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. 2021 Aug 6:2021:1857464.
doi: 10.1155/2021/1857464. eCollection 2021.

Liquiritin from Radix Glycyrrhizae Protects Cardiac Mitochondria from Hypoxia/Reoxygenation Damage

Vu Thi Thu  1   2 , Ngo Thi Hai Yen  1 , Nguyen Thi Ha Ly  3
Affiliations

Liquiritin from Radix Glycyrrhizae Protects Cardiac Mitochondria from Hypoxia/Reoxygenation Damage

Vu Thi Thu et al. J Anal Methods Chem. .

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.

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Conflict of interest statement

The authors report no conflicts of interest.

Figures

Figure 1
Figure 1
Structure of liquiritin (LIQ) from Radix Glycyrrhizae.
Figure 2
Figure 2
Experimental design and cell viability assay. (a) HR model and LIQ treatment. ((b)-(c)) The graph indicates H9C2 cell viability under different conditions and treatments. Con: normal control; HR: hypoxia/reoxygenation; LIQ: HR + liquiritin (LIQ); NEC: HR + NecroX-5 (10 μM); LIQ60: HR + LIQ at a dose of 60 μM; ∗p < 0.05 vs. Con; #p < 0.05 vs. HR; &p < 0.05 vs. LIQ at doses of 1.2, 12, 300, and 600 μM, n = 6 for each group.
Figure 3
Figure 3
Mitochondrial mass in H9C2 cells subjected to different treatments. (a) Representative images of H9C2 cells stained with MitoTracker Green. (b) The graph indicates MitoTracker green fluorescence intensity per cell (○, open circle) of the representative images. (c) The graph indicates the total MitoTracker green intensity. (d) Representative images of H9C2 cells stained with MitoTracker red. (e) The graph indicates MitoTracker red fluorescence intensity per cell (○, open circle) of the representative images. (f) The graph indicates the total MitoTracker red intensity. AU: arbitrary unit; Con: normal control; HR: hypoxia/reoxygenation; NEC: HR + NecroX-5; LIQ60: HR + LIQ at a dose of 60 μM; ∗p < 0.05 vs. Con; #p < 0.05 vs. HR; n = 3; scale bar 100 μm.
Figure 4
Figure 4
Mitochondrial function in H9C2 cells subjected to different treatments. (a) Representative images of H9C2 cells stained with tetramethylrhodamine ethyl ester (TMRE). (b) The graph indicates fluorescence intensity per cell (○, open circle) of the representative images. (c) The graph indicates the total TMRE intensity; AU: arbitrary unit; Con: normal control; HR: hypoxia/reoxygenation; NEC: HR + NecroX-5; LIQ60: HR + LIQ at a dose of 60 μM; ∗p < 0.01 vs. Con; #p < 0.05 vs. HR; n = 3; scale bar 100 μm.
Figure 5
Figure 5
Mitochondrial oxidative stress in H9C2 cells subjected to different treatments. (a) Representative images of H9C2 cells stained with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA). (b) The graph indicates fluorescence intensity per cell (○, open circle) of the representative images. (c) The graph indicates the total CM-H2DCFDA intensity; AU: arbitrary unit; Con: normal control; HR: hypoxia/reoxygenation; NEC: HR + NecroX-5; LIQ60: HR + LIQ at a dose of 60 μM; ∗p < 0.05 vs. Con; #p < 0.05 vs. HR; n = 3 ÷ 5; scale bar 100 μm.
Figure 6
Figure 6
Mitochondrial calcium level in H9C2 cells subjected to different treatments. (a) Representative images of H9C2 cells stained with Rhod-2 AM. (b) The graph indicates fluorescence intensity per cell (○, open circle) of the representative images. (c) The graph indicates the total Rhod-2 AM intensity; AU: arbitrary unit; Con: normal control; HR: hypoxia/reoxygenation; NEC: HR + NecroX-5; LIQ60: HR + LIQ at a dose of 60 μM; ∗p < 0.05 vs. Con; #p < 0.05 vs. HR; n = 3; scale bar 100 μm.
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