Effects of the Nrf2 Protein Modulator Salvianolic Acid A Alone or Combined with Metformin on Diabetes-associated Macrovascular and Renal Injury

Abstract

Nuclear factor E2-related factor 2 (Nrf2) is considered a promising target against diabetic complications such as cardiovascular diseases and diabetic nephropathy. Herein, we investigated the effects of a potential Nrf2 modulator, salvianolic acid A (SAA), which is a natural polyphenol, on diabetes-associated macrovascular and renal injuries in streptozotocin-induced diabetic mice. Given that lowering glucose is the first objective of diabetic patients, we also examined the effects of SAA combined with metformin (MET) on both complications. Our results showed that SAA significantly increased the macrovascular relaxation response to acetylcholine and sodium nitroprusside in diabetic mice. Interestingly, treatment with SAA alone only provided minor protection against renal injury, as reflected by minor improvements in impaired renal function and structure, despite significantly reduced oxidative stress observed in the diabetic kidney. We demonstrated that decreased oxidative stress and NF-κB p65 expression were associated with SAA-induced expression of Nrf2-responsive antioxidant enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase (quinone) 1 (NQO-1), and glutathione peroxidase-1 (GPx-1) in vivo or in vitro, which suggested that SAA was a potential Nrf2 modulator. More significantly, compared with treatment with either SAA or MET alone, we found that their combination provided further protection against the macrovascular and renal injury, which was at least partly due to therapeutic activation of both MET-mediated AMP-activated protein kinase and SAA-mediated Nrf2/antioxidant-response element pathways. These findings suggested that polyphenol Nrf2 modulators, especially combined with drugs activating AMP-activated protein kinase, including hypoglycemic drugs, are worthy of further investigation to combat diabetic complications.

Keywords: Nuclear factor 2 (erythroid-derived 2-like factor) (NFE2L2) (Nrf2); diabetes; diabetic macrovascular injury; diabetic nephropathy; metformin; oxidative stress; salvianolic acid A.

FIGURE 1.

Impaired vascular function and structure were improved by SAA, MET, or a combination. Isometric tension studies were performed in diabetic or non-diabetic ICR mice and aorta relaxation in response to endothelium-dependent ACh and endothelium-independent SNP. Data were representative of 6–10 mice per group (A). H&E-stained aortas in mice were examined at ×400 (B). Data were expressed as mean ± S.E. *, p < 0.05; **, p < 0.01 versus ND; #, p < 0.05 versus D; γ, p < 0.05 at the same dose points of ACh and SNP. ND, non-diabetic mice; D, diabetic mice; D+MET, diabetic mice treated with metformin; D+SAA, diabetic mice treated with SAA; D+MET+SAA, diabetic mice treated with SAA combined with metformin; ACh, acetylcholine; SNP, sodium nitroprusside.

FIGURE 2.

Expression of VCAM-1 was decreased and HO-1 was increased by SAA and SAA combined with MET. HUVECs were treated with NG, Man, HG, HG + MET, HG + SAA, or HG + MET + SAA for 48 h. Man was used as an osmotic control. The expression of HO-1 and VCAM-1 in cell lysates was analyzed by Western blotting (A), and the quantitation of HO-1 and VCAM-1 is shown in B and C, respectively. Data were expressed as mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus NG; #, p < 0.05; ##, p < 0.01 versus HG, γ, p < 0.05. NG, 5.5 mm normal glucose; HG, 25 mm high glucose; Man, 19.5 mm mannitol; MET, 2 mm metformin; SAA, 5 μm salvianolic acid A.

FIGURE 3.

Impaired kidney function and structure in diabetic mice were improved by SAA in combination with MET. Proteinuria, expressed as urinary protein excreted relative to urinary creatinine, was decreased after SAA treatment in combination with MET (A). SAA in combination with MET significantly reduced kidney index, which was indicative as kidney weight (mg)/body weight (g) (B and C). PAS-stained glomeruli are shown (D). SAA in combination with MET significantly reduced glomerular size, glomerular PAS area, and GSI in STZ-induced ICR mice, respectively (E–G). PAS-stained tissue was examined at ×400. Data were expressed as mean ± S.E. n = 6–10 per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus ND; #, p < 0.05; ##, p < 0.01 versus D; γ, p < 0.05. ND, non-diabetic mice; D, diabetic mice; D+MET, diabetic mice treated with metformin; D+SAA, diabetic mice treated with SAA; D+MET+SAA, diabetic mice treated with SAA combined with metformin; GSI, glomerulosclerosis index.

FIGURE 4.

Diabetes-associated renal fibrosis was attenuated by SAA treatment in combination with MET. Fixed kidney tissues were stained with Masson's trichrome, and fibrosis areas were stained blue (A, top panel, ×200). Renal fibrosis-associated collagen I and α-SMA expression were examined using immunohistochemistry, and representative images (×200) are shown (A). The quantitative analysis (B) showed that the blue area in Masson's trichrome and expression of collagen I and α-SMA were significantly reduced by SAA in combination with MET. Data were mean ± S.E. n = 5 to 6 per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus ND; #, p < 0.05; ##, p < 0.01 versus D. ND, non-diabetic mice; D, diabetic mice; D+MET, diabetic mice treated with metformin; D+SAA, diabetic mice treated with SAA; D+MET+SAA, diabetic mice treated with SAA combined with metformin.

FIGURE 5.

Attenuation of oxidative stress by SAA or SAA in combination with MET was linked to the up-regulation of antioxidant enzymes. Oxidative stress in the kidney cortex was examined by NT immune staining in paraformaldehyde-fixed kidney and DHE fluorescent dye in kidney cryosections (A). The quantitative results in NT and DHE staining were assessed by Image-Pro Plus 6.0 (B–D). The protein level for NOX-4 was examined by Western blotting with its quantitation shown (E). Similarly, protein levels of several antioxidant enzymes, HO-1, NQO-1, and GPx-1 are shown (F–H). Data were mean ± S.E. n = 6–10 per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus ND; #, p < 0.05; ##, p < 0.01 versus D. γ, p < 0.05. ND, non-diabetic mice; D, diabetic mice; D+MET, diabetic mice treated with metformin; D+SAA, diabetic mice treated with SAA; D+MET+SAA, diabetic mice treated with SAA combined with metformin.

FIGURE 6.

Effects of SAA, MET, or a combination on renal tubulointerstitial inflammation and survival of STZ-induced diabetic mice. The renal pathological changes (at ×200) were assessed by H&E (HE) staining (A, left panel). The score of renal tubulointerstitial inflammation is presented (B). Ly6G, a marker of granulocytes, was examined using immunohistochemistry (A, right panel), and the quantitation is shown in C. The MCP-1 level in the kidney was measured by ELISA (D), and NF-κB protein level was also examined by Western blotting (E). One hundred and forty days after STZ treatment, animal survival was analyzed using the Kaplan-Meier survival analysis (F). Data were mean ± S.E. n = 6–10 per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus ND; #, p < 0.05 versus D; ##, p < 0.01 versus D. ND, non-diabetic mice; D, diabetic mice; D+MET, diabetic mice treated with metformin; D+SAA, diabetic mice treated with SAA; D+MET+SAA, diabetic mice treated with SAA combined with metformin.

FIGURE 7.

Combination of SAA and MET inhibited HG-induced harmful effects in HK-2 cells through MET-mediated AMPK activation and SAA-induced HO-1 up-regulation. Expression of AMPK and p-AMPK in the diabetic kidney was analyzed by Western blotting, showing that MET activated AMPK (A) (n = 6 mice per group). HK-2 cells were treated with NG, Man, HG, HG + MET, HG + SAA, HG + MET + SAA for the indicated times. After a 12-h incubation, cell lysates were used to detect AMPK and p-AMPK expression by Western blotting, and the quantitation showed metformin-activated AMPK (B). After a 24-h incubation, HO-1 expression was analyzed by Western blotting, which showed that SAA induced HO-1 up-regulation (C). After a 48-h incubation, the α-SMA and p65 expression were analyzed by Western blotting, with their expression showing a trend toward further decreases under the SAA treatment in combination with MET (D). Similarly, HK-2 cells were treated with the above agents, or a combination for 48 h, and then evaluated by fluorescent microscopy after DHE and DCFH-DA treatments for 30 min (E, ×200). Results were mean ± S.E. (B–E, n = 3 independent experiments). *, p < 0.05; **, p < 0.01; ***, p < 0.001. NG, 5.5 mm normal glucose; HG, 25 mm high glucose; Man, 19.5 mm mannitol; MET, 2 mm metformin; SAA, 5 μm salvianolic acid A.

FIGURE 8.

SAA reduced NF-κB expression and ROS levels in HG-exposed HK-2 cells through Nrf2-mediated HO-1 up-regulation. HK-2 cells were treated with 1, 5, and 20 μm SAA for 4 h. The total cytoplasmic and nuclear Nrf2 proteins were analyzed by Western blotting (A). Nrf2 DNA binding activity was analyzed by firefly luciferase activity following normalization against Renilla luciferase activity and presented as fold Nrf2 DNA binding activity relative to its basal levels in HK-2 cells (B, n = 3). HK-2 cells were transfected with Nrf2 siRNA or SCR siRNA and then were treated with or without 5 μm SAA in the presence of NG or HG for 48 h. Cell lysates were used to detect expression of Nrf2, HO-1, and p65 (C, n = 3) and DHE and DCFH-DA fluorescence intensities were evaluated by fluorescent microscopy (D, ×200, n = 3). NG, 5.5 mm normal glucose; HG, 25 mm high glucose; SCR siRNA, negative scrambled siRNA. Results were mean ± S.E. *, p < 0.05; **, p < 0.01.

FIGURE 9.

Schematic diagram showing that treatment with the combination of SAA and MET alleviates diabetes-associated vasculopathy and nephropathy. SAA, salvianolic acid A; MET, metformin; Nrf2, nuclear factor E2-related factor 2; AMPK, AMP-activated protein kinase; ROS, reactive oxygen species; α-SMA, α-smooth muscle actin; p65, nuclear factor NF-κB p65 subunit; VCAM-1, vascular cell adhesion molecule-1; MCP-1, monocyte chemoattractant protein-1.