Figures & data
Figure 1 Chromatographic fingerprint analysis and cell viability assay of Chinese herbal formula B401.
Notes: (A) HPLC fingerprint of the herbal formula B401. Characteristic peaks of B401, that is, ginsenoside Rb1 from Panax ginseng, formononetin from Astragalus membranaceus, 5-HMF from Rehmannia glutinosa, ferulic acid from Ligustri fructus, and wedelolactone from Eclipta prostrate, were identified and marked at the corresponding peaks in the fingerprint. (B) Cell viabilities of RA-induced SH-SY5Y cells in the absence (Ctrl) (n=6) or presence of B401 at the indicated doses (n=6 for each treatment). Values are mean ± SEM (P>0.01, one-way ANOVA).
Abbreviations: 5-HMF, 5-hydroxymethylfurfural; AU, arbitrary perfusion units; Ctrl, control; HPLC, high-performance liquid chromatography; RA, retinoic acid; SEM, standard error of the mean.
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 the accelerating mode of rotorod test at indicated speeds 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, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons post-test).
Abbreviations: ANOVA, analysis of variance; Mn, manganese; rpm, revolutions per minute; SEM, standard error of the mean.
Figure 3 Expression levels of D1R and DA were increased significantly in the brain tissues of Mn-treated mice with oral B401 treatment.
Notes: (A) IHC staining shows that the expression of D1R was reduced in the substantia nigra and the striatum of Mn-treated mice than in mice given sham treatment, but was increased in mice administered oral B401 treatment. Scale bar: 30 µm. (B) Western blotting analysis shows the following: (a) the expression levels of D1R and DA in the whole brain tissues of sham-treated, B401-treated, B401 + Mn-treated, and Mn-treated mice; (b) quantified D1R and DA levels were decreased significantly in the brain of Mn-treated mice than in those given sham treatment (P<0.01), but were increased significantly in mice 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).
Abbreviations: ANOVA, analysis of variance; D1R, dopaminergic D1 receptors; DA, dopamine β hydroxylase; IHC, immunohistochemistry; Mn, manganese, SEM, standard error of the mean.
Figure 4 Expression levels of BDNF were increased significantly in the brain tissue of Mn-treated mice with oral B401 treatment.
Notes: (A) IHC staining shows that the expression levels of BDNF were decreased in the substantia nigra and the striatum of Mn-treated mice than in mice given sham treatment, but were increased in mice under oral B401 treatment. Scale bar: 30 µm. (B) Western blotting analysis shows the following: (a) the expression levels of BDNF in the whole brain tissue of sham-treated, B401-treated, B401 + Mn-treated, and Mn-treated mice; (b) quantified BDNF levels were decreased significantly in the brain of Mn-treated mice than in those given sham treatment (P<0.01), but were increased significantly in mice 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, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons post-test).
Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; IHC, immunohistochemistry; Mn, manganese; SEM, standard error of the mean.
Figure 5 Expression levels of ROS were significantly decreased in the blood of Mn-treated mice under oral B401 treatment.
Notes: (A) Blood ROS levels were compared in mice under sham, B401, B401 plus Mn, and Mn treatments by chemiluminescence analysis. Blood ROS level was obviously increased in mice under Mn treatment, but decreased with B401 treatment. (B) The quantified ROS levels were increased significantly in the blood of Mn-treated mice than in those given sham treatment (P<0.01), but were decreased significantly with 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).
Abbreviations: ANOVA, analysis of variance; ECL, electrochemiluminescence; Mn, manganese; ROS, reactive oxygen species; SEM, standard error of the mean.
Figure 6 Expression levels of antioxidative stress-related SOD2 were increased significantly in the brain tissues of Mn-treated mice under oral B401 treatment.
Notes: (A) IHC staining shows that the expression of SOD2 decreased significantly in the substantia nigra and the striatum of Mn-treated mice than in those given sham treatment (P<0.01), but increased significantly with oral B401 treatment (P<0.01). Scale bar: 30 µm. (B) Western blotting analysis shows the following: (a) the expression levels of SOD2 in the whole brain tissue of sham-treated, B401-treated, B401 + Mn-treated, and Mn-treated mice; (b) quantified SOD2 levels were decreased significantly in the brain of Mn-treated mice than in those given sham treatment (P<0.01), but increased significantly with 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).
Abbreviations: ANOVA, analysis of variance; IHC, immunohistochemistry; Mn, manganese; SEM, standard error of the mean; SOD2, superoxide dismutase 2.
Figure 7 Expression levels of oxidative stress-related 3-NT were decreased significantly in the brain tissues of Mn-treated mice under oral B401 treatment.
Notes: (A) IHC staining shows that the expression levels of 3-NT were increased significantly in the substantia nigra and the striatum of Mn-treated mice than in those given sham treatment (P<0.01), but were decreased significantly in mice under oral B401 treatment (P<0.01). Scale bar: 30 µm. (B) Western blotting analysis shows the following: (a) the expression levels of 3-NT in the whole brain tissues of sham-treated, B401-treated, B401 + Mn-treated, and Mn-treated mice; (b) quantified 3-NT levels were increased significantly in the brain of Mn-treated mice than in those given sham treatment (P<0.01), but were decreased significantly in mice given 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).
Abbreviations: 3-NT, 3-nitrotyrosine; ANOVA, analysis of variance; IHC, immunohistochemistry; Mn, manganese; SEM, standard error of the mean.
Figure 8 The ratio of antiapoptosis-related Bcl-2/BAX was increased significantly in the brain tissues of Mn-treated mice under oral B401 treatment.
Notes: (A) IHC staining shows that the expression of Bcl-2 was decreased significantly in the substantia nigra and the striatum of Mn-treated mice than in those given sham treatment (P<0.01), but was increased significantly in mice under oral B401 treatment (P<0.01). Furthermore, the expression of BAX was increased significantly in the substantia nigra and the striatum of Mn-treated mice than in those given sham treatment (P<0.01), but was decreased significantly in mice under oral B401 treatment (P<0.01). Scale bar: 30 µm. (B) Western blotting analysis shows the following: (a) the expression levels of Bcl-2 and BAX in the whole brain tissues of sham-treated, B401-treated, B401 + Mn-treated, and Mn-treated mice; (b) the ratio of Bcl-2/BAX was decreased significantly in the brain of Mn-treated mice than in those given sham treatment (P<0.01), but was increased significantly in mice 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, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons post-test).
Abbreviations: ANOVA, analysis of variance; BAX, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; IHC, immunohistochemistry; Mn, manganese; SEM, standard error of the mean.
Figure 9 Expression levels of apoptosis-related Casp 3 were decreased significantly in the brain tissues of Mn-treated mice under oral B401 treatment.
Notes: (A) IHC staining shows that the expression of Casp 3 was increased significantly in the substantia nigra and the striatum of Mn-treated mice than in those given sham treatment (P<0.01), but was decreased significantly in mice given oral B401 treatment (P<0.01). Scale bar: 30 µm. (B) Western blotting analysis shows the following: (a) the expression levels of Casp 3 and c-Casp 3 in the whole brain tissues of sham-treated, B401-treated, B401 + Mn-treated, and Mn-treated mice; (b) quantified Casp 3 and c-Casp 3 levels were increased significantly in the brain of Mn-treated mice than in those given sham treatment (P<0.01), but were decreased significantly in mice 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).
Abbreviations: ANOVA, analysis of variance; Casp 3, caspase 3; c-Casp 3, cleaved caspase 3; IHC, immunohistochemistry; Mn, manganese; SEM, standard error of the mean.
Figure 10 The schematic diagram illustrates the possible neuroprotective pathways of B401 treatment in the brain tissues of mice with Mn-induced neurotoxicity.
Notes: Oral treatment of the herbal formula B401 Mn-treated mice enhanced neuroprotection by increasing the expression levels of BDNF, dopaminergic receptors, antioxidative SOD2, and antiapoptosis Bcl-2, while suppressing ROS production, the expression levels of oxidation-related 3-NT, and apoptosis-related BAX and caspase 3 in the brain, especially in the substantia nigra and the striatum. It also improved their motor coordination.
Abbreviations: 3-NT, 3-nitrotyrosine; BAX, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; BDNF, brain-derived neurotrophic factor; Mn, manganese; ROS, reactive oxygen species; SOD2, superoxide dismutase 2.
