Hemodynamics indices
The therapeutic effects of HG/[6]-GR on heart function was evaluated by assessing the hemodynamics indices. Compared with the control group, DOX could substantially decrease the LVSP and +dp/dtmax value while significantly increase the LVEDP and -dp/dtmax value, indicating that the model of CHF was successfully prepared. However, compared with the DOX group, DH, HG, [6]-GR, and HG/[6]-GR could dramatically increase the levels of LVSP and +dp/dtmax and decrease the LVEDP and -dp/dtmax value. The order of the therapeutic effects was HG/[6]-GR > HG > [6]-GR. Also, compared with HG and [6]-GR used alone, HG/[6]-GR group had a more superior effect on increasing heart function (Table 2).
Table 2 Effects of HG/[6]-GR on hemodynamic indices in rats.
Group
|
LVSP (mmHg)
|
LVEDP (mmHg)
|
+dp/dtmax (mmHg/s)
|
-dp/dtmax (mmHg/s)
|
C
|
140.56 ± 12.75
|
-12.97 ± 2.85
|
7506.87 ± 668.49
|
-5330.51 ± 884.92
|
DOX
|
51.57 ± 8.38**
|
10.54± 1.83**
|
2313.95 ± 416.24**
|
-1386.08 ± 172.61**
|
DH
|
132.04 ± 10.48##
|
-7.40 ± 0.89##
|
8579.56 ± 873.29##
|
-6757.02 ± 601.40##
|
HG
|
112.30 ± 10.91##
|
-1.47 ± 2.59##
|
9034.06 ± 426.66##
|
-6778.09 ± 291.51##
|
[6]-GR
|
100.91 ± 12.02##
|
7.34± 0.86##
|
5029.14 ± 256.68##
|
-3729.03 ± 104.06##
|
HG/[6]-GR
|
125.25 ± 7.92##abb
|
-4.07 ± 0.93##abb
|
10263.24 ± 1056.67##aabb
|
-8428.85 ± 399.13##aabb
|
Notes: Compared with the control group, **P < 0.01; compared with the DOX group, ##P < 0.01; compared with the HG group, aP < 0.05, aaP < 0.01; compared with the [6]-GR group, bbP < 0.01.
Myocardial biomarkers
Serum levels of myocardial biomarkers were included in Figure 1. Compared with the control group, serum levels of BNP, NT-proBNP, LDH, CK-MB, and AST in the DOX group were significantly increased (P<0.01) while serum levels of ATP, ATPase, NAD, and NADH were decreased in the DOX group, which indicated the damage of heart function and energy metabolism disorder in DOX group. However, compared with the DOX group, HG and [6]-GR could reduce the serum concentrations of BNP, NT-proBNP, LDH, CK-MB, and AST, but increase the serum levels of ATP, ATPase, NAD, and NADH. Notably, these biomarkers were substantially changed in DH and HG/[6]-GR (P<0.01) group compared with the DOX group. Furthermore, HG/[6]-GR group was almost equal to the DH group, which markedly decreased the serum levels of BNP, NT-proBNP, LDH, CK-MB, and AST but increased the levels of ATP, ATPase, NAD, and NADH compare with HG or [6]-GR used alone (P<0.05, P<0.01). Thus, [6]-GR might enhance the therapeutic role of HG in the treatment of CHF.
Histopathological changes
The histopathological results showed the degree of damage in each group. After administration, the rats in DOX group had pathological changes such as widening and breaking of myocardial tissue space, vacuolar degeneration, edema, and necrosis of myocardial cells (Figure 2B). Compared with the DOX group, the histopathology of HG and [6]-GR group was improved, but some rats still had widened and broken myocardial tissue space, vacuolation and degeneration of myocardial cells (Figure 2D, 2E), while DH and HG/[6]-GR group showed significant improvement in cardiac pathology, less vacuolation, edema, necrosis, atrophy and other pathological changes of myocardial cells (Figure 2C, 2F).
Detection of cardiomyocyte apoptosis
TUNEL staining was used to detect the therapeutic effect of HG/[6]-GR on DOX induced cardiomyocyte apoptosis and its recovery. As shown in Figure 3, compared with the control group, the TUNEL positive proportion of cardiomyocytes in DOX treatment group increased significantly, indicating that DOX could cause cardiomyocyte apoptosis. In contrast, HG and [6]-GR used alone could reduce the apoptosis rate of cardiomyocytes in varying degrees. Moreover, HG/[6]-GR had a significant inhibitory effect on cardiomyocyte apoptosis, indicating that HG combined with [6]-GR had a synergistic anti-apoptotic effects. These results showed that HG/[6]-GR had a significant protective effect on CHF myocardial tissue. The results clearly showed that the HG/[6]-GR could effectively alleviate CHF in rats.
Metabolic profile analysis
The metabolic profile analysis of serum samples was performed using UHPLC-Q-TOF/MS both in the positive and negative electrospray ionization (ESI) modes. PCA analysis was performed to assess alterations in the metabolome of each group. In the PCA score plot (Figure 4A, 4B), the control groups and DOX groups were clearly divided into two clusters. In addition, the HG/[6]-GR and HG groups were significantly separated from DOX group and closer to the control group, especially the HG/[6]-GR group. Furthermore, to maximize the difference of metabolic profiles, OPLS-DA analysis was carried out subsequently (Figure 4 C, D). The results showed that the OPLS-DA models were verified by the class permutation and all these models had predictive ability with an R2Y (cum), and Q2 (cum). The corresponding value had been marked in the Figure 4C-4F. The R2Y (cum) and Q2Y (cum) were 0.999 and 0.992 in ESI+ mode, 0.998 and 0.971 in the ESI- mode, respectively. Also, the OPLS-DA model was performed based on the DOX and HG/[6]-GR group (Figure 4E, 4F). The DOX group could be clearly separated from the HG/[6]-GR group. The R2Y (cum) and Q2Y (cum) were 1 and 0.99 in the ESI+ mode, 0.997 and 0.963 in the ESI- mode, respectively. In addition, metabolic profile analysis between the DOX and HG or [6]-GR group in the positive mode and negative mode was also performed (Figure 5A-5D). Scatter plots of the control and DOX group, DOX and HG/[6]-GR group were shown in Figure 5E-5H, and scatter plots of the DOX and HG group, DOX and [6]-GR group were shown in Figure 5I-5L.
Identification and quantification of potential biomarkers
Next, differential metabolites in CHF treatment were identified. The variables that substantially contributed to the clustering and identification were identified when their VIP values ≥ 1.0 and |p(corr)| values ≥ 0.58 in S-plots. Finally, eight potential metabolites were expressed at significant levels and identified as biomarkers for the treatment of CHF. The basic information of these potential biomarkers was summarized in Table 3 with their corresponding name, formula, mass (m/z), retention time (min), and ratio changes (significance). Next, the mechanism of action of HG/[6]-GR on DOX induced CHF and the changes of eight possible metabolites were assessed and discussed. Compared with the control group, DOX substantially decreased peak area of acetylphosphate, 3-carboxy-1-hydroxypropylthiamine diphosphate, coenzyme A, PE(O-18:1(1Z)/20:4(5Z,8Z,11Z,14Z)), oleic acid, anslysoPC(18:1(9Z)) (Figure 6A-6E, 6G), but increase the peak area of PC(16:0/16:0) (Figure 6F) and palmitic acid (Figure 6H). Conversely, HG/[6]-GR could reverse these changes and decrease the peak area of PC(16:0/16:0) and palmitic acid. Notably, most of the metabolites indicated the formation of mitochondrial energy metabolism substrate. Besides, to determine the distribution and differences between groups, the clustering heat map and PCA were constructed based on the potential biomarker data (Figure 7B). Overall, the results indicated that HG/[6]-GR had obvious therapeutic effects on DOX-induced CHF. Especially, the curative effect of HG/[6]-GR group was better than that of HG and [6]-GR used alone (Figure 6).
Table 3 Identified metabolites of the serum sample from different groups.
No
|
Compound Name
|
Formula
|
Mass (m/z)
|
Retention time (min)
|
Ratio changes (significance)
|
Control/DOX
|
HG/[6]-GR/DOX
|
1
|
Acetylphosphate
|
C2H5O5P
|
139.9872
|
20.01
|
4.57**
|
3.98##
|
2
|
3-Carboxy-1-hydroxypropylthiamine diphosphate
|
C16H25N4O10P2S
|
527.0692
|
13.79
|
1.46**
|
1.48##
|
3
|
Coenzyme A
|
C21H36N7O16P3S
|
767.1152
|
11.45
|
3.95**
|
3.54##
|
4
|
Palmitic acid
|
C16H32O2
|
256.2402
|
16.37
|
0.39**
|
0.54##
|
5
|
PE(O-18:1(1Z)/20:4(5Z,8Z,11Z,14Z))
|
C37H66NO8P
|
683.4375
|
20.46
|
3.57**
|
3.05##
|
6
|
Oleic acid
|
C18H34O2
|
287.2819
|
7.84
|
3.30**
|
3.10##
|
7
|
LysoPC(18:1(9Z))
|
C24H51NO6P
|
480.3086
|
15.87
|
1.85**
|
1.75##
|
8
|
PC(16:0/16:0)
|
C46H83NO8P
|
808.5856
|
15.33
|
0.43**
|
0.64##
|
Notes: Compared with the control group, **P < 0.01; compared with the DOX group,
Pathway analysis of CHF treatment
To explore the possible pathway of HG/[6]-GR and DOX intervention in CHF, the KEGG ID of endogenous metabolites was imported into the MetaboAnalyst 4.0 system for the pathway analysis and visualization. The results showed that CHF-related metabolites were responsible for energy metabolism pathway, including glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids, fatty acid degradation, linoleic acid metabolism, alpha-Linolenic acid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, pantothenate and CoA biosynthesis, citrate cycle (TCA cycle), pyruvate metabolism, arachidonic acid metabolism, fatty acid elongation, fatty acid biosynthesis (Figure 7A). The match status, p value, -log(p) and the impact of each metabolic pathway were listed in Table 4. In addition, the relationship among metabolic pathways and metabolites was shown in the Figure 7C. The recovery trend of metabolites showed that the therapeutic effect of HG/[6]-GR on heart was related to the above eight metabolic biomarkers and twelve metabolic pathways. These results were consistent with the biochemical parameters and histological examination.
Table 4 Results of integrating pathway analysis with MetaboAnalyst 4.0.
No
|
Pathway Name
|
Match Status
|
p
|
-log(p)
|
Impact
|
1
|
Glycerophospholipid metabolism
|
3/36
|
0.00064401
|
7.3478
|
0.21631
|
2
|
Biosynthesis of unsaturated fatty acids
|
2/36
|
0.014161
|
4.2573
|
0
|
3
|
Fatty acid degradation
|
2/39
|
0.016523
|
4.103
|
0.12404
|
4
|
Linoleic acid metabolism
|
1/5
|
0.026263
|
3.6396
|
0
|
5
|
Alpha-Linolenic acid metabolism
|
1/13
|
0.067028
|
2.7026
|
0
|
6
|
Glycosylphosphatidylinositol (GPI)-anchor biosynthesis
|
1/14
|
0.072017
|
2.6308
|
0.00399
|
7
|
Pantothenate and CoA biosynthesis
|
1/19
|
0.096614
|
2.337
|
0.175
|
8
|
Citrate cycle (TCA cycle)
|
1/20
|
0.10146
|
2.288
|
0.08764
|
9
|
Pyruvate metabolism
|
1/22
|
0.1111
|
2.1973
|
0
|
10
|
Arachidonic acid metabolism
|
1/36
|
0.17603
|
1.7371
|
0
|
11
|
Fatty acid elongation
|
1/39
|
0.18939
|
1.6639
|
0
|
12
|
Fatty acid biosynthesis
|
1/47
|
0.2241
|
1.4957
|
0.01472
|