Puerarin mitigated LPS-ATP or HG-primed endothelial cells damage and diabetes-associated cardiovascular disease via ROS-NLRP3 signalling

Abstract

The occurrence and development of diabetic vascular diseases are closely linked to inflammation-induced endothelial dysfunction. Puerarin (Pue), the primary component of Pueraria lobata, possesses potent anti-inflammatory properties. However, its vasoprotective role remains elusive. Therefore, we investigated whether Pue can effectively protect against vascular damage induced by diabetes. In the study, Pue ameliorated lipopolysaccharide-adenosine triphosphate (LPS-ATP) or HG-primed cytotoxicity and apoptosis, while inhibited reactive oxygen species (ROS)-mediated NLR family pyrin domain containing 3 (NLRP3) inflammasome in HUVECs, as evidenced by significantly decreased ROS level, NOX4, Caspase-1 activity and expression of NLRP3, GSDMD, cleaved caspase-1, IL-1β and IL-18. Meanwhile, ROS inducer CoCI₂ efficiently weakened the effects of Pue against LPS-ATP-primed pyroptosis. In addition, NLRP3 knockdown notably enhanced Pue's ability to suppress pyroptosis in LPS-ATP-primed HUVECs, whereas overexpression of NLRP3 reversed the inhibitory effects of Pue. Furthermore, Pue inhibited the expression of ROS and NLRP3 inflammasome-associated proteins on the aorta in type 2 diabetes mellitus rats. Our findings indicated that Pue might ameliorate LPS-ATP or HG-primed damage in HUVECs by inactivating the ROS-NLRP3 signalling pathway.

Keywords: Puerarin; ROS; diabetic vasculopathy; pyroptosis.

FIGURE 1

Pue attenuated LPS‐ATP‐primed HUVECs apoptosis HUVECs were pre‐treated with varying concentration of Pue for 2 h, then primed with 500 ng/mL LPS for 24 h and then stimulated with 5 mM ATP for 30 min. (A, B) The cell viability were detected using CCK8 assay kit. (C) The LDH release was measured using LDH assay kit. (D) The cell growth was examined by EdU assay kit. (E) Annexin FITC‐PI assay was performed to measure cell apoptosis. (F) Mitochondrial membrane potential was examined using JC‐1 assay kit. (G) The ratio of p‐eNOS‐eNOS were detected using western blot. Data are expressed as means ± SD. *p < 0.05.

FIGURE 2

Pue repressed LPS‐ATP‐primed HUVECs pyroptosis. HUVECs were pre‐treated with varying concentrations of Pue for 2 h, then primed with 500 ng/mL LPS for 24 h and then stimulated with 5 mM ATP for 30 min (A) The activity of Caspase‐1 was tested using Caspase‐1 activity assay kit. (B–F) Western blot were performed to detect the protein expression of NLRP3, GSDMD, Cleaved Caspase‐1, Caspase‐1, IL‐1β and IL‐18. (G–K) The mRNA expression of NLRP3, GSDMD, Cleaved Caspase‐1, IL‐1β and IL‐18 were measured using RT‐PCR. (L, M) The release of IL‐1β and IL‐18 in cell supernatant were examined using ELISA kits. (N) The expression of NLRP3 was examined using immunofluorescence. Scale = 7.5 μm. Data are expressed as means ± SD. *p < 0.05.

FIGURE 3

Pue repressed NLRP3 inflammasome activation and pyroptosis by repressing ROS generation. HUVECs were pre‐treated with varying concentrations of Pue or 500 CoCI₂ for 2 h before primed with HUVECs (A) DCFH‐DA probe was subjected to examine the production of ROS. (B) The expression of NOX4 was detected using western blot. (C, D) The release of LDH and Caspase‐1 activity were measured by detection kits. (E–I) The levels of pyroptosis‐associated protein containing NLRP3, Cleaved Caspase‐1‐Caspase‐1, GSDMD, IL‐1β, and IL‐18 were determined by western blot. (J, K) The release of IL‐1β and IL‐18 in cell supernatant were determined by ELISA kit. Data are expressed as means ± SD. *p < 0.05.

FIGURE 4

Pue alleviated pyroptosis dependent on NLRP3 inflammasome. HUVECs were transfected with NLRP3 siRNA for 24 h, then exposed to Pue for 2 h and following primed with 500 ng/mL LPS for 24 h and eventually stimulated with 5 mM ATP for 30 min. (A, B) The release of LDH and Caspase‐1 activity were measured by detection kits. (C–G) The levels of pyroptosis‐associated proteins containing NLRP3, Cleaved Caspase‐1‐Caspase‐1, GSDMD, IL‐1β and IL‐18 were determined by western blot. (H, I) The release of IL‐1β and IL‐18 in cell supernatant were determined by ELISA kits. (J) The expression of NLRP3 was examined using immunofluorescence. Scale = 7.5 μm. Data are expressed as means ± SD. *p < 0.05.

FIGURE 5

Pue treatment attenuated apoptosis in the aorta of T2DM rats T2DM rats were administrated with Pue (100 mg/kg/day or 150 mg/kg/day, i.g.) for 4 weeks (A) Schematic of the study design depicting 6‐week‐old male rats. (B) H&E staining was performed to detect aorta wall (Scale bar = 100 μm) (C, D) Immunohistochemistry staining and western blot were subjected to measure the expression of Bax‐Bcl‐2 in aorta of T2DM rats. (E) The samples were sectioned and processed using a TUNEL staining kit. (Scale bar = 50 μm) (F) Immunofluorescence staining was employed to detect the expression of CD31 (Scale bar = 100 μm). Data are expressed as means ± SD. *p < 0.05.

FIGURE 6

Pue suppressed pyroptosis in the aorta of T2DM rats T2DM rats were administrated with Pue (100 mg/kg/day or 150 mg/kg/day, i.g.) for 4 weeks (A) The activity of Caspase‐1 was measured by Caspase‐1 activity assay kit. (B) Immunohistochemistry staining were used to measure the expression level of NLRP3 in aorta wall. (C) Western blot was performed to detect the expression level of NLRP3, GSDMD, Cleaved Caspase‐1, Caspase‐1, IL‐1β and IL‐18 (D) The release of IL‐1β and IL‐18 in the serum samples were determined by ELISA kit. (E) Frozen aortic sections were incubated with 5 μmol/L DHE for 20 min and analysed using fluorescence microscopy (Scale bar = 100 μm). Data are expressed as means ± SEM. *p < 0.05.