β-Caryophyllene as a Potential Protective Agent Against Myocardial Injury: The Role of Toll-Like Receptors

Abstract

Myocardial infarction (MI) remains one of the major causes of mortality around the world. A possible mechanism involved in myocardial infarction is the engagement of Toll-like receptors (TLRs). This study was intended to discover the prospective cardioprotective actions of β-caryophyllene, a natural sesquiterpene, to ameliorate isoproterenol (ISO)-induced myocardial infarction through HSP-60/TLR/MyD88/NFκB pathway. β-Caryophyllene (100 or 200 mg/kg/day orally) was administered for 21 days then MI was induced via ISO (85 mg/kg, subcutaneous) on 20th and 21st days. The results indicated that ISO induced a significant infarcted area associated with several alterations in the electrocardiogram (ECG) and blood pressure (BP) indices and caused an increase in numerous cardiac indicators such as creatine phosphokinase (CPK), creatine kinase-myocardial bound (CK-MB), lactate dehydrogenase (LDH), and cardiac tropinine T (cTnT). In addition, ISO significantly amplified heat shock protein 60 (HSP-60) and other inflammatory markers, such as TNF-α, IL-Iβ, and NFκB, and affected TLR2 and TLR4 expression and their adaptor proteins; Myeloid differentiation primary response 88 (MYD88), and TIR-domain-containing adapter-inducing interferon-β (TRIF). On the other hand, consumption of β-caryophyllene significantly reversed the infarcted size, ECG and BP alterations, ameliorated the ISO elevation in cardiac indicators; it also notably diminished HSP-60, and subsequently TLR2, TLR4, MYD88, and TRIF expression, with a substantial reduction in inflammatory mediator levels. This study revealed the cardioprotective effect of β-caryophyllene against MI through inhibiting HSP-60/TLR/MyD88/NFκB signaling pathways.

Keywords: MyD88; heat shock protein 60; isoproterenol; toll-like receptors; β-caryophyllene.

Figure 1

β-Caryophyllene (BCP) structure.

Figure 2

Representative pictures of the TTC-stained rat-heart slices showing the effects of β-caryophyllene treatment (100, 200 mg/kg) for 21 days on myocardial infarction size in ISO-induced myocardial infarction. All values were expressed as mean ± SD (n = 6). ISO: isoproterenol; BCP: β-Caryophyllene; TTC: triphenyl tetrazolium chloride. # indicates statistically significant from normal group, * indicates statistically significant from isoproterenol group, @ indicates statistically significant from β-caryophyllene (100 mg/kg) group (p < 0.05) using one way ANOVA followed by Tukey’s test as a post-hoc analysis.

Figure 3

Representative traces of the electrocardiographic (ECG) showing the effects of β-caryophyllene treatment (100, 200 mg/kg) for 21 days on ECG components in ISO-induced myocardial infarction. All values were expressed as mean  ±  SD (n = 6). ISO: isoproterenol and BCP: β-Caryophyllene. # indicates statistically significant from normal group, * indicates statistically significant from isoproterenol group, @ indicates statistically significant from β-caryophyllene (100 mg/kg) group (p < 0.05) using one way ANOVA followed by Tukey’s test as a post-hoc analysis.

Figure 4

Upper section: effects of β-caryophyllene treatment (100, 200 mg/kg) for 21 days on Blood Pressure (BP) indices in ISO-induced myocardial infarction: (A) SAP; (B) DAP; (C) MAP; (D) HR. Lower section: shows representative computerized traces of the β-caryophyllene effects on BP. All values were expressed as mean ± SD (n = 6). ISO: isoproterenol; BCP: β-Caryophyllene, SAP: systolic arterial pressure; DAP: diastolic arterial pressure; MAP: mean arterial pressure; and HR: heart rate. # indicates statistically significant from normal group, * indicates statistically significant from ISO group, @ indicates statistically significant from BCP (100 mg/kg) group (p < 0.05) using one-way ANOVA followed by Tukey’s test as a post hoc analysis.

Figure 5

Effects of β-caryophyllene treatment (100, 200 mg/kg) for 21 days on cardiac marker enzymes in ISO-induced myocardial infarction: (A) CPK, (B) CK-MB, (C) LDH, and (D) cTnT. All values were expressed as mean ± SD (n = 6). ISO: isoproterenol; BCP: β-Caryophyllene; CPK: Creatine Phosphokinase; CK-MB: Creatine Kinase-Myocardial Bound; LDH: Lactate dehydrogenase and cTnT: Cardiac Troponin T. # indicates statistically significant from normal group, * indicates statistically significant from isoproterenol group, @ indicates statistically significant from β-caryophyllene (100 mg/kg) group (p < 0.05) using one-way ANOVA followed by Tukey’s test as a post-hoc analysis.

Figure 6

Effects of β-caryophyllene treatment (100, 200 mg/kg) for 21 days on the TLR pathway in ISO-induced myocardial infarction; (A) TLR2 mRNA, (B) TLR4 mRNA, (C) MyD88 mRNA, and (D) TRIF mRNA expression levels. All values were expressed as mean ± SD. ISO: isoproterenol; BCP: β-Caryophyllene and TLR: Toll-like Receptors. # indicates statistically significant from normal group, * indicates statistically significant from isoproterenol group, @ indicates statistically significant from β-caryophyllene (100 mg/kg) group (p < 0.05) using one-way ANOVA followed by Tukey’s test as a post-hoc analysis.

Figure 7

Effects of β-caryophyllene treatment (100, 200 mg/kg) for 21 days on TLR pathway protein content in ISO-induced myocardial infarction: (A) TLR2, (B) TLR4, (C) MyD88, and (D) TRIF protein content. Upper section: representative immunoblots of TLR4, TLR2, MyD88, and TRIF protein content in the normal control heart tissues and in the hearts after isoproterenol-induced myocardial infarction in the absence or presence of pre-treatment with the graded doses of β-caryophyllene. Lower section: Densitometric analysis of TLR4, TLR2, MyD88, and TRIF. Values are mean ± S.E.M (n = 6) for the ratio of TLR4, TLR2, MyD88, and TRIF to β-actin. ISO: isoproterenol and BCP: β-Caryophyllene; # indicates statistically significant from normal group, * indicates statistically significant from isoproterenol group, @ indicates statistically significant from β-caryophyllene (100 mg/kg) group (p < 0.05) using one-way ANOVA followed by Tukey’s test as a post-hoc analysis.

Figure 8

Effects of β-caryophyllene treatment (100, 200 mg/kg) for 21 days on HSP-60 and inflammatory markers in ISO-induced myocardial infarction: (A) HSP-60, (B) TNF-α, (C) IL-1β, and (D) NF- κB. All values were expressed as mean ± SD (n = 6). ISO: isoproterenol; BCP: β-Caryophyllene; HSP-60: Heat shock protein 60; TNF-α: Tumor necrosis factor–alpha; IL-Iβ: Interleukin-1β and NFκB: nuclear factor-κB. # indicates statistically significant from normal group, * indicates statistically significant from isoproterenol group, @ indicates statistically significant from the β-caryophyllene (100 mg/kg) group (p < 0.05) using one-way ANOVA followed by Tukey’s test as a post-hoc analysis.

Figure 9

Suggested roll of β-caryophyllene on Toll-like receptor signaling. HSP-60: Heat shock protein 60; TLR2: Toll-like receptor 2; MYD88: Myeloid differentiation primary response gene 88; TRIF: TIR-domain-containing adapter-inducing interferon-β; and NFκB: Nuclear Factor-κB.