Cistanche Glycosides Attenuate Prostatic Fibrosis in Benign Prostate Hyperplasia By Regulating MiRNA‑483‑3p

 

ABSTRACT

Objective: To investigate the ameliorative effects of cistanche glycosides on prostatic fibrosis in benign prostatic hyperplasia (BPH) and the mechanism involving regulation of microRNA (miRNA)-483-3p.

Methods: Male mice were randomly assigned to five groups: normal control, BPH model control, finasteride, cistanche glycosides, and cistanche glycosides + miRNA‑483‑3p antagonist. The BPH model was established by bilateral orchiectomy followed by daily subcutaneous injection of testosterone propionate. After 30 days, prostatic collagen deposition was evaluated by Masson's trichrome and Sirius Red staining, and hydroxyproline (Hyp) content was measured. Prostatic mRNA levels of transforming growth factor‑β1 (TGF‑β1), mitogen‑activated protein kinase 2 (MK2), and mitogen‑activated protein kinase kinase 6 (MKK6), as well as miRNA‑483‑3p levels, were determined by quantitative real-time PCR (qPCR). Prostatic protein levels of TGF‑β1, MK2, phosphorylated MK2 (p‑MK2), MKK6, and phosphorylated MKK6 (p‑MKK6) were assessed by Western blotting. The direct binding of miRNA‑483‑3p to MK2 was examined using a luciferase reporter assay.

Results: Relative to the normal control group, the BPH model control group showed significantly increased prostatic collagen deposition; elevated mRNA levels of TGF‑β1, MK2, and MKK6; and higher protein levels of TGF‑β1, p‑MK2, and p‑MKK6 (all P < 0.01), while miRNA‑483‑3p levels were significantly decreased (P < 0.01). Compared with the BPH model control group, cistanche glycosides upregulated miRNA‑483‑3p, reduced the mRNA levels of TGF‑β1, MK2, and MKK6, lowered the protein levels of TGF‑β1, p‑MK2, and p‑MKK6, and alleviated prostatic collagen deposition. Notably, co-administration of the miRNA‑483‑3p antagomir with cistanche glycosides significantly decreased miRNA‑483‑3p, increased collagen deposition, and elevated mRNA levels of TGF‑β1, MK2, and MKK6 as well as protein levels of TGF‑β1, p‑MK2, and p‑MKK6 compared with cistanche glycosides alone (all P < 0.01). The luciferase assay demonstrated that miRNA‑483‑3p directly targets MK2.

Conclusion: Cistanche glycosides attenuate prostatic fibrosis in BPH by regulating miRNA‑483‑3p and inhibiting MK2 signaling.

Keywords: Cistanche glycosides; miRNA‑483‑3p; prostate; fibrosis; benign prostatic hyperplasia

 

 

 

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Introduction

Benign prostatic hyperplasia (BPH) is a common condition in middle-aged and older men, with prevalence increasing with age. Reports indicate that the prevalence of BPH exceeds 50% among men over 50 years old. BPH and the associated lower urinary tract symptoms (LUTS) impose substantial burdens on quality of life and long-term healthcare costs. Clinically, 5α-reductase inhibitors and α‑adrenergic receptor blockers are recommended pharmacotherapies. However, α‑blockers do not reduce prostate volume, and 25–30% of patients show no improvement in LUTS with 5α-reductase inhibitors; moreover, 5–7% may even worsen. Although surgical innovations such as thulium laser prostatectomy are increasingly utilized compared with transurethral resection of the prostate, postoperative LUTS still occur at rates exceeding 23%.

Current evidence suggests three major pathobiological processes in BPH: glandular hyperplasia, smooth muscle contraction, and prostatic fibrosis. These processes act independently or synergistically to drive clinical progression. BPH commonly (though not invariably) coexists with LUTS; fibrosis, characterized by excessive extracellular matrix deposition, compromises urethral flexibility within the prostate and contributes to urinary obstruction. Yet, therapeutic options specifically targeting organ fibrosis remain limited.

Cistanche (Cistanche spp.) is a traditional medicinal and nutritional plant. Its characteristic phenylethanoid glycosides (collectively referred to here as cistanche glycosides, such as echinacoside and acteoside) exhibit broad bioactivities, including antimicrobial, hypolipidemic, antiallergic, cardiotonic, antidiabetic, antitumor, neuroprotective, antioxidant, and immunomodulatory effects. Accumulating evidence indicates anti-fibrotic actions of these glycosides in organs such as the kidney, lung, heart, and liver. However, their effects on prostatic fibrosis have not been reported. This study examined the impact of cistanche glycosides on prostatic fibrosis and explored the underlying mechanisms, focusing on the miRNA‑483‑3p/MK2 axis relevant to extracellular matrix remodeling.

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Materials and Methods

Materials

 

miRNA‑483‑3p antagomir (chemically modified): purchased from Shanghai GenePharma Co., Ltd. (Batch No.: 12953).

Testosterone propionate injection: Tianjin Jinyao Pharmaceutical Co., Ltd. (Batch No.: 150822).

Cistanche glycosides: Shanghai Aladdin Biochemical Technology Co., Ltd. (purity ≥98%, Batch No.: M14062). Note for nutraceutical developers: material corresponds to standardized phenylethanoid glycoside fraction; ensure specification by HPLC content (e.g., total PEGLs, echinacoside/acteoside).

Finasteride tablets: MSD (Hangzhou) Pharmaceutical Co., Ltd. (Batch No.: U011856; 5 mg/tablet).

Luciferase reporter assay kit (Batch No.: E2920) and psiCHECK‑2 vector (Batch No.: C8021): Promega (USA).

qPCR detection kit: KAPA Biosystems (Batch No.: KM4101).

Hydroxyproline (Hyp) assay kit: Nanjing Jiancheng Bioengineering Institute (Batch No.: 20190626).

Primers for TGF‑β1, MK2, and MKK6 were synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd.

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Animals

Seven-week-old specific pathogen-free (SPF) male C57BL/6 mice (22–24 g) were obtained from Huazhong Agricultural University (Production License No.: SCXK Hubei 2015‑0019). Animals were housed at 23 ± 2°C, 50 ± 5% humidity, under a 12 h light/12 h dark cycle.

 

Equipment

 

StepOnePlus real-time PCR system (Applied Biosystems, USA).

Luminoskan 5200110 microplate luminometer (Thermo Fisher Scientific, USA).

Experimental Design and BPH Model
After 1 week of acclimatization, mice were randomly allocated (random number table) into five groups (n = 6 per group): normal control, BPH model control, finasteride, cistanche glycosides, and cistanche glycosides + miRNA‑483‑3p antagomir. The BPH model was induced by surgical castration plus daily subcutaneous injection of testosterone propionate at 7.5 mg·kg⁻¹ for 30 days. The finasteride group received oral finasteride 1 mg·kg⁻¹ daily (dissolved in 0.5% CMC‑Na). The cistanche glycosides group received oral cistanche glycosides 100 mg·kg⁻¹ daily. The combination group received oral cistanche glycosides 100 mg·kg⁻¹ daily plus tail vein injection of miRNA‑483‑3p antagomir at 10 mg·kg⁻¹ every 5 days following the manufacturer's instructions. Normal and BPH model control groups received an equal volume of 0.5% CMC‑Na orally.

After 30 days, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg·kg⁻¹) and euthanized by cervical dislocation. Prostates were excised; portions were fixed and embedded for Masson's trichrome and Sirius Red staining, and fresh tissues were processed for qPCR, Western blotting, and biochemical assays. Animal procedures complied with the Hubei Provincial Regulations on Laboratory Animal Management (Operator Qualification No.: TY20190784).

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Assessment of Prostatic Fibrosis

Collagen deposition was evaluated by Sirius Red and Masson's trichrome staining. Hyp content in prostate tissue was quantified per kit instructions. For each slide, three random fields were analyzed using Image‑Pro Plus 6.0 to calculate the percentage of positive stained area.

Evaluation of MK2 Pathway Activity

qPCR: Prostatic mRNA levels of MK2, TGF‑β1, and MKK6 were measured. Primer sequences:

MK2 (264 bp) forward 5'-GGAAGTGCCTGCTGAT-3', reverse 5'-AGTTGTGACTGGTGGTTT-3';

TGF‑β1 (153 bp) forward 5'-AGGAGACGGAATACAGGG-3', reverse 5'-ATGAGGAGCAGGAAGGG-3';

MKK6 (146 bp) forward 5'-CCTCAGACCAGTTCCAC-3', reverse 5'-CGCATCTTCTCCACCA-3'.

Western blot: Protein levels of TGF‑β1, MK2, p‑MK2, MKK6, and p‑MKK6 were determined following previously established protocols.

Measurement of miRNA‑483‑3p
miRNA‑483‑3p primers: forward 5'-GGGTCACTCCTCCCCT-3', reverse 5'-AACTGGTGTCGTGGAGTCGGC-3'; RT primer 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAGACGGG-3'. qPCR was performed per kit instructions.

 

Luciferase Reporter Assay

The MK2 3'UTR was cloned into the psiCHECK‑2 vector to construct wild-type (WT) and mutant (MUT) reporter plasmids. HEK‑293T cells were co-transfected with miRNA‑483‑3p and the reporter plasmids using Lipofectamine 3000; control cells received empty vector. After 24 h, luciferase activity was measured according to the kit protocol.

Statistical Analysis
Data were analyzed using SPSS 17.0. One-way ANOVA was applied after confirmation of normality and homogeneity of variance. Results are expressed as mean ± SD. LSD post hoc tests were used for multiple comparisons. Differences were considered statistically significant at P < 0.05.

 

Results

Compared with the normal control group, the BPH model control group exhibited marked increases in prostatic collagen deposition; elevated mRNA expressions of TGF‑β1, MK2, and MKK6; and higher protein levels of TGF‑β1, p‑MK2, and p‑MKK6 (all P < 0.01). miRNA‑483‑3p levels were significantly reduced (P < 0.01).

Relative to the BPH model control, cistanche glycosides significantly upregulated miRNA‑483‑3p, decreased the mRNA levels of TGF‑β1, MK2, and MKK6, lowered the protein levels of TGF‑β1, p‑MK2, and p‑MKK6, and mitigated prostatic collagen deposition.

Compared with cistanche glycosides alone, the combination of cistanche glycosides with the miRNA‑483‑3p antagomir significantly decreased miRNA‑483‑3p and increased collagen deposition, plus mRNA levels of TGF‑β1, MK2, and MKK6, as well as protein levels of TGF‑β1, p‑MK2, and p‑MKK6 (all P < 0.01).

The luciferase reporter assay confirmed that miRNA‑483‑3p directly targets MK2.

 

Discussion (brief, nutraceutical-focused)

This study indicates that cistanche glycosides attenuate prostatic fibrosis in a castration/testosterone-induced BPH model by upregulating miRNA‑483‑3p and suppressing MK2 pathway activity downstream of TGF‑β signaling. Given the limitations of current BPH pharmacotherapies in addressing fibrotic remodeling, cistanche glycosides may represent a complementary nutraceutical strategy aimed at extracellular matrix balance.

For dietary supplement development, considerations include:

Standardization: define total phenylethanoid glycosides and marker compounds (e.g., echinacoside ≥20%, acteoside ≥10% by HPLC).

Dose translation: 100 mg·kg⁻¹ in mice approximates 8–12 mg·kg⁻¹ human equivalent dose (HED) using body surface area scaling, roughly 500–800 mg/day for a 60–70 kg adult; clinical validation is required.

Quality and safety: ensure identity (botanical source, contaminants, heavy metals, pesticides), and assess potential interactions with α‑blockers or 5α‑reductase inhibitors.

Claims: structure/function claims should focus on "supporting urinary flow and prostate tissue balance" and avoid disease-treatment language without clinical evidence.

 

Conclusions

Cistanche glycosides alleviate prostatic fibrosis in BPH by regulating miRNA‑483‑3p and inhibiting MK2 signaling. These findings support further translational research on standardized cistanche extracts as adjunctive nutraceuticals for prostate health.

 

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 81703594).

Author Information
Yang Chuhao (1991–), female, Wuhan, Hubei, clinical pharmacist, M.S.; main research area: clinical pharmacy. ORCID: 0000-0002-8921-5758; Tel: +86-27-82440854.