Oral treatment with herbal formula B307 alleviates cardiac failure in aging R6/2 mice with Huntington’s disease via suppressing oxidative stress, inflammation, and apoptosis

Figures & data

Figure 1 Chromatographic fingerprint analysis and SH-SY5Y cell viabilities of the herbal formula B307.

Notes: (A) HPLC characteristic peaks of B307: (a) Ginsenosides Rb1 (from Panax ginseng), (b) schizandrin, gomisin A (from Schisandra chinensis), (c) rosmarinic acid, salvianolic acid B, tanshinone IIA (from Salvia miltiorrhiza), and (d) methylophiopogonanone B (from Liriope spicata); they were identified and marked at the corresponding peaks in the fingerprint. (B) Cell viabilities of RA-induced SH-SY5Y cells in the absence (Ctrl) or presence of B307 at indicated doses. The herbal formula B307 has no cytotoxicity in SH-SY5Y cells under treatment at a dose of less than 50 mg/mL (IC₅₀ of B307 >50 mg/mL).
Abbreviations: AU, arbitrary perfusion units; HPLC, high-performance liquid chromatography; RA, retinoic acid.

Figure 2 Life span, body weight, and motor ability of R6/2 HD mice and their WT under oral B307 and sham treatments.

Notes: (A) Survival durations of R6/2 HD mice were shorter than their WT but were increased under oral B307 treatment. (B) Body weight of R6/2 HD mice was significantly reduced as compared to their WT from 10 weeks of age and thereafter, while it had significantly increased under oral B307 from 10 weeks of age and thereafter. Furthermore, brain (cerebrum plus cerebellum) weight in the R6/2 HD mice had been significantly reduced as compared to their WT from 10 weeks of age and thereafter, while it had significantly increased under oral B307 from 10 weeks of age and thereafter. There were six mice per each group. (C) Latencies to fall in the R6/2 HD mice identified by rotarod testing were significantly lower than their WT from 10 weeks of age and thereafter, while they were significantly increased under oral B307 from 10 weeks of age and thereafter. There were six mice per each group. (D) Heart weight in R6/2 HD mice had been significantly reduced as compared to their WT (N=6) from 10 weeks of age and thereafter, while they were significantly increased under oral B307 from 10 weeks of age and thereafter. There were six mice per each group. Both H&E staining and IHC staining of mHtt aggregation were illustrated in the plot. Expressions of myocarditis (marked by arrows in H&E staining) and mHtt aggregation (marked by arrows in IHC staining) were remarkable in comparison to their WT but were reduced under oral B307 treatment. Values are mean ± SEM (**P<0.01, *P<0.05, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: WT, wild-type littermate controls; HD, Huntington’s disease; H&E, hematoxylin and eosin; mHtt, mutant huntingtin; IHC, immunohistochemical; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 3 Cardiac performance in R6/2 HD mice under oral B307 and sham treatments, and of their WT.

Notes: (A) Echocardiographic evidence shows systolic and diastolic cardiac performances in R6/2 HD mice under oral B307 and sham treatments, and in their WT. (B) Quantified cardiac performances in (a) left ventricular fractional shortening, (b) ejection fraction, (c) maximal mitral valve blood flow velocity, (d) maximal aortic valve blood, and (e) stroke volume of R6/2 HD mice were significantly reduced in comparison to their WT, while they were significantly enhanced under oral B307 treatment. There were six mice per each group. Values are mean ± SEM (**P<0.01, *P<0.05, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: WT, wild-type littermate controls; HD, Huntington’s disease; MV, mitral valve; AV, aortic valve; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 4 Expressions of 3-NT and 4-HNE, two markers of oxidative stress, and SOD2, a marker of antioxidative stress, in the heart tissue of R6/2 HD mice under oral B307 treatment, and of their WT.

Notes: (A) IHC staining illustrates that expressions of 3-NT and 4-HNE (shown in brown) were remarkably higher in comparison to their WT but were reduced under oral B307 treatment. Further expressions of SOD2 (shown in brown) were weaker than their WT but were enhanced under oral B307 treatment. Scale bars: 25 μm. (B) Western blotting analysis shows the following: (a) Cardiac expression levels of 3-NT, SOD2, and 4-HNE in R6/2 HD mice under sham and oral B307 treatments, and of their WT. (b) Quantified cardiac 3-NT and 4-HNE levels in R6/2 HD mice were significantly enhanced in comparison with their WT but were significantly weaker under oral B307 treatment. Further quantified cardiac SOD2 levels in R6/2 HD mice were significantly weaker than their WT but were significantly enhanced under oral B307 treatment. There were six mice per each group. Values are mean ± SEM (**P<0.01, *P<0.05, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: WT, wild-type littermate controls; HD, Huntington’s disease; 3-NT, neurotrophin-3; SOD2, superoxide dismutase 2; 4-HNE, 4-hydroxynonenal; IHC, immunohistochemical; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 5 Expression of TNF-α, a marker of inflammation, in the heart tissue of R6/2 HD mice under oral B307 treatment, and of their WT.

Notes: (A) IHC staining illustrates that expressions of TNF-α (shown in brown) were remarkably higher in comparison to their WT but were reduced under oral B307 treatment. Scale bars: 25 μm. (B) Western blotting analysis shows the following: (a) Cardiac expression levels of TNF-α in R6/2 HD mice under sham and oral B307 treatments, and of their WT. (b) Quantified cardiac TNF-α levels in R6/2 HD mice were significantly enhanced in comparison to their WT but were significantly weaker under oral B307 treatment. There were six mice per each group. Values are mean ± SEM (**P<0.01, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: WT, wild-type littermate controls; HD, Huntington’s disease; TNF-α, tumor necrosis factor alpha; IHC, immunohistochemical; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 6 Expressions of Bcl-2, a marker of anti-apoptosis, and Bax and Cyto-C, two markers of apoptosis, in the heart tissue of R6/2 HD mice under oral B307 treatment.

Notes: (A) IHC staining illustrates that expressions of Bcl-2 (shown in brown) were weaker than their WT but were enhanced under oral B307 treatment. Further expressions of Bax and Cyto-C (shown in brown) were remarkable in comparison to their WT but were reduced under oral B307 treatment. Scale bars: 25 μm. (B) Western blotting analysis shows the following: (a) Cardiac expression levels of Bcl-2, Bax, and Cyto-C in R6/2 HD mice under sham and oral B307 treatments, and of their WT. (b) Quantified cardiac Bcl-2 levels in R6/2 HD mice were significantly weaker than their WT but were significantly enhanced under oral B307 treatment. Further quantified cardiac Bax and Cyto-C levels in R6/2 HD mice were significantly enhanced in comparison to their WT but were significantly weaker under oral B307 treatment. There were six mice per each group. Values are mean ± SEM (**P<0.01, *P<0.05, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).

Abbreviations: WT, wild-type littermate controls; HD, Huntington’s disease; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Cyto-C, cytochrome C; IHC, immunohistochemical; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 7 Expressions of calpain and caspase 12, two markers of ER stress-related apoptosis, in the heart tissue of R6/2 HD mice under oral B307 treatment, and of their WT.

Notes: (A) IHC staining illustrates that expressions of calpain and caspase 12 (shown in brown) were remarkable in comparison to their WT but were reduced under oral B307 treatment. Scale bars: 25 μm. (B) Western blotting analysis shows the following: (a) Cardiac expression levels of calpain and caspase 12 in R6/2 HD mice under sham and oral B307 treatments, and of their WT. (b) Quantified cardiac calpain and caspase 12 levels in R6/2 HD mice were significantly enhanced in comparison to their WT but were significantly weaker under oral B307 treatment. There were six mice per each group. Values are mean ± SEM (**P<0.01, *P<0.05, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).

Abbreviations: WT, wild-type littermate controls; HD, Huntington’s disease; ER, endoplasmic reticulum; IHC, immunohistochemical; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 8 Expressions of caspase 9 and caspase 3, two markers of apoptosis, in the heart tissue of R6/2 HD mice under oral B307 treatment and of their WT.

Notes: (A) IHC staining illustrates that expressions of caspase 9 and caspase 3 (shown in brown) were remarkable in comparison to their WT but were reduced under oral B307 treatment. Scale bars: 25 μm. (B, C) Western blotting analysis shows the following: (a) Cardiac expression levels of caspase 9 and caspase 3 in R6/2 HD mice under sham and oral B307 treatments, and of their WT. (b) Quantified cardiac caspase 9 and caspase 3 levels in R6/2 HD mice were significantly enhanced in comparison to their WT but were significantly weaker under oral B307 treatment. There were six mice per each group. Values are mean ± SEM (**P<0.01, *P<0.05, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest).
Abbreviations: WT, wild-type littermate controls; HD, Huntington’s disease; IHC, immunohistochemical; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 9 The schematic diagram illustrates the possible cardioprotective pathways under oral B307 treatment.

Notes: Cardioprotection in R6/2 HD mice under B307 treatment may be promoted via attenuating oxidative stress (marked by increasing expression of SOD2), anti-apoptosis (marked by increasing expression of Bcl-2), reducing mHtt aggregation, oxidative stress (marked by reducing expressions of 3-NT and 4-HNE), inflammation (marked by reducing expression of TNF-α), ER stress-related apoptosis (marked by reducing expressions of calpain and caspase 12), and mitochondrial dysfunction-related apoptosis (marked by reducing expressions of Bax, Cyto-C, caspase 9, and caspase 3) in heart tissue.
Abbreviations: mHtt, mutant huntingtin; SOD2, superoxide dismutase 2; ROS, reactive oxygen species; 3-NT, neurotrophin-3; 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; Cyto-C, cytochrome C; ER, endoplasmic reticulum; WT, wild-type littermate controls; HD, Huntington’s disease.