSH levels in each group (F = 11.00, P < 0.000). (c) Serum LH levels in each group (F = 22.54, P < 0.000), n = 3, all bar graphs are presented as mean ± SD.

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Effects of ICT on uterine morphology in female PD model mice following OVX

Following OVX, estrogen levels decrease, leading to uterine atrophy. Compared to the sham surgery group, ICT treatment after MPTP administration attenuated uterine atrophy, whereas the estrogen inhibitor (ICI182780) exacerbated it (Fig. 3a,b). Representative images of WB-quantified ER and α-syn proteins (Fig. 3c). When comparing the ICT group to the MPTP group, the ICT group showed an upregulation of ER expression and a downregulation of α-syn expression. In comparison with the ICT group, the ICI182780 group demonstrated a downregulation of ER expression and an upregulation of α-syn expression. In the ICT group, ER expression increased compared to the MPTP group, while the ICI group showed reduced ER expression (Fig. 3d). α-syn expression was increased in the MPTP group but was reduced following ICT intervention. With ICI182780 intervention, the pathological damage of PD worsened (Fig. 3e). Bilateral OVX decreases estrogen levels and reduces ER expression, uterine atrophy, and α-syn damage. ICT estrogen mimic therapy alleviated both uterine and brain damage.

Fig. 3
figure 3

Effects of ICT on Uterine Morphology and α-syn, ER in Female PD Model Mice OVX. (a) Representative images of uteri in each group. (b) Cross-sectional images of uterine bodies stained with hematoxylin and eosin (H&E) in each group, were observed under an optical microscope. Scale bar = 20 μm, 100 μm. (c) Representative bands. (d) α-syn relative expression level (F = 22.98, P < 0.000) (e) ER, a relative expression level (F = 8.321, P = 0.001) n = 3, all bar graphs are presented as mean ± SD.

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ICT protection against castration-induced midbrain and striatal damage in female PD model mice

The striatum is a major projection target of dopaminergic neurons, and the level of tyrosine hydroxylase (TH) in this region directly reflects alterations in dopaminergic function 31. Dopaminergic nerve terminals are more sensitive to damage from toxic factors, and TH expression in the striatum can reflect early neuronal functional impairment 32. To further verify the neuroprotective effects of ICT, we analyzed the intergroup differences in TH expression in the midbrain and striatum using immunohistochemistry and immunofluorescence. Compared with the sham group, the MPTP group exhibited reduced TH fluorescence intensity, while the ICT group showed increased TH fluorescence intensity compared to the MPTP group. Furthermore, the ICI182780 group exhibited decreased TH fluorescence intensity compared to the ICT group (Fig. 4a,c). Simultaneously, the number of TH-positive neurons in the striatum was examined using immunohistochemistry. Compared with the sham group, the MPTP group showed a reduction in TH-positive neurons, while the ICT group exhibited an increase in TH-positive neurons compared to the MPTP group. In contrast, the ICI182780 group showed a decrease in TH-positive neurons compared to the ICT group (Fig. 4b,d). The results of midbrain immunofluorescence and striatal immunohistochemistry were consistent.

Thus, we infer that the PD model was successfully induced by MPTP. Moreover, ICT demonstrated neuroprotective effects in the ovariectomized female mouse model of Parkinson’s disease by mitigating TH.

Fig. 4
figure 4

ICT protection against castration-induced midbrain and striatal damage in female PD model mice. (a) Representative image of brain TH in immunofluorescence (Scale bar = 50 μm). (b) Representative image of striatal TH-positive cells in immunohistochemistry (Scale bar = 100 μm,20 μm). (c) Summary image of brain TH in immunofluorescence (F = 18.85, P < 0.000) (d) Summary image of TH-positive neurons in immunohistochemistry (F = 35.36, P < 0.000), n = 3, all bar graphs are presented as mean ± SD.

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Impact of ICT on the PI3K/Akt signaling pathway in PD model mice OVX

Representative images of WB-quantified proteins PI3K, Akt, p-PI3K (p85), and p-Akt (Ser473) (Fig. 5a). Compared to the sham-operated group, both the MPTP and ICI182780 groups showed decreased expression of p-Akt (Ser473)/Akt, whereas the ICT group exhibited increased expression of p-Akt (Ser473)/Akt (Fig. 5b). The expression of p-PI3K (p85)/PI3K was decreased in the MPTP and ICI182780 groups, while ICT increased the expression of p-PI3K (p85)/PI3K (Fig. 5c). This suggests that the estrogenic effects of ICT might be mediated through the activation of the ER/PI3K/Akt signaling pathway, enhancing the phosphorylation of p-Akt (Ser473) and p-PI3K (p85), thereby exerting ICT’s estrogenic neuroprotective effects to achieve anti-PD outcomes.

Fig. 5
figure 5

Impact of ICT on the phosphorylation of the PI3K/Akt signaling pathway in MPTP-induced PD model mice OVX. (a) Representative bands. (b) p-Akt (Ser473)/Akt (F = 30.74, P < 0.000). (c) p-PI3K (p85)/PI3K (F = 10.45, P = 0.001), n = 3, all bar graphs are presented as mean ± SD.

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Impact of ICT on apoptotic protein expression in the midbrain of PD model mice OVX

To assess the impact of ICT on apoptotic proteins in mice treated with MPTP, the expression of apoptosis-related proteins in different experimental groups was analyzed. Representative images of WB-quantified Bcl-2, Bax, cleaved Caspase-3, and pro-Caspase-3 proteins (Fig. 6a). As shown in Fig. 5b, compared to the Sham group, the expression of pro-Caspase-3 was reduced in the MPTP model group and the ICI182780 group. Compared to the MPTP group, pro-Caspase-3 expression increased in the ICT group (Fig. 6b). Conversely, compared to the MPTP group, cleaved Caspase-3 protein was reduced in the ICT group but increased under ICI182780 intervention (Fig. 6c). Compared to the MPTP group, the expression of the anti-apoptotic protein Bcl-2 increased in the ICT group, whereas it was less in the ICI182780 group (Fig. 6d). Compared to the sham-operated group, the pro-apoptotic protein Bax increased in both the MPTP group and the ICI182780 group; compared to the MPTP group, Bax expression decreased in the ICT group (Fig. 6e). Compared to the MPTP group, the ratio of Bax/Bcl-2 was lower in the ICT group, showing a difference (Fig. 6f). These results suggest that ICT can reduce cell apoptosis, protect neuronal cells, and exert anti-PD effects.

Fig. 6
figure 6

Impact of ICT on apoptotic protein expression in the midbrain of MPTP-Induced PD model mice OVX. (a) Representative bands for Bax. (b ~ f) in sequence pro-Caspase-3(F = 11.09, P = 0.001)、cleaved Caspase-3 (F = 6.597, P = 0.002), Bcl-2 (F = 16.87, P < 0.000), Bax.

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(F = 5.602, P = 0.005), Bax/Bcl-2(P = 0.004), n = 3, all bar graphs are presented as mean ± SD.

The ameliorative effect of ICT on osteoporosis in MPTP-induced OVX PD model mice

Due to abnormal posture and gait, PD patients have a higher incidence of fractures and osteoporosis. The decrease in bone mineral density (BMD) and osteoporosis post-menopause are associated with an increased risk of PD. Effective treatment and management of BMD can effectively prevent and treat PD post-menopause33.

Aniline Blue staining of the distal femur (Fig. 7a) We observed chondrocytes in the distal femur of model mice through Aniline Blue staining. Chondrocytes are the primary cells of cartilage tissue, responsible for synthesizing and maintaining the cartilage matrix. Compared to the sham surgery group, the MPTP group exhibited a reduction in chondrocyte numbers. In contrast, the ICT group showed an increase in chondrocytes compared to the MPTP group, with a regular arrangement of cells. When comparing the ICT group to the ICI182780 group, a decrease in chondrocyte numbers and irregular cell arrangement were noted in the ICI182780 group.

Therefore, we infer that ICT exerts estrogenic effects, promoting the formation of chondrocytes, enhancing the cartilage matrix, and regulating bone metabolism to alleviate osteoporosis. Dual-energy X-ray densitometry showed that compared to the MPTP group, the ICT group exhibited increased BMD. Compared to the sham surgery, the MPTP, and ICI182780 groups showed a significant decrease in BMD (Fig. 7b). The results indicate that ICT has a therapeutic effect on osteoporosis in MPTP-induced post-menopausal PD model mice, and ICI182780 successfully blocks the estrogenic effection of ICT.

Fig. 7
figure 7

The Ameliorative Effect of ICT on Osteoporosis in MPTP-Induced OVX PD Model Mice. (a) Left femur aniline blue staining (the red arrow indicates chondrocytes, with the image magnified, x40, x400) (b) Dual-energy X-ray BMD (F = 9.822, P = 0.0017), n = 4, all bar graphs are presented as mean ± SD.

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Discussion

The estrogen signaling pathway is a key mechanism in neuroprotection34,35. With increasing age, there is a significant decline in estrogen levels in postmenopausal women12. Hormone replacement therapy (HRT) is commonly used to alleviate symptoms after menopause8. HRT functions through ER modulation in tissues, aiming to preserve the beneficial effects on various bodily systems12. Clinically, estrogen and progesterone therapy often raise concerns about the risks of breast cancer and cardiovascular diseases, questioning the effectiveness of HRT36. Therefore, our study aims to elucidate the protective mechanisms of ICT in postmenopausal PD, offering the potential for developing safer hormone replacement therapy strategies. These strategies aim to reduce risks associated with HRT while preserving its benefits for the health of postmenopausal women.

We focused on establishing an effective postmenopausal PD model to understand its pathological mechanisms. We used bilateral OVX in female mice to simulate the postmenopausal state, validating the model through measurements of serum E2, FSH, and LH levels, midbrain α-syn protein expression, and histological evaluation of uterine tissue and vaginal cells. We investigated the estrogen-like effects of ICT on this model and its neuroprotective mechanisms, observing decreased serum E2 levels and increased FSH and LH levels post-OVX, along with uterine atrophy and altered midbrain α-syn protein expression. Exploration linked ICT’s neuroprotective effects to the ER-mediated PI3K/Akt signaling pathway, demonstrating increased protein expression and pronounced phosphorylation at the p-PI3K (p85) and p-Akt (Ser473) sites. When ER was blocked, the expression of proteins in this pathway significantly decreased, further confirming the ER-mediated action of ICT. This may be associated with the PI3K/Akt pathway, which can enhance cellular metabolism, and promote cell proliferation and survival15,37. We also examined the expression changes of apoptosis-related proteins that ICT could reduce MPTP-induced apoptosis of midbrain neurons by increasing the expression of pro-Caspase-3 and Bcl-2 proteins while decreasing the expression of cleaved Caspase-3 and Bax proteins. ICT may regulate apoptotic proteins through the PI3K/Akt signaling cascade, thereby exerting neuroprotective effects38,39. We observed a significant decrease in BMD in mice after OVX, while BMD significantly increased after ICT intervention. Combining behavioral results, we speculate that ICT alleviates symptoms of osteoporosis. This is consistent with the increased risk of PD in postmenopausal women with lower BMD40,41.

Our findings consistently suggest that ICT may possess estrogen-like effects, activating the ER-mediated PI3K/Akt signaling pathway, promoting phosphorylation at relevant sites, modulating apoptosis-related protein expression, and exerting neuroprotection. This may be attributed to the flavonoid estrogenic components within ICT, which, through anti-apoptotic mechanisms and increased neuronal support, contribute to the preservation of the nigrostriatal DA pathway, inhibiting α-syn and oligomerization, and promoting neuronal protection42,43. While our in vivo experiments validate the neuroprotective potential of ICT, due to significant individual variability in animal models, the data is not perfect. In the comparisons depicted in Figs. 1c, 2a, 3d, 4c, and 5b,f, a trend of variation is observed between the Sham group and the MPTP group; however, no statistically significant differences were detected. This may be attributed to the OVX PD model in this study, in which the MPTP group is influenced by low estrogen levels, resulting in non-significant variations. In the comparisons presented in Figs. 1b,c, 2b,c, and 5c,e,f, despite ICI182780 exhibiting inhibitory effects on ER, no statistically significant differences were noted when compared to the ICT group. In the comparisons shown in Figs. 1c, 2a, 3d, 4c, and 5b,f, changes were observed between the Sham group and the MPTP group; however, no statistically significant differences were detected. This might be related to the OVX PD model used in this study, where the MPTP group was influenced by low estrogen levels, resulting in no significant changes being observed. In the comparisons shown in Fig. 1b,c, 2b,c, and 5c,e,f, although ICI182780 exhibited inhibitory effects on ER, no statistically significant differences were found compared to the ICT group. The ER/PI3K/Akt pathway is an intracellular signaling mechanism of estrogen, and ICI182780 primarily exerts its effects by inhibiting this intracellular estrogen signaling pathway. Additionally, estrogen may exert protective effects through extracellular signaling pathways such as ERK, JNK and MAPK44,45.

Furthermore, the observed results may be attributed to large individual differences in the in vivo animal model, hormonal environment variations, potential neurotoxic effects of penicillin, and environmental stress factors. A limited number of animal models and insufficient data repetitions may have contributed to these findings. External factors such as drug absorption and environmental temperature could further impact drug efficacy. Lastly, regarding the ICT intervention time setting, we referred to similar studies and selected a median time point for reference46,47,48. In future studies, we plan and improve the scientific rigor of the experiment.

Further investigations through in vitro experiments are warranted to elucidate its mechanisms of action, Providing a more comprehensive perspective on the neuroprotective mechanisms of ICT. Given that ERα and ERβ may play distinct roles in neuroprotection and exhibit tissue distribution variances49, refine our research, our future studies will consider utilizing ERα and ERβ-specific agonists or antagonists, as well as employing cell and animal models with knockout or overexpression of the respective receptor subtypes, to investigate the impact of ICT on these receptor subtypes.

Additionally, we should consider other signal pathways relevant to neuroprotection, such as MAPK, and ERK, and their interactions. ICT’s effects on these signaling pathway networks can be achieved through phosphorylated Protein arrays or high-throughput sequencing technologies. Despite the lack of in vitro experimental validation, we acknowledge these limitations. Considering the multifactorial influences on the onset of PD after menopause, including hormonal changes35, dietary factors50, and industrial pollution51, future research should be more comprehensive and closely aligned with the clinical conditions of postmenopausal PD patients20.

Conclusion

ICT can activate the ER/PI3K/Akt estrogen signaling pathway, alleviating motor impairments in OVX PD model mice. Concurrently, ICT reduces cellular apoptosis and mitigates osteoporosis in these models, providing evidence for the potential of ICT in treating post-menopausal PD. This evidence supports further investigation into ICT as a therapeutic strategy for neurological and skeletal disorders associated with estrogen deficiency.