Magnolol reduces myocardial injury induced by renal ischemia and reperfusion

Fig. 1. The chemical structure of magnolol and the experimental protocol. A, The chemical structure of magnolol. B, The experimental protocol. The rats in group 1 underwent the same surgical procedures as the rats in the other five groups, and an intravenous infusion of normal saline (1 mL/kg) for 10 min, without bilateral renal artery occlusion (BRAO) or reperfusion (BRAR). The rats in group 2 received an intravenous infusion of normal saline (1 mL/kg) for 10 min. The rats in group 3 received an intravenous administration of 1 mL/kg of 4 × 10−3% (v/v) propylene glycol (PG) for 10 min. The rats in group 4 received an intravenous administration of 0.001 mg/kg of magnolol for 10 min. The rats in group 5 received an intravenous administration of 0.003 mg/kg of magnolol for 10 min. The rats in group 6 received an intravenous administration of 0.006 mg/kg of magnolol for 10 min. Ten minutes after the above pretreatments, all rats except those in group 1 underwent a 60-min BRAO and a 24-h BRAR. Because there have not been any previous reports like our current study, there is no reference substance or treatment that has been previously established as having any effect when tested by the assay…

Fig. 2. Acute kidney injury induced by renal ischemia and reperfusion. A, The blood urea nitrogen level. B, The creatinine level. C, Representative photomicrographs of hematoxylin and eosin staining of kidney sections (×200 magnification). The renal glomeruli and tubules of the kidneys of the rats in group 1 appear normal. Necrosis of renal glomeruli (red arrows) and tubules (black arrows) was found in the kidneys of the rats in groups 2 and 3. Dilatation of Bowman’s capsule (red arrowheads) and necrosis of renal tubules were present in the kidneys of the rats in group 4. Dilatation of Bowman’s capsule and renal tubules (black arrowheads) were found in the kidneys of the rats in groups 5 and 6. D, Histological injury scoring of the renal glomeruli. E, Histological injury scoring of the renal tubules; *p < 0.001 vs group 1; $p < 0.05 vs group 1; #p < 0.001 vs group 2; ◆p < 0.05 vs group 2; §p < 0.001 vs group 3; ☆p < 0.05 vs group 3; ★p < 0.001 vs group 4; and ◎p < 0.01 vs group 4. Blood from six rats from each group was collected for analysis of blood urea nitrogen and creatinine levels at the end of the experiments. The kidneys of four rats from group 1 and 5 rats each from group 2…

Fig. 3. Biochemical analysis of myocardial injury. A, The creatine phosphokinase level. B, The cardiac troponin I level. C, The lactate dehydrogenase level. *p < 0.001 vs group 1; +p < 0.01 vs group 1; $p < 0.05 vs group 1; #p < 0.001 vs group 2; &p < 0.01 vs group 2; ※p < 0.01 vs group 3; ◎p < 0.01 vs group 4; and ◇p < 0.05 vs group 4. Blood from six rats from each group was collected for analysis at the end of the experiments.

Fig. 4. Echocardiographic evaluation of the left ventricular function. A, Representative M-mode echocardiograms. B, The left ventricular ejection fraction (LVEF). C, The left ventricular fractional shortening (LVFS). *p < 0.001 vs group 1; +p < 0.01 vs group 1; $p < 0.05 vs group 1; #p < 0.001 vs group 2; &p < 0.01 vs group 2; §p < 0.001 vs group 3; ★p < 0.001 vs group 4; ◎p < 0.01 vs group 4; and ◇p < 0.05 vs group 4. Five rats from each group received the transthoracic echocardiography examination after 24-h bilateral renal artery reperfusion.

Fig. 5. Histological examination of myocardial injury. A, Representative photomicrographs of hematoxylin and eosin staining of myocardial sections (×200 magnification). The myocardium in the left ventricles of the rats in group 1 appeared normal. Renal I/R resulted in interstitial edema, myocardial cell swelling, and disruption of myocardial fibers in group 2. Administration of magnolol at doses of 0.003 and 0.006 mg/kg ameliorated the histological changes in the myocardium induced by renal I/R (groups 5 and 6). B, Histological injury scoring of the myocardium. $p < 0.05 vs group 1; ◆p < 0.05 vs group 2. The left ventricles of four rats from group 1 and 5 rats each from group 2 through group 6 were used for histological examination of myocardial injury at the end of the experiments.

Fig. 6. Analysis of apoptosis in the myocardium. A, Representative photomicrographs of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining of the myocardium (×400 magnification). Dark brown staining (red arrows) indicates TUNEL-positive nuclei. B, The percentage of TUNEL-positive nuclei. C, Assay of activated caspase-3 in the myocardium. A representative Western blot of activated caspase-3 is shown (top). The density of activated caspase-3 was analyzed using arbitrary units (AU; bottom). *p < 0.001 vs group 1; $p < 0.05 vs group 1; #p < 0.001 vs group 2; §p < 0.001 vs group 3; ※p < 0.01 vs group 3; ★p < 0.001 vs group 4; and ◎p < 0.01 vs group 4. The left ventricles of four rats from group 1 and five rats each from group 2 through group 6 were used for TUNEL staining. The left ventricles of four rats from each group were used for the Western blot analysis of activated caspase-3.

Fig. 7. Analysis of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-10. A, Serum level of tumor necrosis factor-α (TNF-α). B, Serum level of interleukin-1β. C, Serum level of interleukin-6. D, Serum level of interleukin-10. E, Assay of TNF-α in the myocardium. A representative Western blot of TNF-α is shown (top). The density of TNF-α was analyzed using arbitrary units (AU; bottom). F, Correlation between the TNF-α level in the serum and the myocardium (r = 0.900, p < 0.01). *p < 0.001 vs group 1; $p < 0.05 vs group 1; #p < 0.001 vs group 2; &p < 0.01 vs group 2; ◆p < 0.05 vs group 2; §p < 0.001 vs group 3; ※p < 0.01 vs group 3; ★p < 0.001 vs group 4; ◎p < 0.01 vs group 4; ◇p < 0.05 vs group 4; and ▼p < 0.01 vs group 5. Blood was collected from six rats in each group to determine the serum levels of TNF-α, interleukin-1β, interleukin-6, and interleukin-10. The left ventricles of four rats from each group were used for the Western blot analysis of TNF-α in the myocardium.

Fig. 8. Western blot analysis of Bcl-2, Bax, Akt, ERK, p38, and JNK. A, Bcl-2 assay. B, Bax assay. C, Phospho-Akt (p-Akt) and total Akt (t-Akt) assay. D, Phospho-extracellular signal-regulated kinases 1 and 2 (p-ERK) and total ERK (t-ERK) assay. E, Phospho-p38 (p-p38) and total p38 (t-p38) assay. F, Phospho-c-Jun N-terminal kinase (p-JNK) and total JNK (t-JNK) assay. Representative Western blots of Bcl-2, Bax, p-Akt, t-Akt, p-ERK, t-ERK, p-p38, t-p38, p-JNK, and t-JNK are shown (top). The densities of Bcl-2, Bax, p-Akt, p-ERK, p-p38, and p-JNK bands were analyzed using arbitrary units (AU; bottom). *p < 0.001 vs group 1; $p < 0.05 vs group 1; #p < 0.001 vs group 2; &p < 0.01 vs group 2; §p < 0.001 vs group 3; ※p < 0.01 vs group 3; ★p < 0.001 vs group 4; and ◇p < 0.05 vs group 4. The left ventricles of 4 rats from each group were used for the Western blot analysis of Bcl-2, Bax, p-Akt, t-Akt, p-ERK, t-ERK, p-p38, t-p38, p-JNK, and t-JNK in the myocardium.