Author Contributions
Conceptualization, M.M. and A.B.A.B.; methodology, J.B.S.; software, J.B.S.; validation, V.U.N., Z.A.O. and Z.Z.; formal analysis, J.B.S.; investigation, J.B.S., V.U.N., Z.A.O., A.B.A.B. and Z.Z.; writing—original draft preparation, J.B.S., V.U.N., Z.A.O., A.B.A.B. and Z.Z.; writing—review and editing, J.B.S., V.U.N., Z.A.O., A.B.A.B. and Z.Z.; visualization, J.B.S., and M.M.; supervision, M.M. and A.B.A.B.; project administration, M.M., and A.B.A.B.; funding acquisition, M.M. and A.B.A.B. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Representative photomicrographs of H&E staining of testes in (a) C, (b) Ob, (c), Ob + BB (d) Ob + OR groups. C: control; Ob: obese rats; Ob + BB: obese + bee bread (6 weeks after induction of obesity); and Ob + OR: obese + orlistat (6 weeks after induction of obesity). The seminiferous tubules in the Ob group were collapsed showing decreased amount of sperm and germ cells (red arrows) compared to C, Ob + BB and Ob + OR groups (c,d) which showed increased amount of sperm and germ cells as well as normal size seminiferous tubules (black arrows) (magnification of panel above: ×100, scale bar = 200 µm; magnification of panel below: ×400, scale bar = 50 µm). (e) Seminiferous tubule with germ cell loss, (f) seminiferous tubular diameter, and (g) seminiferous epithelial height are quantitative data, values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob (one-way ANOVA followed by Tukey post-hoc test).
Figure 1.
Representative photomicrographs of H&E staining of testes in (a) C, (b) Ob, (c), Ob + BB (d) Ob + OR groups. C: control; Ob: obese rats; Ob + BB: obese + bee bread (6 weeks after induction of obesity); and Ob + OR: obese + orlistat (6 weeks after induction of obesity). The seminiferous tubules in the Ob group were collapsed showing decreased amount of sperm and germ cells (red arrows) compared to C, Ob + BB and Ob + OR groups (c,d) which showed increased amount of sperm and germ cells as well as normal size seminiferous tubules (black arrows) (magnification of panel above: ×100, scale bar = 200 µm; magnification of panel below: ×400, scale bar = 50 µm). (e) Seminiferous tubule with germ cell loss, (f) seminiferous tubular diameter, and (g) seminiferous epithelial height are quantitative data, values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob (one-way ANOVA followed by Tukey post-hoc test).
Figure 2.
Effects of bee bread on the mRNA levels of oxidative stress markers; (a) Gpx, (b) Cat, (c) Sod, and (d) Nrf2 in the testes of obese rats. Gpx: glutathione peroxidase, Cat: catalase, Sod: superoxide dismutase, Nrf2: nuclear factor erythroid 2-related factor 2, C: control, Ob: obese rats, Ob + BB: obese + bee bread (6 weeks after induction of obesity), and Ob + OR: Obese + orlistat (6 weeks after induction of obesity). Values are shown mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 2.
Effects of bee bread on the mRNA levels of oxidative stress markers; (a) Gpx, (b) Cat, (c) Sod, and (d) Nrf2 in the testes of obese rats. Gpx: glutathione peroxidase, Cat: catalase, Sod: superoxide dismutase, Nrf2: nuclear factor erythroid 2-related factor 2, C: control, Ob: obese rats, Ob + BB: obese + bee bread (6 weeks after induction of obesity), and Ob + OR: Obese + orlistat (6 weeks after induction of obesity). Values are shown mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 3.
Effects of bee bread on the mRNA levels of inflammatory markers; (a) Inos, (b) Tnf-α, (c) Nf-κB, (d) Il-1β and (e) Il-10 in the testes of obese rats. Inos: inducible nitric oxide synthase, Il: interleukin, Nf-κB: nuclear factor kappa B, Tnf-α: tumour necrotic factor-alpha, C: control, Ob: obese rats, Ob + BB: obese + bee bread (6 weeks after induction of obesity), and Ob + OR: Obese + orlistat (6 weeks after induction of obesity). Values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob, c p < 0.05 vs. Ob + BB (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 3.
Effects of bee bread on the mRNA levels of inflammatory markers; (a) Inos, (b) Tnf-α, (c) Nf-κB, (d) Il-1β and (e) Il-10 in the testes of obese rats. Inos: inducible nitric oxide synthase, Il: interleukin, Nf-κB: nuclear factor kappa B, Tnf-α: tumour necrotic factor-alpha, C: control, Ob: obese rats, Ob + BB: obese + bee bread (6 weeks after induction of obesity), and Ob + OR: Obese + orlistat (6 weeks after induction of obesity). Values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob, c p < 0.05 vs. Ob + BB (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 4.
Effects of bee bread on immunoexpression of NF-κB, TNF-α, IL-1β and IL-10 proteins in the testes of obese rats. For each protein, C, Ob, Ob + BB and Ob + OR groups are represented by (a–d), respectively, while (e) represents the negative control (PBS). Staining using immunohistochemistry protocol showed seminiferous tubules with increased expressions of NF-κB, TNF-α and IL-1β, and decreased expression of IL-10 in Ob group, compared to C, Ob + BB and Ob + OR groups. Quantitative data are shown in (f) NF-κB, (g) TNF-α, (h) IL-1β and (i) IL-10. Values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 4.
Effects of bee bread on immunoexpression of NF-κB, TNF-α, IL-1β and IL-10 proteins in the testes of obese rats. For each protein, C, Ob, Ob + BB and Ob + OR groups are represented by (a–d), respectively, while (e) represents the negative control (PBS). Staining using immunohistochemistry protocol showed seminiferous tubules with increased expressions of NF-κB, TNF-α and IL-1β, and decreased expression of IL-10 in Ob group, compared to C, Ob + BB and Ob + OR groups. Quantitative data are shown in (f) NF-κB, (g) TNF-α, (h) IL-1β and (i) IL-10. Values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 5.
Effects of bee bread on the mRNA levels of apoptosis-related genes; (a) Bax, (b) Bcl-2, (c) Bax/Bcl2 ratio, (d) p53, (e) caspase-9, (f) caspase-8 and (g) caspase-3 in the testes of obese rats. p53: tumour suppressor, Bax: beta cell lymphoma 2 apoprotein X, Bcl2: beta cell lymphoma 2. Values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob, c p < 0.05 vs. Ob + BB (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 5.
Effects of bee bread on the mRNA levels of apoptosis-related genes; (a) Bax, (b) Bcl-2, (c) Bax/Bcl2 ratio, (d) p53, (e) caspase-9, (f) caspase-8 and (g) caspase-3 in the testes of obese rats. p53: tumour suppressor, Bax: beta cell lymphoma 2 apoprotein X, Bcl2: beta cell lymphoma 2. Values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob, c p < 0.05 vs. Ob + BB (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 6.
Effects of bee bread on immunoexpression of cleaved caspase-3 protein and proliferating cell nuclear antigen (PCNA) in the testes of obese rats. For each protein, C, Ob, Ob + BB, and Ob + OR groups are represented by (a–d), respectively, while (e) represents the negative control (PBS). Staining using immunohistochemistry protocol showed seminiferous tubules with increased cleaved caspase-3 and decreased PCNA levels in Ob group compared to C, Ob + BB, and Ob + OR groups (magnification = ×100, scale bar = 200 µm). Quantitative data are shown in (f) Caspase-3 staining intensity, and (g) PCNA-positive germ cells. Values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 6.
Effects of bee bread on immunoexpression of cleaved caspase-3 protein and proliferating cell nuclear antigen (PCNA) in the testes of obese rats. For each protein, C, Ob, Ob + BB, and Ob + OR groups are represented by (a–d), respectively, while (e) represents the negative control (PBS). Staining using immunohistochemistry protocol showed seminiferous tubules with increased cleaved caspase-3 and decreased PCNA levels in Ob group compared to C, Ob + BB, and Ob + OR groups (magnification = ×100, scale bar = 200 µm). Quantitative data are shown in (f) Caspase-3 staining intensity, and (g) PCNA-positive germ cells. Values are mean ± SD, n = 6. a p < 0.05 vs. C, b p < 0.05 vs. Ob (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 7.
Schematic representation of therapeutic effects of bee bread on testicular-derived oxidative stress, inflammation, and apoptosis. HFD: high-fat diet, ROS reactive oxygen species.
Figure 7.
Schematic representation of therapeutic effects of bee bread on testicular-derived oxidative stress, inflammation, and apoptosis. HFD: high-fat diet, ROS reactive oxygen species.
Table 1.
Sequence for Primers in tabular form.
Table 1.
Sequence for Primers in tabular form.
| | Primer Sequence (5′–3′) | |
---|
Gene | Accession Number | Forward | Reverse | Amplicon Size (bp) |
---|
CAT | NM_012520.2 | ACAACTCCCAGAAGCCTAAGAATG | GCTTTTCCCTTGGCAGCTATG | 76 |
Caspase-8 | NM_022277.1 | GTTCTCTCAGTTGCCTTTCTCC | GGCCAGTCCGCCAAAGTTTA | 90 |
IL-10 | NM_012854.2 | TTGAACCACCCGGCATCTAC | CCAAGGAGTTGCTCCCGTTA | 91 |
Inos | XM_006246949.3 | CAGCCCTCAGAGTACAACGAT | CAGCAGGCACACGCAATGAT | 91 |
SOD | X05634.1 | CGAGCATGGGTTCCATGTC | CTGGACCGCCATGTTTCTTAG | 101 |
IL-1b | NM_031512.2 | GACTTCACCATGGAACCCGT | GGAGACTGCCCATTCTCGAC | 104 |
p53 | NG_005120.4 | CTACTAAGGTCGTGAGACGCTGCC | TCAGCATACAGGTTTCCTTCCACC | 106 |
Nrf2 | NM_031789.1 | CAGGTTGCCCACATTCCCAA | ATATCCAGGGCAAGCGACTCAT | 110 |
Bax | U49729.1 | CGCGTGGTTGCCCTCTTCTACTTT | CAAGCAGCCGCTCACGGAGGA | 129 |
Bcl-2 | NM_016993.1 | ATCGCTCTGTGGATGACTGAGTAC | AGAGACAGCCAGGAGAAATCAAAC | 134 |
GPx | NM_030826.4 | GGAGAATGGCAAGAATGAAGA | CCGCAGGAAGGTAAAGAG | 139 |
TNF-a | NM_012675.3 | ACTGAACTTCGGGGTGATCG | GCTTGGTGGTTTGCTACGAC | 153 |
NF-kB(p65) | NM_199267.2 | CGCGGGGACTATGACTTGAA | AGTTCCGGTTTACTCGGCAG | 163 |
Caspase-9 | NM_031632 | CTGAGCCAGATGCTGTCCCATA | CCAAGGTCTCGATGTACCAGGAA | 168 |
GAPDH | NM_017008 | TCACCACCATGGAGAAGGC | GCTAAGCAGTTGGTGGTGCA | 169 |
Caspase-3 | NM_012922 | AAGATACCAGTGGAGGCCGACTTC | GGGAGAAGGACTCAAATTCCGTGG | 199 |
Table 2.
Body weight and related parameters in all groups.
Table 2.
Body weight and related parameters in all groups.
Parameter | C | Ob | Ob + BB | Ob + OR |
---|
Initial body weight | 242.20 ± 34.47 | 256.10 ± 47.24 | 271.90 ± 14.14 | 253.50 ± 36.20 |
Final body weight (g) | 386.80 ± 40.11 | 444.90 ± 38.62 a | 411.90 ± 36.22 | 405.30 ± 41.25 |
Mean weight gain (g) | 144.70 ± 53.51 | 188.90 ± 44.80 a | 140.10 ± 32.46 b | 151.80 ± 19.18 |
Mean daily weight gain (g/day) | 1.72 ± 0.64 | 2.25 ± 0.53 | 1.67 ± 0.39 | 1.81 ± 0.23 |
Table 3.
Energy intake, feed consumption, Lee obesity index, and Body mass index in all groups.
Table 3.
Energy intake, feed consumption, Lee obesity index, and Body mass index in all groups.
Parameter | C | Ob | Ob + BB | Ob + OR |
---|
Lee obesity index | 305.90 ± 5.88 | 323.10 ± 8.91 a | 304.60 ± 8.12 b | 309.10 ± 6.01 b |
BMI (gcm−1) | 0.71 ± 0.04 | 0.83 ± 0.06 a | 0.72 ± 0.04 b | 0.73 ± 0.05 b |
Total feed consumption (g) | 1822.00 ± 125.80 | 1647.00 ± 266.20 | 1560.00 ± 171.40 | 1628.00 ± 232.80 |
Mean food consumption (g) | 21.69 ± 1.49 | 19.61 ± 3.17 | 18.57 ± 2.04 | 19.38 ± 2.77 |
Energy intake (kcal/day) | 69.15 ± 4.77 | 101.30 ± 16.37 a | 95.91 ± 10.53 a | 100.10 ± 14.31 a |
Table 4.
Reproductive organs and epididymal fat weights in all groups.
Table 4.
Reproductive organs and epididymal fat weights in all groups.
Parameter | | C | Ob | Ob + BB | Ob + OR |
---|
Testis ≠ | AW (g) | 3.75 ± 0.26 | 3.34 ± 0.58 a | 3.52 ± 0.29 | 3.57 ± 0.57 |
| RW (%) | 1.00 ± 0.09 | 0.79 ± 0.19 a | 0.82 ± 0.11 | 0.76 ± 0.17 a |
Epididymis ≠ | AW (g) | 1.52 ± 0.28 | 1.32 ± 0.29 | 1.51 ± 0.22 | 1.44 ± 1.17 |
| RW (%) | 0.40 ± 0.07 | 0.31 ± 0.09 | 0.35 ± 0.05 | 0.31 ± 0.06 a |
Prostate | AW (g) | 1.22 ± 0.43 | 1.52 ± 0.98 | 1.53 ± 0.61 | 1.68 ± 0.40 |
| RW (%) | 0.31 ± 0.11 | 0.34 ± 0.20 | 0.34 ± 0.12 | 0.35 ± 0.09 |
Seminal Vessicle | AW(g) | 1.76 ± 0.30 | 1.97 ± 0.57 | 2.11 ± 0.41 | 2.00 ± 0.52 |
| RW (%) | 0.47 ± 0.08 | 0.45 ± 0.11 | 0.48 ± 0.06 | 0.42 ± 0.12 |
Penis | AW (g) | 0.27 ± 0.04 | 0.24 ± 0.03 a | 0.27 ± 0.05 | 0.30 ± 0.03 b |
| RW (%) | 0.07 ± 0.01 | 0.06 ± 0.01 | 0.06 ± 0.01 | 0.06 ± 0.01 |
Epididymal Fat | AW (g) | 3.77 ± 1.33 | 10.38 ± 4.27 a | 7.87 ± 5.89 b | 9.54 ± 3.92 a |
| RW (%) | 0.99 ± 0.28 | 2.33 ± 0.74 a | 1.74 ± 1.08 | 1.92 ± 0.54 |
Table 5.
Lipid profile parameters in all groups.
Table 5.
Lipid profile parameters in all groups.
Parameter | C | Ob | Ob + BB | Ob + OR |
---|
TC (mmol/L) | 1.61 ± 0.18 | 2.36 ± 0.76 a | 1.89 ± 0.20 | 2.11 ± 0.17 |
TG (mmol/L) | 0.50 ± 0.07 | 0.93 ± 0.09 a | 0.71 ± 0.15 | 0.93 ± 0.28 a |
HDL-C (mmol/L) | 0.42 ± 0.10 | 0.22 ± 0.15 a | 0.40 ± 0.04 b | 0.37 ± 0.09 |
LDL-C (mmol/L) | 0.63 ± 0.05 | 1.56 ± 0.15 a | 1.25 ± 0.14 a,b | 1.35 ± 0.21 a |
Table 6.
Oxidant–antioxidant Parameters in the testis in all groups.
Table 6.
Oxidant–antioxidant Parameters in the testis in all groups.
Parameters | C | Ob | Ob + BB | Ob + OR |
---|
CAT activity (unit/mg protein) | 32.88 ± 3.92 | 14.86 ± 3.29 a | 25.80 ± 3.11 a,b | 23.12 ± 1.63 a,b |
GPx activity (unit/mg protein) | 43.60 ± 2.19 | 20.13 ± 1.88 a | 41.30 ± 3.14 b | 37.79 ± 1.80 a,b,c |
GR activity (unit/mg protein) | 43.92 ± 2.62 | 16.99 ± 1.79 a | 46.71 ± 6.53 b | 42.26 ± 3.77 b |
GSH (mmol GSH Eq/mg protein) | 10.73 ± 1.60 | 19.76 ± 5.41 a | 13.94 ± 2.38 b | 13.83 ± 5.00 b |
GST activity (unit/mg protein) | 257.20 ± 26.93 | 151.40 ± 17.32 a | 272.2 ± 24.37 b | 250.70 ± 32.41 b |
iNOS activity (ng/mL protein) | 1.80 ± 0.63 | 7.31 ± 1.06 a | 3.01 ± 0.79 a,b | 1.80 ± 0.51 b |
MDA (nmol/mg protein) | 1.27 ± 0.12 | 12.01 ± 0.76 a | 1.70 ± 0.28 b | 1.38 ± 0.33 b |
SOD activity (unit/mg protein) | 2.78 ± 0.12 | 0.29 ± 0.05 a | 2.31 ± 0.08 b | 2.31 ± 0.09 b |
TAC (nmol uric acid Eq/mg protein) | 137.90 ± 4.64 | 63.64 ± 6.01 a | 131.30 ± 10.85 b | 128.70 ± 6.74 b |