Discussion
In the present studies, we address the functional roles of glycolysis in
mediating cardioprotective responses of pinocembrin. We demonstrated
that (i) pinocembrin delivered at the onset of reperfusion
(postconditioning) significantly improved post-ischemic myocardial
function and reduced infarct size after I/R ex vivo ; (ii)
pinocembrin pretreatment significantly protected mouse hearts from acute
myocardial I/R injury in vivo ; (iii) those protection is at least
partially related to enhanced glycolysis by pinocembrin in the I/R
cardiomyocytes; and (iv) the cardioprotective effects of pinocembrin are
mediated by the activation of PFKFB3. These results extend previous
findings indicating the cardioprotection of pinocembrin against I/R
injury and reveal the new mechanisms of pinocembrin in the
cardioprotection.
It would be an attractive treatment principle to reduce the infarct size
through pharmaceutical intervention to assist classic reperfusion
intervention (Morel et al., 2012). Pinocembrin is a potential
cardiovascular drug with potential neuroprotective effects on transient
and long-term ischemic stroke in rats (Wu et al., 2013). Also, previous
study indicated that administration of pinocembrin before myocardial
ischemia improved LV function (Lungkaphin et al., 2015). Pharmacological
postconditioning is easier to implement and has therapeutically
potential in both clinical and experimental setting, which avoids the
potential injury induced by ischemic conditioning, therefore has good
clinical application prospects (Heusch, 2015). Therefore, our study aims
to explore the cardioprotective effects of pinocembrin postconditioning.
The ex vivo results showed that pinocembrin from 10 to 100 µM
delivered at the first 5 minutes of reperfusion remarkably improved
post-ischemic myocardial function and attenuates cell death in a
concentration-dependent manner. Furthermore, in vivo mouse
myocardial I/R injury model was prepared and pinocembrin
postconditioning was fulfilled by an intraperitoneal injection of
pinocembrin (5 mg/kg and 10 mg/kg body weight) 5 min before reperfusion.
Cardiac function, serum LDH activity and cTnT content, and infarct size
were significantly improved with pinocembrin treatment. These data are
consistent with the observation of cardioprotective effects of
pinocembrin preconditioning others reported.
Various metabolic abnormalities occur during myocardial I/R, such as
increased fatty acid oxidation and decreased glucose oxidation
(Fillmore, Mori, & Lopaschuk, 2014). This phenomenon is related to
uncoupling of mitochondrial respiration, increased proton leakage, ROS
formation and, more importantly, increased myocardial oxygen consumption
(Boudina & Abel, 2006). During myocardial I/R, the metabolic shift
aimed at increasing glucose oxidation have proved to be beneficial.
Although some therapeutic strategies have tried to reverse this
metabolic imbalance, there is still no approved treatment regimen so far
(Jaswal, Keung, Wang, Ussher, & Lopaschuk, 2011; Kantor, Lucien, Kozak,
& Lopaschuk, 2000; Taniguchi et al., 2001). In addition, strategies
aimed at increasing clinical glucose consumption have different results
and have not yet reached routine clinical practice. Looking for drugs
that can safely induce the transfer of cellular energy metabolism and a
better understanding of the protective mechanisms of increased glucose
oxidation may facilitate transfer to the clinic. Our results show that
increasing glycolysis is responsible for pinocembrin induced protective
effects. This is consistent with previous studies that metabolic shift
towards increased glycolysis protects the heart from I/R injury
(Nadtochiy et al., 2018). Moreover, PFKFB3 expression is notably
upregulated during I/R, which directs cellular glucose metabolism from
PPP to aerobic glycolysis (Li et al., 2019). Our data shown that PFKFB3
was significantly upregulated with pinocembrin treatment and specific
inhibitor of PFKFB3 abolished pinocembrin-afforded protective effects in
cardiomyocytes. Most importantly, knock down of PFKFB3 in myocardium
using AAV9 reversed the cardioprotection of pinocembrin
postconditioning. Previous studies have shown that the main reason
responsible for the protection offered by the metabolic shift is the
fact that glycolysis is able to produce two molecules of ATP without
need for oxygen, so that uncoupling between mitochondrial full glucose
oxidation and glycolysis leads to increased cardiac efficiency
(Lopaschuk, 2017). However, potential protective mechanisms of the
metabolic shift are largely unknown. Further studies are needed to
investigate how pinocembrin regulate the glycolysis and search for its
putative downstream targets.
In summary, our findings demonstrate that compound pinocembrin exhibits
significant protective effects on cardiac I/R injury when delivered at
the beginning of or before reperfusion in ex vivo rat andin vivo mouse models through enhancing glycolysis. Pinocembrin
may be considered as an effective lead compound for large animal
experiments, and is expected to be used in clinical research of acute
myocardial infarction
Acknowledgements:
This work was supported by the Project of National Natural Science
Foundation of China [grant numbers 81700354, 81970229].
Author contributions:
ZYJ and GXF performed and analysed the experiments. ZYJ wrote the first
draft of the manuscript. WGQ participated in the analysis of the
experiments. LJR revised the manuscript. ZYJ and LJR contributed to the
experimental design and revised the manuscript.
Conflict of interest: No conflicts of interest.
Declaration of transparency and scientific rigour
This Declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical research
as stated in the BJP guidelines for Design & Analysis, Immunoblotting
and Immunochemistry, and Animal Experimentation, and as recommended by
funding agencies, publishers and other organisations engaged with
supporting research.