The Anti-inflammatory Effect of Baicalin on Hypoxia/Reoxygenation and TNF-α Induced Injury in Cultural Rat Cardiomyocytes Lin Lin 1 , Xiao-dong Wu 1, *, Andrew K Davey 2 and Jiping Wang 2, * 1 Department of Pharmacology, School of Basic Medicine, Southeast University, Nanjing 210009, PR China 2 Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia The aim of present study was to investigate the effect of baicalin on hypoxia/reoxygenation (H/R) injury in cardiomyocytes and the mechanisms involved, particularly in relation to cytokines. The cardiomyocytes for the H/R groups were placed into a hypoxic chamber for 12 h and then underwent reoxygenation for 1 h. The cells in the TNF-α groups were administered 100 ng/mL rrTNF-α and incubated for 13 h under normal conditions. The cells in the baicalin pretreatment groups were administered 10 μM baicalin 30 min prior to exposure to H/R or TNF-α. It was observed that pretreatment with baicalin (10 μM) significantly reduced the cell damage and death induced by H/R or TNF-α. In the culture medium of the H/R cells, the SOD activity increased, while TNF-α was decreased by baicalin. The levels of IL-6 in culture medium for H/R or TNF-α treated cells were suppressed by baicalin pretreatment. In contrast, the levels of IL-10 in culture medium for H/R or TNF-α treated cells were significantly elevated by baicalin. Moreover, baicalin inhibited the nuclear translocation of NF-κB induced by H/R or TNF-α. In conclusion, baicalin may protect cardiomyocytes from H/R injury through an anti-inflammatory mechanism. Copyright © 2009 John Wiley & Sons, Ltd. Keywords: baicalin; hypoxia/reoxygenation; cardiomyocytes; TNF-α; IL-6; inflammation. INTRODUCTION Myocardial ischemic injury results from severe impair- ment of the coronary blood supply due to thrombosis or acute alterations of coronary atherosclerotic plaques (Buja, 2005; Frangogiannis et al., 2002). Myocardial ischemia is a severe stress that leads to the loss of car- diomyocytes. The cell loss may be attributed to apopto- sis. However, the exact mechanisms involved in the loss of myocytes are only partially defined and this limits therapeutic opportunities for rescuing the cells (Szegezdi et al., 2006). The first experimental evidence of inflam- mation causing myocardial injury was shown using anti-inflammatory strategies in animal models with myocardial ischemia and reperfusion (Frangogiannis et al., 2002). Recently numerous clinical and experimen- tal studies proved that immuno-inflammatory response to injury caused by hypoxia and reoxygenation (H/R) in the infarcted myocardium is associated with the induction of cytokines including tumor necrosis factor- α (TNF-α) and interleukin-6 (IL-6) (Kosmala et al., 2005). TNF-α, a key cytokine in the pathogenesis of H/R injury (Pevni et al., 2008), induces other inflam- matory cytokines such as IL-6 (Bartoccioni et al., 1994), and mediates the activation of transcription factors such as NF-κB. NF-κB is critical in the induction of cytokines and immunoregulatory proteins and is pivotal in the inflammatory response (Chandrasekar et al., 2000; Wong et al., 2005; Hall et al., 2006). IL-10 is an anti-inflammatory cytokine which protects cardiac function through inhibition of TNF-α production and down regulation of IL-6 mRNA (Wang et al., 1995). Scutellaria baicalensis Georgi (Huang Qui), a Chinese traditional medicinal herb, is widely used as an anti- inflammatory, antibacterial, and hepatoprotective drug. Baicalin (BA, 7-glucuronic acid,5,6-dihydroxy-flavone, Fig.1) (Li et al., 2000), a flavonoid compound extracted from Huang Qui, possesses antioxidant properties and free radical scavenging activity (Chou et al., 2003; Woo et al., 2005) and has been used in the treatment of a variety of inflammatory diseases such as bronchitis, nephritis, hepatitis, asthma, atopic dermati- tis (Li et al., 2000) and in carrageenan-evoked thermal hyperalgesia (Chou et al., 2003). Baicalin can also inhibit reverse transcriptase activity in HIV-1-infected cells and interfere with the interaction of HIV-1 envelope proteins and chemokine receptors (Dongyan et al., 2000). Previously, we reported the protective effect of BA on myocardial ischemia in the isolated rat heart and isoproterenol-induced myocardial ischemia in rats (Zhang and Wu, 2007). The mechanism of action may be due to reduced NF-κB leading to decreased TNF-α and IL-6 (Zhang and Wu, 2007). However, whether BA plays a direct role against the inflammation induced by cardiomyocytes in H/R injury has not been clarified. In this study, we hypothesized that BA may attenuate the expression of inflammatory cytokines such as TNF-α and IL-6 in vitro. To test this, we determined the effects of BA on the levels of TNF-α, IL-6 and IL-10 in cul- tured cardiomyocytes activated by H/R and examined the ultrastructure of the cardiomyocytes. To investigate the damage induced by oxidative stress during H/R, SOD leakage from cells was measured. * Correspondence to: Xiao-Dong Wu, 87 Dingjiaqiao Rd, Nanjing 210009, PR China. E-mail: robertwxd@yahoo.com.cn; Jiping Wang, Reid Building, UniSA City East Campus, Adelaide, SA 5000, Australia. E-mail: jiping.wang@unisa.edu.au Received 05 March 2009 Revised 24 June 2009 Copyright © 2009 John Wiley & Sons, Ltd. Accepted 06 August 2009 PHYTOTHERAPY RESEARCH Phytother. Res. 24: 429–437 (2010) Published online 13 October 2009 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ptr.3003