Objective:

2

To investigate the ameliorative effects of quercetin (QE) on spermatogenic function and elucidate the underlying molecular mechanisms

in vivo

.

Methods:

2

Thirty male C57BL/6 mice (6–8 weeks old) were randomly divided into 5 groups using a random number (

n

=6 per group): control

triptolide (TP) model (0.1 mg/kg per day)

and different doses of quercetin (QE) treatment groups (25

50

and 100 mg/kg per day

intragastrically). Except for controls

all mice received TP to induce spermatogenic impairment

with concurrent QE administration in treatment groups. The intervention lasted 35 days

covering 1 complete spermatogenic cycle

and mice were euthanized on day 38. Histopathological damage and apoptosis in spermatogenic cells were evaluated using hematoxylin and eosin (H

&

E) staining

TUNEL assay

and Western blot analysis for Bcl-2

Bax

and cleaved caspase-9. Blood-testis barrier (BTB) integrity was assessed by immunofluorescence and Western blot for tight junction proteins

including zonula occludens-1 (ZO-1) and junctional adhesion molecule A (JAMA). The PI3K/AKT signaling pathway was investigated through Western blot analysis of PI3K

AKT

and phosphorylated AKT (p-AKT). Network pharmacology and molecular docking simulations were performed to predict QE's molecular mechanisms

followed by experimental verification.

Results:

2

QE treatment significantly ameliorated TP-induced testicular damage

increased spermatogenic epithelial thickness and spermatogonial tubule diameter

and decreased apoptosis of spermatogenic cells (

P

<

0.05 or

P

<

0.01). QE also improved the distribution and expression of key BTB proteins

including ZO-1 and JAMA (

P

<

0.05 or

P

<

0.01). Network pharmacology and molecular docking studies suggested that QE influences the PI3K-AKT signaling pathway

which was confirmed by increased AKT phosphorylation levels observed in Western blot results (

P

<

0.05 or

P

<

0.01).

Conclusions:

2

QE can mitigate TP-induced spermatogenic dysfunction

reduce apoptosis of spermatogenic cells

and preserve BTB structural integrity by upregulating the PI3K-AKT signaling pathway. QE may be a potential therapeutic agent for treating TP-induced spermatogenic disorders.