Research Article: Regulation of Heat Shock Proteins 27 and 70, p-Akt/p-eNOS and MAPKs by Naringin Dampens Myocardial Injury and Dysfunction In Vivo after Ischemia/Reperfusion

Date Published: December 6, 2013

Publisher: Public Library of Science

Author(s): Neha Rani, Saurabh Bharti, Mansi Manchanda, T. C. Nag, Ruma Ray, S. S. Chauhan, Santosh Kumari, Dharamvir Singh Arya, Rajesh Gopalrao Katare.


Naringin has antioxidant properties that could improve redox-sensitive myocardial ischemia reperfusion (IR) injury. This study was designed to investigate whether naringin restores the myocardial damage and dysfunction in vivo after IR and the mechanisms underlying its cardioprotective effects. Naringin (20–80 mg/kg/day, p.o.) or saline were administered to rats for 14 days and the myocardial IR injury was induced on 15th day by occluding the left anterior descending coronary artery for 45 min and subsequent reperfusion for 60 min. Post-IR rats exhibited pronounced cardiac dysfunction as evidenced by significantly decreased mean arterial pressure, heart rate, +LVdP/dtmax (inotropic state), -LVdP/dtmax (lusitropic state) and increased left ventricular end diastolic pressure as compared to sham group, which was improved by naringin. Further, on histopathological and ultrastructural assessments myocardium and myocytes appeared more normal in structure and the infarct size was reduced significantly in naringin 40 and 80 mg/kg/day group. This amelioration of post-IR-associated cardiac injury by naringin was accompanied by increased nitric oxide (NO) bioavailability, decreased NO inactivation to nitrotyrosine, amplified protein expressions of Hsp27, Hsp70, β-catenin and increased p-eNOS/eNOS, p-Akt/Akt, and p-ERK/ERK ratio. In addition, IR-induced TNF-α/IKK-β/NF-κB upregulation and JNK phosphorylation were significantly attenuated by naringin. Moreover, western blotting and immunohistochemistry analysis of apoptotic signaling pathway further established naringin cardioprotective potential as it upregulated Bcl-2 expression and downregulated Bax and Caspase-3 expression with reduced TUNEL positivity. Naringin also normalized the cardiac injury markers (lactate dehydrogenase and creatine kinase-MB), endogenous antioxidant activities (superoxide dismutase, reduced glutathione and glutathione peroxidase) and lipid peroxidation levels. Thus, naringin restored IR injury by preserving myocardial structural integrity and regulating Hsp27, Hsp70, p-eNOS/p-Akt/p-ERK signaling and inflammatory response.

Partial Text

After an acute myocardial infarction (MI), early reperfusion therapy either by thrombolysis or primary percutaneous coronary intervention seems to be the most practical and widely practiced approach for myocardial salvage [1]. Nevertheless, reperfusion itself may paradoxically intensify further injury to ischemic myocardium, termed as “Ischemia-reperfusion (IR) injury” [1,2]. Several recent studies show that exaggerated production of reactive oxygen species (ROS) along with decreased endogenous antioxidant defense such as heat shock proteins (Hsp’s) are involved in the pathogenesis of myocardial IR injury [3-5]. Likewise, a large body of evidence demonstrates that over expression of Hsp27 and Hsp70 protects against IR-induced cardiac dysfunction and increases the resistance of the heart to ischemic injury [4,5]. Furthermore, in ischemia-reperfused hearts, ROS impaired cardiac functioning by decreasing NO bioavailability and/or by decreasing endothelial nitric oxide synthase (eNOS) expression/activity [6,7]. Of note, IkB kinase/nuclear factor-kappa B (IKK-β/NF-κB) and mitogen-activated protein kinases (MAPKs) including c-Jun N-terminal kinase (JNK) pathways are activated and extracellular signal-regulated kinase (ERK) suppressed in IR heart leading to apoptosis and necrosis of myocytes [6-9].

In this paper we focused on the cardioprotective potential of naringin and its underlying molecular mechanism/s. Our results identify naringin as a cardioprotective agent as it reduced myocardial infarct size; preserved myocyte structural integrity and decreased LVEDP and tended to improve ±LVdP/dtmaxin vivo after IR. This myocardial salvaging effect of naringin is primarily mediated through attenuation of oxidative stress by improving endogenous antioxidant defense system via amplified expression of β-catenin, Hsp27 and Hsp70 and interruption of inflammatory signaling through TNF-α/IKK-β/NF-κB suppression. Interestingly, naringin increased Akt and eNOS phosphorylation and augmented myocardial NO production. Importantly, regulation of MAPKs including inhibition of JNK, as well as activation of ERK by naringin was also involved in attenuation of IR injury. Further, naringin anti-oxidant and anti-inflammatory finding firmly corroborated with its anti-apoptotic potential as it precludes myocyte apoptotic cell death through increasing Bcl-2 expression and decreasing TUNEL positivity, Bax and caspase-3 expression. Thus, our findings identify a novel mechanism whereby naringin could be an effective candidate against IR induced myocardial damage and dysfunction.