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Lagerstroemia speciosa extract inhibit TNF-induced activation of nuclear factor-κB in rat cardiomyocyte H9c2 cells
Journal of Ethnopharmacology 128 (2010) 254–256
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Lagerstroemia speciosa extract inhibit TNF-induced activation of nuclear factor-B in rat cardiomyocyte H9c2 cells Haruyo Ichikawa a,∗ , Hisato Yagi a , Takeshi Tanaka a , Jong-Chol Cyong a,b , Tomoh Masaki a a International Research and Educational Institute for Integrated Medical Sciences (IREIIMS), Tokyo Women’s Medical University, 8-1, Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan b Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kita-adachi-gun, Saitama 362-0806, Japan
a r t i c l e
i n f o
Article history: Received 7 September 2009 Received in revised form 5 December 2009 Accepted 17 December 2009 Available online 4 January 2010 Keywords: Lythraceae Lagerstroemia speciosa Cardiomyocyte hypertrophy TNF NF-B
a b s t r a c t Aim of the study: Lagerstroemia speciosa has been used as a folk medicine among people with diabetes in the Philippines. It is known to exhibit antidiabetic, antiobesity, and glucose transport activities through mechanisms not well defined. Diabetes leads to cardiomyocyte hypertrophy in association with an upregulation of vasoactive factors and activation of nuclear factor (NF)-B and activating protein-1. We therefore investigated the effect of Lagerstroemia speciosa on the activation of NF-B as a key mediator of cardiomyocyte hypertrophy, in rat cardiomyocyte H9c2 cells. Materials and methods: Water extract of Lagerstroemia speciosa (Lythraceae family) was prepared. H9c2 cells were used for treatment of Lagerstroemia speciosa extract with/without tumor necrosis factor (TNF). To examine NF-B’s activation, we performed an electrophoretic mobility shift assay (EMSA). Results: The activation of NF-B by TNF was completely blocked by a Lagerstroemia speciosa extract in a dose- and time-dependent manner in H9c2 cells. Conclusion: Overall, our results indicate that Lagerstroemia speciosa can inhibit DNA-binding of NF-B. This may explain its possible inhibition of diabetes-induced caridomyocyte hypertrophy. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The three characteristic metabolic disturbances in diabetics are hyperlipidemia, and early hyperinsulinemia followed by pancreatic -cell failure, leading to hyperglycemia. Hyperinsulinemia is an important trigger of cardiac hypertrophy in diabetic cardiomyopathy (Ilercil et al., 2002; Poornima et al., 2006). There are at least three cellular mechanisms whereby hyperinsulinemia mediates cardiomyocyte hypertrophy: (1) a PI3K/Akt-1 pathway, (2) a MAP kinase/extracellular signal-regulated kinase pathway, and (3) a GSK-3/NFAT pathway (Morisco et al., 2001). Recent publications also indicated that diabetes leads to myocardial hypertrophy in association with an upregulation of vasoactive factors such as endothelin-1 and activation of redox-sensitive transcription factors such as NF-B and activating protein-1 (Chen et al., 2003). Leaves of Lagerstroemia speciosa L. have been used in Southeast Asia as a folk medicine for the treatment of diabetes and kidney diseases (Liu et al., 2001). Several studies have indicated a beneficial role for Lagerstroemia speciosa (especially, corosolic acid) in terms of
antiobesity (Suzuki et al., 1999) and antidiabetes (Klein et al., 2007), and a hypoglycemic effect (Kakuda et al., 1996). Treatment with Lagerstroemia speciosa reduced blood glucose and insulin levels in diabetic KK-AY mice (Hong and Jai Maeng, 2004). Thus, it is possible that Lagerstroemia speciosa mediates anti-cardiac hypertrophy through a mechanism that is not well defined. NF-B is an inducible transcription factor that is activated by various carcinogens, inflammatory stimuli, and growth factors and controls the expression of genes linked with survival, proliferation, invasion, and metastasis of tumors (Aggarwal, 2004). Recent studies by several investigators have also implicated NF-B’s activation in the cardiac hypertrophic response, as modeled in cultured cardiac myocytes (Purcell et al., 2001). Because the NF-B activation pathway has been closely linked with cardiac hypertrophy, it is possible that Lagerstroemia speciosa acts through the modulation of this pathway. In the present study, Lagerstroemia speciosa extract abrogated the TNF-induced activation of NF-B in rat cardiomyocyte H9c2 cells. 2. Materials and methods
Abbreviations: NF-B, nuclear factor-B; TNF, tumor necrosis factor; EMSA, electrophoretic mobility shift assay. ∗ Corresponding author. Tel.: +81 3 3353 8111x24007; fax: +81 3 3353 3088. E-mail address: [email protected] (H. Ichikawa). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.12.033
2.1. Reagents Recombinant human TNF was obtained from R&D system (Minneapolis, MN). Dulbecco’s modified Eagle’s medium nutri-
H. Ichikawa et al. / Journal of Ethnopharmacology 128 (2010) 254–256
ent mixture F-12 HAM (DMEM/F-12) was purchased from Sigma–Aldrich Inc. (St. Louis, MO). Antibiotic Antimycotic, and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY). Antibodies against NF-Bp50 and NF-Bp65 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 2.2. Preparation of the Lagerstroemia speciosa extract Dried leaves of Lagerstroemia speciosa (Lythraceae family) as 100% (Code# J4571104434313) (3 g) obtained from LIV Laboratories Corp. (Tokyo, Japan) and were boiled with 47 ml of distilled water for 10 min. This crude extract was centrifuged (1610 g × 5 min) and the supernatant was filtered (0.2 m). The filtered extract was freeze dried and dissolved with distilled water as a 0.1 g/ml stock solution. The extract was stored at −20 ◦ C prior to use. 2.3. Cell lines H9c2 (rat cardiomyocyte) cells were obtained from American Type Culture Collection (Bethesda, MD). H9c2 cells were cultured in DMEM/F-12 supplemented with 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin, and 250 ng/ml amphotericin B. 2.4. Cell culture Cell culturing was conducted as reported (Hwang et al., 2006). For the study of cellular hypertrophy, cells were seeded at a density of 5 × 106 cells per 100 × 20 mm dish and cultured for 24 h in DMEM/F-12 containing 10% FBS. The cells were washed with serum-free medium (DMEM/F-12 without serum) and then treated with indicated concentrations of the Lagerstroemia speciosa extract under serum-free conditions for 24 h. Cells were treated with 50 ng/ml TNF in medium (DMEM/F12 with 0.5% FBS) with or without a fresh supply of the extract. Cells were incubated for the periods indicated at 37 ◦ C: a humidified atmosphere containing 5% CO2 . 2.5. Electrophoretic mobility shift assays for NF-B To examine NF-B’s activation, we performed an electrophoretic mobility shift assay (EMSA) as described (Ichikawa et al., 2005). Briefly, cells were washed with ice-cold phosphatebuffered saline and suspended in 0.4 ml of lysis buffer (10 mM HEPES, pH 7.9, 10 mM KC1, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, 2 g/ml aprotinin, and 0.5 mg/ml benzamidine). The cells were allowed to swell on ice for 35 min, after which 25 l of 10% Nonidet P-40 was added. The tubes were then agitated on a vortex for 30 s every 10 min and microcentrifuged for 30 s. The nuclear pellets were resuspended in 25 l of ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2.0 pg/ml leupeptin, 2.0 g/ml aprotinin, and 0.5 mg/ml benzamidine), and the tubes were incubated on ice for 30 min with intermittent agitation. This nuclear extract was microcentrifuged for 5 min at 4 ◦ C, and the supernatant was frozen at −80 ◦ C. EMSAs were performed by incubating 10 g of nuclear extract with 16 fmol of 32 P-end-labeled, 45-mer double-stranded NF-B oligonucleotides from the human immunodeficiency virus long terminal repeat (5 TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3 ; boldface indicates NF-B binding sites) in the presence of 0.5 g of poly (dI–dC), as a nonspecific competitor, in a binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% Nonidet P-40, 5% glycerol, and 50 mM NaCl) for 30 min at 37 ◦ C. The DNA–protein complex formed was separated from the free
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oligonucleotide on a 6.6% native polyacrylamide gel using a buffer containing 50 mM Tris, 200 mM glycine, and 1 mM EDTA, pH 8.5. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with the antibody against either p50 or p65 of NF-B for 15 min at 37 ◦ C before the complex was analyzed by EMSA. The NF-B band was supershifted to a higher molecular mass only when the antibody against either p50 or p65 of NF-B was used. When an excess of unlabeled NF-B oligonucleotide was added to the reaction mixture, it competed for binding sites and no binding with the labeled oligonucleotide was observed, suggesting that the complex formed was specific. To confirm the specificity further, binding was carried out with a labeled mutant oligonucleotide in which the conserved GGG required for binding NF-B was mutated to CTC. No band for NF-B was observed, suggesting that the complex formed required the conserved GGG for binding (Fig. 1C). The dried gels were visualized, and radioactive bands were quantified with a FLA5100 fluoro image analyzer (FUJIFILM, Tokyo, Japan) using Multigauge software (FUJIFILM, Tokyo, Japan). Fold increase is based on the control value. 3. Results The goal of this study was to investigate the mechanism by which the Lagerstroemia speciosa extract exhibits anti-hypertrophic effects, by examining its effects on the activation of NF-B. 3.1. Lagerstroemia speciosa extract inhibits NF-B’s activation TNF is a potent activator of NF-B, and its mechanism of action has been well established; therefore, we determined the effects of the Lagerstroemia speciosa extract on the TNF-induced activation of NF-B. In rat cardiomyocyte H9c2 cells, the extract suppressed NF-B’s activation in a dose-dependent manner, and this inhibition was observed at a concentration as low as 0.25 mg/ml: TNF-induced activation was completely suppressed after 4 h at 0.5 mg/ml of the Lagerstroemia speciosa extract (Fig. 1A). Next, we determined the minimum exposure time required for the extract to inhibit the TNF-mediated activation of NF-B. The cells were exposed to the inhibitor for 0.5, 1, 2, or, 4 h and then treated with TNF for 30 min. We found that the Lagerstroemia speciosa extract started to inhibit NF-B’s activation after only 1 h and almost completely suppressed it after 4 h (Fig. 1B). However, under the same conditions, the extract alone (in the absence of TNF) had no effect. At this concentration of Lagerstroemia speciosa extract used for 4 h, cell viability was >90%. To determine which NF-B subunits are responsible for the shifted bands, we performed a supershift assay by adding NF-B subunit-specific antibodies to the binding reactions. As shown in Fig. 1C, the band visualized in TNF-treated cells was indeed NF-B (see also Materials and methods and Fig. 1C). 4. Discussion We found that Lagerstroemia speciosa extract inhibited the TNF-induced activation of NF-B in rat H9c2 cardiomyocytes. Scientists have identified the different components of Lagerstroemia speciosa responsible for its activity. Leaves contains unique chemical constituents such as lagertannins (Takahashi et al., 1976), lagerstroemin (Hosoyama et al., 2003), corosolic acid (Hou et al., 2009), ursolic acid (Hou et al., 2009), oleanolic acid (Hou et al., 2009). Oleanolic acid and ursolic acid have been known to have inhibitory effects on NF-B’s activation in cancer cells (Shishodia et al., 2003; Suh et al., 2007). However, the effect of the other compounds on NF-B is unclear. Several investigators have reported
256
H. Ichikawa et al. / Journal of Ethnopharmacology 128 (2010) 254–256
in rat H9c2 cardiomyocytes. However, studies in vivo are needed to validate these findings. Acknowledgments This project was supported by the Encouraging Development of Strategic Research Centers, Special Coordination Funds for Promoting Science and Technology, Ministry of Education, Culture, Sports, Science and Technology, Japan. References
Fig. 1. Inhibition of NF-B’s activation by the Lagerstroemia speciosa extract. (A) The Lagerstroemia speciosa extract (LS) suppresses TNF-induced activation of NF-B in a dose-dependent manner in H9c2 cells. Cells were incubated with different concentrations of LS for 4 h and then with 50 ng/ml of TNF for 30 min. Nuclear extracts were prepared and assayed for NF-B activity by EMSA. (B) H9c2 cells were preincubated with 0.5 mg/ml of LS for the periods indicated, treated with 50 ng/ml of TNF for 30 min, and subjected to EMSA to determine for NF-B activity. (C) NF-B is composed of p65 and p50 subunits. Nuclear extracts from untreated or TNF-treated cells were incubated with the indicated antibodies, unlabeled NF-B oligo-probe, or mutant oligo-probe and then assayed for NF-B activity by EMSA.
an effect of corosolic acid on diabetes (Klein et al., 2007). It is possible that corosolic acid, oleanolic acid and ursolic acid play a major role in the effect of Lagerstroemia speciosa on diabetes and diabetes-induced cardiac myopathies like cardiomyocyte hypertrophy. Alternatively, new factors in the extract of Lagerstroemia speciosa leaves may act as NF-B inhibitors in our system. This is an important topic for future study. Overall, our results demonstrated that the Lagerstroemia speciosa extract suppressed the TNF-induced activation of NF-B
Aggarwal, B.B., 2004. Nuclear factor-kappaB: the enemy within. Cancer Cell 6, 203–208. Chen, S., Khan, Z.A., Cukiernik, M., Chakrabarti, S., 2003. Differential activation of NFkappa B and AP-1 in increased fibronectin synthesis in target organs of diabetic complications. American Journal of Physiolgy Endocrinology and Metabolism 284, E1089–E1097. Hong, H., Jai Maeng, W., 2004. Effects of malted barley extract and banaba extract on blood glucose levels in genetically diabetic mice. Journal of Medicinal Food 7, 487–490. Hosoyama, H., Sugimoto, A., Suzuki, Y., Sakane, I., Kakuda, T., 2003. Isolation and quantitative analysis of the alpha-amylase inhibitor in Lagerstroemia speciosa (L.) Pers. (Banaba). Yakugaku Zasshi 123, 599–605. Hou, W., Li, Y., Zhang, Q., Wei, X., Peng, A., Chen, L., Wei, Y., 2009. Triterpene acids isolated from Lagerstroemia speciosa leaves as alpha-glucosidase inhibitors. Phytotherpy Research 23, 614–618. Hwang, G.S., Oh, K.S., Koo, H.N., Seo, H.W., You, K.H., Lee, B.H., 2006. Effects of KR31378, a novel ATP-sensitive potassium channel activator, on hypertrophy of H9c2 cells and on cardiac dysfunction in rats with congestive heart failure. European Journal of Pharmacology 540, 131–138. Ichikawa, H., Takada, Y., Murakami, A., Aggarwal, B.B., 2005. Identification of a novel blocker of I kappa B alpha kinase that enhances cellular apoptosis and inhibits cellular invasion through suppression of NF-kappa B-regulated gene products. Journal of Immunology 174, 7383–7392. Ilercil, A., Devereux, R.B., Roman, M.J., Paranicas, M., O’Grady, M.J., Lee, E.T., Welty, T.K., Fabsitz, R.R., Howard, B.V., 2002. Associations of insulin levels with left ventricular structure and function in American Indians: the strong heart study. Diabetes 51, 1543–1547. Kakuda, T., Sakane, I., Takihara, T., Ozaki, Y., Takeuchi, H., Kuroyanagi, M., 1996. Hypoglycemic effect of extracts from Lagerstroemia speciosa L. leaves in genetically diabetic KK-AY mice. Bioscience Biotechnolohy and Biochemistry 60, 204–208. Klein, G., Kim, J., Himmeldirk, K., Cao, Y., Chen, X., 2007. Antidiabetes and anti-obesity activity of Lagerstroemia speciosa. Evidence Based Complementary Alternative Medicine 4, 401–407. Liu, F., Kim, J., Li, Y., Liu, X., Li, J., Chen, X., 2001. An extract of Lagerstroemia speciosa L. has insulin-like glucose uptake-stimulatory and adipocyte differentiationinhibitory activities in 3T3-L1 cells. Journal of Nutrition 131, 2242– 2247. Morisco, C., Seta, K., Hardt, S.E., Lee, Y., Vatner, S.F., Sadoshima, J., 2001. Glycogen synthase kinase 3beta regulates GATA4 in cardiac myocytes. Journal of Biological Chemistry 276, 28586–28597. Poornima, I.G., Parikh, P., Shannon, R.P., 2006. Diabetic cardiomyopathy: the search for a unifying hypothesis. Circulation Research 98, 596–605. Purcell, N.H., Tang, G., Yu, C., Mercurio, F., DiDonato, J.A., Lin, A., 2001. Activation of NF-kappa B is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes. Proceedings of the National Academy of Sciences of the United States of America 98, 6668–6673. Shishodia, S., Majumdar, S., Banerjee, S., Aggarwal, B.B., 2003. Ursolic acid inhibits nuclear factor-kappaB activation induced by carcinogenic agents through suppression of IkappaBalpha kinase and p65 phosphorylation: correlation with down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1. Cancer Research 63, 4375–4383. Suh, S.J., Jin, U.H., Kim, K.W., Son, J.K., Lee, S.H., Son, K.H., Chang, H.W., Lee, Y.C., Kim, C.H., 2007. Triterpenoid saponin, oleanolic acid 3-O-beta-dglucopyranosyl(1→3)-alpha-l-rhamnopyranosyl(1→2)-alpha-l-ar abinopyranoside (OA) from Aralia elata inhibits LPS-induced nitric oxide production by down-regulated NF-kappaB in raw 264.7 cells. Archives of Biochemistry and Biophysics 467, 227–233. Suzuki, Y., Unno, T., Ushitani, M., Hayashi, K., Kakuda, T., 1999. Antiobesity activity of extracts from Lagerstroemia speciosa L. leaves on female KK-Ay mice. Journal of Nutritional Science and Vitaminology (Tokyo) 45, 791–795. Takahashi, M., Osawa, K., Ueda, J., Yamamoto, F., Tsai, C.T., 1976. The components of the plants of Lagerstroemia genus. III. On the structure of the new tannin “lagertannin” from the leaves of Lagerstroemia speciosa (L.) Pers. (author’s transl). Yakugaku Zasshi 96, 984–987.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Lagerstroemia speciosa extract inhibit TNF-induced activation of nuclear factor-B in rat cardiomyocyte H9c2 cells Haruyo Ichikawa a,∗ , Hisato Yagi a , Takeshi Tanaka a , Jong-Chol Cyong a,b , Tomoh Masaki a a International Research and Educational Institute for Integrated Medical Sciences (IREIIMS), Tokyo Women’s Medical University, 8-1, Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan b Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kita-adachi-gun, Saitama 362-0806, Japan
a r t i c l e
i n f o
Article history: Received 7 September 2009 Received in revised form 5 December 2009 Accepted 17 December 2009 Available online 4 January 2010 Keywords: Lythraceae Lagerstroemia speciosa Cardiomyocyte hypertrophy TNF NF-B
a b s t r a c t Aim of the study: Lagerstroemia speciosa has been used as a folk medicine among people with diabetes in the Philippines. It is known to exhibit antidiabetic, antiobesity, and glucose transport activities through mechanisms not well defined. Diabetes leads to cardiomyocyte hypertrophy in association with an upregulation of vasoactive factors and activation of nuclear factor (NF)-B and activating protein-1. We therefore investigated the effect of Lagerstroemia speciosa on the activation of NF-B as a key mediator of cardiomyocyte hypertrophy, in rat cardiomyocyte H9c2 cells. Materials and methods: Water extract of Lagerstroemia speciosa (Lythraceae family) was prepared. H9c2 cells were used for treatment of Lagerstroemia speciosa extract with/without tumor necrosis factor (TNF). To examine NF-B’s activation, we performed an electrophoretic mobility shift assay (EMSA). Results: The activation of NF-B by TNF was completely blocked by a Lagerstroemia speciosa extract in a dose- and time-dependent manner in H9c2 cells. Conclusion: Overall, our results indicate that Lagerstroemia speciosa can inhibit DNA-binding of NF-B. This may explain its possible inhibition of diabetes-induced caridomyocyte hypertrophy. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The three characteristic metabolic disturbances in diabetics are hyperlipidemia, and early hyperinsulinemia followed by pancreatic -cell failure, leading to hyperglycemia. Hyperinsulinemia is an important trigger of cardiac hypertrophy in diabetic cardiomyopathy (Ilercil et al., 2002; Poornima et al., 2006). There are at least three cellular mechanisms whereby hyperinsulinemia mediates cardiomyocyte hypertrophy: (1) a PI3K/Akt-1 pathway, (2) a MAP kinase/extracellular signal-regulated kinase pathway, and (3) a GSK-3/NFAT pathway (Morisco et al., 2001). Recent publications also indicated that diabetes leads to myocardial hypertrophy in association with an upregulation of vasoactive factors such as endothelin-1 and activation of redox-sensitive transcription factors such as NF-B and activating protein-1 (Chen et al., 2003). Leaves of Lagerstroemia speciosa L. have been used in Southeast Asia as a folk medicine for the treatment of diabetes and kidney diseases (Liu et al., 2001). Several studies have indicated a beneficial role for Lagerstroemia speciosa (especially, corosolic acid) in terms of
antiobesity (Suzuki et al., 1999) and antidiabetes (Klein et al., 2007), and a hypoglycemic effect (Kakuda et al., 1996). Treatment with Lagerstroemia speciosa reduced blood glucose and insulin levels in diabetic KK-AY mice (Hong and Jai Maeng, 2004). Thus, it is possible that Lagerstroemia speciosa mediates anti-cardiac hypertrophy through a mechanism that is not well defined. NF-B is an inducible transcription factor that is activated by various carcinogens, inflammatory stimuli, and growth factors and controls the expression of genes linked with survival, proliferation, invasion, and metastasis of tumors (Aggarwal, 2004). Recent studies by several investigators have also implicated NF-B’s activation in the cardiac hypertrophic response, as modeled in cultured cardiac myocytes (Purcell et al., 2001). Because the NF-B activation pathway has been closely linked with cardiac hypertrophy, it is possible that Lagerstroemia speciosa acts through the modulation of this pathway. In the present study, Lagerstroemia speciosa extract abrogated the TNF-induced activation of NF-B in rat cardiomyocyte H9c2 cells. 2. Materials and methods
Abbreviations: NF-B, nuclear factor-B; TNF, tumor necrosis factor; EMSA, electrophoretic mobility shift assay. ∗ Corresponding author. Tel.: +81 3 3353 8111x24007; fax: +81 3 3353 3088. E-mail address: [email protected] (H. Ichikawa). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.12.033
2.1. Reagents Recombinant human TNF was obtained from R&D system (Minneapolis, MN). Dulbecco’s modified Eagle’s medium nutri-
H. Ichikawa et al. / Journal of Ethnopharmacology 128 (2010) 254–256
ent mixture F-12 HAM (DMEM/F-12) was purchased from Sigma–Aldrich Inc. (St. Louis, MO). Antibiotic Antimycotic, and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY). Antibodies against NF-Bp50 and NF-Bp65 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 2.2. Preparation of the Lagerstroemia speciosa extract Dried leaves of Lagerstroemia speciosa (Lythraceae family) as 100% (Code# J4571104434313) (3 g) obtained from LIV Laboratories Corp. (Tokyo, Japan) and were boiled with 47 ml of distilled water for 10 min. This crude extract was centrifuged (1610 g × 5 min) and the supernatant was filtered (0.2 m). The filtered extract was freeze dried and dissolved with distilled water as a 0.1 g/ml stock solution. The extract was stored at −20 ◦ C prior to use. 2.3. Cell lines H9c2 (rat cardiomyocyte) cells were obtained from American Type Culture Collection (Bethesda, MD). H9c2 cells were cultured in DMEM/F-12 supplemented with 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin, and 250 ng/ml amphotericin B. 2.4. Cell culture Cell culturing was conducted as reported (Hwang et al., 2006). For the study of cellular hypertrophy, cells were seeded at a density of 5 × 106 cells per 100 × 20 mm dish and cultured for 24 h in DMEM/F-12 containing 10% FBS. The cells were washed with serum-free medium (DMEM/F-12 without serum) and then treated with indicated concentrations of the Lagerstroemia speciosa extract under serum-free conditions for 24 h. Cells were treated with 50 ng/ml TNF in medium (DMEM/F12 with 0.5% FBS) with or without a fresh supply of the extract. Cells were incubated for the periods indicated at 37 ◦ C: a humidified atmosphere containing 5% CO2 . 2.5. Electrophoretic mobility shift assays for NF-B To examine NF-B’s activation, we performed an electrophoretic mobility shift assay (EMSA) as described (Ichikawa et al., 2005). Briefly, cells were washed with ice-cold phosphatebuffered saline and suspended in 0.4 ml of lysis buffer (10 mM HEPES, pH 7.9, 10 mM KC1, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, 2 g/ml aprotinin, and 0.5 mg/ml benzamidine). The cells were allowed to swell on ice for 35 min, after which 25 l of 10% Nonidet P-40 was added. The tubes were then agitated on a vortex for 30 s every 10 min and microcentrifuged for 30 s. The nuclear pellets were resuspended in 25 l of ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2.0 pg/ml leupeptin, 2.0 g/ml aprotinin, and 0.5 mg/ml benzamidine), and the tubes were incubated on ice for 30 min with intermittent agitation. This nuclear extract was microcentrifuged for 5 min at 4 ◦ C, and the supernatant was frozen at −80 ◦ C. EMSAs were performed by incubating 10 g of nuclear extract with 16 fmol of 32 P-end-labeled, 45-mer double-stranded NF-B oligonucleotides from the human immunodeficiency virus long terminal repeat (5 TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3 ; boldface indicates NF-B binding sites) in the presence of 0.5 g of poly (dI–dC), as a nonspecific competitor, in a binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% Nonidet P-40, 5% glycerol, and 50 mM NaCl) for 30 min at 37 ◦ C. The DNA–protein complex formed was separated from the free
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oligonucleotide on a 6.6% native polyacrylamide gel using a buffer containing 50 mM Tris, 200 mM glycine, and 1 mM EDTA, pH 8.5. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with the antibody against either p50 or p65 of NF-B for 15 min at 37 ◦ C before the complex was analyzed by EMSA. The NF-B band was supershifted to a higher molecular mass only when the antibody against either p50 or p65 of NF-B was used. When an excess of unlabeled NF-B oligonucleotide was added to the reaction mixture, it competed for binding sites and no binding with the labeled oligonucleotide was observed, suggesting that the complex formed was specific. To confirm the specificity further, binding was carried out with a labeled mutant oligonucleotide in which the conserved GGG required for binding NF-B was mutated to CTC. No band for NF-B was observed, suggesting that the complex formed required the conserved GGG for binding (Fig. 1C). The dried gels were visualized, and radioactive bands were quantified with a FLA5100 fluoro image analyzer (FUJIFILM, Tokyo, Japan) using Multigauge software (FUJIFILM, Tokyo, Japan). Fold increase is based on the control value. 3. Results The goal of this study was to investigate the mechanism by which the Lagerstroemia speciosa extract exhibits anti-hypertrophic effects, by examining its effects on the activation of NF-B. 3.1. Lagerstroemia speciosa extract inhibits NF-B’s activation TNF is a potent activator of NF-B, and its mechanism of action has been well established; therefore, we determined the effects of the Lagerstroemia speciosa extract on the TNF-induced activation of NF-B. In rat cardiomyocyte H9c2 cells, the extract suppressed NF-B’s activation in a dose-dependent manner, and this inhibition was observed at a concentration as low as 0.25 mg/ml: TNF-induced activation was completely suppressed after 4 h at 0.5 mg/ml of the Lagerstroemia speciosa extract (Fig. 1A). Next, we determined the minimum exposure time required for the extract to inhibit the TNF-mediated activation of NF-B. The cells were exposed to the inhibitor for 0.5, 1, 2, or, 4 h and then treated with TNF for 30 min. We found that the Lagerstroemia speciosa extract started to inhibit NF-B’s activation after only 1 h and almost completely suppressed it after 4 h (Fig. 1B). However, under the same conditions, the extract alone (in the absence of TNF) had no effect. At this concentration of Lagerstroemia speciosa extract used for 4 h, cell viability was >90%. To determine which NF-B subunits are responsible for the shifted bands, we performed a supershift assay by adding NF-B subunit-specific antibodies to the binding reactions. As shown in Fig. 1C, the band visualized in TNF-treated cells was indeed NF-B (see also Materials and methods and Fig. 1C). 4. Discussion We found that Lagerstroemia speciosa extract inhibited the TNF-induced activation of NF-B in rat H9c2 cardiomyocytes. Scientists have identified the different components of Lagerstroemia speciosa responsible for its activity. Leaves contains unique chemical constituents such as lagertannins (Takahashi et al., 1976), lagerstroemin (Hosoyama et al., 2003), corosolic acid (Hou et al., 2009), ursolic acid (Hou et al., 2009), oleanolic acid (Hou et al., 2009). Oleanolic acid and ursolic acid have been known to have inhibitory effects on NF-B’s activation in cancer cells (Shishodia et al., 2003; Suh et al., 2007). However, the effect of the other compounds on NF-B is unclear. Several investigators have reported
256
H. Ichikawa et al. / Journal of Ethnopharmacology 128 (2010) 254–256
in rat H9c2 cardiomyocytes. However, studies in vivo are needed to validate these findings. Acknowledgments This project was supported by the Encouraging Development of Strategic Research Centers, Special Coordination Funds for Promoting Science and Technology, Ministry of Education, Culture, Sports, Science and Technology, Japan. References
Fig. 1. Inhibition of NF-B’s activation by the Lagerstroemia speciosa extract. (A) The Lagerstroemia speciosa extract (LS) suppresses TNF-induced activation of NF-B in a dose-dependent manner in H9c2 cells. Cells were incubated with different concentrations of LS for 4 h and then with 50 ng/ml of TNF for 30 min. Nuclear extracts were prepared and assayed for NF-B activity by EMSA. (B) H9c2 cells were preincubated with 0.5 mg/ml of LS for the periods indicated, treated with 50 ng/ml of TNF for 30 min, and subjected to EMSA to determine for NF-B activity. (C) NF-B is composed of p65 and p50 subunits. Nuclear extracts from untreated or TNF-treated cells were incubated with the indicated antibodies, unlabeled NF-B oligo-probe, or mutant oligo-probe and then assayed for NF-B activity by EMSA.
an effect of corosolic acid on diabetes (Klein et al., 2007). It is possible that corosolic acid, oleanolic acid and ursolic acid play a major role in the effect of Lagerstroemia speciosa on diabetes and diabetes-induced cardiac myopathies like cardiomyocyte hypertrophy. Alternatively, new factors in the extract of Lagerstroemia speciosa leaves may act as NF-B inhibitors in our system. This is an important topic for future study. Overall, our results demonstrated that the Lagerstroemia speciosa extract suppressed the TNF-induced activation of NF-B
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