Oral treatment with herbal formula B401 alleviates penile toxicity in aging mice with manganism

Figures & data

Figure 1 Chromatographic fingerprint analysis for the Chinese herbal formula B401.

Notes: HPLC fingerprint of the herbal formula B401. Characteristic peaks of B401, ie, A: ginsenosides Rb1 (C54H92O23; MW: 1109.3 g/mol; from Panax ginseng) and oleanolic acid (C30H48O3; MW: 456.7 g/mol; from Ligustri fructus); B: formononetin (C16H12O4; MW: 268.3 g/mol; from Astragalus membranaceus); C: 5-hydroxymethylfurfural (C6H6O3; MW: 126.1 g/mol; from Rehmannia glutinosa); D: ferulic acid (C10H10O4; MW: 194.2 g/mol; from Ligustri fructus); E: wedelolactone (C16H10O7; MW: 314.3 g/mol; from Eclipta prostrata), were identified and marked at the corresponding peaks in the fingerprint.
Abbreviations: AU, arbitrary perfusion units; 5-HMF, 5-hydroxymethylfurfural; HPLC, high-performance liquid chromatography; MW, molecular weight.

Figure 2 Cavernosal nitric oxide (NO) levels and H&E staining in Mn-treated mice with and without oral B401 treatment.

Notes: (A) Cavernosal NO levels measured by NO electrochemical sensor meter; (a) surgical technique used for NO monitoring in vivo; and (b) quantified cavernosal NO levels in Mn-treated mice under oral B401 treatment (B401+Mn, n=6) were significantly higher than those in Mn-treated mice without oral B401 treatment (Mn, n=6). Values are mean ± SEM (*P<0.05, one-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest). (B) Cavernosal H&E staining in (a) Mn-treated mice under oral B401 treatment (B401+Mn) and (b) cavernosal tissue in Mn-treated mice without oral B401 treatment (Mn).
Abbreviations: ANOVA, analysis of variance; H&E, hematoxylin and eosin; NO, nitric oxide; SEM, standard error of the mean.

Figure 3 Cavernosal levels of nitric oxide synthases in Mn-treated mice with and without oral B401 treatment.

Notes: (A) Immunohistochemical staining illustrated that expression levels of cavernosal eNOS of (a, B401+Mn) Mn-treated mice under oral B401 treatment were visibly enhanced in comparison with the (b, Mn) Mn-treated mice without oral B401 treatment. (B) Western blotting analysis shows the following: (a) cavernosal expression levels of nNOS and eNOS of Mn-treated mice with and without oral B401 treatment and (b) quantified cavernosal nNOS and eNOS levels of Mn-treated mice with oral B401 treatment (B401+Mn, n=6) were significantly enhanced in comparison with the same in Mn-treated mice without oral B401 treatment (Mn, n=6). Values are mean ± SEM (**P<0.01, one-way ANOVA followed by Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 4 Cavernosal levels of VEGF, catalase, and SOD2 among Mn-treated mice with or without oral B401 treatment and among the sham mice.

Notes: Western blotting analysis shows the following: (A) Cavernosal expression levels of VEGF, catalase, and SOD2 among Mn-treated mice with or without oral B401 treatment and among the sham mice. (B) Quantified cavernosal VEGF, catalase, and SOD2 levels in Mn-treated mice (Mn, n=6) were significantly lower than those in the sham mice (Sham, n=6), while they were significantly enhanced under oral B401 treatment (B401+Mn, n=6). Values are mean ± SEM (**P<0.01, *P<0.05, one-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: VEGF, vascular endothelial growth factor; SOD2, superoxide dismutase 2; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 5 Cavernosal levels of 4-HNE, an inducer of oxidative stress, in Mn-treated mice with and without oral B401 treatment.

Notes: Western blotting analysis shows the following: (A) Cavernosal expression levels of 4-HNE in Mn-treated mice with and without oral B401 treatment. (B) Quantified cavernosal 4-HNE levels of Mn-treated mice under oral B401 treatment (B401+Mn, n=6) were significantly lower than those in the Mn-treated mice without oral B401 treatment (Mn, n=6). Values are mean ± SEM (**P<0.01, one-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: 4-HNE, 4-hydroxynonenal; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 6 Cavernosal levels of TNF-α, a marker of inflammation, in Mn-treated mice with and without oral B401 treatment.

Notes: Western blotting analysis shows the following: (A) Cavernosal expression levels of TNF-α in Mn-treated mice with and without oral B401 treatment. (B) Quantified cavernosal TNF-α levels of Mn-treated mice under oral B401 treatment (B401+Mn, n=6) were significantly lower than those in the Mn-treated mice without oral B401 treatment (Mn, n=6). Values are mean ± SEM (**P<0.01, one-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: TNF-α, tumor necrosis factor alpha; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 7 Cavernosal levels of antiapoptotic Bcl-2 and apoptosis inducers c-PARP, Bax, and Cyt-C in Mn-treated mice with and without oral B401 treatment.

Notes: Western blotting analysis shows the following: (A) Cavernosal expression levels of c-PARP, Bcl-2, Bax, and Cyt-C in Mn-treated mice with and without oral B401 treatment. (B) The quantified ratio of cavernosal Bcl-2/Bax in Mn-treated mice under oral B401 treatment (B401+Mn, n=6) was significantly enhanced in comparison with the Mn-treated mice without oral B401 treatment (Mn, n=6). Quantified cavernosal c-PARP and Cyt-C levels in Mn-treated mice under oral B401 treatment (B401+Mn, n=6) were significantly lower compared with the Mn-treated mice without oral B401 treatment (Mn, n=6). Values are mean ± SEM (**P<0.01, *P<0.05, one-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; c-PARP, cleaved poly(adenosine diphosphate-ribose) polymerase; Cyt-C, cytochrome c; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 8 Cavernosal levels of apoptosis inducers caspase-12 and caspase-3 among Mn-treated mice with and without oral B401 treatment, as well as among the sham mice.

Notes: Western blotting analysis shows the following: (A) Cavernosal expression levels of caspase-12 and caspase-3 among Mn-treated mice with and without oral B401 treatment, as well as among the sham mice. (B) Quantified cavernosal caspase-12 and caspase-3 levels in Mn-treated mice (Mn, n=6) were significantly higher than those in the sham mice (Sham, n=6), while being significantly reduced under oral B401 treatment (B401+Mn, n=6). Values are mean ± SEM (**P<0.01, *P<0.05, one-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 9 The schematic diagram illustrates that oral B401 treatment may alleviate penile toxicity in Mn-treated mice by enhancing both synthesis of nitric oxide and angiogenesis, while reducing oxidative stress, inflammation, and apoptosis in the corpus cavernosum.

Abbreviations: NO, nitric oxide; nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase; VEGF, vascular endothelial growth factor; SOD2, superoxide dismutase 2; 4-HNE, 4-hydroxynonenal; TNF-α, tumor necrosis factor alpha; ER, endoplasmic reticulum; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; c-PARP, cleaved poly(adenosine diphosphate-ribose) polymerase; Cyto-C, cytochrome C; ATP, adenosine triphosphate.