Astragaloside IV enhances cardioprotection of remote ischemic conditioning after acute myocardial infarction in rats

Abstract

Background: Remote ischemic conditioning (RIC) has been shown to be a practical method for protecting the heart from ischemic/reperfusion (I/R) injury. In the present study, we investigated whether or not the combination of RIC and Astragaloside IV (AS-IV) could improve cardioprotection against acute myocardial infarction (AMI)-induced heart failure (HF) when compared with individual treatments.

Material and methods: A rat model of AMI was established via permanent ligation of the left anterior descending coronary artery (LAD). Postoperatively, the rats were randomly grouped into a sham group (n=10), a model group (n=15), an AS-IV alone group (n=15), an RIC alone group (n=15) and a combined treatment group (AS-IV+RIC; n=15). All treatments were administered for 2 weeks.

Results: After treatment for 2 weeks, the survival rate was improved, the cardiac function was preserved and the infarcted size was limited in AS-IV alone and RIC alone treatment groups compared to the model group, whereas the combined treatment yielded the most optimal protective effects. Additional studies suggested that AS-IV enhanced the cardioprotective effects of RIC by alleviating myocardial fibrosis, suppressing inflammation, attenuating apoptosis and ameliorating impairment of the myocardial ultrastructural.

Conclusion: AS-IV enhances the cardioprotective effects of RIC against AMI-induced HF and ventricular remodeling, which represents a potential therapeutic approach for preserving cardiac function and improving the prognosis of AMI.

Keywords: Astragaloside IV; acute myocardial infarction; cardioprotection; remote ischemic conditioning; ventricular remodeling.

Figure 1

Survival rate and the heart/body weight ratio after treatment for 2 weeks. A: Kaplan-Meier analysis indicated the survival rates of the rats after AMI. The combination treatment group exhibited a trend towards improved overall survival rate 2 weeks after the induction of AMI, but differences did not reach statistical significance (log-rank: P=0.0638). B: Analysis of the heart weight/body weight ratio, data are expressed as the mean ± SD. **: P<0.01 versus sham group, ##: P<0.01 versus model group, $: P<0.05 versus combined treatment group.

Figure 2

Cardiac function at the end of treatment. (A) M-mode echocardiographic images of the rats in all groups. The analysis of LVIDd (B), LVIDs (C), LVEF (D), and LVFS (E) 2 weeks after treatment was also conducted, which revealed that LVIDd and LVIDs were decreased, whereas LVEF and LVFS improved in the combined treatment group compared with the model group and the individual treatment groups. Data are expressed as the mean ± SD. **: P<0.01 versus sham group, #: P<0.05 versus model group, ##: P<0.01 versus model group, $: P<0.05 versus combined treatment group, $$: P<0.01 versus combined treatment group.

Figure 3

The measurement of infarcted size. A: Post-euthanasia, each rat heart was cut into five slices and stained with triphenyltetrazolium chloride (TTC). The red region represents the survival myocardium, whereas the pale region represents the infarcted site. B: The analysis of the infarcted size in each group. The combined treatment exerted a more vigorous infarct-sparing effect than the individual treatments. Data are expressed as the mean ± SD. ##: P<0.01 versus sham group, **: P<0.01 versus combined treatment group.

Figure 4

Evaluation of the pathological changes and interstitial fibrosis at 400× magnification. A: Representative images of H&E and Masson’s trichrome staining of the hearts from rats with AMI. Necrotic tissue and poorly arranged cardiac myocytes were noted following H&E staining. In the Masson’s trichrome stained sections, blue represents the collagen fiber. B: The quantitative analysis of areas of interstitial fibrosis after treatment. The rats in the combined treatment group exhibited the least pathological changes and a minimal ratio of fibrotic area to total area of connective and myocardial tissue compared with the individual treatment groups. Data are expressed as the mean ± SD. ##: P<0.01 versus sham group, **: P<0.01 versus combined treatment group, $$: P<0.01 versus combined treatment group.

Figure 5

The expression of inflammatory factors after treatment. A: Western blotting analysis of the expression of TLR4 and its downstream protein NF-κB with β-actin as the internal control. B: Quantified western blot results of TLR4, which was normalized to the housekeeping gene β-actin. C: Quantified western blot results of NF-κB, which was normalized to the housekeeping gene β-actin. TLR4 and NF-κB were downregulated in all treatment groups to varying degrees, but the combined treatment yielded optimal anti-inflammatory effects. Data are expressed as the mean ± SD. **: P<0.01 versus the sham group, ##: P<0.01 versus the model group, $: P<0.05 versus the combined treatment group, and $$: P<0.01 versus the combined treatment group.

Figure 6

Assessment of myocardial apoptosis after treatment. (A) Representative graph of TUNEL/DAPI immunofluorescence at 400× magnification 2 weeks after AMI. The green fluorescence represents TUNEL-positive cells, whereas the blue fluorescence represents DAPI-positive cells. (B) The ratio of myocardial apoptosis (TUNEL-positive cells/DAPI-positive cells). Apoptosis was significantly inhibited by the combined treatment. (C) Expression of the apoptosis-related proteins Bcl-2 and Bax were measured by western blotting analysis, β-actin served as the internal control. Quantified western blot results of Bcl-2 (D), Bax (E), and the Bcl-2/Bax ratio (F) are provided. The combined treatment led to the downregulation of Bax and the upregulation of Bcl-2, which in turn led to an increased ratio of Bcl-2/Bax compared with the untreated and individual treatment groups. Data are expressed as the mean ± SD. **: P<0.01 versus the sham group, ##: P<0.01 versus the model group, and $$: P<0.01 versus the combined treatment group.

Figure 7

Observation of the ultrastructure performed by TEM at 25000× magnification. (A) The sham group. The myofilaments were neatly arranged and normal mitochondria were observed. (B) The model group. The structure of the myofilaments was barely visible, the mitochondria were markedly swollen, the mitochondrial cristae were fractured (arrows), and the fusion of the mitochondria (arrowheads) was likewise detected. The AS-IV (C) and RIC groups (D) exhibited fewer swollen mitochondria, and the structure of the myofilaments was somewhat improved. (E) The combined treatment group. The injury to the mitochondria and myofilaments was drastically reduced.