Abstract
Korean Red Ginseng (KRG) is an herbal oriental medicine known to alleviate cardiovascular dysfunction. To analysis the expression of diabetic cardiac complication-associated genes in db/db mice, we studied the cardiac gene expression following KRG treatment. In result, a total of 585 genes were found to be changed in db/db mice. Among the changed expression, 245 genes were found to 2-fold upregulated, and 340 genes were 2-fold downregulated. In addition, the changed gene expressions were ameliorated by KRG. In conclusion, KRG may be possible to normalize cardiac gene expressions in db/db mice.
Keywords: Panax ginseng, Diabetes mellitus, Diabetic cardiac complication, Microarray, Gene profiling
The prevalence of diabetes mellitus (DM) at all ages was estimated at 2.8% in 2000 and 4.4% in 2030. DM could lead to an increase in diabetic cardiac complications (DCC), becoming global public health threats [1,2]. However, there are no confirmed remedy for DCC. Traditionally, Korean Red Ginseng (KRG, Panax ginseng Meyer) is used as a principal herbal medicine in Far East Asia. The major biological functions of KRG are known to be helpful in cardiovascular diseases because of its pharmacological activities such as anti-inflammatory, antioxidant, and ameliorative effects on blood flow and cardiac function [[3], [4], [5], [6], [7]]. Yet, there is no a sure proof showing the benefits of cardiovascular effect of KRG in type 2 diabetic db/db mice. Considering these facts, we determined to study the expression of DCC-associated genes in db/db mice. Further, we assessed the reversal of this expression following KRG treatment.
In the present study, the systematic analysis of hemodynamics and gene expression profiling was performed in the heart of type II diabetic db/db mice before and after KRG treatment. Six-week-old, male, db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J strain) and db/+ mice were purchased from Orient, Korea. The blood glucose levels in db/db mice were significantly higher than in db/+ mice (596.3 ± 85.4 and 253.7 ± 8.4, respectively). After acclimatization, animals were divided randomly into 5 groups (n = 9): normal control group (N/C), 200 mg/kg KRG alone group (200KRG-alone), KRG-untreated db/db group (db/db), 100 mg/kg KRG-treated db/db group (db/db+100KRG), and 200 mg/kg KRG-treated db/db group (db/db+200KRG). All animals were housed under standard temperature (22 ± 1°C), humidity (55 ± 5%), and light cycle conditions (12 h light/dark cycle). Administration of KRG was conducted by feeding the chow mixed with KRG for 16 weeks. The dose of KRG was selected following a preliminary experiment administrated at various doses. KRG powder was purchased from the Korea Ginseng Corporation (Daejeon, Korea). At a two-week interval for a total of 16 weeks, biochemical and hemodynamic study were performed. At the end of the experimentation, microarray gene expression profiling was analyzed on the extracted RNA samples (Fig. 1). The Principles of Laboratory Animal Care were followed according to the Guidelines for Institutional Animal Care and Use Committees (IACUC) of Jeonbuk National University (Jeonju, Korea). For all studies, data are expressed as mean ± standard error of mean. Comparison between the groups was analyzed by Student's t-tests and one-way analysis of variance. Significance was statistically considered at p < 0.05.
Fig. 1.
Experimental protocol. All animals divided randomly into 5 groups (n = 9, respectively): N/C, 200 mg/kg KRG, db/db, db/db+100 mg/kg KRG and db/db+200 mg/kg KRG. At a two-week interval for a total of 16 weeks, biochemical and hemodynamic study were performed. At the end of the experimentation, microarray gene expression profiling was analyzed on the extracted RNA samples. N/C, normal control; KRG, Korean Red Ginseng.
Above all, we assessed the cardiovascular function of KRG by hemodynamic data such as heart rate (HR), left ventricle peak systolic pressure (LVSP), maximal rates of developed left ventricular pressure (+dP/dtmax), minimal rates of developed left ventricular pressure (−dP/dtmax), fractional shortening (FS) and ejection fraction (EF). We found that HR revealed no significant difference in each group (Fig. 2A). Functional parameters such as LVSP, +dP/dtmax and −dP/dtmax were increased in db/db group after 10 weeks compared with in N/C groups. Whereas, db/db+100KRG group reduced the level of LVSP and −dP/dtmax, after 16 and 12 weeks, respectively. Similarly db/db+200KRG group reduced the level of LVSP, +dP/dtmax and −dP/dtmax, after 12, 10, and 8 weeks, respectively (Fig. 2B–D).
Fig. 2.
The effect of KRG on echocardiographic evaluation to assess the cardiovascular function by hemodynamic data such as (A) heart rate, (B) peak systolic pressure, (C) +dP/dtmax and (D) –dP/dtmax. Data are presented as means ± SE; #p < 0.05, ##p < 0.01 compared N/C; ∗p < 0.05, ∗∗p < 0.01 compared with db/db control. N/C, normal control; +dP/dtmax, maximal rates of developed left ventricular pressure; –dP/dtmax, minimal rates of developed left ventricular pressure; KRG, Korean Red Ginseng; SE, standard error.
Currently, it was well known that FS and EF are the most precious function index for clinical application [8]. As revealed in Fig. 3A, there are no remarkable differences in FS among each groups until 6 weeks. But db/db group decreased the level of FS since 8 weeks. 200KRG-alone group was shown no changes, which indicating that KRG does not show any side effects. The FS value was 47.6 ± 4.69%, 49.6 ± 4.01%, and 51.7 ± 3.71% in 8 weeks; 47.7 ± 4.02%, 49.7 ± 4.22%, and 49.4 ± 4.15 in 10 weeks; 46.9 ± 3.969, 49.9 ± 3.98%, and 51.7 ± 3.98% in 12 weeks; 45.4 ± 4.02%, 48.5 ± 3.18%, and 49.9 ± 3.87% in 14 weeks; 43.6 ± 4.81%, 47.1 ± 4.03%, and 47.1 ± 3.99% in 16 weeks, for db/db, db/db+100KRG and db/db+200KRG, respectively (Fig. 3A). Similarly, as revealed in Fig. 3B no significant differences were observed in EF among each group until 6 weeks. However, db/db group decreased the average level of EF since 8 weeks. 200KRG-alone group was also shown no changes. The EF value were 89.1 ± 4.2%, 87.6 ± 3.4%, and 91.6 ± 2.9% in 8 weeks; 82.6 ± 4.7%, 83.2 ± 3.7%, and 90.6 ± 2.8% in 10 weeks; 82.7 ± 3.9%, 82.9 ± 3.9%, and 87.6 ± 2.7% in 12 weeks; 78.5 ± 3.2%, 81.4 ± 3.2%, and 85.6 ± 3.1% in 14 weeks; 79.4 ± 4.3%, 81.6 ± 3.0%, and 85.9 ± 3.4% in 16 weeks, for db/db, db/db+100KRG and db/db+200KRG, respectively. After all, administration of KRG inhibited the decreases of EF and FS levels. These facts propose that KRG has a potential to reduce the cardiac ventricular dysfunction in db/db mice.
Fig. 3.
The effect of KRG on echocardiographic evaluations to assess the cardiovascular function by hemodynamics such as (A) fractional shortening and (B) ejection fraction, and activities such as (C) troponin T and (D) MPO in the heart tissue. Data are presented as means ± SE; #p < 0.05, ##p < 0.01 compared N/C; ∗p < 0.05, ∗∗p < 0.01 compared with db/db control. N/C, normal control; MPO, myeloperoxidase; KRG, Korean Red Ginseng; SE, standard error.
Meanwhile, the cardiac troponin T (cTnT) levels and myeloperoxidase (MPO) activity of all groups were shown in Fig. 3C and D. Regarding cTnI activity, 200KRG-alone group did not show significant changes compared with the N/C group. The cTnT levels in db/db group were increased after 16 weeks (0.57 ± 0.0221 μg/l) compared with those in N/C groups (0.54 ± 0.0270 μg/l). While those in db/db+200KRG group decreased (0.52 ± 0.0310 μg/l) compared with those in db/db group (Fig. 3C). In an assay of neutrophil infiltration, the MPO activity in db/db group was increased (6.9 ± 0.32 nmol/mg for 12 weeks and 7.1 ± 0.57 nmol/mg for 16 weeks) compared with that in the N/C (5.9 ± 0.21 nmol/mg for 12 weeks and 6.17 ± 0.22 nmol/mg for 16 weeks) and 200KRG-alone group (6.26 ± 0.22 nmol/mg for 12 weeks and 6.31 ± 0.20 nmol/mg for 16 weeks). While that in db/db+200KRG group led to decrease (6.2 ± 0.54 nmol/mg for 12 weeks and 6.4 ± 0.51 nmol/mg for 16 weeks) compared with that in db/db group (Fig. 3D).
For investigation of global gene expression, total RNAs extracted from mouse cardiac tissue were analyzed using the whole mice genome microarray. We performed hierarchical clustering to get a rough estimate of the number of changed genes of db/db group, db/db+100KRG group and db/db+200KRG group as compared with N/C. In Fig. 4, gene expression levels of N/C are shown in black color as baselines. Red color indicates gene overexpression and green color indicates gene underexpression compared to N/C. A total of 585 genes were differentially changed in the db/db group: 245 genes showed 2-fold upregulated expression and 340 genes showed 2-fold downregulated expression. In the db/db+100KRG group, a total of 578 genes were differentially changed: 224 genes were upregulated and 354 genes were downregulated. In the db/db+200KRG group, a total of 557 genes were differentially changed: 287 genes were upregulated and 270 genes were downregulated (Fig. 4A). Hierarchical clustering of apoptosis, inflammation and stress response genes were shown in Fig. 4–D, respectively. We observed a relieved pattern of the number of differentially expressed genes (DEGs) in the db/db group by KRG treatment. Also, DEGs were divided into the functions: cell apoptosis, behavior, adhesion, differentiation, migration, proliferation, growth, homeostasis, immune response, inflammation, lipid metabolism, stress response, signaling transduction, transcription, translation and transport. DEGs in each category are shown in the graph, based on the number of changed genes in Fig. 5. Some genes changed in db/db mice expressed a relieved pattern after 100KRG or 200KRG treatment. Among them, we showed a list of several genes related to apoptosis, inflammation and stress response (Table 1, Table 2, Table 3). The list of genes is as follows: apoptosis-related genes such as Bcl3, Cideb, Gadd45b, Myc, Prune2, Thbs1, Sfrp2 and Snai2; inflammation-related genes such as C5ar1, Ccl12, Cxc11, S100a9, Sbno2 and Nt5e; stress response-related genes such as Slc27a1, Fos, Ugt1, Pfkfb1 and Fmod. The expression levels of these genes in db/db mice were upregulated or downregulated as compared to those from N/C, while they were alleviated in KRG-treated groups. It indicates that the changed gene expressions were ameliorated by the administration of KRG in dose-dependent fashion.
Fig. 4.
Hierarchical clustering image showing (A) the differential gene expression patterns of total genes, (B) apoptotic genes, (C) inflammatory genes and (D) stress response genes in N/C group, db/db group, db/db+100KRG group and db/db+200KRG group, respectively. Gene expression levels of N/C are shown in black color as baselines. Red color indicates gene overexpression and green color indicates gene underexpression compared to N/C. N/C, normal control; db/db, KRG-untreated db/db group; db/db+100KRG, 100 mg/kg KRG-treated db/db group; db/db+200KRG, 200 mg/kg KRG-treated db/db group; KRG, Korean Red Ginseng.
Fig. 5.
Functional classification of differential gene expression divided into the functions: cell apoptosis, behavior, adhesion, differentiation, migration, proliferation, growth, homeostasis, immune response, inflammation, lipid metabolism, stress response, signaling transduction, transcription, translation and transport. Changed genes in each category are shown based on the number of genes. KRG, Korean Red Ginseng.
Table 1.
List of genes related with apoptosis showing reversed expression by the administration of KRG in db/db mice
| Gene symbol | Description | Fold change (db/db) | Fold change (db/db+100KRG) | Fold Change (db/db+200KRG) |
|---|---|---|---|---|
| Bcl3 | B-cell leukemia/lymphoma 3 | 2.1668 | 1.4606 | 1.1840 |
| Cideb | Cell death-inducing DNA fragmentation factor, alpha subunit-like effector B | 2.0333 | 1.9911 | 1.9025 |
| Gadd45b | Growth arrest and DNA-damage-inducible 45 beta | 2.2262 | 1.4778 | 1.4152 |
| Myc | Myelocytomatosis oncogene | 3.0172 | 2.5677 | 1.9027 |
| Prune2 | Prune homolog 2 (Drosophila) | 3.8508 | 3.1494 | 2.7030 |
| Thbs1 | Thrombospondin 1 | 10.4962 | 9.6363 | 6.9609 |
| Sfrp2 | Secreted frizzled-related protein 2 | 0.3653 | 0.4420 | 0.5760 |
| Snai2 | Snail homolog 2 (Drosophila) | 0.4028 | 0.4867 | 0.6122 |
db/db: KRG-untreated db/db group, db/db+100KRG: 100 mg/kg KRG-treated db/db group, db/db+200KRG: 200 mg/kg KRG-treated db/db group
Table 2.
List of genes related with inflammation showing reversed expression by the administration of KRG in db/db mice
| Gene symbol | Description | Fold change (db/db) | Fold change (db/db+100KRG) | Fold change (db/db+200KRG) |
|---|---|---|---|---|
| C5ar1 | Complement component 5a receptor 1 | 2.1100 | 1.8013 | 1.2296 |
| Ccl12 | Chemokine (C–C motif) ligand 12 | 2.0020 | 1.9699 | 0.5957 |
| Cxcl1 | Chemokine (C-X-C motif) ligand 1 | 5.11323 | 4.2344 | 4.65223 |
| S100a9 | S100 calcium binding protein A9 (calgranulin B) | 10.2298 | 4.2848 | 7.8296 |
| Sbno2 | Strawberry notch homolog 2 (Drosophila) | 3.1278 | 1.0981 | 1.3114 |
| Nt5e | 5′ nucleotidase, ecto | 0.4777 | 0.5253 | 0.8077 |
| Sfrp2 | Secreted frizzled-related protein 2 | 0.3653 | 0.4420 | 0.5760 |
| Snai2 | Snail homolog 2 (Drosophila) | 0.4028 | 0.4867 | 0.6122 |
db/db: KRG-untreated db/db group, db/db+100KRG: 100 mg/kg KRG-treated db/db group, db/db+200KRG: 200 mg/kg KRG-treated db/db group
Table 3.
List of genes related with stress response showing reversed expression by the administration of KRG in db/db mice
| Gene symbol | Description | Fold change (db/db) | Fold change (db/db+100KRG) | Fold change (db/db+200KRG) |
|---|---|---|---|---|
| Slc27a1 | Solute carrier family 27 (fatty acid transporter), member 1 | 6.7424 | 5.9982 | 2.5752 |
| Fos | FBJ osteosarcoma oncogene | 3.2937 | 2.1530 | 1.5849 |
| Ugt1a1, -1a2, -1a5, -1a6a, -1a6b, -1a7c, -1a9, -1a10 | UDP glucuronosyltransferase 1 family, polypeptide A1, A2, A5, A6A, A6B, A7C, A9, A10 | 2.4950 | 2.4371 | 2.2896 |
| Pfkfb1 | 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 | 2.1084 | 1.1797 | 1.1569 |
| Fmod | Fibromodulin | 3.1993 | 1.4630 | 1.4492 |
db/db: KRG-untreated db/db group, db/db+100KRG: 100 mg/kg KRG-treated db/db group, db/db+200KRG: 200 mg/kg KRG-treated db/db group
In conclusion, we revealed KRG may normalize gene expressions caused by DCC in type 2 diabetic mice model. These data showed that KRG plays a role in various biological pathways in DCC and that some of genes associated with these pathways are sufficiently responsive to KRG. That means KRG have therapeutic effects on DCC by adjusting the expression levels of genes.
Conflict of interest
The authors report no conflicts of interest.
Acknowledgements
This work was supported by the 2013 grant from the Korean Society of Ginseng funded by Korea Ginseng Corporation.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jgr.2020.06.001.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
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