Noise-Induced Vascular Dysfunction, Oxidative Stress, and Inflammation Are Improved by Pharmacological Modulation of the NRF2/HO-1 Axis

Abstract

Vascular oxidative stress, inflammation, and subsequent endothelial dysfunction are consequences of traditional cardiovascular risk factors, all of which contribute to cardiovascular disease. Environmental stressors, such as traffic noise and air pollution, may also facilitate the development and progression of cardiovascular and metabolic diseases. In our previous studies, we investigated the influence of aircraft noise exposure on molecular mechanisms, identifying oxidative stress and inflammation as central players in mediating vascular function. The present study investigates the role of heme oxygenase-1 (HO-1) as an antioxidant response preventing vascular consequences following exposure to aircraft noise. C57BL/6J mice were treated with the HO-1 inducer hemin (25 mg/kg i.p.) or the NRF2 activator dimethyl fumarate (DMF, 20 mg/kg p.o.). During therapy, the animals were exposed to noise at a maximum sound pressure level of 85 dB(A) and a mean sound pressure level of 72 dB(A). Our data showed a marked protective effect of both treatments on animals exposed to noise for 4 days by normalization of arterial hypertension and vascular dysfunction in the noise-exposed groups. We observed a partial normalization of noise-triggered oxidative stress and inflammation by hemin and DMF therapy, which was associated with HO-1 induction. The present study identifies possible new targets for the mitigation of the adverse health effects caused by environmental noise exposure. Since natural dietary constituents can achieve HO-1 and NRF2 induction, these pathways represent promising targets for preventive measures.

Keywords: NRF2; aircraft noise exposure; endothelial dysfunction; environmental risk factors; heme oxygenase-1; inflammation; oxidative stress.

Figure 1

Scheme for treatments and noise exposure. Male C57BL/6J mice were acclimatized to blood pressure measurement, and baseline measurements were taken prior to treatment with hemin or DMF. Hemin was administered once every 2 days during aircraft noise exposure via intraperitoneal injection (IP) injection (25 mg/kg). DMF was administered via daily gavage at a dose of 20 mg/kg. Created with BioRender.com.

Figure 2

Induction of HO-1 and activation of NRF2 by treatments with hemin or DMF. (A) mRNA expression of Hmox1 in cardiac tissue was measured via quantitative RT-PCR. (B) HO-1 protein expression was measured in heart tissue (representative Western blots below the densitometry). (C) Quantification and representative chromatograms of bilirubin levels in plasma as measured by HPLC analysis of bilirubin formation and expressed as changes to untreated control. Data points from (A) are measurements from 6–13 individual animals, (B) represents 9–18 individual samples (each pooled from 2 to 4 mice), and (C) represents 5–15 individual samples; * represents p < 0.05 vs. untreated control; ⁺ represents p < 0.05 vs. + Noise.

Figure 3

Blood pressure in mice treated with inducer of HO-1 and activator of NRF2 and exposed to noise. (A,E) The time courses of systolic blood pressure over the span of the hemin and DMF treatments. (C,G) The respective time courses of diastolic blood pressure over the treatment periods. (B,D,F,H) Systolic and diastolic arterial blood pressure measured on the final day of the treatments. Data points are measurements from individual samples; n = 8–10. * represents p < 0.05 vs. untreated control; ⁺ represents p < 0.05 vs. + Noise.

Figure 4

Vascular function in mice treated with inducer of HO-1 and activator of NRF2 and exposed to noise. (A,B) Potassium chloride (KCl, 80 mM)- or prostaglandin F2α (PGF2α, 3 µM)-induced vasoconstriction. (C,D) Endothelium-dependent (ACh) and independent (GTN) relaxation of thoracic aortic rings was measured by isometric tension method. (E) Quantification of maximum relaxation of all groups. Data points are measurements from individual samples, n = 7–16; * represents p < 0.05 vs. untreated control; ⁺ represents p < 0.05 vs. + Noise.

Figure 5

Markers of inflammation show an attenuation in the presence of both treatments. (A,B) Densitometry and representative dot blot of IL-6-positive proteins in plasma as well as IL-6 mRNA by quantitative RT-PCR in the heart. (C) mRNA expression of CD68 in cardiac tissue was measured via quantitative RT-PCR. Data points from (A) are measurements from 8–18 individual animals, and (B,C) represent 4–8 individual samples (each pooled from two mice). * represents p < 0.05 vs. untreated controls; ⁺ represents p < 0.05 vs. + Noise; ^$ represents p < 0.05 vs. DMF.

Figure 6

Vascular reactive oxygen species (ROS) formation is decreased by HO-1/NRF2 inducer or activator in noise-exposed mice. (A–C) Dihydroethidium stainings of aortic, cardiac, and cortical cryosections and their representative photomicrographs show ROS formation as red fluorescence and autofluorescence from aortic laminae as green. A, adventitia; E, endothelium; M, media. Scale bars indicate 100 µm, and a magnification of 20× was used. Data points from (A–C) are measurements from 6–14 individual animals; * represents p < 0.05 vs. untreated controls; ⁺ represents p < 0.05 vs. + Noise.

Figure 7

Oxidative stress and sources of ROS are decreased by HO-1/NRF2 inducer or activator in noise-exposed mice. (A,B) Densitometry and representative dot blots of 3-NT-positive proteins in heart tissue and 4-HNE-positive proteins in plasma. eNOS mRNA via quantitative RT-PCR. Data points from (A,B) represent 4–13 individual animals, and (C) represents 4–10 individual samples, * represents p < 0.05 vs. untreated controls; ⁺ represents p < 0.05 vs. + Noise.

Figure 8

Mechanistic scheme of adverse cardiovascular effects of noise exposure and protection by HO-1/NRF2 induction or activation. Heme or hemin are involved in the dynamic exchange of Bach1 and Nrf2 in the Maf transcription factor network. Enzymatic degradation of heme is catalyzed by HO-1, leading to the formation of biliverdin, carbon monoxide, and ferrous iron. DMF and its primary intestinal metabolite, monomethyl fumarate (MMF), can bind to the cysteine residues of the NRF2/KEAP-1 complex in the cytoplasm or reduce glutathione (GSH) levels. As a consequence, GSH metabolism may affect the oxidative clearance and increase ROS. Besides Hmox1, NRF2 also regulates other antioxidant and cytoprotective genes encoding for superoxide dismutases, peroxiredoxins, and others. The effects studied in the present study are marked with red color and roman numbers (I,II,III,IV). We found the upregulation of HO-1 and higher plasma levels of bilirubin in response to hemin and DMF treatments (I). Markers of inflammation (IL-6, CD68) and oxidative stress (4-HNE, 3-NT) (II) and aortic, cerebral, and cardiac ROS formation (DHE fluorescence) were suppressed (III), leading to subsequent improvement of blood pressure and endothelial function (ACh response) (IV). Created with BioRender.com.