The present study assessed the therapeutic potentials of troxerutin in mitigation of MI/R damage in isolated rat heart. By evaluating clinically relevant end points, this study proved that preadministration of troxerutin is able to reduce cardiac infarct size and LDH release following MI/R compared with the untreated I/R myocardium. Besides, this study documented that the positive effects of troxerutin preconditioning may be mediated in part by its effect on inflammation. In more details, troxerutin preconditioning showed potent anti-inflammatory and cardioprotective activity via decreasing TNF-α and IL-1β levels. Also, troxerutin was found to suppress inflammatory response by reducing the activity of TLR4/NF-κB signaling pathway. In addition, our results showed that the beneficial effect of troxerutin was reversed by using 5-HD, as a mitoKATP channel blocker. So it can be said that troxorutein may exert its cardioprotective effect under MI/R injury by the opening of mitoKATP channels.
In previous experimental studies of MI/R models, the beneficial properties of troxerutin have been evaluated. However, in order to achieve clinical goals in future, we need to widely study the effects of troxerutin on cellular signalings. For that reason, several experimental studies have assessed cardioprotective effects of troxerutin by evaluation of important cellular signalings, but the mechanism of troxerutin on inflammation via TLR4/NF-κB signaling pathway in MI/R damage has never been studied in depth. It seems that specific modulation of TLR4/NF-κB signaling pathway by troxerutin might be a novel strategy to increase survival under MI/R injury in the future. It should be noted that the results obtained from the present study can lead to the better understanding of cardioprotective activity of troxerutin as a natural bioflavonoid in MI/R injury. The most significant observation of the current work is that troxerutin preconditioning reduced I/R injury, which was confirmed by reduction of cardiac infarct size and LDH levels. It means that pretreatment with troxerutin can increase cardiac resistance to I/R injury. Our work demonstrated that cardioprotective effects of troxerutin in I/R injury were accompanied by its beneficial effects on inflammation through reduction of inflammatory cytokines such as TNF-α and IL-1β levels, as well as reduction of TLR4 and NF-κB expressions.
The specific pathophysiological mechanisms underlying MI/R injury are complex and needs further investigations; however, several studies demonstrated that inflammatory response takes part in MI/R injury. It has been proven that an important protein which is involved in the inflammatory response of MI/R damage is TLR4 . In particular, TLR4 triggers the cytokines releasing, neutrophil activation, and elevation in ROS following MI/R injury [22–24]. TLR4 plays its role via MyD88 pathway, and NF-κB can be activated through this dependant pathway, so the associated inflammatory cytokines will be released. It has been proven that the NF-κB pathway can be activated under myocardial ischemia, and can be further activated under reperfusion. So it has a decisive role in MI/R injury and participates in the stress reaction and tissue damage [10, 25]. After translocation of NF-κB to the nucleus within the first few minutes following reperfusion, NF-κB promotes cell dysfunction and death by induction of pro-inflammatory and pro-apoptotic genes expression . Therefore, inhibition of TLR4/NF-κB signaling pathway can be considered as an important therapeutic target for attenuation of MI/R injury. In consistent with previous studies, our data showed that I/R led to the increase in TLR4 and NF-κB expressions, and this accelerated inflammatory response causes a decrease in cardiac resistance to reperfusion injury. Interestingly, preadministration of troxerutin for 4 weeks could alleviate myocardial I/R-induced elevation of TLR4 and NF-κB expressions, and ultimately protect the heart against I/R damage. So it can be said that troxerutin has potent anti-inflammatory property via reducing the activity of TLR4-NF-κB pathway.
TNF-α and IL-1β play the key role in MI/R injury via causing cardiac dysfunction and cardiomyocyte necrosis and apoptosis . It has been demonstrated that activation of myocardial TNF-α and TNF receptor have complex role in MI/R damage and protection from it. Excessive expression of TNF-α and stimulation of cardiomyocyte TNF receptor type 1 can lead to cardiac cell death, fibrosis, hypertrophy and contractile dysfunction. Whereas, lower expression of TNF-α and stimulation of cardiomyocyte TNF receptor type 2 are protective . Our study revealed that I/R caused a significant increase in the myocardial levels of TNF-α and IL-1β, which was associated with the increased cardiac injury in comparison with healthy hearts. However, pretreatment with troxerutin caused decreased in the myocardial levels of TNF-α and IL-1β. Thus, positive effect of troxerutin on myocardial I/R damage may be attributed to the reduction of inflammatory cytokines such as TNF-α and IL-1β via decreasing the activity of TLR4-NF-κB signaling pathway.
Studies have demonstrated that mitoKATP channel, which is considered as end effector of cardioprotective pathways, has cardioprotective activity in MI/R injury by regulation of the mitochondrial matrix volume, and decreasing in both calcium uptake by mitochondria and mitochondrial membrane potential . It has been suggested that the mitoKATP channel opening decreases pro-apoptotic mediators and improves mitochondrial function , while administration of 5-HD, as a mitoKATP channel blocker, reverses the reducing effect of preconditioning and postconditioning on infarct size . In the present study, we showed that the anti-inflammatory effects of troxerutin were reversed when the hearts were perfused with a K-H solution containing 5-HD, 20 min before the ischemia. So it can be said that the positive effects of troxerutin may partly be mediated through the mitoKATP channels opening, and probably there is an association between the activation of mitoKATP channels and inhibitory effect of troxerutin on inflammatory mediators under MI/R injury, but needs to be investigated in depth. In addition, it seems that the activation of mitoKATP channels by troxerutin can protect the functional integrity of mitochondria, prevent the mitochondria swelling, and inhibit the mitochondrial permeability transition pore (mPTP) opening, leading to the decreased production and releasing of inflammatory mediators from the mitochondria in MI/R condition.
Conforming to our study, many previous researches demonstrated that troxerutin has protective effects against cardiovascular diseases, and oxidative and inflammatory damages in numerous organs [16–19, 29, 30]. Troxerutin has been shown to inhibit the endoplasmic reticulum stress pathway and reduce intracellular inflammatory proteins and enzyme activities including COX2, JNK1/IKKb, iNOS, and NF-κB in specific tissues of healthy rats [20, 30–32]. Moreover, in our previous works, we have indicated that troxerutin preconditioning could protect the diabetic I/R hearts by prevention of myocardial apoptosis through glycogen synthase kinase-3β (GSK-3β) phosphorylation . In addition, we proved that troxerutin preconditioning had protective effects in doxorubicin-induced cardiotoxicity in rats via anti-oxidative activities and restoration of mitochondrial function and the expression profiles of cardiac SIRT-1/PGC-1α/NRF-2 network .