Didymin, a natural flavonoid, relieves the progression of myocardial infarction via inhibiting the NLR family pyrin domain containing 3 inflammasome

Abstract

Context: Globally, the morbidity and mortality of cardiovascular diseases remain high. Didymin, a flavonoid glycoside, has long been used as a dietary antioxidant.

Objective: To determine the role of didymin in myocardial infarction (MI), and its possible myocardial protective mechanism.

Materials and methods: C57/BL6 mice (aged 6-8 weeks, n = 40) were divided into five groups: sham group, ischaemia-reperfusion (I/R) group, I/R + didymin (1 mg/kg) group, I/R + didymin (2 mg/kg) group and I/R + didymin (4 mg/kg) group. Didymin was administered intragastrically daily before I/R for 5 consecutive days. H9C2 cells were divided into five groups: control group, H/R group, H/R + didymin (3 μM) group, H/R + didymin (10 μM) group and H/R + didymin (30 μM) group. H9C2 cells were treated with didymin for 24 h before hypoxia/reoxygenation (H/R).

Results: In vivo, didymin reduced the pathological damage and fibrosis of myocardial tissues, decreased the levels of lactate dehydrogenase, creatine kinase, connective tissue growth factor, collagen I and collagen III. Moreover, didymin reduced myocardial apoptosis, inhibited NLRP3, ASC and caspase-1 expression, and alleviated the inflammatory response. In vitro, didymin reduced MI, apoptosis, inflammation and the levels of NLRP3, ASC and caspase-1 in H9C2.

Discussion and conclusions: Didymin prevented the deterioration of MI by inhibiting NLRP3 inflammasome in vivo and in vitro, and may be a potential natural drug for the treatment of MI. Our study provides the scientific basis for further research of didymin.

Keywords: Ischaemia–reperfusion; NOD-like receptor protein 3; fibrosis.

Figure 1.

Didymin relieved myocardial infarction injury after I/R. (A) The echocardiography of mice in indicated groups; (B, C) the change of ultrasonic indexes; (D, E) the change of haemodynamics indexes; (F, G) the levels of LDH and CK in myocardial tissue. **p < 0.01 vs. sham group; ^##p < 0.01 vs. I/R group.

Figure 2.

Didymin relieved cardiac fibrosis caused by myocardial infarction. (A) H&E staining of myocardial tissues; (B) Masson staining of myocardial tissues; (C–F) Western blot was used to detect fibrotic factors in myocardial tissue. **p < 0.01 vs. sham group; ^#p < 0.05, ^##p < 0.01 vs. I/R group.

Figure 3.

Didymin reduced apoptosis induced by myocardial infarction. (A) TUNEL staining of myocardial tissues; (B–D) Western blot was used to measure the levels of apoptosis factors in myocardial tissue; **p < 0.01 vs. sham group; ^##p < 0.01 vs. I/R group.

Figure 4.

Didymin relieved inflammation caused by myocardial infarction. (A) The levels of inflammatory factors in myocardial tissue were measured by ELISA; (B) PCR was used to measure inflammatory factors in myocardial tissue. **p < 0.01 vs. sham group; ^##p < 0.01 vs. I/R group.

Figure 5.

Didymin relieves myocardial infarction by inhibiting NLRP3 inflammasome. (A–F) The expression of NLRP3, ASC, caspase-1, IL-1β and IL-18 in myocardial tissue was determined by western blot. **p < 0.01 vs. sham group; ^#p < 0.05, ^##p < 0.01 vs. I/R group.

Figure 6.

Didymin alleviates H/R induced cardiomyocyte injury by inhibiting NLRP3 inflammasome. (A) Cell viability was measured by MTT; (B) the LDH level in H9C2 was detected via commercial kits; (C, D) cell apoptosis was detected by flow cytometry; (E–J) the expression of related proteins was detected by western blot. **p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01 vs. H/R group.