In the analysis of the research results, because there was no difference between the results of the control group and the results of the sham group, we do not report the results of the control group.
3, 1. Body and heart Weight
As shown in Table 2, after eight weeks, the OD rats had a significantly decreased body weight (BW) compared with the sham group (p < 0.05). However, HW to BW ratio in the OD rats was significantly higher than those within the other groups. Table 2 also shows that the HW to BW ratio was significantly different in the ODS and ODSG groups compared with sham and ODG groups.
Table 2
Effects of ovariectomy, diabetes and exercise on body and heart weight of different studied groups
|
BW final ( g )
|
HW ( mg )
|
HW/ BW (mg / g )
|
Sham
|
261.87 ± 4.87
|
896 ± 0.02
|
3.42 ± 0.133
|
OVX
|
273.88 ± 2. 55
|
967 ± 0.02
|
3.53 ± 0.016
|
OD
|
196.00 ± 4.0*
|
867 ± 0.02
|
4.42 ± 0.235*
|
ODS
|
259.88 ± 3.54
|
983 ± 0.03
|
3.78 ± 0.252**, #
|
ODG
|
260.87 ± 4.37
|
869 ± 0.01
|
3.33 ± 0.116
|
ODGS
|
259.50 ± 5.41
|
971 ± 0.03
|
3.74 ± 0.184**, #
|
OVX: ovariectomized group, OD: ovariectomized with 8-weeks diabetic group. ODS: ovariectomized diabetic group with 8-weeks swimming training. ODG: ovariectomized diabetic group with 8-weeks genistein administration. ODGS: ovariectomized diabetic group with 8-weeks swimming training and genistein administration. |
Data are expressed as mean ± SEM. |
*p < 0.05 vs. Sham, OVX, ODG, ODS & ODGS; |
**p < 0.05 vs. Sham; |
#p < 0.05 vs. ODG. |
3, 2. Biochemically results
Glucose homeostasis parameters are shown in Table 3. A significant elevation in fasting blood glucose (FBG) level was observed in OD rats when compared to sham, OVX, and OD groups (p < 0.05). Moreover, FBG was increased in the ODS rats compared to the sham, O, and OD group (p < 0.05). Furthermore, OD rats showed a significant increase in insulin levels compared to sham, O, and ODS groups and ODS rats compared to O and sham rats (p < 0.05). Moreover, plasma glycosylated hemoglobin (HbA1c) increased in the OD group compared to sham, O, and ODS groups (p < 0.05), although this elevation was not statistically significant.
Table 3
Parameters of glucose homeostasis of different studied groups at the end of the study
|
Fasting blood glucose (mg/dl)
|
Serum insulin (µIU/mL)
|
Plasma glycosylated hemoglobin (%)
|
Sham
|
98.20 ± 2.13
|
4.3 ± 1.8
|
4.380 ± 0.57
|
OVX
|
109.00 ± 1.87
|
13.2 ± 1.1**
|
4.920 ± 0.63
|
OD
|
269.00 ± 13.26*
|
28.9 ± 1.5*
|
5.960 ± 0.60
|
ODS
|
160.00 ± 10.83 #
|
24.9 ± 1.6*
|
4.620 ± 0.67
|
ODG
|
155.80 ± 9.85
|
22.9 ± 1.3*
|
4.780 ± 0. 51
|
ODGS
|
110.80 ± 9.01
|
9.3 ± 1.7
|
4.540 ± 0.50
|
OVX: ovariectomized group, OD: ovariectomized with 8-weeks diabetic group. ODS: ovariectomized diabetic group with 8-weeks swimming training. ODG: ovariectomized diabetic group with 8-weeks genistein administration. ODGS: ovariectomized diabetic group with 8-weeks swimming training and genistein administration. |
Data are expressed as mean ± SEM. |
*p < 0.05 vs. Sham & OVX; |
**p < 0.05 vs. Sham & OD; |
# p < 0.05 vs. Sham, OVX & OD. |
The plasma lipid profiles are shown in Fig. 1. The triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL) levels of O and OD rats were significantly higher. In contrast, high-density lipoprotein (HDL) was significantly lower than sham control rats (p < 0.05). No significant change was observed in other groups TG, TC, LDL, and HDL compared with correspondence control groups.
3, 3. Effect of genistein treatment and/or swimming training in diabetic ovariectomized rats on total antioxidant capacity, total oxidant status, and oxidative stress index
The heart homogenates total antioxidant capacity (TAC) levels are shown in Fig. 2A. In heart homogenates TAC values, a significant decrease was observed in OVX and OD groups compared to the sham control group (p < 0.001). An increasing effect was observed in heart homogenates of OD rats receiving swimming training compared with the OD group (p < 0.001). A significant increase in TAC was recorded in genistein treated OD rats as compared with OVX (p < 0.05) and OD (p < 0.001) group. The TAC value of heart tissues in the ODGS group was more significant in comparison with OVX (p < 0.001) and OD (p < 0.001) group.
The heart homogenates total oxidant status (TOS) are shown in Fig. 2B. Heart total oxidant status in the OVX (p < 0.01) and OD (p < 0.001) rats increased significantly as compared to the sham control group (p < 0.01 and p < 0.05, respectively). The heart TOS in ODS (p < 0.001 as compared with OD group), ODG (p < 0.001 as compared with OD group and p < 0.05 as compared with OVX group) and also ODGS. (p < 0.001 as compared with OD group and p < 0.01 as compared with OVX group)-treated rats were significantly reduced.
Moreover, oxidative stress index (OSI, Fig. 2C) were increased in the OVX and OD rats (compared to the sham group, p < 0.001) and decreased in ODS (p < 0.001 as compared with OD group), ODG (p < 0.001 as compared with OD group and p < 0.01 as compared with OVX group) and ODGS. (p < 0.001 as compared with OD and OVX groups) groups.
3, 4. Effects of genistein treatment and/or swimming training in diabetic ovariectomized rats on lipid peroxidation (MDA concentration).
Figure 3 shows the results of heart tissues MDA concentrations. The MDA level of OVX (p < 0.001) and OD (p < 0.001) groups were significantly higher than the sham control group. In addition, ODS (p < 0.001 as compared with OD group), ODG (P < 0.05 as compared with OVX and p < 0.001 as compared with OD group) and ODGS (p < 0.01 as compared with OVX and p < 0.001 as compared with OD group) treated rats showed lower MDA concentrations.
3, 5. Effect of genistein treatment and/or swimming training in diabetic ovariectomized rats on Bax, Bcl2, IGF-1, NF-κB, and IGF-1protein concentrations
The concentration of IGF-1 protein was assessed in rat heart tissues by the ELISA method and shown in Fig. 4A. Ovariectomy leads to decreased IGF-1 protein concentration in the heart tissues compared with the sham group (p < 0.001). In ovariectomized rats, a diabetic condition caused a reduction in IGF-1 protein concentrations (p < 0.001). In the heart tissues of ODS (p < 0.001 as compared with OVX), ODG (p < 0.001 as compared with OVX and p < 0.01 as compared with OD group) and ODGS. (p < 0.001 as compared with OVX and p < 0.01 as compared with OD group) animal group significant increase in the IGF-1 level was observed.
The protein concentration of Bcl-2 was assessed in rat heart tissues by ELISA (Fig. 4B). Compared with the sham and OD group, ovariectomy (OVX group) decreased BCL-2 level in the heart tissues (p < 0.001). A significant decrease in Bcl-2 level was observed in the OD compared to the sham group (p < 0.001). Protein concentration of Bcl-2 in the heart tissues was higher in the ODS (p < 0.001 as compared with OVX), ODG (p < 0.001 as compared with OVX and p < 0.001 as compared with OD group), and also ODGS (p < 0.001 as compared with OVX and p < 0.001 as compared with OD group) groups.
The concentration of Bax protein was assessed in rat heart tissues by the ELISA method (Fig. 4C). As shown in Fig. 4C, ovariectomy resulted in a significant increase in Bax protein levels of the heart tissues when compared with sham and OD groups (p < 0.001). Further analysis indicated that the concentration of Bax was increased in the OD animals compared with the sham group (p < 0.001). Moreover, in ODS (p < 0.001 as compared with OVX), ODG (p < 0.001 as compared with OVX and p < 0.001 as compared with OD group) and ODGS. (p < 0.001 as compared with OVX and p < 0.001 as compared with OD group) groups, the protein concentration of Bax in the heart tissues was lower.
As shown in Fig. 4D and 4E, NF-κB and TNF- α protein concentrations were markedly increased in heart tissues of OVFX and OD groups in comparison with sham groups (p < 0.001). However, genistein (p < 0.001 as compared with OVX and p < 0.001 as compared with OD group) and exercise (p < 0.001 as compared with OD group) treatment alone or in combination (p < 0.001 as compared with OVX and p < 0.001 as compared with OD group) reduces the NF-κB and TNF- α protein concentrations.
3, 6. Effect of genistein treatment and/or swimming training in diabetic ovariectomized rats on miRNA-133 expression
The level of heart miRNA-133 expression under the treatments was examined by real-time RT-PCR (Fig. 5). As shown in Fig. 5, the miRNA-133 expression was significantly decreased in the heart tissues of ovariectomized rats compared with the sham control group (p < 0.001). Moreover, compared with OVX and sham rats, expression levels of miRNA-133 were lower in the heart tissues of OD rats (p < 0.001). An increase in the level of heart miRNA-133 expression also was observed in ODS (p < 0.001 as compared with OVX), ODG (p < 0.001 as compared with OVX and p < 0.001 as compared with OD group), and also ODSG (p < 0.001 as compared with OVX and p < 0.001 as compared with OD group) groups.
3, 7. Histological results
Table 4 and Fig. 6A-L demonstrated the histological changes of routine and OVX, OD, ODS, ODG, and ODSG rats. Genistein or swimming treatment caused decreased myocardial fibrosis, leukocyte infiltration, and disorganization myocardial. Based on Fig. 6A, cardiac muscle cells were incorporated in standard features of myocytes, including a cross-striated banding model, nucleus with oval, vesicular, and centrally placed pale staining appearance intercalated disks found at the interface among the neighboring cardiac muscle cells in the sham animal group. Furthermore, in the OVX animal group, the sarcoplasm of myocyte was striped and acidophilus and was organized on an unvarying basis. However, this study specified that numerous variations such as structural protection integrity were comparable with variations observed in the sham animal group. Other conclusions revealed an intensification of dense fibrosis and necrotic cell compactness and outstanding importance in the number of connective tissue elements in the OVX group (Fig. 6B). Histological changes in the heart of OD were similar to the OVX animal group but significantly worse (Fig. 6C). The findings of this study also declared that the genistein administration (Fig. 6D) or swimming training (Fig. 6E) improved a series of variations, including the reduced tendency in the fibrotic area and necrotic cell density and normalized quantity of connective tissue in the ODG and ODS animal group. However, in the ODGS animal group, further, the improvement of variations, including the reduced tendency in the fibrotic area and necrotic cell density and normalized quantity of connective tissue, were seen (Fig. 6F). Besides, the histological evaluations of heart tissue in PAS analysis showed a constant and homogeneous granule of glycogen in the sarcoplasm of cardiomyocytes in the sham animal group (Fig. 6G). However, fragmentation and irregular accumulation of glycogen granules were seen in the OVX animal group (Fig. 6H), and these changes in the heart of the OD animal group were worse (Fig. 6I). Genistein administration (Fig. 6J) or swimming training (Fig. 6K) decreased fragmentation of glycogen granules in these groups compared with OVX and OD animal groups in the sarcoplasm of cardiomyocytes. Also, in the ODGS group, further, improvement was observed and further decreased fragmentation of glycogen granules in the sarcoplasm of cardiomyocytes (Fig. 6L).
Table 4
Histological changes of the heart (H&E) in different studied groups
|
Sham
|
OVX
|
OD
|
ODS
|
ODG
|
ODGS
|
Cardiac fibers hypertrophy
|
0
|
6.60 ± 0.50*
|
9.20 ± 0.37
|
4.00 ± 0.44**
|
3.90 ± 0.49**
|
1.50 ± 0.39
|
Myocardial fibrosis
|
0
|
5.20 ± 0.58*
|
9.00 ± 0.31
|
3.40 ± 0.81**
|
3.20 ± 0.70**
|
1.34 ± 0.51
|
Leukocyte Infiltration
|
0
|
5.00 ± 0.37*
|
7.80 ± 0.24
|
2.20 ± 0.37**
|
2.10 ± 0.47**
|
1.77 ± 0.26
|
Disorganization Myocardial
fibers disarray
|
0
|
5.40 ± 0.60*
|
9.40 ± 0.40
|
2.80 ± 0.58**
|
2.70 ± 0.49**
|
1.57 ± 0.23
|
Cardiomyocyte vacuolation
|
0
|
5.00 ± 0.31*
|
7.80 ± 0.37
|
2.20 ± 0.58**
|
2.10 ± 0.43**
|
1.31 ± 0.13
|
Cardiomyocyte degeneration
|
0
|
4.80 ± 0.37*
|
8.60 ± 0.50
|
4.40 ± 0.50**
|
4.30 ± 0.48**
|
1.42 ± 0.35
|
Cardiac nucleus eccentric
|
0
|
5.00 ± 0.44*
|
9.00 ± 0.44
|
4.00 ± 0.70**
|
3.90 ± 0.65**
|
1.40 ± 0.44
|
OVX: ovariectomized group, OD: ovariectomized with 8-weeks diabetic group. ODS: ovariectomized diabetic group with 8-weeks swimming training. ODG: ovariectomized diabetic group with 8-weeks genistein administration. ODGS: ovariectomized diabetic group with 8-weeks swimming training and genistein administration. A minimum of ten fields for each heart slide were examined and assigned for severity of injury based on numbers of 10 microscopic field. Data is expressed as mean ± SEM. |
* p < 0.05 vs. Sham & OD; |
** p < 0.05 vs. Sham & OVX & OD & ODGS. |