Astragaloside-IV protects H9C2(2-1) cardiomyocytes from high glucose-induced injury via miR-34a-mediated autophagy pathway

Figures & data

Figure 1. AS-IV is a triterpenoid saponin extracted from Astragalus membranaceus. The molecular formula is C₄₁H₆₈O₁₄.

Figure 2. (a) The effect of different concentrations of AS-IV on the viability of HG-damaged H9C2(2-1) cell. X axis: normal group (NG, 5 mM), HG group (HG, 33 mM) and HG combined with different AS-IV concentrations; Y axis: H9C2(2-1) cell viability, absorbance (OD 450 value) by CCK-8 assays; ***p < .001. (b) The effect of miR-34a and AS-IV on the viability of HG-damaged H9C2(2-1) cells. X axis: different intervention groups of H9C2(2-1) cells; Y axis: H9C2(2-1) cell viability, absorbance (OD450 value) by CCK-8 assays; *p < .05, **p < .01.

Figure 3. The effect of AS-IV on cell morphology, autophagy flow and autophagosome in HG-damaged H9C2(2-1) cells. (a–f) Changes in cell morphology under different conditions. (g) Representative images of LC3-GFP adenovirus infection monitored the autophagy flow in H9C2(2-1) cells under fluorescent inverted microscope (500× magnification). (h) Representative images of autophagosome obtained by electron microscope. The red arrows refer to the autophagosomes (First row：Scale bar 1 μm, 18500× magnification; Second row：Scale bar 200 nm, 68000× magnification). (i) Statistical graph of LC3-GFP dots/cell for each group of H9C2(2-1) cells. Each group counts autophagosomes in at least 20 cells. Data are shown as mean ± SEM. ***p < .001, ****p < .0001.

Figure 4. The effect of AS-IV on oxidative stress factors in HG-damaged H9C2(2-1) cells. X axis: different intervention groups of H9C2(2-1) cells; Y axis: LDH, MDA and SOD contents in cell culture supernatant; *p < .05, **p < .01, ***p < .001.

Table 1. Effect of AS-IV on oxidative stress index of H9C2(2-1) cell injury induced by high glucose (Mean ± SEM).

Group	SOD(U/ml)	MDA(nmol/ml)	LDH(U/L)
Normal group (NG)	6.15 ± 0.53	12.09 ± 0.25	95.43 ± 0.82
High glucose group (HG)	4.80 ± 0.09*	19.34 ± 0.79***	151.63 ± 0.27***
HG + AS-IV group	5.74 ± 0.21^##	15.00 ± 0.13^###	96.24 ± 3.22^###
HG + miR-34a-inhibitor group	5.64 ± 0.13^###	13.49 ± 0.71^###	113.35 ± 10.86^##
HG + AS-IV + miR-34a-inhibitor group	6.16 ± 0.10^###	14.94 ± 0.17^###	99.64 ± 4.56^###

*p<.05, ***p<.001 vs. normal group (NG).

^##p<.01, ^###p<.001 vs. high glucose group (HG).

Figure 5. The effect of AS-IV on mRNA expression of autophagy-related factors in HG-damaged H9C2(2-1) cells. X axis: different intervention groups of H9C2(2-1) cells; Y axis: relative mRNA quantity of target gene was compared with internal reference gene GAPDH; **p < .01, ***p < .001.

Figure 6. The effect of AS-IV on autophagy-related factor protein levels. (a,b) Western blot analysis showing the levels of the autophagy-related factors LC3I, LC3II, P62, Bcl-2, pAKT, AKT, Sirt1 and caspase3. Tubulin was used as loading control. (c,d) Histograms showed the relative proteins expression of LC3II/LC3I ratio and P62 in each intervention group; *p < .05, **p < .01, ***p < .001.