Rosmarinic acid protects against hypertensive nephropathy by inhibiting NADPH oxidase/ROS/NLRP3 inflammasome pathway

Abstract

Objective To investigate the protective effect of rosmarinic acid (RA) on hypertensive nephropathy in mice and its underlying mechanism. Methods Thirty male C57BL/6 mice (6~8 weeks) were randomly divided into 3 groups: control group, AngⅡ group (Angiotensin II, 1.46 mg·kg-1·d-1) and AngⅡ+RA group (AngiotensinⅡ1.46 ·kg-1·d-1 + RA50 mg·kg-1·d-1), with 10 mice in each group, and were intervened for 4 weeks. Blood pressure, urinary microalbumin, serum creatinine and blood urea nitrogen were detected. Immunofluorescence assay and Masson staining were performed respectively to observe the macrophage infiltration and the severity of renal fibrosis. In addition, the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was determined using NADP+/NADPH test kit; and the level of reactive oxygen species (ROS) in renal tissue was tested by dihydroethidium (DHE) staining. Finally, the expression levels of NADPH oxidase subunits (p22phox, p47phox) and the following proteins were detected by RT-qPCR and western blotting: nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), Caspase-1, IL-1β and IL-18. Results As compared with the control group, the indicators of blood pressure, urinary microalbumin, serum creatinine and blood urea nitrogen, as well as inflammatory reaction and fibrosis severity of kidney were all significantly increased in AngⅡgroup (P < 0.05). However, RA treatment significantly reversed all the above changes (P < 0.05). Meanwhile, RA also decreased the expression of NADPH oxidase subunits (p22phox and p47phox) and the activity of NADPH oxidase(P < 0.05). The level of ROS in renal tissue and the expression of NLRP3, ASC, Caspase-1, IL-1β and IL-18 were all reduced as well in AngⅡ+RA group. Conclusion RA may play a protective role in hypertensive nephropathy by inhibiting the activation of NADPH oxidase/ROS/NLRP3 inflammasome pathway.

Keywords

• rosmarinic acid
• hypertensive nephropathy
• nlrp3 inflammasome
• nadph oxidase
• reactive oxygen species