Study On The Therapeutic Effects And Mechanisms Of Traditional Chinese Medicine Monomers On A Rat Model Of Late-Onset Hypogonadism

 

Abstract

Objective
To evaluate the therapeutic effects and mechanisms of three traditional Chinese medicine (TCM) monomers-icariin, verbascoside, and ginsenoside Rg1-on late-onset hypogonadism (LOH) in a rat model, and to explore their potential as natural interventions for age-related testosterone decline in middle-aged males.

Methods
A late-onset hypogonadism (LOH) model was established in aged male Sprague-Dawley rats via intraperitoneal injection of cyclophosphamide, simulating androgen deficiency. Three TCM monomers-icariin, verbascoside, and ginsenoside Rg1-were administered orally for 60 days.

At the end of the treatment, body weight and the weights of reproductive organs (testes, epididymis, seminal vesicles, etc.) were measured. Histopathological changes in testicular tissue were assessed using hematoxylin-eosin (H&E) staining. Serum levels of FSH, LH, and testosterone were tested via ELISA. The expression of androgen receptor (AR) protein and mRNA was evaluated using Western blot and quantitative real-time PCR (qRT-PCR).

 

 

 

Results

Ginsenoside Rg1 showed the most significant improvement in body weight and reproductive organ weight (P < 0.01).

Icariin significantly increased the weight of the testes, epididymis, and seminal vesicles (P < 0.01).

Verbascoside moderately improved seminal vesicle weight (P < 0.05).

All three TCM monomers effectively reversed testicular degeneration in LOH rats, restoring seminiferous tubule structure and increasing the number of spermatogenic cells and mature sperm.

All monomer groups demonstrated the ability to lower serum FSH and LH levels while raising serum testosterone. The icariin group showed the most significant endocrine improvements (P < 0.01).

Expression levels of androgen receptor (AR) in both protein and mRNA were significantly upregulated in all treated groups. The effect of icariin was more pronounced than testosterone propionate, indicating a strong regulatory effect on AR signaling.

 

Conclusion

This study suggests that ginsenoside Rg1 is most effective in improving body and organ weight in LOH rats, while icariin shows the strongest effect in boosting testosterone production and enhancing AR expression. All three monomers-icariin, verbascoside, and ginsenoside Rg1-demonstrated protective effects against testicular degeneration and support male reproductive endocrine function, offering promising natural alternatives for the management of age-related hypogonadism.

 

Keywords

Late-onset hypogonadism (LOH); Icariin; Verbascoside; Ginsenoside Rg1; Traditional Chinese Medicine; Natural testosterone booster; Aging male syndrome; Androgen receptor expression; Herbal therapy for male health

Click the pic to learn more about the TCM herb for sexual health

 

 

Background

Late-onset hypogonadism (LOH) in middle-aged and elderly men is closely associated with age-related decline in androgen levels, particularly testosterone. A 2021 population-based epidemiological study conducted across six representative provinces in China, involving 6,296 men aged 40–79, revealed the following prevalence rates of LOH:

3.2% in men in their 50s

11.2% in their 60s

24.0% in their 70s [1]

LOH is characterized by reduced serum testosterone along with a constellation of androgen deficiency symptoms such as:

Low libido

Erectile dysfunction

Depression and irritability

Cognitive decline and memory loss

These symptoms can significantly impair physical and mental well-being, as well as quality of life [2–3].

Currently, testosterone replacement therapy (TRT) is the mainstream treatment for LOH and may temporarily alleviate symptoms. However, concerns about cardiovascular risk, prostate health, and long-term safety remain [4]. Therefore, there is growing interest in exploring natural, safer alternatives, especially from Traditional Chinese Medicine (TCM).

This study aims to evaluate the therapeutic effects and mechanisms of three TCM monomers-icariin, verbascoside, and ginsenoside Rg1-using a rat model of LOH.

 

1. Materials and Methods

1.1 Experimental Animals

Sixty 15-month-old male Sprague-Dawley (SD) rats (700–800g) were obtained in January 2022 from the Animal Experimental Center of China Three Gorges University. License numbers:

Animal Certificate: No. 42010200007997

Production License: SCXK (E) 2022-0012

Rats were housed in a controlled environment (temperature: 19–23°C, relative humidity: 55%–65%), with a natural light-dark cycle and free access to food and water. After 1 week of acclimatization, experiments began.

1.2 Reagents and Instruments

Cyclophosphamide (Nanjing Jiancheng)

Icariin (Nanjing Guangrun Biotech)

Ginsenoside Rg1 (Shanghai Macklin)

Verbascoside (Shanghai Zhenzhun Biotech)

AR antibody (Santa Cruz, USA)

β-actin antibody (Sigma, USA)

H&E staining kits (Shanghai Yiji Industrial)

ELISA kits for serum FSH, LH, testosterone (Shanghai Meilian)

qRT-PCR reagents (SYBR Green I) (Shanghai Julu Biotech)

Instruments: Thermo microplate reader, Olympus optical microscope, LI-COR Odyssey imaging system, ABI qRT-PCR machine

 

1.3 Animal Grouping and LOH Model Induction

Rats were randomly assigned to 6 groups (n = 10 per group):

Control group:

Intraperitoneal (i.p.) injection of 0.9% saline for 5 days

Oral saline (1 mL/100g) for 60 days

Model group:

i.p. cyclophosphamide 20 mg/kg/day for 5 days

Followed by oral saline for 60 days

Testosterone propionate (positive control):

i.p. cyclophosphamide (same as above)

Subcutaneous injection of testosterone propionate 5 mg/kg/day for 7 days

Icariin group:

i.p. cyclophosphamide (same)

Oral icariin 200 mg/kg/day for 60 days

Verbascoside group:

i.p. cyclophosphamide (same)

Oral verbascoside 200 mg/kg/day for 60 days

Ginsenoside Rg1 group:

i.p. cyclophosphamide (same)

Oral ginsenoside Rg1 200 mg/kg/day for 60 days

All treatments were administered between 8:00–11:30 AM. Assessments were performed 24 hours after the final dose.

 

1.4 Body Weight and Reproductive Organ Index

After weighing, rats were euthanized by cervical dislocation. The testes, epididymis, and seminal vesicles were dissected and weighed.

One testis from each rat was fixed in 4% paraformaldehyde for 72 hours, dehydrated with graded alcohol, paraffin-embedded, sectioned (5 μm), and stained with Hematoxylin-Eosin (H&E). Morphological changes were observed under light microscopy.

 

1.5 Serum Hormone Assays

Rats were anesthetized with 3% pentobarbital sodium, and blood was collected via cardiac puncture. Serum was separated by centrifugation at 3000 rpm for 10 min at 4°C. ELISA kits were used to measure FSH, LH, and testosterone levels according to the manufacturer's protocols.

 

1.6 AR Protein Expression (Western Blot)

Testicular tissue was lysed and protein concentration measured. Equal amounts of protein were resolved by 10% SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies against AR and β-actin (1:1000) overnight. Secondary antibody incubation was followed by chemiluminescent detection. ImageJ was used for analysis.

 

1.7 AR mRNA Expression (qRT-PCR)

Total RNA was extracted from testicular tissue. cDNA was synthesized via reverse transcription. qRT-PCR was performed using the SYBR Green method.

AR primers:

Forward: 5′-CAGGGACCA CGTTTTACCCA-3′

Reverse: 5′-TTCCCTTCCGCAGCTCTTTT-3′

β-actin primers (internal control):

Forward: 5′-CCACCATGTACCCAGGCATT-3′

Reverse: 5′-CGGACTCATCGTACTCCTGC-3′

Thermal cycling: 95°C for 15 min (pre-denaturation), followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed using the 2^-ΔΔCt method.

 

1.8 Statistical Analysis

Data were analyzed using SPSS 26.0.

Results are presented as mean ± standard deviation (x̄ ± s)

t-test was used for two-group comparisons

One-way ANOVA was used for multiple group comparisons

P < 0.05 was considered statistically significant