Atorvastatin improves hyperlipidemia-induced erectile dysfunction via PKA-associated eNOS phosphorylation at the Ser633 site

Figures

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  Figure 1. Atorvastatin’s effects on lipid metabolism and oxidative stress in HLED rats. (A) Representative liver H&E staining images from each group (n = 3, magnification × 10, scale bar = 250 μm), with boxes highlight areas of fatty degeneration in hepatocytes (magnification × 40; scale bar = 100 μm). (B) Serum levels of GLU, HDL, LDL, TC, and TG in each group (n = 6). (C) Body-weight changes in each group during the experimental period (n = 6). (D-F) serum levels of ALT activity, AST activity, and TBIL concentration in each group (n = 6). (G and H) serum levels of ROS and ox-LDL in each group (n = 6). Data are presented as mean ± SD. Exact P values are indicated in the corresponding panels. ALT, alanine aminotransferase; AST, aspartate aminotransferase; GLU, glucose; H&E, hematoxylin and eosin; HDL, high-density lipoprotein; HLED, hyperlipidemia-induced erectile dysfunction; LDL, low-density lipoprotein; ox-LDL, oxidized low-density lipoprotein; TC, total cholesterol; TG, triglyceride; ROS, reactive oxygen species.

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  Figure 2. Atorvastatin’s effects on corpus cavernosum fibrosis in HLED rats. (A) Representative Masson’s trichrome staining images of penile tissue from each group (n = 3, magnification × 4, scale bar = 1 mm), with boxes better visualize the distribution of collagen fibers and smooth muscle bundles (magnification × 40; scale bar = 100 μm). (B and C) quantification of cavernous collagen volume fraction and smooth muscle/collagen area ratio for each group (n = 3). (D-G) representative western blots and corresponding quantitative analysis of α-SMA and collagen I protein levels in penile tissue from each group (n = 4). Data are presented as mean ± SD. Exact P values are indicated in the corresponding panels. HLED, hyperlipidemia-induced erectile dysfunction.

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  Figure 3. Atorvastatin’s effects on erectile function in HLED rats. (A) Frequency of APO-induced erections in each group during a 30-min observation period (n = 6). Response rate was defined as the percentage of rats showing at least one APO-induced erectile response. Erection frequency is presented as median (IQR) and was analyzed as discrete count data using the Kruskal–Wallis test followed by Dunn’s multiple-comparison test. (B and C) representative images and analysis of ICPmax/MAP ratio in each group (n = 6). (D) Penile tissue cGMP levels in each group (n = 6). For panels C and D, data are presented as mean ± SD. Exact P values are indicated in the corresponding panels. APO, apomorphine; cGMP, cyclic guanosine monophosphate; HLED, hyperlipidemia-induced erectile dysfunction; IQR, interquartile range; MAP, mean arterial pressure.

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  Figure 4. Atorvastatin’s modulation of the PKA/eNOS pathway in penile tissue. (A-D) representative western blots showing levels of p-eNOS Ser1177, p-eNOS Ser633, p-eNOS Thr495, total eNOS, p-PKA Thr197, and total PKA in penile tissue from each group (n = 4). (E-H) quantitative analysis of eNOS phosphorylation at Ser1177, Ser633, and Thr495 (normalized to total eNOS), and PKA Thr197 phosphorylation (normalized to total PKA) in each group (n = 4). Data are presented as mean ± SD. Exact P values are indicated in the corresponding panels. eNOS, endothelial nitric oxide synthase; PKA, protein kinase A.

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  Figure 5. Atorvastatin’s protective effects against ox-LDL–induced lipotoxicity in RCECs. (A) RCECs viability under increasing concentrations of atorvastatin (n = 6). (B) RCECs viability under increasing concentrations of ox-LDL, with 20 μg/mL ox-LDL selected as the optimal concentration for subsequent modeling (n = 6). (C) Effects of atorvastatin on ox-LDL–induced cytotoxicity in RCECs, with the most pronounced protective effect observed at 2 μM (n = 6). (D) Representative oil red O staining images for each group (n = 3, magnification × 20, scale bar = 100 μm). (E and F) representative DCFH-DA fluorescence images of RCECs and quantitative analysis of ROS MFI for each group. Nuclei counterstained with Hoechst (n = 6, magnification × 20, scale bar = 50 μm). Data are presented as mean ± SD. Exact P values are indicated in the corresponding panels. ox-LDL, oxidized low-density lipoprotein; RCECs, rat corpus cavernosum endothelial cells; ROS, reactive oxygen species.

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  Figure 6. Atorvastatin’s effects on ox-LDL–induced endothelial dysfunction in RCECs. (A) Representative images of Transwell migration assay and tube formation assay for each group (n = 3, magnification × 10, scale bar = 200 μm). (B) Quantification of migrated cells in Transwell migration assay for each group (n = 3, 5 random fields per image were selected for quantification and averaged). (C and D) quantification of master junctions and segments in tube formation assay for each group (n = 3). (E and F) relative quantitative western blot analysis of eNOS protein level for each group (n = 4). Data are presented as mean ± SD. Exact P values are indicated in the corresponding panels. eNOS, endothelial nitric oxide synthase; ox-LDL, oxidized low-density lipoprotein; RCECs, rat corpus cavernosum endothelial cells.

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  Figure 7. Atorvastatin’s modulation of the PKA/eNOS pathway in RCECs. (A) Representative immunofluorescence images of p-eNOS Ser1177, p-eNOS Ser633, and p-PKA Thr197 for each group. Nuclei counterstained with DAPI (n = 3, magnification × 40, scale bar = 20 μm). (B-D) quantification of MFI for p-eNOS Ser1177, p-eNOS Ser633, and p-PKA Thr197 for each group (n = 3). Data are presented as mean ± SD. Exact P values are indicated in the corresponding panels. eNOS, endothelial nitric oxide synthase; PKA, protein kinase A; RCECs, rat corpus cavernosum endothelial cells