Fig 1.
D-gal-induced aging led to tight junction damage in SCs in vitro.
(A) The viability of SCs was evaluated after exposure to D-gal at concentrations of 0, 5, 10, 20, 40, and 80 mg/mL. (B) Following a 48-h treatment with D-gal concentrations of 0, 5, 10, 20, and 40 mg/mL, Western blotting was performed to assess the expressions of Claudin-4, Claudin-7, p21, ZO-1, p16, and Occludin proteins in SCs. (C) Cellular immunofluorescence (scale bar = 50 μm) was utilized to examine and quantify the expressions of p21, Claudin-4, Occludin, and ZO-1 in SCs treated with varying D-gal concentrations (0, 5, 10, 20, and 40 mg/mL). Data is presented as the mean ±standard error of the mean (SEM). The data are statistically significant between different letters(p<0.05).
Fig 2.
Curcumin restores D-gal-induced SCs TJ damage in vitro.
(A) The survival rate of SCs was determined after exposure to curcumin at concentrations of 0, 5, 10, 20, and 40 μM. (B) SCs were incubated with curcumin at concentrations of 0, 5, 10, and 20 μM in the presence of 40 mg/mL D-gal for 48 h, followed by Western blotting analysis to evaluate the expressions of Claudin-4, Claudin-7, ZO-1, and Occludin proteins. (C) A cellular immunofluorescence assay (scale bar = 50 μm) was performed to assess and quantify the expressions of Claudin-4, Claudin-7, Occludin, and ZO-1 in SCs co-treated with curcumin at concentrations of 0, 5, 10, and 20 μM and 40 mg/mL D-gal. Data is presented as the mean ±standard error of the mean (SEM). The data are statistically significant between different letters(p<0.05).
Fig 3.
D-gal-induced aging leads to reduced autophagy levels in SCs, which are restored by curcumin in vitro.
(A) Protein expression and quantification of Beclin1 and LC3 in SCs treated with different concentrations of D-gal (0, 5, 10, 20, 40 mg/mL) by Western blotting. (B) The expression and quantification of Beclin1 and LC3 proteins in SCs were evaluated after co-treatment with a range of curcumin concentrations (0, 5, 10, and 20 μM) along with a constant 40 mg/mL D-gal by using Western blotting. (C) The formation of autophagosomes was visualized in SCs following their co-treatment with the different series of curcumin concentrations (0, 5, 10, and 20 μM) in the presence of 40 mg/mL D-gal. Data is presented as the mean ±standard error of the mean (SEM). The data are statistically significant between different letters (p<0.05).
Fig 4.
Curcumin restores D-gal-induced SCs TJ injury through autophagy activation in vitro.
(A) Protein expression and quantification of Beclin1, LC3, Clauin-4, Claudin-7, Occludin and ZO-1 in SCs after D-gal (40 mg/mL) and curcumin (20 µM)/CQ (50 µM) treatment by Western blotting. (B) Cellular immunofluorescence assay for protein expression and quantification of LC3, Claudin-4, Occludin, and ZO-1 in SCs after treatment with D-gal (40 mg/mL) and curcumin (20 µM)/CQ (50 µM). Data is presented as the mean ±standard error of the mean (SEM). The data are statistically significant between different letters(p<0.05).
Fig 5.
Curcumin restores aging-induced disruption of the BTB in mice by activating autophagy in mouse testes injected with D-gal.
The mice were assigned to the control group (administered 200 mg/kg saline), model group (administered 200 mg/kg D-gal), curcumin-treated group (administered 200 mg/kg curcumin + 200 mg/kg D-gal), and rapamycin group (administered 200 mg/kg D-gal + 2 mg/kg RAPA). (A) Western blotting was utilized to detect and quantify the expressions of proteins including p21, p16, Beclin1, LC3, Claudin-4, Claudin-7, Occludin, and ZO-1. (B) Hematoxylin and eosin (H&E) staining was conducted to examine the histological alterations within the testes. The upper panels display the modifications in the seminiferous tubules at a 100× magnification (scale bar = 200 μm), while the lower panels present a closer view at a 400× magnification (scale bar = 50 μm). (C) Transmission electron microscopy (TEM) was employed to visualize autophagic structures in the mouse testis (scale bar = 1 μm), arrows indicate autophagosomes. (D) Tissue immunofluorescence for protein expression and quantification of LC3, Claudin-4, Claudin-7, Occludin, and ZO-1. Data is presented as the mean ±standard error of the mean (SEM). The data are statistically significant between different letters (p<0.05).
Fig 6.
Activation of the AMPK/mTOR signaling pathway correlates with curcumin-activated autophagy to restore D-gal-induced tight junction damage in SCs.
(A) Protein expression and quantification of p -AMPK/AMPK and p-mTOR/mTOR after co-treatment of SCs with different concentrations of curcumin (0, 5, 10, and 20 μM) and 40 mg/mL D-gal by Western blotting. (B) Protein expression and quantification of Beclin1, LC3, Claudin-4, Claudin-7, Occludin, and ZO-1 in SCs after treatment with D-gal (40 mg/mL) and curcumin (20 µM)/CC (10 µM) by Western blotting. (C) Cellular immunofluorescence assay (scale bar = 50 μm) for protein expression and quantification of Beclin1, LC3, Claudin-4, Claudin-7, Occludin, and ZO-1 in SCs after D-gal (40 mg/mL) and curcumin (20 µM)/CC (10 µM) treatment. Data is presented as the mean ±standard error of the mean (SEM). The data are statistically significant between different letters (p<0.05).
Fig 7.
Schematic diagram of the mechanism by which curcumin ameliorates aging-induced TJ damage in SCs.
Image abstract: In mouse SCs, D-gal-induced aging leads to a severe lack of cellular autophagy as well as tight junction damage. Curcumin ameliorates aging-induced TJ damage in mouse SCs by activating AMPK/mTOR-mediated autophagy.