Korean J Physiol Pharmacol.  2021 Jan;25(1):27-38. 10.4196/kjpp.2021.25.1.27.

Gamma-aminobutyric acid-salt attenuated high cholesterol/high salt diet induced hypertension in mice

• Son M
• Oh S
• Lee HS
• Choi J
• Lee BJ
• Park JH
• Park CH
• Son KH
• Byun K

• KMID: 2509652
• DOI: http://doi.org/10.4196/kjpp.2021.25.1.27

Abstract

Excessive salt intake induces hypertension, but several gamma-aminobutyric acid (GABA) supplements have been shown to reduce blood pressure. GABAsalt, a fermented salt by L. brevis BJ20 containing GABA was prepared through the post-fermentation with refined salt and the fermented GABA extract. We evaluated the effect of GABA-salt on hypertension in a high salt, high cholesterol diet induced mouse model. We analyzed type 1 macrophage (M1) polarization, the expression of M1 related cytokines, GABA receptor expression, endothelial cell (EC) dysfunction, vascular smooth muscle cell (VSMC) proliferation, and medial thicknesses in mice model. GABA-salt attenuated diet-induced blood pressure increases, M1 polarization, and TNF-α and inducible nitric oxide synthase (NOS) levels in mouse aortas, and in salt treated macrophages in vitro. Furthermore, GABA-salt induced higher GABA_B receptor and endothelial NOS (eNOS) and eNOS phosphorylation levels than those observed in salt treated ECs. In addition, GABA-salt attenuated EC dysfunction by decreasing the levels of adhesion molecules (E-selectin, Intercellular Adhesion Molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) and of von Willebrand Factor and reduced EC death. GABA-salt also reduced diet-induced reductions in the levels of eNOS, phosphorylated eNOS, VSMC proliferation and medial thickening in mouse aortic tissues, and attenuated Endothelin-1 levels in salt treated VSMCs. In summary, GABA-salt reduced high salt, high cholesterol diet induced hypertension in our mouse model by reducing M1 polarization, EC dysfunction, and VSMC proliferation.

Keyword

Blood pressure; Endothelial cells; Gamma-aminobutyric acid (GABA)-salt; Macrophage polarization; Smooth muscle cells

Figure

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  Fig. 1 Characteristics of gamma-aminobutyric acid (GABA)-salt. The HPLC chromatogram shows the (A) base salt and (B) GABA standard and (C) GABA-salt peak. Retention time of GABA standard (B) is 12.28 min and its time of GABA-salt (C) is 12.29 min.

• 

  Fig. 2 Regulation of blood pressure, water intake, urine volume and renal function of gamma-aminobutyric acid (GABA)-salt in high cholesterol (HCD) diet-induced mouse model. Blood pressures were measured at 3, 4 and 5 weeks. (A–C) Graphs of the blood pressures of mice fed a normal diet (ND, circle), a high cholesterol diet and normal saline (HCD/salt, square) diet, or a high cholesterol and GABA-salt (HCD/GABA-salt, triangle) diet showing; (A) systolic blood pressures, (B) diastolic blood pressures, and (C) mean arterial blood pressures. (D) Water intake and (E) urine volume were measured at 5 weeks and blood chemistry tests including (F) serum creatinine and (G) BUN show kidney function in ND group, HCD/salt group and HCD/GABA-salt group. BUN, blood urea nitrogen. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. ND; and $p < 0.05, $$p < 0.01, and $$$p < 0.001 vs. HCD/Salt.

• 

  Fig. 3 Inhibitory effects of gamma-aminobutyric acid (GABA)-salt on expressions of type 1/type 2 macrophage (M1/M2) and associated cytokines in high cholesterol diet (HCD)-induced mouse model. (A) Immunohistochemistry images showing M1 macrophages (CD86; brown, arrows). Scale bar = 40 µm. (B) The qRT-PCR graph showing mRNA expressions of M1 (CD86) macrophage marker. (C) The qRT-PCR graph showing the mRNA expressions of M1 related cytokines including TNF-α and iNOS. (D) Immunohistochemistry images showing M2 macrophages (CD206; brown, arrow). Scale bar = 40 µm. (E) The qRT-PCR graph showing mRNA expressions of M2 (CD206) macrophage marker. (F) The qRT-PCR graph showing the mRNA expressions of M2 related cytokines including TGF-β and Arg-1. NS, not significant compared to HCD/salt. *p < 0.05, and **p < 0.01 vs. normal diet (ND) and $p < 0.05, and $$p < 0.01 vs. HCD/salt.

• 

  Fig. 4 Inhibitory effects of gamma-aminobutyric acid (GABA)-salt on the expressions of type 1 macrophages (M1) and associated cytokines in vitro. (A, B) qRT-PCR graph showing the mRNA expressions of M1 and M2 macrophage marker in media, salt or GABA-salt treated Raw 264.7 cells. (C, D) qRT-PCR graph showing the mRNA expressions of M1 (TNF-α) and M2 (TGF-β) macrophage marker in media, salt or GABA-salt treated Raw 264.7 cells. (E, F) qRT-PCR graph showing the mRNA expressions of M1 (iNOS) and M2 (Arg-1) macrophage marker in media, salt or GABA-salt treated Raw 264.7 cells. NS, not significant compared to salt. *p < 0.05 vs. media; $p < 0.05 vs. salt.

• 

  Fig. 5 Effects of gamma-aminobutyric acid (GABA)-salt on the expression and function of GABAB receptor in endothelial cell (ECs). To identify changes in GABAB receptors and in peNOS and eNOS, media, salt or GABA-salt treated ECs and aorta of hypertensive mice were subjected to qRT-PCR and immunofluorescence. (A) The qRT-PCR graph showing the mRNA expressions of GABAB receptor in media, salt or GABA-salt treated SVEC 4–10. (B, C) Immunofluorescent images showing eNOS and peNOS (green, nuclei; blue) expressions in (B) SVEC 4–10 and in (C) the aorta of hypertensive mice (brown, arrows). Scale bar = 50 µm. (D) Immunofluorescent images showing the expression of the adherent proteins such as E-selectin, VCAM-1, ICAM-1 and vWF (green, nuclei; blue) in the aorta of hypertensive mice. Scale bar = 50 µm. (E, F) Apoptotic cells are detected by TUNEL analysis. The fluorescent images and graph showing apoptotic cell in the aorta of hypertensive mice. Scale bar = 50 µm. ICAM-1, Intercellular Adhesion Molecule-1; VCAM-1, vascular cell adhesion molecule-1; vWF, von Willebrand Factor. *p < 0.05, and ***p < 0.001 vs. culture media or normal diet (ND); and $p < 0.05, and $$$p < 0.001 vs. salt or high cholesterol diet (HCD)/salt.

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  Fig. 6 Effects of gamma-aminobutyric acid (GABA)-salt on the expression of GABAB receptor and its function in vascular smooth muscle cells (VSMCs). (A) The qRT-PCR graph showing the mRNA expressions of GABAB receptor in media, salt or GABA-salt treated mouse vascular smooth muscle cell (MOVAS). (B) Immunofluorescent images showing Endothelin-1 (ET-1) (green, nuclei; blue) expressions in media, salt or GABA-salt treated MOVAS. Scale bar = 50 µm. (C, D) Immunohistochemistry staining image and graph showing proliferating cell nuclear antigen (PCNA), as proliferating cell marker, positive cell (brown, arrows) in hypertensive mice. Scale bar = 50 µm. H&E staining image and graph showing changes of vessel thicknesses in hypertensive mice. Scale bar = 500 µm. *p < 0.05, and **p < 0.01 vs. culture media or normal diet (ND); $p < 0.05 vs. salt or high cholesterol diet (HCD)/salt.

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