Kaempferol inhibits cardiomyocyte pyroptosis via promoting O-GlcNAcylation of GSDME and improved acute myocardial infarction

Abstract

Acute myocardial infarction (AMI) is a leading fatal cardiovascular disease and poses a major threat to human health. Pyroptosis, an inflammation-related programmed cell death, plays a critical role in the progression of AMI. Kaempferol is a natural flavonoid compound with a variety of pharmacological effects, which exerts a significant cardioprotective function. The role of O-GlcNAcylation, a post-translation modification, has received attention in diseases including AMI. In this research, we explored the therapeutic potential of Kaempferol to AMI due to its well-known cardioprotective effect, including its antioxidant and anti-inflammatory properties. Hypoxia/reoxygenation (H/R) model was adopted to provoke myocardial injury and AMI mice model was established. Our findings indicated that H/R lessened cell viability and contributed to the release of LDH, IL-1β and IL-18, cell pyroptosis rate, and the expression of NLRP3, active caspase 1 and GSDMD-N-terminal domain (GSDMD-N). Kaempferol mitigated myocardial damage caused by H/R through repressing cell pyroptosis. Besides, we discovered that Kaempferol restored the levels of O-GlcNAcylation by regulating the activity of OGT (O-GlcNAc transferase) and OGA (O-GlcNAcase) in H/R-treated H9c2 cells. Notably, molecular docking revealed the binding relationship between Kaempferol and OGT. Further, we proved that knockdown of OGT abrogated the function of Kaempferol in H/R-induced pyroptosis. In AMI mice, Kaempferol relieved the myocardial tissue injury and decreased the NLRP3 and GSDME-N protein levels. More importantly, our results illustrated that OGT was responsible for the O-GlcNAcylation of GSDME at T94 site and acted as an inducing factor for GSDME phosphorylation. Namely, this study validated that Kaempferol facilitated GSDME O-GlcNAcylation to inhibit H/R-induced pyroptosis in an OGT-dependent manner.

Keywords: Acute myocardial infarction; GSDME; Kaempferol; O-GlcNAcylation; Pyroptosis.

Fig. 1

Kaempferol decreased cell proliferation following H/R treatment. (A) The molecular structural formula of Kaempferol. (B) Flow chart of cell experimental design. (C) H9c2 cells were induced by H/R and treated with 0, 5, 10, and 20 µM Kae, and then CCK-8 assay was performed to evaluate cell viability. CON, control; H/R, hypoxia/reoxygenation; Kae, Kaempferol. ^**P < 0.01

Fig. 2

Kaempferol restrained H/R-induced cell pyroptosis. For identifying the effects of Kaempferol on cell pyroptosis, H9c2 cells stimulated with H/R to induce injury, and were administered with 20 µM Kaempferol (A) LDH release was measured with LDH detection assay. The concentrations of (B) IL-1β and (C) IL-18 were examined by ELISA assays. (D) Flow cytometry was carried out to analyze cell pyroptosis rate. (E) Western blot analysis of the expression of pyroptosis markers NLRP3, caspase 1-p20 and GSDMD-N. CON, control; H/R, hypoxia/reoxygenation; Kae, Kaempferol; LDH, lactate dehydrogenase; IL-1β, interleukin-1β; IL-18, interleukin-18; PI, propidium iodide; GSDMD-N, GSDMD-N-terminal domain. ^**P < 0.01. ^*P < 0.05

Fig. 3

Kaempferol promoted OGT-mediated O-GlcNAcylation in H/R-treated cardiomyocytes. (A) Following different treatments, western blot was conducted to detect O-GlcNAcylation level and the expression of OGT and OGA. (B-D) Molecular docking results confirmed the strong hydrogen bonding between Kaempferol and OGT. (E, F) SPR analysis was performed to verify the binding between Kaempferol and OGT. CON, control; H/R, hypoxia/reoxygenation; Kae, Kaempferol; SPR, Surface plasmon resonance. ^**P < 0.01. ^*P < 0.05. ns: no significance

Fig. 4

Knockdown of OGT reversed the inhibitory effects of Kaempferol on H/R-induced pyroptosis. (A, B) The RT-qPCR and western blot detection of OGT expression in H9c2 cells transfected with si-nc or si-OGT#1/2. (C) LDH activity and the expression of (D) IL-1β and (E) IL-18 were examined by using corresponding kits. (F) The percent of pyroptotic cells was estimated by flow cytometry following different treatments. (G) The role of OGT in Kaempferol-mediated pyroptosis was also validated by western blot. si-OGT, small interfering RNA targeting OGT; si-nc, siRNA negative control; LDH, lactate dehydrogenase; IL-1β, interleukin-1β; IL-18, interleukin-18; PI, propidium iodide; GSDMD-N, GSDMD-N-terminal domain. ^*P < 0.05, ^**P < 0.01

Fig. 5

OGT functioned as a suppressing factor for GSDME cleavage via increasing its O-GlcNAcylation. (A) Western blot was applied to identify the impacts of OGT overexpression and knockdown on the OGT and total O-GlcNAc levels, as well as O-GlcNAcylation modifications of NLRP3, caspase 1, GSDMD and GSDME. (B) The O-GlcNAcylation sites of GSDME predicted by GlycoMine website. (C, D) To ascertain the site of O-GlcNAcylation modification on GSDME, potential sites were mutated and then OGT, total O-GlcNAc, GSDME O-GlcNAcylation were assessed by western blot. (E) The phosphorylated and cleaved GSDME levels mediated by OGT were also determined with western blot. RL-2, O-GlcNAc; si-nc, small interfering RNA negative control; si-OGT, small interfering RNA targeting OGT; WT, wild-type; p-GSDME(Thr6), phosphorylation of GSDME at Thr6 site; GSDME-N, GSDME-N-terminal domain. ^**P < 0.01. ^*P < 0.05. ns: no significance

Fig. 6

Kaempferol alleviated the AMI progression in vivo. (A) LDH, (B) CK-MB and (C) cTnI levels in the myocardial tissues were detected by kits. (D) EF and (E) FS were measured using transthoracic echocardiography to evaluate the cardiac function of mice. (F) H&E staining of myocardial tissues. Scale bar: 100 μm. magnification: 40×. (G) Protein levels of OGT, p-GSDME(Thr6), GSDME-N and NLRP3 in the myocardial tissues were detected by western blot. (H) The protein levels were quantified by grey analysis. LDH, lactate dehydrogenase; CK-MB, creatine kinase MB isoenzyme; cTnI, cardiac troponin I; EF, ejection fraction; FS, fractional shortening; H&E, Hematoxylin-Eosin; p-GSDME(Thr6), phosphorylation of GSDME at Thr6 site; GSDME-N, GSDME-N-terminal domain. ^**P < 0.01. ^*P < 0.05