Astragalus polysaccharides protect cardiac stem and progenitor cells by the inhibition of oxidative stress-mediated apoptosis in diabetic hearts

Figures & data

Table 1 Antibodies for cardiac immature cells (including cardiac stem cells, cardiac progenitors, cardiac precursor cells, and amplifying cells)

Cell markers	Function	Antibody labeling
1. CSPC Epitope
C-kit	Stem cell factor receptor	Conjugated primary Ab
2. Hematopoietic Markers
TER-119	Erythroid marker	Conjugated primary Ab
GATA-1	Differentiation of blood cells	Conjugated primary Ab
GATA-2	Differentiation of blood cells	Conjugated primary Ab
CH20	B-lymphocyte marker	Conjugated primary Ab
CD45RO	T-lymphocyte subset marker	Conjugated primary Ab
CD8	T-lymphocyte subset marker	Conjugated primary Ab
CD45	HSC/EC antigen	Conjugated primary Ab
CD34	Pan-myeloid marker	Conjugated primary Ab
3. Transcription factors of cardiac cell lineages
Ets1	Differentiation of ECs	Conjugated primary Ab
MEF2C	Differentiation of cardiomyocytes	Conjugated primary Ab
GATA-4	Differentiation of cardiomyocytes	Conjugated primary Ab
GATA-5	Regulator of Nkx2.5	Conjugated primary Ab
GATA-6	Differentiation of VSMCs	Conjugated primary Ab
4. Structural proteins of cardiac cell lineages
α-sarcomeric actin	Contractile protein of cardiomyocytes	Conjugated primary Ab
Desmin	Intermediate filament of muscle	Conjugated primary Ab
Connexin 43	Electrical coupling	Primary/secondary Ab
Cardiac myosin	Contractile protein of cardiomyocytes	Conjugated primary Ab
N-cadherin	Mechanical coupling	Primary/secondary Ab
TGFβ1 receptor	VSMC receptor	Conjugated primary Ab
α-SM actin	Contractile protein of VSMCs	Conjugated primary Ab
flk 1	VEGF2 receptor in ECs	Conjugated primary Ab
VWF	Factor VIII receptor in ECs	Conjugated primary Ab

Abbreviations: Ab, antibody; CSPC, cardiac stem/progenitor cells; ECs, endothelial cells; HSC, hematopoietic stem cells; α-SM, α smooth muscle; TGFβ1, transforming growth factor β1; VEGF2, vascular endothelial growth factor 2; VSMCs, vascular smooth muscle cells; VWF, Von Willebrand factor.

Table 2 APS inhibited hyperglycemia and weight loss in diabetes

	Ctrl	DM	APS-DM	SOD2+/−	APS-SOD2+/−	DM-SOD2+/−	APS-DM-SOD2+/−
Body weight, 8 weeks (g)	28.1±0.74	27.1±0.82	28.4±0.64	27.8±0.98	28.6±0.88	26.9±1.5	28.5±0.98
Fasting blood glucose, 8 weeks (mmol/L)	5.6±0.1	17.5±0.4a	5.8±0.2b	5.4±0.1	5.8±0.2	18±0.4a	6±0.2c
Body weight, 20 weeks (g)	33.8±0.68	23.2±0.84a	33.1±0.52b	34.6±0.86	35.2±0.42	22.4±0.9a	34.8±0.24c
Blood glucose (nonfasting), 20 weeks (mmol/L)	6.2±0.3	24.7±0.72a	6.4±0.1b	6.1±0.2	6.2±0.2	25.8±0.82a	6.5±0.1c
Glycated hemoglobin, 20 weeks (% standard)	4.8±0.3	9.8±0.7a	4.7±0.3b	4.7±0.2	4.6±0.1	9.6±0.6a	4.9±0.2c

Notes: C57BJ/6J and SOD2+/− mice were treated with streptozotocin injection at 6 weeks to induce diabetes. APS was administered continuously for the following 12 weeks. C57BJ/6J mice were taken as the control. Mice were weighed, and blood glucose and glycated hemoglobin were tested at 8 and 20 weeks. Values are mean ± SEM.

a P<0.05 vs C57BJ/6J control mice (n=8 per group),

b P<0.05 vs diabetic mice,

c P<0.05 vs diabetic SOD2+/− mice.

Abbreviations: APS, astragalus polysaccharides; APS-DM, diabetic mice with APS treatment; APS-DM-SOD2+/−, diabetic SOD2+/− mice with APS treatment; Ctrl, control C57BJ/6J mice; DM-SOD2+/−, diabetic SOD2+/− mice; DM, diabetic mice.

Table 3 APS protected heart function in diabetes

	Ctrl	DM	APS-DM	SOD2+/−	APS-SOD2+/−	DM-SOD2+/−	APS-DM-SOD2+/−
LV systolic pressure (mmHg)	96.1±2.1	80.5±3.0a	95.2±2.2b	81.6±2.3a	96.6±1.8c	80.5±3.6a	94.8±1.2d
LV fractional shortening (%)	58±2.8	34±1.4a	54±2.4b	32±1.3a	53±2.3c	30±1.0a	50±2.0d
LV end-diastolic pressure (mmHg)	7.4±1.2	11.6±3.2a	7.6±1.6b	11.1±2.4a	7.0±1.0c	12.0±1.8a	7.2±0.9d
LV dP/dt-max (mmHg/s)	8,142±477	5,010±602a	8,158±352b	5,125±424a	8,124±245c	5,000±612a	8,010±312d
LV dP/dt-min (mmHg/s)	6,640±573	4,242±325a	6,502±477b	4,412±288a	6,260±498c	4,018±250a	6,312±388d

Notes: LV hemodynamics were evaluated in all mice at 16 weeks. Values are mean ± SEM.

a P<0.05 vs C57BJ/6J control mice (n=6 per group),

b P<0.05 vs diabetic mice,

c P<0.05 vs SOD2+/− mice and

d P<0.05 vs diabetic SOD2+/− mice.

Abbreviations: APS-DM, diabetic mice with APS treatment; APS-DM-SOD2+/−, diabetic SOD2+/− mice with APS treatment; Ctrl, control C57BJ/6J mice; DM, diabetic mice; DM-SOD2+/−, diabetic SOD2+/− mice; LV, left ventricle.

Figure 1 APS protect cardiac function in diabetes.

Note: Representative echocardiography images of hearts from diabetic or nondi-abetic SOD2+/− mice with or without APS treatment (n=6 per group).
Abbreviations: APS, astragalus polysaccharides; APS-DM, diabetic mice with APS treatment; APS-DM-SOD2+/−, diabetic SOD2+/− mice with APS treatment; Ctrl, C57BJ/6J mice were taken as the normal control; DM, diabetic mice; DM-SOD2+/−, diabetic SOD2+/− mice; SOD2+/−, nondiabetic SOD2+/− mice.

Figure 2 APS increased the abundance of CSPCs in diabetes.

Notes: Left ventricle was obtained from all mice after killing (n=6 per group). CSPCs were recognized by staining for the cell surface marker c-kit, and the c-kit-positive and lineage-negative cells were characterized and sorted by flow cytometry. (A) The number of CSPCs in left ventricle. Ventricular CSPCs were quantified utilizing the Schwartz–Saltykov methodology. Values are mean ± SEM. ^!P<0.05 vs diabetic mice, ^#P<0.05 vs SOD2+/− mice, ^☨P<0.05 vs diabetic SOD2+/− mice, and *P<0.05 vs C57BJ/6J control mice. (B) Flow cytometry analysis for the identification of CSPCs. C-kit-positive and lineage-negative cells were gated from total population extracted from the left ventricle and the percentage of CSPC population was analyzed. (C) Confocal microscopy images showing c-kit-positive (blue) and lineage-negative CSPCs by IHC staining for the sections of left ventricle. (D) Confocal microscopy images showing c-kit-positive (blue) and lineage-negative CSPCs by IHC staining for the isolated ventricular CSPCs (original magnification: ×8 million).
Abbreviations: APS, astragalus polysaccharides; APS-DM, diabetic mice with APS treatment; APS-DM-SOD2+/−, diabetic SOD2+/− mice with APS treatment; Ctrl, C57BJ/6J mice were taken as the normal control; DM, diabetic mice; DM-SOD2+/−, diabetic SOD2+/− mice; IHC, immunohistochemistry; SOD2+/−, nondiabetic SOD2+/− mice.

Figure 3 APS inhibited the apoptosis of CSPCs in diabetes.

Notes: Left ventricle was obtained from all mice after killing (n=6 per group). CSPCs were characterized and sorted by flow cytometry. Hairpin oligonucleotide probes were employed for the detection of cell apoptosis. (A) CSPC apoptosis in left ventricle detected by hairpin 1; Apoptotic CSPCs were quantified utilizing the Schwartz–Saltykov methodology. Values are mean ± SEM. ^!P<0.05 vs diabetic mice, ^#P<0.05 vs SOD2+/− mice, ^☨P<0.05 vs diabetic SOD2+/− mice, and *P<0.05 vs C57BJ/6J control mice. (B) Flow cytometry analysis for the identification of CSPC apoptosis. Hairpin-1-positive cells were gated from total CSPC population extracted from the left ventricle and the percentage of apoptotic CSPC population was analyzed. (C) Confocal microscopy images showing hairpin 1-positive (red) CSPCs in left ventricle. CSPCs were recognized by c-kit labeling (blue) (original magnification: ×10 million).
Abbreviations: APS, astragalus polysaccharides; APS-DM, diabetic mice with APS treatment; APS-DM-SOD2+/−, diabetic SOD2+/− mice with APS treatment; CSPCs, cardiac stem and progenitor cells; Ctrl, C57BJ/6J mice were taken as the normal control; DM, diabetic mice; DM-SOD2+/−, diabetic SOD2+/− mice; SOD2+/−, nondiabetic SOD2+/− mice.

Figure 4 APS enhanced the proliferation of CSPCs in diabetes.

Notes: Left ventricle was obtained from all mice after killing (n=6 per group). CSPCs were characterized and sorted by flow cytometry. The expression of Ki67 was determined for the cell proliferation. (A) CSPC proliferation in left ventricle detected by Ki67. Proliferating CSPCs were quantified utilizing the Schwartz–Saltykov methodology. Values are mean ± SEM. ^!P<0.05 vs diabetic mice, ^#P<0.05 vs SOD2+/− mice, ^☨P<0.05 vs diabetic SOD2+/− mice, and *P<0.05 vs C57BJ/6J control mice. (B) Flow cytometry analysis for the identification of CSPC proliferation. Ki67-positive cells were gated from total CSPC population extracted from the left ventricle and the percentage of proliferating CSPC population was analyzed. (C) Confocal microscopy images showing Ki67-positive (red) CSPCs in ventricle. CSPCs were recognized by c-kit labeling (blue) (original magnification: ×8 million).
Abbreviations: APS, astragalus polysaccharides; APS-DM, diabetic mice with APS treatment; APS-DM-SOD2+/−, diabetic SOD2+/− mice with APS treatment; CSPCs, cardiac stem and progenitor cells; Ctrl, C57BJ/6J mice were taken as the normal control; DM, diabetic mice; DM-SOD2+/−, diabetic SOD2+/− mice; SOD2+/−, nondiabetic SOD2+/− mice.

Figure 5 APS inhibited ROS formation and oxidative damage of CSPCs in diabetes.

Notes: Left ventricle was obtained from all mice after killing (n=6 per group). The c-kit-positive and lineage-negative cells were characterized and sorted by flow cytometry. CM-H₂DCFDA was employed to measure the ROS formation, and the intracellular H₂O₂ and ⋅OH concentrations were analyzed utilizing InSpeak Microscopy Image Intensity Calibration microspheres and ImagePro analysis software. Nitrotyrosine and 8-OH-dG antibodies were employed to detect oxidative damage to cytoplasmic proteins and oxidative stress in the nuclei, respectively. (A) ROS formation in CSPCs in left ventricle. (B) Oxidative stress in the nuclei in CSPCs of left ventricle. (C) Oxidative damage to cytoplasmic proteins in CSPCs of left ventricle. Values are mean ± SEM. ^!P<0.05 vs diabetic mice, ^#P<0.05 vs SOD2+/− mice, ^☨P<0.05 vs diabetic SOD2+/− mice, and *P<0.05 vs C57BJ/6J control mice.
Abbreviations: APS, astragalus polysaccharides; APS-DM, diabetic mice with APS treatment; APS-DM-SOD2+/−, diabetic SOD2+/− mice with APS treatment; CM-H₂DCFDA, 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; CSPCs, cardiac stem and progenitor cells; Ctrl, C57BJ/6J mice were taken as the normal control; DM, diabetic mice; DM-SOD2+/−, diabetic SOD2+/− mice; H₂O₂, hydrogen peroxide; ⋅OH, hydroxyl radicals; ROS, reactive oxygen species; SOD2+/−, nondiabetic SOD2+/− mice.

Figure 6 APS enhanced SOD2 enzyme activities and protein levels of CSPCs in diabetes.

Notes: Left ventricle was obtained from all mice after killing (n=6 per group). The c-kit-positive and lineage-negative cells were characterized and sorted by flow cytometry. SOD activity was measured by the extent of reduction in the appearance of nitroblue tetrazolium-diformazan utilizing the Superoxide Dismutase Assay Kit. Total SOD and SOD2 activities in U/mg protein were calculated, and SOD1 activity was determined by subtracting the SOD2 activity from the total SOD activity. SOD protein levels were analyzed by Western blotting. Membranes were stripped and reprobed for glyceraldehyde 3-phosphate dehydrogenase as a loading control. Protein content was measured and normalized as a ratio relative to the normal control. (A) SOD1 and SOD2 enzyme activities in CSPCs. (B) Protein levels of SOD1 and SOD2 in CSPCs. (C) Representative autoradiographs of Western blots. Values are mean ± SEM. ^!P<0.05 vs diabetic mice, ^#P<0.05 vs SOD2+/− mice, ^☨P<0.05 vs diabetic SOD2+/− mice, and *P<0.05 vs C57BJ/6J control mice.
Abbreviations: APS, astragalus polysaccharides; APS-DM, diabetic mice with APS treatment; APS-DM-SOD2+/−, diabetic SOD2+/− mice with APS treatment; CSPCs, cardiac stem and progenitor cells; Ctrl, C57BJ/6J mice were taken as the normal control; DM, diabetic mice; DM-SOD2+/−, diabetic SOD2+/− mice; SOD2+/−, nondiabetic SOD2+/− mice.