Non-Invasive Local Acoustic Therapy Ameliorates Diabetic Heart Fibrosis by Suppressing ACE-Mediated Oxidative Stress and Inflammation in Cardiac Fibroblasts

Purpose The extent of myocardial fibrosis is closely related to the prognosis of diabetic cardiomyopathy (DCM). Low-intensity pulsed ultrasound (LIPUS) has been reported to have multiple biological effects. However, the effect of LIPUS on diabetic heart fibrosis remains unclear. The present study aimed to investigate the effect of LIPUS on diabetic heart fibrosis and explore its underlying mechanisms. Methods and Results High glucose (HG) was applied to cultured neonatal rat cardiac fibroblasts (NRCFs) to mimic the in vivo hyperglycemia microenvironment. LIPUS (19.30 mW/cm(2) to 77.20 mW/cm(2)) dose-dependently inhibited HG-induced fibrotic response in NRCFs. Also, LIPUS downregulated NADPH oxidase 4 (NOX4)-associated oxidative stress and nod-like receptor protein-3 (NLRP3) inflammasome activation in NRCFs. In vivo, diabetes in mice was induced with streptozotocin (STZ). Mice in the LIPUS group and STZ + LIPUS group were treated with LIPUS (77.20 mW/cm(2)) twice a week for 12 weeks and then euthanized at 12 weeks or 24 weeks post-diabetes. Treatment with LIPUS significantly ameliorated the progression of cardiac fibrosis (Masson staining 6.5 +/- 2.3% vs. 2.8 +/- 1.5%, P < 0.001) and dysfunction (E/A ratio 1.35 +/- 0.14 vs. 1.59 +/- 0.11, P < 0.05), as well as NOX4-associated oxidative stress (relative expression fold of NOX4 1.43 +/- 0.12 vs. 1.07 +/- 0.10, P < 0.01; relative DHE fluorescence 1.51 +/- 0.13 vs. 1.28 +/- 0.06, P < 0.05) and NLRP3 inflammasome activation (relative expression fold of NLRP3 1.57 +/- 0.12 vs. 1.05 +/- 0.16, P < 0.01), at 12 weeks post-diabetes. At 24 weeks post-diabetes, the heart function in diabetic mice treated with LIPUS was still significantly better than untreated diabetic mice (E/A ratio 1.08 +/- 0.12 vs. 1.49 +/- 0.14, P < 0.001). Further exploration revealed that LIPUS significantly attenuated the upregulated angiotensin-converting enzyme (ACE) and angiotensin II (AngII), in both HG-induced NRCFs and diabetic hearts (relative expression of ACE in myocardium 3.77 +/- 0.55 vs. 1.07 +/- 0.13, P < 0.001; AngII in myocardium 115.5 +/- 21.77 ng/ml vs. 84.28 +/- 9.03 ng/ml, P < 0.01). Captopril, an ACE inhibitor, inhibited NOX4-associated oxidative stress and NLRP3 inflammasome activation in both HG-induced NRCFs and diabetic hearts. Conclusion Our results indicate that non-invasive local LIPUS therapy attenuated heart fibrosis and dysfunction in diabetic mice and the effect could be largely preserved at least 12 weeks after suspending LIPUS stimulation. LIPUS ameliorated diabetic heart fibrosis by inhibiting ACE-mediated NOX4-associated oxidative stress and NLRP3 inflammasome activation in cardiac fibroblasts. Our study may provide a novel therapeutic approach to hamper the progression of diabetic heart fibrosis.