Journal of Cellular Biochemistry 93:162–172 (2004) Paeoniflorin Induces Apoptosis of Lymphocytes Through a Redox-Linked Mechanism Hideo Tsuboi, 1 Khaled Hossain, 2 Anwarul A. Akhand, 3 Kozue Takeda, 1 Jun Du, 4 Muhaimin Rifa’i, 1 Yan Dai, 1 Akemi Hayakawa, 5 Haruhiko Suzuki, 1 and Izumi Nakashima 1 * 1 Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan 2 Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi 6205, Bangladesh 3 Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka 1000, Bangladesh 4 Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya 461-8673, Japan 5 Equipment Center for Research and Education, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan Abstract Paeoniflorin (PF), isolated from paeony root, has been used as a herbal medicine for more than 1,200 years in China, Korea, and Japan for its anti-allergic, anti-inflamatory, and immunoregulatory effects. In this study, we found that PF induces apoptosis in both murine T-lineage cells and human T-cell leukemia Jurkat cells. This apoptosis was mediated through the reduction of mitochondrial membrane potential, activation of caspase, and fragmentation of DNA. Interestingly, PF induced generation of reactive oxygen species (ROS) and a reducing agent, dithiothreitol (DTT), and a ROS scavenger, N-acetyl cysteine (NAC), successfully attenuated the PF-induced apoptosis. Additionally, PF induced the phosphorylation of three mitogen-activated protein (MAP) family kinases, extracellular signal-regulated kinase, c-Jun amino-terminal kinase (JNK), and p38 MAP kinase. Curcumin, an anti-oxidant and JNK inhibitor, inhibited PF- induced apoptosis, suggesting the possible involvement of curcumin-sensitive JNK or other redox-sensitive elements in PF-induced apoptosis. These results partially explain the action mechanism of PF-containing paeony root as a herbal medicine. J. Cell. Biochem. 93: 162 – 172, 2004. ß 2004 Wiley-Liss, Inc. Key words: paeoniflorin; paeony root; apoptosis; reactive oxygen species (ROS); redox Paeony root (Paeoniae radix; Shakuyaku in Japanese) is one of the most well-known herbs in China, Korea, and Japan and has been used as a medicine for more than 1,200 years. Several ingredients have been extracted from the paeony root, including oxypaeoniflorin, albiflorin, ben- zoylpaeoniflorin, and paeoniflorin (PF) [Shibata et al., 1963; Kaneda et al., 1972; Nishizawa et al., 1979]. Among them, PF, a glucoside, is known to be one of the principle bioactive components of paeony root [Takagi and Harada, 1969a]. PF has been reported to have immunoregulatory [Liang et al., 1990], anti-allergic [Yamahara et al., 1982], anti-inflammatory [Yamahara et al., 1982], cognition-enhancing [Ohta et al., 1993], neuromuscular-blocking [Kimura et al., 1984, 1986], anti-convulsant [Atef et al., 1999], anti- hyperglycemic [Hsu et al., 1997], anti-coagulant [Ishida et al., 1987], and sedative [Takagi and Harada, 1969b] effects. Preparations of more than 40 kampo (Chinese and Japanese tradi- tional) medicines contain paeony root. Although paeony root or PF as its principle component is one of the main constituents of many kampo medicines, the mechanism by which it elicits immunoregulatory action has not been eluci- dated. In this study, we found that PF induces apoptosis in both murine T-lymphocytes and human T-cell leukemia Jurkat cells through a redox-linked mechanism. PF-induced apoptosis is mediated by the activation of caspase followed ß 2004 Wiley-Liss, Inc. *Correspondence to: Dr. Izumi Nakashima, Department of Immunology, Nagoya University Graduate School of Med- icine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: inakashi@med.nagoya-u.ac.jp Received 1 July 2003; Accepted 3 March 2004 DOI 10.1002/jcb.20134 Published online 29 June 2004 in Wiley InterScience (www.interscience.wiley.com).