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Published on: March 11, 2018
Guilu Erxian oral liquid mitigates oxidative damage in spermatogonial cells via miR-6739-5p modulation and PI3K/AKT
Zefeng Sun1, Xinrong Fan2, Zhenquan Liu1
1Beijing University of Chinese Medicine, Beijing.
Abstract:
Oxidative stress is a major contributor to male infertility, particularly oligoasthenozoospermia. This study aimed to investigate the cytoprotective mechanism of Guilu Erxian Oral Liquid (GLEX) against H₂O₂-induced oxidative damage in spermatogonial cells, focusing on miR-6739-5p regulation and activation of the PI3K/AKT pathway using histocytochemical approaches. An oxidative stress model was established in rat spermatogonial stem cells (SSCs) with 250 µM H₂O₂. Cell proliferation, apoptosis, reactive oxygen species (ROS) accumulation, and DNA oxidative damage were assessed using EdU incorporation, flow cytometry, immunofluorescence, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA. Expression of miR-6739-5p and Phosphatidylinositol 3-Kinase/Protein Kinase B (PI3K/AKT) pathway components (PIK3CA, p-PI3K, p-AKT) was evaluated by RT-qPCR and Western blotting. The interaction between miR-6739-5p and PIK3CA was confirmed via dual-luciferase reporter assay. The cytoprotective effects of GLEX were examined through pre-treatment and quantified using histochemical and cytological markers. H₂O₂ treatment significantly impaired cell viability, increased apoptosis and ROS production, and upregulated miR-6739-5p. Overexpression of miR-6739-5p exacerbated damage, while silencing reversed it and restored PI3K/AKT signaling. GLEX pretreatment effectively reduced miR-6739-5p expression, restored cell viability, suppressed oxidative and inflammatory markers (ROS, 8-OHdG, TNF-α, IL-1β), and enhanced PI3K/AKT activation. These effects were comparable to PI3K pathway activation. GLEX confers histocytochemical protection to spermatogonial cells under oxidative stress by downregulating miR-6739-5p and activating the PI3K/AKT pathway. This study highlights a novel regulatory mechanism and supports GLEX as a potential therapeutic agent for oxidative stress-associated male infertility.
Insights
Guilu Erxian Oral Liquid (GLEX) protects male reproductive cells from oxidative stress by downregulating miR-6739-5p and activating the PI3K/AKT pathway, offering potential for treating infertility.
Area of Science:
- Reproductive Biology
- Molecular Biology
- Cell Biology
Background:
- Oxidative stress is a key factor in male infertility, specifically oligoasthenozoospermia.
- Spermatogonial stem cells (SSCs) are vulnerable to oxidative damage, impacting sperm production.
- Understanding molecular mechanisms underlying oxidative stress in SSCs is crucial for developing treatments.
Purpose of the Study:
- To investigate the cytoprotective effects of Guilu Erxian Oral Liquid (GLEX) on H₂O₂-induced oxidative damage in rat SSCs.
- To elucidate the role of miR-6739-5p and the PI3K/AKT pathway in GLEX's protective mechanism.
- To assess GLEX's potential as a therapeutic agent for oxidative stress-related male infertility.
Main Methods:
- Establishment of an oxidative stress model in rat SSCs using hydrogen peroxide (H₂O₂).
- Assessment of cell viability, apoptosis, reactive oxygen species (ROS), and DNA damage (8-hydroxy-2'-deoxyguanosine).
- Evaluation of miR-6739-5p and PI3K/AKT pathway component expression via RT-qPCR and Western blotting; dual-luciferase reporter assay confirmed molecular interactions.
Main Results:
- H₂O₂ exposure increased apoptosis, ROS, and miR-6739-5p expression while decreasing cell viability.
- miR-6739-5p overexpression worsened damage, whereas its silencing reversed damage and restored PI3K/AKT signaling.
- GLEX pretreatment reduced miR-6739-5p, improved cell viability, decreased oxidative/inflammatory markers, and activated the PI3K/AKT pathway.
Conclusions:
- GLEX provides histocytochemical protection to spermatogonial cells against oxidative stress.
- The protective mechanism involves downregulating miR-6739-5p and activating the PI3K/AKT pathway.
- GLEX demonstrates potential as a therapeutic agent for male infertility linked to oxidative stress.
