Abstract
Purpose: To investigate the effect of punicalagin, an ellagitannin present in pomegranates, on intracerebral haemorrhage (ICH)-induced inflammatory responses and oxidative stress, and also unravel the underlying mechanism(s) of action.
 Methods: Collagenase type IV (0.2 U) was used to induce ICH in adult male Sprague-Dawley rats. Punicalagin was given to the rats at doses of 25, 50, and 75 mg/kg body weight via oral gavage for 15 days before ICH induction. The animals were sacrificed 24h following induction of ICH, and their brains were excised immediately and used for analysis. Histological changes were determined with Haematoxylin and Eosin (H&E) staining. Permeability to blood-brain barrier (BBB) was determined by quantifying the extent of extravasation of Evan Blue (EB). Protein expressions of HO-1/Nrf-2/ARE and NF-κB signaling were assayed using immunoblotting and RT-PCR. Levels of reactive oxygen species (ROS) and serum levels of cytokines were also determined.
 Results: Punicalagin treatment reduced inflammatory cell infiltration and cell damage, improved brain tissue architecture and BBB integrity. The punicalagin treatment increased the activities of antioxidant enzymes, and enhanced antioxidant status via activation of Nrf-2/ARE/HO-1 signaling pathway (p < 0.05). The treatment upregulated the expressions of HO-1 to 174 %, relative to 127 % in ICH control rats. Furthermore, it enhanced NF-κB levels and reversed the ICH injury-induced upregulations of IL-6, IL-18 and IL-1β.
 Conclusion: These findings indicate that punicalagin exerts neuroprotective effect in rats after experimental ICH through regulation of theHO-1/Nrf-2/ARE signaling pathway. Thus, punicalagin has therapeutic potential for ICH.
 Keywords: Brain injury, Haemoxygenase-1, Intracerebral haemorrhage, Inflammatory responses, Nrf2/ARE signalling, Punicalagin
Highlights
Spontaneous intracerebral haemorrhage (ICH) is a type of stroke with increasing incidence and high mortality and morbidity [1]
The membranes were washed with Tris-buffered saline and Tween- 20 (TBST) and incubated overnight at 4 oC with primary antibodies against NF-κb-p65, Heme oxygenase (HO)-1, Nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H quinone dehydrogenase 1(NQO1), and inducible nitric oxide synthase, TNFα (1:1000, Cell Signaling Technology), as well as β-actin, COX-2, IκBα, IKKβ, IKKα, p-IκBα, p-IKKβ and p-IKKα (1:1000, Santa Cruz Biotechnology, USA).Following incubation, the membranes were washed with TBST and further incubated with secondary antibody for 60 min at room temperature
Brain water content measured at 24 h following ICH induction revealed significant (p < 0.05) brain oedema (Figure 1) in the ICH control group, relative to normal control
Summary
Spontaneous intracerebral haemorrhage (ICH) is a type of stroke with increasing incidence and high mortality and morbidity [1]. Heme oxygenase (HO), the ratelimiting enzyme in heme catabolism, induces HO-1, the inducible form of HO, improves the integrity of BBB, and reduces neurological impairments [9] Inflammatory mediators such as chemokines, cytokines, and prostaglandins released at the perifocal tissues by activated inflammatory cells following ICH, trigger secondary brain injuries [10]. The membranes were washed with Tris-buffered saline and Tween- 20 (TBST) and incubated overnight at 4 oC with primary antibodies against NF-κb-p65, HO-1, Nrf, NAD(P)H quinone dehydrogenase 1(NQO1), and inducible nitric oxide synthase (iNOS), TNFα (1:1000, Cell Signaling Technology), as well as β-actin, COX-2, IκBα, IKKβ, IKKα, p-IκBα, p-IKKβ and p-IKKα (1:1000, Santa Cruz Biotechnology, USA).Following incubation, the membranes were washed with TBST and further incubated with secondary antibody (horseradish peroxidaseconjugated; 1:2000, Santa Cruz Biotechnology) for 60 min at room temperature. Values of p < 0.05 were regarded as statistically significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
