The effect of Astragalus polysaccharides on attenuation of diabetic cardiomyopathy through inhibiting the extrinsic and intrinsic apoptotic pathways in high glucose -stimulated H9C2 cells

Fig. 1. a Cell viabilities in cells treated with various concentrations of glucose for 24 h. MTT assay was used to measure cell viability. Data were expressed as Mean ± SD. * P < 0.05 versus control. ** P < 0.01 versus control. b. Cell viabilities in cells treated with 5.5 mmol/L glucose group, 5.5 mmol/L glucose with 33 mmol/L mannitol and 33 mmol/L glucose groups for 24 h. MTT assay was used to measure cell viability. Data were expressed as Mean ± SD. ** P < 0.01 versus control

Fig. 2. Cell viabilities in cells treated with 5.5 mmol/L glucose, 33 mmol/L glucose or 33 mmol/L glucose in combination with different concentrations of APS (0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL) for 24 h. MTT assay was used to measure cell viability. Data were expressed as Mean ± SD. ** P < 0.01 versus control. # P < 0.05 versus 33 mmol/L glucose group. ## P < 0.01 versus 33 mmol/L glucose group

Fig. 3. Cell apoptotic rates measured by flow cytometry using annexin V-FITC/PI staining. H9C2 cells were pre-incubated with/without APS for 24 h and then treated with 33 mmol/L glucose for 24 h. Cells were harvested and detected with a flow cytometer. a. 5.5 mmol/L glucose group. b. 33 mmol/L glucose group. c. 33 mmol/L glucose and 0.8 mg/mL APS group. Data were expressed as Mean ± SD. ** P < 0.01 versus 5.5 mmol/L glucose group; ## P < 0.01 versus 33 mmol/L glucose group

Fig. 4. APS modulated the mRNA expression levels of extrinsic apoptotic pathway-related factors. H9C2 cells were pre-incubated with/without APS for 24 h and then treated with 33 mmol/L glucose for 24 h. The expression of GAPDH was measured as an internal control. a. the relative mRNA expression levels of caspase 8 in different groups. b. the relative mRNA expression levels of caspase 3 in different groups. Data were expressed as Mean ± SD. ** P < 0.01 versus 5.5 mmol/L glucose group; # P < 0.05 versus 33 mmol/L glucose group. ## P < 0.01 versus 33 mmol/L glucose group

Fig. 5. APS modulated the mRNA expression levels of intrinsic apoptotic pathway-related factors. H9C2 cells were pre-incubated with/without APS for 24 h and then treated with 33 mmol/L glucose for 24 h. The expression of GAPDH was measured as an internal control. a. the relative mRNA expression levels of caspase 9 in different groups. b. the relative mRNA expression levels of cytochrome C in different groups. Data were expressed as Mean ± SD. ** P < 0.01 versus 5.5 mmol/L glucose group; # P < 0.05 versus 33 mmol/L glucose group. ## P < 0.01 versus 33 mmol/L glucose group

Fig. 6. APS modulated the mRNA expression levels of Bax and Bcl-2. H9C2 cells were pre-incubated with/without APS for 24 h and then treated with 33 mmol/L glucose for 24 h. The expression of GAPDH was measured as an internal control. a. the relative mRNA expression levels of Bax in different groups. b. the relative mRNA expression levels of Bcl-2 in different groups. Data were expressed as Mean ± SD. ** P < 0.01 versus 5.5 mmol/L glucose group; ## P < 0.01 versus 33 mmol/L glucose group

Fig. 7. APS modulated apoptosis-related proteins in the extrinsic pathways. H9C2 cells were pre-incubated with/without APS for 24 h and then treated with 33 mmol/L glucose for 24 h. The expression of GAPDH was measured as an internal control for the cytoplasmic fraction. a. Semi-quantitative expressions of cleaved caspase 3 in different groups. b. Semi-quantitative expressions of caspase 8 in different groups. Data were expressed as Mean ± SD. ** P < 0.01 versus 5.5 mmol/L glucose group; ## P < 0.01 versus 33 mmol/L glucose group

Fig. 8. APS modulated apoptosis-related proteins in the intrinsic pathways. H9C2 cells were pre-incubated with/without APS for 24 h and then treated with 33 mmol/L glucose for 24 h. The expression of GAPDH was measured as an internal control for the cytoplasmic fraction. The expression of VDAC was measured as an internal control for the mitochondrial fraction. a. Semi-quantitative expressions of cleaved caspase 9 in different groups. b. Semi-quantitative expressions of mitochondrial cytochrome C in different groups. c. Semi-quantitative expressions of cytoplasmic cytochrome C in different groups. Data were expressed as Mean ± SD. * P < 0.05 versus 5.5 mmol/L glucose group; ** P < 0.01 versus 5.5 mmol/L glucose group; # P < 0.05 versus 33 mmol/L glucose group; ## P < 0.01 versus 33 mmol/L glucose group

Fig. 9. APS modulated apoptosis-related proteins of the Bcl-2 family. H9C2 cells were pre-incubated with/without APS for 24 h and then treated with 33 mmol/L glucose for 24 h. The expression of VDAC was measured as an internal control for the mitochondrial proteins. a. Semi-quantitative expressions of mitochondrial Bax in different groups. b. Semi-quantitative expressions of mitochondrial Bcl-2 in different groups. Data were expressed as Mean ± SD. ** P < 0.01 versus 5.5 mmol/L glucose group; # P < 0.05 versus 33 mmol/L glucose group