In this study, we evaluated the effects of the Chinese herbal formula B401 in the cavernosal tissues of Mn-treated mice. Our results demonstrated that oral treatment of the herbal formula B401 effectively enhances synthesis of nitric oxide (Figures 10 and 11) and angiogenesis (Figure 12), and reduces oxidative stress (Figures 13 and 14), inflammation (Figure 15), and apoptosis (Figures 16 and 17) in penile corpus cavernosum of Mn-treated mice.
We demonstrated ingredients of the herbal formula B401 as shown in Figure 1A, the extract from Panax ginseng has been proven to reduce oxidative stress (Chen et al., 2003) and inflammation (Lee et al., 2011; Lin et al., 2007; Yu et al., 2000). Furthermore, Rg1 from Panax ginseng can induce rapid nitric oxide production from eNOS via the PI3K/Akt pathway (Leung et al., 2006). The extract from Astragalus membranaceus has been reported to reduce oxidative stress (Ji et al., 2014) and enhanced nitric oxide production (Meng et al., 2007). The extract from Angelica sinensis has been reported to have angiogenic effects in zebrafish via enhancing VEGF mRNA expression (Lam et al., 2008). The extract from Rehmannia glutinosa has long been used in age-related diseases and its therapeutic efficacy via attenuating oxidative damage in the brain of mice (Zhang et al., 2008b). The extract from Ligustri fructus has been used to protect against hydrogen
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peroxide-induced oxidative damage (Ju et al., 2012). The extract from Eclipta prostrata has been has been reported to have cerebroprotective and antioxidant effect via reducing global cerebral ischemia in rat (Mansoorali et al., 2012). Taken together, the herbal formula B401 might have protective effect via enhancing nitric oxide and VEGF production, and reducing oxidative stress and inflammation. As for traditional Chinese medicine, we know that none of these herbs have much therapeutic value. To use any of them alone may encounter problems. That is why we use combined herbal formula to treat penile toxicity of mice with acute manganism.
As suggested in the Introduction, exposure to excessive Mn exposuremay lead to erectile dysfunction in human and other animal models (Emara et al., 1971; Mena et al., 1967; Rodier, 1995; Lauwerys et al., 1985; Chandra et al., 1973; Seth et al., 1973). The nitric oxide play a key mediator in penile erection (Rajfer et al., 1992). Impaired nitric oxide formation may be considered pathological mechanisms in erectile dysfunction (Kim et al., 1991). Nitric oxide can be synthesized and released from non-adrenergic, non-cholinergic nerve endings by specific nNOS and from the endothelium by eNOS (Burnett, 1997). It has been suggested that rapid, brief activation of nNOS initiates the erectile process, whereas PI3-kinase/Akt-dependent phosphorylation and activation of eNOS by augmented blood flow and endothelial shear stress lead to sustained nitric oxide production and maximal erection (Hurt et al., 2002). Inhibition of nNOS attenuated erectile responses (Ignarro et al., 1990). Thus, it is very likely that physiologic penile erection is mediated by both nNOS and eNOS. Furthermore, it has been suggested that VEGF induced penile erection and corrected alterations in eNOS phosphorylation (Musicki et al., 2005). Excess expression of VEGF may induce synthesis eNOS and iNOS in the penis of rats (Lin et al., 2002). As suggested from our observations in this study, we found that Mn-treated mice with oral B401 treatment (ICR mice) were significantly reduced synthesis of nitric oxide (Figure 10) and expression levels of nNOS,
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eNOS (Figure 11) and VEGF (Figure 12) in penile corpus cavernosum. Taken together, excessive Mn exposure may lead to erectile dysfunction via inhibiting nitric oxide-mediated signaling pathways. In Taiwan, the Chinese herbal formula B401 has been developed as a health supplement for promoting blood circulation and enhancing brain function. In this study, we observed that synthesis of nitric oxide, and expression levels of nNOS, eNOS and VEGF were significantly enhanced in penile corpus cavernosum of Mn-treated mice under oral the herbal formula B401treatment (Figures 10-12). Thus, we assumed that B401 might may have protective effects on penile toxicity of mice with acute manganism via promoting nitric oxide-mediated signaling pathways.
Several evidences have indicated that oxidative stress, mitochondrial dysfunction, inflammation and apoptosis were involved in underlying pathological mechanisms of excessive Mn-induced neurodegeneration (Milatovic et al., 2009). As suggested from our results, we found that ICR mice were significantly reduced SOD2, catalase (Figure 13), and Bcl-2 (Figure 16) in penile corpus cavernosum, but increased expression levels of 4-HNE (Figure 14), TNF-α (Figure 15), Bax, Cyt-C( Cyt. C) (Figure 16), caspase-12 and caspase-3 (Figure 17) in penile corpus cavernosum. Previous studies have suggested that oxidative stress plays a causative role in erectile dysfunction (Agarwal et al., 2006;
Jeremy et al., 2000). Enhanced expression of 4-HNE, an oxidative marker, has been observed in a rat model of radiation-induced erectile dysfunction (Kimura et al., 2012).
Oxidative stress and inflammation lead to endothelium dysfunction that plays a key event in the pathophysiology of erectile dysfunction (Berk et al., 2001). Administration of the antioxidants catalase and SOD2 may restore diabetes-induced oxidative stress and erectile dysfunction (Kawakami et al., 2009). Inflammatory conditions were also observed in patients with erectile dysfunction via increasing plasma levels of TNF-α (Giugliano et al., 2004). Furthermore, apoptosis is a downstream event in erectile dysfunction (Yamanaka et al., 2003). The Bcl-2 protein plays an important role in
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inhibiting apoptosis, while the Bax is a pro-apoptotic member that releases Cyt-C from the mitochondria (Jürgensmeier et al., 1998; Reed, 1994). The PARP is cleaved by caspase-3 that is a key executor of apoptosis (Boulares et al., 1999; Jiang et al., 2001). In this study, we observed that expression levels of anti-oxidative catalase and SOD2, and anti-apoptotic Bcl-2 were significantly enhanced, but expression levels of 4-HNE, c-PARP, Bax, Cyt-C, caspase-12 and caspase-3 were significantly reduced in penile corpus cavernosum of Mn-treated mice under oral the herbal formula B401treatment (Figures 12-16). In other words, B401 might may have protective effects on penile toxicity of ICR mice with acute manganism via reducing oxidative stress, mitochondrial dysfunction, inflammation and apoptosis.
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CHAPTER 5
CONCLUSION
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Here we used oral treatment with the herbal formula B401 as alternative medical applications in remission of Mn-induced neurotoxicity. As summarized in Figure 18, we found that Mn-treated mice with oral B401treatment significantly improves the motor coordination, reduces blood ROS, and enhances expression levels of dopaminergic receptors, dopamine and BDNF, but suppressing oxidative stress (by up-regulating SOD2 expressions, and down-regulating 3-NT expressions) and apoptosis (by up-regulating Bcl-2/BAX ratio, and down-regulating caspase 3 expressions) in the brain, especially in the substantia nigra and the striatum. As summarized in Figure 19, we have suggested that oral B401 treatment could alleviate penile toxicity of excess Mn-treated mice via enhancing synthesis of nitric oxide and angiogenesis, and reducing oxidative stress, inflammation, and apotosis in corpus cavernosum. We suggested that the herbal formula B401 might be developed as a potential health supplement for ameliorating Mn-induced neurotoxicity, and it is highly possible that the herbal formula B401 may also be useful dietotherapic supplement for penile toxicity or dysfunction.
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FIGURES
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Figure 1 Chromatographic fingerprint analysis and cell viability assay of Chinese herbal formula B401.
Notes: (A) High-performance liquid chromatography (HPLC) fingerprint of the herbal formula B401. Characteristic peaks of B401, i.e. ginsenosides Rb1 from Panax ginseng, formononetin from Astragalus membranaceus, 5-HMF from Rehmannia glutinosa, ferulic acid from Ligustri Fructus, and wedelolactone from Eclipta prostrata were identified and marked at the corresponding peaks in the fingerprint. (B) Cell viabilities of retinoic acid (RA)-induced SH-SY5Y cells in the absence (Ctrl) (n=6) or presence of the B401 at indicated doses (n=6 for each treatment). Values are mean ± SEM (p>0.01).
Abbreviations: AU, arbitrary perfusion units; 5-HMF, 5-hydroxymethylfurfural; Ctrl, control; HPLC, high-performance liquid chromatography; SEM, standard error of the mean; ANOVA, analysis of variance.
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Figure 2 Motor coordination of Mn-treated mice was significantly enhanced under oral B401 treatment .
Notes: Fall time of mice under sham, B401, B401 plus Mn, and Mn treatments was compared in accelerating mode of rotorod test at indicated speed of 5, 10, and 20 rpm (n=8 for each treatment). Fall time of mice was significantly decreased under Mn treatment (Sham vs Mn, p <0.01). In addition, fall time of Mn-treated mice was
significantly increased under B401 treatment (Mn vs B401+Mn, p<0.01). Values are mean ± SEM (**p<0.01, * p<0.05, two-way ANOVA followed by a
Student-Newman-Keuls multiple comparisons posttest).
Abbreviations: Mn, manganese; rpm, revolutions per minute; SEM, standard error of the mean; ANOVA, analysis of variance.
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Figure 3 Expression levels of dopaminergic D1 receptors and dopamine β hydroxylase in the brain tissue of Mn-treated mice were increased significantly under oral B401 treatment.
Notes: (A) IHC staining shows that the expressions of dopaminergic D1 receptors in the substantia nigra and the striatum of Mn-treated mice were reduced than those given the sham treatment, but were increased under oral B401 treatment. Scale bars: 30 µm. (B) Western blotting analysis shows the following: (a) expression levels of D1R and dopamine β hydroxylase (DA) in whole brain tissue of sham-treated, B401-treated, B401+Mn-treated, and Mn-treated mice. (b) Quantified D1R and DA levels in the brain of Mn-treated mice were decreased significantly than those given the sham treatment (p<0.01), but were increased significantly under oral B401 treatment (p<0.01). The number of sham-treated, B401-treated, B401+Mn-treated, and Mn-treated mice was eight for each group. Values are mean ± SEM (**p<0.01, *p<0.05, Two-way ANOVA followed by a Student-Newman-Keuls multiple comparisons post-test).
Notes: (A) IHC staining shows that the expressions of dopaminergic D1 receptors in the substantia nigra and the striatum of Mn-treated mice were reduced than those given the sham treatment, but were increased under oral B401 treatment. Scale bars: 30 µm. (B) Western blotting analysis shows the following: (a) expression levels of D1R and dopamine β hydroxylase (DA) in whole brain tissue of sham-treated, B401-treated, B401+Mn-treated, and Mn-treated mice. (b) Quantified D1R and DA levels in the brain of Mn-treated mice were decreased significantly than those given the sham treatment (p<0.01), but were increased significantly under oral B401 treatment (p<0.01). The number of sham-treated, B401-treated, B401+Mn-treated, and Mn-treated mice was eight for each group. Values are mean ± SEM (**p<0.01, *p<0.05, Two-way ANOVA followed by a Student-Newman-Keuls multiple comparisons post-test).