Korean J Physiol Pharmacol.  2023 Mar;27(2):143-155. 10.4196/kjpp.2023.27.2.143.

Resveratrol pretreatment alleviates NLRP3 inflammasomemediated cardiomyocyte pyroptosis by targeting TLR4/MyD88/ NF-κB signaling cascade in coronary microembolization-induced myocardial damage

• Luo CJ
• Li T
• Li HL
• Zhou Y
• Li L

• KMID: 2539569
• DOI: http://doi.org/10.4196/kjpp.2023.27.2.143

Abstract

Percutaneous coronary intervention and acute coronary syndrome are both closely tied to the frequently occurring complication of coronary microembolization (CME). Resveratrol (RES) has been shown to have a substantial cardioprotective influence in a variety of cardiac diseases, though its function and potential mechanistic involvement in CME are still unclear. The forty Sprague–Dawley rats were divided into four groups randomly: CME, CME + RES (25 mg/kg), CME + RES (50 mg/kg), and sham (10 rats per group). The CME model was developed. Echocardiography, levels of myocardial injury markers in the serum, and histopathology of the myocardium were used to assess the function of the cardiac muscle. For the detection of the signaling of TLR4/MyD88/NF-κB along with the expression of pyroptosisrelated molecules, ELISA, qRT-PCR, immunofluorescence, and Western blotting were used, among other techniques. The findings revealed that myocardial injury and pyroptosis occurred in the myocardium following CME, with a decreased function of cardiac, increased levels of serum myocardial injury markers, increased area of microinfarct, as well as a rise in the expression levels of pyroptosis-related molecules. In addition to this, pretreatment with resveratrol reduced the severity of myocardial injury after CME by improving cardiac dysfunction, decreasing serum myocardial injury markers, decreasing microinfarct area, and decreasing cardiomyocyte pyroptosis, primarily by blocking the signaling of TLR4/MyD88/NF-κB and also reducing the NLRP3 inflammasome activation. Resveratrol may be able to alleviate CME-induced myocardial pyroptosis and cardiac dysfunction by impeding the activation of NLRP3 inflammasome and the signaling pathway of TLR4/MyD88/NF-κB.

Keyword

Coronary microembolization; NLRP3; Pyroptosis; Resveratrol; TLR4/MyD88/NF-κB

Figure

• 

  Fig. 1 Cardiac function-based indices of rats were measured by echocardiography. (A) Representative M-mode images of each group. (B–E) LVEF, LVFS, LVEDd, and LVESd in each group (n = 8 per group). Low-dose: CME + low-dose RES (25 mg/kg), High-dose: CME + high-dose RES (50 mg/kg). Data were presented as the mean ± SD. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVEDd, left ventricular end-diastolic diameter; LVESd, left ventricular end-systolic diameter; CME, coronary microembolization; RES, resveratrol. *p < 0.05, compared with Sham group; #p < 0.05, compared with CME group; +p < 0.05, compared with Low-dose group.

• 

  Fig. 2 Resveratrol reduced the serum levels of myocardial injury markers. (A) cTnI levels in serum. (B) CK-MB levels in serum. (C) LDH levels in serum (n = 8 per group). Low-dose: CME + low-dose RES (25 mg/kg), High-dose: CME + high-dose RES (50 mg/kg). Data were presented as the mean ± SD. cTnI, cardiac troponin I; CK-MB, creatine kinase myocardial band isoenzyme; LDH, lactate dehydrogenase; CME, coronary microembolization; RES, resveratrol. *p < 0.05, compared with Sham group; #p < 0.05, compared with CME group; +p < 0.05, compared with Low-dose group.

• 

  Fig. 3 Histopathological examination of myocardial tissues through H&E and HBFP staining. (A) Representative H&E-stained myocardial sections from each group (×200 magnification; bar = 100 μm). The arrows indicate the microspheres. (B) Representative images of HBFP staining (×200 magnification; bar = 100 μm). The arrows indicate the microinfarct area. (C) The percentage of micro-infarct area in each group (n = 8 per group). Low-dose: CME + low-dose RES (25 mg/kg), High-dose: CME + high-dose RES (50 mg/kg). Data were presented as the mean ± SD. CME, coronary microembolization; RES, resveratrol. *p < 0.05, compared with Sham group; #p < 0.05, compared with CME group; +p < 0.05, compared with Low-dose group.

• 

  Fig. 4 Resveratrol pretreatment attenuated CME-induced mitochondrial injury. (A) Myocardial mitochondrial morphology observed by transmission electron microscopy (magnification ×60,000, scale bar = 500 nm). The black arrows indicate the typical vacuolated degeneration and enlarged mitochondria. (B) ATP concentrations of each group (n = 8 per group). Low-dose: CME + low-dose RES (25 mg/kg), High-dose: CME + high-dose RES (50 mg/kg). Data were presented as the mean ± SD. CME, coronary microembolization; RES, resveratrol. *p < 0.05, compared with Sham group; #p < 0.05, compared with CME group; +p < 0.05, compared with Low-dose group.

• 

  Fig. 5 Resveratrol attenuated CME-induced pyroptosis of cardiomyocytes. (A, B) Serum levels of IL-1β and IL-18 in rats of each group (n = 8 per group). (C) mRNA levels of NLRP3 in each group (n = 8 per group). (D) Representative Western blot bands of NLRP3, ASC, caspase-1 p20, GSDMD-FL, GSDMD-N, IL-1β, and IL-18. (E–J) The relative expression levels of these proteins in each group. GAPDH was used for protein expression normalization (n = 3 per group). Low-dose: CME + low-dose RES (25 mg/kg), High-dose: CME + high-dose RES (50 mg/kg). Data were presented as the mean ± SD. IL, interleukin; NLRP3, nod-like receptor protein 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CME, coronary microembolization; RES, resveratrol. *p < 0.05, compared with Sham group; #p < 0.05, compared with CME group; +p < 0.05, compared with Low-dose group.

• 

  Fig. 6 Immunofluorescence staining of NLRP3 and caspase-1 p20 in cardiac tissues. (A) Representative NLRP3 immunofluorescence staining images of each group. (B) Representative caspase-1 p20 immunofluorescence staining images of each group (magnification ×400, scale bar = 100 μm). Low-dose: CME + low-dose RES (25 mg/kg), High-dose: CME + high-dose RES (50 mg/kg). NLRP3, nod-like receptor protein 3; CME, coronary microembolization; RES, resveratrol.

• 

  Fig. 7 Resveratrol inhibited pyroptosis via the pathway of TLR4/MyD88/NF-κB. (A) Representative Western blot bands of TLR4, MyD88, p-NF-κB p65. (B) The mRNA levels of TLR4 in each group (n = 8 per group). (C–E) Relative protein expression levels in each group. GAPDH was used for protein expression normalization (n = 3 per group). (F) Representative TLR4 immunofluorescence staining in each group (magnification ×400, scale bar = 100 μm). Low-dose: CME + low-dose RES (25 mg/kg), High-dose: CME + high-dose RES (50 mg/kg). Data were presented as the mean ± SD. TLR4, toll-like receptor 4; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor-kappa B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CME, coronary microembolization; RES, resveratrol. *p < 0.05, compared with Sham group; #p < 0.05, compared with CME group; +p < 0.05, compared with Low-dose group.

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