The role of pterostilbene (Pte) in inflammation induced by ischemia/reperfusion is not well understood. The aim of this study was to investigate whether Pte modulates neutrophil accumulation and the induction of tumor necrosis factor-α (TNF-α) in an ischemia/reperfusion (I/R)-injured rat heart model. Rats were randomly exposed to a sham operation, myocardial ischemia/reperfusion (MI/R) alone, MI/R+Pte, MI/R+Pte+L-NAME and MI/R+Pte+ (methylene blue) MB. The results demonstrated that compared with MI/R, Pte reduced the area of myocardial infarction, the levels of myocardial myeloperoxidase, serum creatinine kinase and lactate dehydrogenase, and the production of serum and myocardial TNF-α. These Pte-induced effects were eliminated by the administration of L-NAME, a nitric oxide (NO) synthase inhibitor, and MB, a cyclic guanosine monophosphate (cGMP) inhibitor. In conclusion, Pte produces cardioprotective and anti-inflammatory effects. These effects may be associated with an increase in NO production, the inhibition of neutrophil accumulation, and induction of TNF-α and cGMP signaling pathways in myocardium subjected to MI/R.
The inflammatory reaction induced by ischemia/reperfusion (I/R) is an important process in the development of myocardial ischemia-reperfusion (MI/R) injury (
Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene; Pte) is a naturally-derived compound found primarily in blueberries and
Pte was obtained from Sigma (St. Louis, MO, USA). The myeloperoxidase (MPO) assay kits, creatine kinase (CK) test kits and lactate dehydrogenase (LDH) assay kits were purchased from JianCheng Bioengineering Institute (Nanjing, China). TNF-α enzyme-linked immunosorbent assay kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). L-NAME and methylene blue (MB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A bicinchoninic acid (BCA) protein quantification kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
Fifty adult male Sprague-Dawley rats (250–300 g) were purchased from the Center of Experimental Animal in Jilin University (Changchun, China). All animals used in this study were cared for in accordance with the Guidance for the Care and Use of Laboratory Animals published by the United States National Institute of Health (NIH; publication no. 85-23, revised 1996), and all procedures were approved by the Committee of Experimental Animals of Jilin University.
Male Sprague-Dawley rats (weight, 250–300 g) were anesthetized intraperitoneally with 40 mg/kg sodium pentobarbital (Sigma Aldrich). Myocardial ischemia was induced by exteriorizing the heart with a left thoracic incision followed by a slipknot (5-0 silk; Johnson & Johnson China, Ltd., Shanghai, China) around the left anterior descending (LAD) coronary artery. Following 30 min of ischemia, slipknots were released and animals received 120 min of reperfusion.
Rats were randomly assigned to five experimental groups and there were ten rats in each group. The groups were as follows: Sham group, silk was fed under the LAD coronary artery but the LAD coronary artery was not ligated; MI/R group, the LAD coronary artery was ligated for 30 min and then allowed 120 min reperfusion and was treated with vehicle [0.9% NaCl intravenously (i.v.)]; MI/R + Pte group, 100 μmol/l Pte i.v. was administered 5 min prior to reperfusion; MI/R + Pte + L-NAME group, 1 mmol/l L-NAME i.v., a nitric oxide (NO) synthase inhibitor, was administered 20 min prior to reperfusion. At 15 min post-administration of L-NAME, 100 μmol/l Pte, i.v. was administered; and MI/R + Pte + MB group, 50 μmol/l methylene blue (MB) i.v., a cyclic guanosine monophosphate (cGMP) inhibitor, was administered 20 min prior to reperfusion. At 15 min following treatment with MB, 100 μmol/l Pte, i.v. was administered.
Following reperfusion, myocardial infarct size was determined by means of a double-staining technique and a digital imaging system (Adobe Systems Incorporated, San Jose, CA, USA) (
Following reperfusion, mocardial tissue was maintained at −70°C for preservation. An MPO test kit was employed to detect the level of MPO in the myocardial tissue, according to the manufacturer’s instructions.
Following reperfusion, blood was taken from the carotid artery and kept at room temperature for 30 min at 4°C. Serum was separated by centrifugation at 3,000 × g for 20 min and maintained at −70°C for preservation. A CK test kit was utilized according to the manufacturer’s instructions in order to measure serum CK activity.
Following reperfusion, blood was taken from the carotid artery and kept at room temperature for 30 min. Serum was separated by centrifugation at 3,000 × g for 20 min at 4°C and maintained at −70°C for preservation. The extent of cell injury was monitored by measuring leakage of LDH. An LDH test kit was utilized according to the manufacturer’s instructions in order to measure serum LDH levels.
Following reperfusion, the levels of TNF-α in myocardial tissue homogenate and serum were measured according to the manufacturer’s instructions. A BCA kit was used to detect the protein quantization.
Data are presented as the mean ± standard deviation. The significance of the differences among groups was evaluated by Student’s t-test for unpaired data or Dunnett’s t-test for multiple comparisons, preceded by one-way analysis of variance. SPSS version 13.0 was used for analysis (SPSS, Inc., Chicago, IL, USA) P<0.05 was considered to indicate a statistically significant difference.
MI/R induced an area of infarction in the myocardium. Compared with the MI/R group, Pte reduced the infarcted area in the myocardium significantly. This effect of Pte was eliminated by the administration of L-NAME, a NO synthase inhibitor. In addition, the effect of Pte was significantly attenuated by administration of MB, a cGMP inhibitor (
Neutrophils contain a certain quantity of MPO, which accounts for ~5% of dry cell weight. Thus, the activity of MPO in the myocardium may be considered as an indication of neutrophil infiltration. As shown in
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MI/R injury is known to result in the production of increased levels of TNF-α. Thus, myocardial and serum TNF-α levels were examined. As shown in
The current study found that Pte reduces the inflammatory reaction induced by I/R injury by inhibiting neutrophil infiltration and TNF-α production. In addition, it demonstrated that NO and cGMP may be important mediators of the protective effects of Pte.
The inflammatory reaction is known to be involved in MI/R injury (
TNF-α is secreted predominantly by macrophages. It promotes an inflammatory cascade by increasing the release of other proinflammatory cytokines and influencing neutrophil recruitment (
MI/R injury appears to be induced in part by neutrophil activation. There are a number of mechanisms underlying this effect. Cell damage, caused by the release of oxygen free radicals, proteolytic enzymes and cytotoxic substances leads to increased neutrophil activation. In addition, inflammatory mediators cause vascular endothelial cell damage, increased vascular permeability and edema. Further activation of inflammatory cells leads to further increases in the inflammatory response (
Previous studies have demonstrated an association between neutrophil and MI/R injury. The removal of neutrophils or drug-induced inhibition of neutrophil activity has been shown to reduce MI/R injury (
It is hypothesized that NO production is associated MI/R-induced inflammation (
In conclusion, the present study demonstrated that Pte attenuates inflammation induced by MI/R injury. The protective effects of Pte are associated with inhibition of neutrophil infiltration and TNF-α production, increases in the levels of NO and possible upregulation of the cGMP signaling pathway. The present study provides new insights into the mechanisms involved in the cardioprotective effect of pterostilbene against myocardial ischemia/reperfusion injury, which may be a new clinical therapy for myocardial ischemia/reperfusion injury.
(A) Chemical structure of pterostilbene. (B) Chemical structure of resveratrol. In contrast to resveratrol, pterostilbene contains two methoxy groups, which increase its oral absorption and bioavailability.
Comparison of myocardial infarct area (as a percentage of INF/AAR) in each group. TTC-Evans blue double staining suggested that compared with the MI/R group, Pte significantly reduced the infarct area (white area), while L-NAME and MB eliminated the effect of pterostilbene. *P<0.05 compared with the MI/R group and #P<0.05, compared with the MI/R + Pte group. P<0.0001 between the MI/R and sham group. INF/AAR; infarct area/area at risk; Pte, pterostilbene; MB, methylene blue; TTC, 2,3,5-triphenyltetrazolium chloride; MI/R, myocardial ischemia-reperfusion.
Comparison of MPO activity in each group. Compared with the MI/R group, the MPO activity in the MI/R + Pte group was reduced significantly. L-NAME and MB eliminated the effect of pterostilbene. *P<0.05, compared with the MI/R group and #P<0.05, compared with the MI/R + Pte group. P<0.0001 between the MI/R and sham group. Pte, pterostilbene; MB, methylene blue; MPO, myeloperoxidase; MI/R, myocardial ischemia-reperfusion.
Comparison of serum CK activity in each group. Compared with the MI/R group, Pte reduced the serum CK activity significantly. However, this effect was eliminated by L-NAME and MB. *P<0.05, compared with the MI/R group and #P<0.05, compared with the MI/R + Pte group. P<0.0001 between the MI/R and sham group. Pte, pterostilbene; MB, methylene blue; CK, creatine kinase; MI/R, myocardial ischemia-reperfusion.
Comparison of serum LDH activity in each group. Compared with MI/R group, Pte reduced the serum LDH activity significantly. However, this effect was eliminated by L-NAME and MB. *P<0.05, compared with the MI/R group and #P<0.05, compared with the MI/R + Pte group. P<0.0001 between the MI/R and sham group. Pte, pterostilbene; MB, methylene blue; LDH, lactate dehydrogenase; MI/R, myocardial ischemia-reperfusion.
Comparison of levels of TNF-α in (A) myocardium and (B) serum. Compared with the MI/R group, pterostilbene reduced TNF-α levels significantly. L-NAME and MB eliminated the effect of Pte. *P<0.05, compared with the MI/R group and #P<0.05, compared with the MI/R + Pte group. Pte, pterostilbene; MB, methylene blue; TNF-α, tumor necrosis factor-α; MI/R, myocardial ischemia-reperfusion.