Astragaloside IV protects human cardiomyocytes from hypoxia/reoxygenation injury by regulating miR-101a

Abstract

Astragaloside IV (AS/IV) is one of the extracted components from the traditional Chinese medicine Astragalus which has been demonstrated to have potential capacity for anti-inflammation activity and for treating cardiovascular disease. Our purpose was to determine the function and underlying molecular mechanism of AS/IV in hypoxia/reoxygenation (H/R) injured in cardiomyocytes. Differentially expressed genes (DEGs) were screened using bioinformatic analysis, and the molecular targeting relationship was verified by the dual-luciferase report system. H/R injured cardiomyocytes were employed to explore the effect of AS/IV. QRT-PCR and Western blot analysis were applied to detect the expression of mRNA and proteins, respectively. Additionally, superoxide dismutase (SOD), lactic dehydrogenase (LDH) and MDA (malondialdehyde) levels were detected to determine the oxidative damage. Cell viability was assessed by CCK-8, and flow cytometry was used to evaluate cell apoptosis ratio. TGFBR1 and TLR2 were selected as DEGs. Additionally, AS/IV could enhance cell proliferation and upregulated miR-101a expression, which suppressed TGFBR1 and TLR2 expression in H/R injured cardiomyocytes. Moreover, the results of Western blot exhibited that the downstream genes (p-ERK and p-p38) in the MAPK signaling pathway were suppressed, which meant AS/IV could inhibit this pathway in H/R injured cardiomyocytes. Overall, this study demonstrated AS/IV could attenuate H/R injury in human cardiomyocytes via the miR-101a/TGFBR1/TLR2/MAPK signaling pathway axis, which means that it could serve as a possible alternate for H/R treatment.

Keywords: Astragaloside IV; Hypoxia/reoxygenation injury; MAPK signaling pathway; MiR-101a.

Fig. 1

Effects of AS/IV on H/R in vitro cell experiments. There were 6 groups in these experiments including the con, 0 μM AS/IV+H/R cell, 20 μM AS/IV+H/R cell, 40 μM AS/IV+H/R cell, 80 μM AS/IV+H/R cell and 80 μM AS/IV+H/R+miR-101a inhibitor cell groups. a Efficiency of miR-101a mimic and inhibitor was verified by qRT-PCR. b Relative miR-101a expression was detected by qRT-PCR in these different groups. c Cell survival was detected by CCK-8 in these different groups. d LDH release was detected with an LDH assay kit in these different groups. e SOD activity was detected by DTPA in these different groups. f MDA activity was detected by a microplate spectrophotometer in these different groups. g Flow cytometry detected the apoptosis rate in these different groups. con: Control, H/R: hypoxia/reoxygenation. Data are shown as mean ± SD, *P < 0.05

Fig. 2

Regulating relationship between miR-101a and TGFBR1 in H/R cells. a, b Targeting relationship of miR-101a and TGFBR1-wt/mut groups was detected by luciferase reporter assay.c Relative expression of TGFBR1 regulated by miR-101a mimic was detected by qRT-PCR. d Protein level of TGFBR1 regulated by miR-101a mimic was detected by Western blot. e Relative expression of TGFBR1 in con, si-TGFBR1-1, si-TGFBR1-2, si-TGFBR1-3 and pc-TGFBR1 groups was detected with qRT-PCR. (F) CCK-8 detected cell survival in the con, H/R, H/R+miR-101a mimic, H/R+si-TGFBR1, H/R+miR-101a mimic+TGFBR1 groups. (G) The apoptosis rate was detected by flow cytometry in the con, H/R, H/R+miR-101a mimic, H/R+si-TGFBR1, H/R+miR-101a mimic+TGFBR1 groups. nc: normal control, con: Control, H/R: hypoxia/reoxygenation. Data are shown as mean ± SD, *P < 0.05

Fig. 3

Regulating relationship between miR-101a and TLR2 in H/R cells. a, b Targeting relationship of miR-101a and TLR2-wt/mut groups was detected by a dual-luciferase reporter assay.c Relative expression of TLR2 regulated by miR-101a mimic was detected by qRT-PCR. d Protein level of TLR2 regulated by miR-101a mimic was detected by Western blot. e Relative expression of TLR2 in con, si-TLR2-1, si-TLR2-2, si-TLR2-3 and pc-TLR2 groups was detected with qRT-PCR. f CCK-8 detected cell survival in the con, H/R, H/R+miR-101a mimic, H/R+si-TLR2-1, H/R+miR-101a mimic+TLR2 groups. g Apoptosis rate was detected by flow cytometry in the con, H/R, H/R+miR-101a mimic, H/R+si-TLR2-1, H/R+miR-101a mimic+TLR2 groups. nc: normal control, con: Control, H/R: hypoxia/reoxygenation. Data are shown as mean ± SD, *P < 0.05

Fig. 4

AS/IV protects cell viability and apoptosis through the targeting effect of miR-101a on TGFBR1 and TLR2. a Relative expression of TGFBR1 and TLR2 regulated by AS/IV was detected by qRT-PCR. b CCK-8 detected cell survival in the con, H/R, H/R+AS/IV (80 μm), H/R+AS/IV (80 μm)+miR-101a inhibitor, H/R+AS/IV (80 μm)+TGFBR1, H/R+AS/IV (80 μm)+TLR2 groups. c Apoptosis rate was detected by flow cytometry in the con, H/R, H/R+AS/IV (80 μm), H/R+AS/IV (80 μm)+miR-101a inhibitor, H/R+AS/IV (80 μm)+TGFBR1, H/R+AS/IV (80 μm)+TLR2 groups. con: Control, H/R: hypoxia/reoxygenation. Data are shown as mean ± SD, *P < 0.05

Fig. 5

AS/IV reduces the expression of apoptosis-related proteins by regulating the MAPKs signaling pathway. a–c Expression level of MAPK signaling pathway proteins and apoptosis-related proteins (t-ERK, p-ERK, t-P38, p-P38, Bax, Bcl-2, Cleaved Caspase-3 and Caspase-3) was detected by Western blot in the con, H/R, H/R+AS/IV (80 μm), H/R+AS/IV (80 μm)+miR-101a inhibitor, H/R+AS/IV (80 μm)+TGFBR1, H/R+AS/IV (80 μm)+TLR2 groups. con: Control, H/R: hypoxia/reoxygenation. Data are shown as mean ± SD, *P < 0.05