Abstract
Aims The purpose of this study was to evaluate the protective effect of liquiritin (LIQ) from Radix Glycyrrhizae on cardiac mitochondria against hypoxia/reoxygenation (HR) injury. Methods H9C2 cells were subject to the HR model. LIQ purified from Radix Glycyrrhizae (purity > 95%) was administrated to reoxygenation period. Cell viability, mitochondrial mass, mitochondrial membrane potential, reactive oxygen species, and mitochondrial Ca2⁺ level were then assessed by using Cell Counting kit-8 and suitable fluorescence probe kits. Results LIQ administration remarkably reduced the rate of HR damage via increasing H9C2 cell viability level and preserving mitochondria after HR. Particularly, 60 μM of LIQ posthypoxic treatment markedly reduced cell death in HR-subjected H9C2 cell groups (p < 0.05). Interestingly, posthypoxic treatment of LIQ significantly prevented the loss of mitochondrial membrane potential, the decrease in mitochondrial mass, the increase in reactive oxygen species production, and the elevation of mitochondrial Ca2⁺ level in HR-treated H9C2 cells. Conclusion The present study provides for the first time the cardioprotective of LIQ posthypoxic treatment via reducing H9C2 cell death and protecting cardiac mitochondria against HR damage.
Highlights
As the “powerhouse” and “apoptosis center” in myocytes, mitochondria have been described to play a key role in the pathogenesis of ischemic heart disease [1, 2]
Liquiritin (LIQ) was extracted and purified from the root of Radix Glycyrrhizae based on the bioassay and chromatographic methods described above. e isolated LIQ compound was identified and had the following characteristics: light yellow amorphous powder, purity >95%, and electrospray ionization mass spectrometry: 417.2 [M-H], 453.1 [M + Cl]−, 419.0 [M + H]+ (Figure 1, Supplemental Material 1). e obtained results showed that Radix Glycyrrhizae consists of approximately 0.088 mg/g of the LIQ component, which is consistent with previous reports [35, 37]
H9C2 cells were subjected to different conditions and the cell viabilities were measured using the CCK-8 kit (Figure 2). e results showed that the viabilities were dramatically reduced in the hypoxia/reoxygenation injury (HR)-exposed cells compared to normal cells (Figures 2(a) and 2(b), p < 0.01)
Summary
As the “powerhouse” and “apoptosis center” in myocytes, mitochondria have been described to play a key role in the pathogenesis of ischemic heart disease [1, 2]. After being subjected to different conditions, cells were stained with 0.1 μM tetramethylrhodamine ethyl ester (TMRE; excitation/emission: 535/570 nm, Invitrogen, USA) for 30 min at room temperature They were washed twice with PBS before measuring fluorescence intensity using a microplate reader or ApoTome as previously mentioned [14]. After being subjected to different conditions, cells were stained with 5 μM 2′,7′-dichlorodihydrofluorescein-diacetate (CM-H2DCFDA; ex/em 485/525 nm, Invitrogen, USA) at 37°C for 30 min at room temperature to detect changes in mitochondrial ROS levels. In another experimental set, H2DCFDA-stained cells were captured using the ApoTome and the images were reconstructed from individual tiles (X:6, Y:9) using ZEN Blue 2.5 software (Carl Zeiss). Differences with a p value ≤0.05 were considered significant
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