Fig 1.
DRLE had a cardioprotective effect.
(A) H2O and DRLE (100 mg/kg/d) and (400 mg/kg/d) were orally administered to C57BL/6 mice for 9 d. At Day 4, 60 mg/kg/d of isoproterenol was injected subcutaneously for 5 d. (B) The survival rate of ISO-injected mice treated with H2O and 100 mg/kg/d and 400 mg/kg/d of DRLE. *, P < 0.05 vs. H2O. #, P < 0.05 vs. 100 mg/kg/d (n = 5 per group).
Fig 2.
DRLE reduced ISO-induced ventricular hypertrophy.
Heart and body weight in ISO-induced mice treated with H2O, 100 and 400 mg/kg/d DRLE (A) Heart weight. (B) Body weight. (C) The image of different sized mouse hearts and heart weight/body weight (mg/g) used to estimate the degree of ventricular hypertrophy. *, P < 0.05 vs. H2O (n = 5 per group).
Fig 3.
DRLE alleviated ISO-induced heart injury.
Pathological examination of heart tissues in normal and ISO-induced mice fed with or without DRLE (H&E, 400X). (A) Normal control; (B) 60 mg/kg/d of ISO; (C) 60 mg/kg/d of ISO + 100 mg/kg/d of DRLE (L-DRLE); (D) 60 mg/kg/d of ISO + 400 mg/kg/d DRLE (H-DRLE); (E) 60 mg/kg/d of ISO (mice dead before sacrifice); and (F) 60 mg/kg/d of ISO + 100 mg/kg/d of DRLE (mice dead before sacrifice).
Fig 4.
DRLE prevented ISO-induced elevation of CPK, LDH, GOT, and TNF-α, and elevated serum NO level.
Serum levels of biomarkers and cytokines in normal and ISO-induced mice treated with H2O, 100 and 400 mg/kg/d DRLE. (A) CPK (B) LDH (C) GOT. (D) IL-10 (E) TNF-α (F) NO.*, P < 0.05 vs. H2O; **, P < 0.01 vs. H2O; ***, P < 0.001 vs. H2O (n = 3 to 5 per group).
Fig 5.
DRLE had a direct vasodilatory effect on porcine coronary arterial rings.
Porcine left arterial descending coronary arteries were treated with different DRLE doses of 0.05 mg/mL, 0.5 mg/mL, and 5 mg/mL to observe the direct vasodilatory effect. **, P < 0.01 vs. 0.05 mg/mL; #, P < 0.05 vs. 0.5 mg/mL (n = 3 to 5 per group).
Table 1.
Body Weight and Serum Biochemical Markers in Oral Toxicity Test of DRLE.
Fig 6.
Possible cardioprotective mechanism of DRLE.
Inflammation, oxidation, and NO secretion have been demonstrated to play a critical role in cardiac hypertrophy and myocardial injury. IL-10 can activate STAT3 to inhibit the NF-kB signalling pathway, thereby attenuating cardiac hypertrophy. TNF-alpha is associated with the AKT and JNK pathways in enhancing NF-kB activation to augment cardiac hypertrophy. TNF-alpha can also upregulate ROI expression to enhance myocyte hypertrophy. Another study reported that eNOS upregulation can attenuate myocardial injury. In our study, we first found that DRLE can attenuate TNF-alpha secretion, which might reduce AKT, JNK, and NF-kB signalling pathways, thereby inhibiting cardiac hypertrophy. ROI expression might also be inhibited by DRLE through TNF-alpha stimulation. We assumed that the direct inhibition of ROI is a potential mechanism for inhibiting cardiac hypertrophy, because the antioxidative effect of DRLE has also been reported. Second, we cannot confirm whether DRLE enhances IL-10 expression, because serum IL-10 level was undetected in our experiment. Third, the NO serum level increased in our study, but we could not determine the origin of the NO, and we postulated that eNOS is a possible candidate. Finally, we found that DRLE can dilate the porcine coronary artery directly in vitro.
Fig 7.
Cardioprotective effect of DRLE.
In this study, we found that DRLE can reduce the mortality rate, cardiac hypertrophy and heart injury in ISO-injured mice. Serum CPK, LDH, GOT, and TNF-alpha were also reduced by DRLE administration. Serum NO levels were induced by ISO injection in mice in vivo, and had the vasodilatory effects in vitro.