The neoflavonoid latifolin isolated from dalbergia odorifera attenuates hypoxia and reoxygenation injury by reactive oxygen species scavenging and nrf2 activation in h9c2 cardiomyocyte

R. LIU1, Y. LUO2, L. CHEN2, D. WANG1, F. SHAO1, Y. CUI2. 1School of Pharmacy, Jiangxi University of TCM, Nanchang, CHINA, 2Jiangxi University of TCM, Nanchang, CHINA.

Introduction: Latifolin, as a major neoflavonoid component isolated fromthe heartwood of Dalbergia odorifera T. Chen, possess antioxidant, anti-inflammatory, vasodilatory properties, etc1. This study aimed to investigate the effects of latifolinon the hypoxia and reoxygenation(H/R) injury in H9c2 and the possible molecular mechanisms by activating nuclear factor erythroid 2 related factor 2 (Nrf2)/antioxidant response element (ARE) pathway2.

Method: H9c2 cardiomyocytes pretreated with latifolin were exposed to hypoxia and reoxygenation (H/R). Cell viability and lactate dehydrogenase (LDH) activity in culture medium were determined by MTT and microcolorimetry. The expression and level of intracellular reactive oxygen species (ROS) were measured by DCFH-DA staining and flow cytometry. The expression and location of Nrf2 were identified by Immunocytochemistry. The expression of Nrf2 and HO-1weredetectedby western blotting. Data are given as mean±SD and analysis was performed using one-way ANOVA.

Results:The results showed that latifolin increased the cell viability, reduced LDH activity (Fig.1)and ROS production(Fig.2). Immunocytochemistry and western blotting analysis showed that latifolin increased the expression of HO-1 in cytoplasm and Nrf2 both in cytoplasm and nuclear(Fig.3).

Fig. 1 Effect of latifolin on cell viability and LDH activity. A. cell viability; B. LDH activity. Data were presented as mean ± SD. n=3 per group. *P<0.05, **P< 0.01 vs. control group; #P< 0.05, ##P< 0.01 vs. H/R group.

Fig.2 Effect of latifolin on ROS generation. A. Representative photomicrographs for ROS production as assessed by DCFH-DA staining. B.Intracellular ROS levels were determined by flow cytometry. Data were presented as mean ± SD. n=5 per group. *P<0.05 and **P<0.01 vs. control group; #P< 0.05, ##P< 0.01 vs. H/R group.

Fig. 3 Western blotting analysis of Nrf2 and HO-1 expression. A. Expression of Nrf2 and HO-1 in cytoplasm. B. Expression of Nrf2 in nucleus.

Conclusion: Pretreatment with latifolin attenuated the cell injury,increased the expression of Nrf2 and HO-1.These findings indicated that the anti-oxidative and ROS scavenging activity of latifolin attenuated H/R injurywhich was related to activating Nrf2/ ARE pathway.

References:

1.Yu X, et al. (2007). Food Chem 104: 715-720.

2. Holly K, et al. (2013). Biochemical Pharmacology 85:705-717.