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Transcriptional regulation of corticotrophin releasing factor gene by furocoumarins isolated from seeds of Psoralea corylifolia
Life Sciences 82 (2008) 1117–1121
Contents lists available at ScienceDirect
Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Transcriptional regulation of corticotrophin releasing factor gene by furocoumarins isolated from seeds of Psoralea corylifolia Yangchao Chen a, Yuen-Ting Cheung b, Ling-dong Kong c,⁎, Tzi Bun Ng d, Chunfeng Qiao e, Shi-fu Mo e, Hong-xi Xu e, Hsiang-fu Kung b,⁎ a
Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, China Stanley Ho Center for Emerging Infectious Diseases, The Chinese University of Hong Kong, Hong Kong, China State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, China d Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong, China e Hong Kong Jockey Club Institute of Chinese Medicine, Hong Kong, China b c
A R T I C L E
I N F O
Article history: Received 4 December 2007 Accepted 19 March 2008 Keywords: Depression Antidepressant drug Corticotrophin releasing factor Traditional Chinese medicine
A B S T R A C T Dysregulation of the hypothalamic–pituitary–adrenocortical (HPA) system plays a causal role in the development and course of depression. Clinically effective antidepressant drugs normalize the disturbed activity of the HPA axis by inhibition of corticotrophin releasing factor gene promoter activity. Furocoumarins from Psoralea corylifolia have been demonstrated to possess potent antidepressant properties. In order to ascertain whether these coumarin components directly regulate corticotrophin releasing factor (CRF) gene transcription, we studied their effect on CRF promoter activity using the luciferase reporter assay in Neuro-2A cells. CRF promoter was cloned into firefly luciferase reporter vector and co-transfected into Neuro-2A cells with Renilla luciferase plasmid as internal control. CRF promoter transcription activity was induced by forskolin. We found that one of the components of P. corylifolia, psoralidin, strongly inhibited forskolin-induced CRF promoter activity. We further confirmed that psoralidin suppressed CRF gene transcription by quantitative reverse transcription polymerase chain reaction. Hence, down-regulation of CRF gene transcription by psoralidin may be involved in the molecular mechanism underlying its potent antidepressant effect. © 2008 Elsevier Inc. All rights reserved.
Introduction Major depression is frequently associated with hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis (Holsboer et al., 1985; Linkowski et al., 1985; Erhardt et al., 2006), whose proximal part is mediated by secretion of corticotrophin releasing factor (CRF) from the paraventricular nucleus of the hypothalamus. Antidepressant drugs as clinically effective therapeutics normalize the dysregulated HPA axis partly by decreasing CRF synthesis and secretion (Budziszewska et al., 2004). Many studies attempted to identify herbal medicines and their extracts and evaluate their effectiveness in treating depression (He et al., 2004; Li et al., 2005; Luo et al., 2006; Mantle, 2002; Wu et al., 1999). Our preliminary data suggest that furocoumarins from Psoralea corylifolia possess potent antidepressant properties in an animal model (Kong et al., 2001; Chen et al., 2005; Qiao et al., 2006). Recently, we investigated the antidepressant-like effects of psoralidin isolated
from the seeds of P. corylifolia in the forced swimming test (FST) in mice. Psoralidin significantly decreased immobility time and increased swimming behavior without altering climbing behavior in the mouse FST after oral administration for 3 consecutive days at dosage of 60 mg/kg (Yi et al., 2008). Furthermore, no toxicity was observed in mice after psoralidin treatment, suggesting it an effective yet safe antidepressant drug. Psoralidin also ameliorated the elevations in serum CRF induced by swimming stress in mice. However, the exact molecular mechanism has not been elucidated. In the present investigation, we aim to test the effect of five furocoumarins isolated from P. corylifolia, including psoralen, isopsoralen, psoralidin, psoralenoside and isopsoralenside on transcriptional regulation of CRF gene. The effects of the compounds from P. corylifolia on CRF promoter activity and its mRNA level were investigated to explore the molecular mechanism underlying the antidepressant activity of the medicinal herb. Materials and methods
⁎ Corresponding authors. H. Kung is to be contacted at Stanley Ho Center for Emerging Infectious Diseases, 511A Basic Medical Sciences Building, The Chinese University of Hong Kong, Shatin, Hong Kong, China. Tel.: +852 26037743; fax: +852 29944988. Kong, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, China. E-mail addresses: [email protected] (L. Kong), [email protected] (H. Kung). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.03.014
Chemicals Five furocoumarins (1 psoralenoside, 2 isopsoralenside, 3 psoralidin 4 psoralen, 5 isopsoralen) were isolated from P. corylifolia and purified as described in our previous reports (Yi et al., 2008). The purity
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regarding for drug treatment, N2A cells were transfected with pGL3/ hCRF plasmid, 12 h after transfection, N2A cells were exposed to the five furocoumarins at the indicated concentration for 48 h. Afterwards, N2A cells were incubated with 15 μM forskolin or 0.5% DMSO for another 24 h and luciferase activity was measured. Quantitative real-time RT-PCR (QRT-PCR) The QRT-PCR was carried out using SYBR green QRT-PCR Master Mix, 2-Step kit from ABI. Briefly, total RNA was extracted using Trizol reagent as described by the manufacturer (Invitrogen). The cDNA was generated using oligo dT primer mix and total RNA. The cDNA was PCR amplified using primers specific for mouse GAPDH and CRF. The PCR amplication was carried out in an ABI 7500 machine. The Ct (threshold cycle) value of CRF amplification was normalized to that of GAPDH control. The primers for QRT-PCR were— CRF forward: 5-GCT AAC TTT TTC CGC GTG TTG CTG-3; CRF reverse: 5-GGT GGA AGG TGA GAT CCA GAG AG-3; GAPDH forward: 5-AGG TGA CCG CAT CTT CTT GT-3; GAPDH reverse: 5CTT GCC GTG GGT AGA GTC AT-3. Fig. 1. Chemical structure of five furocoumarins from Psoralea corylifolia.
Determination of cell viability
of the chemicals exceeded 98% as judged by HPLC analysis. Their chemical structures are shown in Fig. 1. Forskolin, fluoxetine hydrochloride and dimethylsulfoxide (DMSO) were purchased from Sigma. All drugs were dissolved in DMSO.
Cell viability was determined using the methylthiazoletetrazolium (MTT) assay. MTT (Sigma) was dissolved in PBS and added to the culture to a final concentration of 0.5 mg/ml and incubated for 3 h. The formazan product was extracted with DMSO and detected with a UV spectrophotometer at 595 nm.
Cell culture and drug treatment
Statistical analysis
The mouse neuroblastoma cell line Neuro-2A (N2A) was purchased from American Type Culture Corporation (ATCC). N2A cells were cultured in Minimum Essential Medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and maintained at 37 °C in a humidified atmosphere of 5% CO2. N2A cells (8 × 104 per well) were seeded in a 96-well culture plate 72 h prior to drug treatment. The five furocoumarins were incubated with the cells for 48 h at the indicated concentration. Afterwards, N2A cells were incubated with 15 μM forskolin or 0.5% DMSO for another 24 h.
The data are presented as means ± SEM of three independent experiments in triplicate wells, and the significance of differences between the means was evaluated using one-way analysis of variance (ANOVA). A value of P b 0.05 was considered to be statistically significant in all cases. Results Effect of furocoumarins from P. corylifolia on forskolin-induced CRF gene promoter activity
Constructs and CRF promoter activity assay Human DNA was extracted from the immortalized hepatocyte cell line LO2 by Wizard SV Genomic DNA Purification System (Promega). The regulatory region of human CRF gene (−663 to +124) was amplified by PCR using as forward primer CRF KpnI 5′ CGC GGT ACC GAG AGA CGT CTC CGG GGG C 3′ (28 bp, KpnI recognition site underlined) and as reverse primer CRF BglII 5′ GCG AGA TCT GGC TCA TAA CTC CTT TAT GTG CTT GC 3′ (35 bp, BglII recognition site underlined). The PCR reaction mixture consisted of 50 ng of human genomic DNA, 2 μl (10 μM) of each primer, 5 μl (10 mM) of dNTPs, 5 μl of 10 × PCR buffer, 2 U of Taq polymerase. The fragment was amplified by initial denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 58 °C for 1 min and 72 °C for 1 min, and final extension at 72 °C for 10 min. Purified PCR fragment was sequenced, digested and then ligated into KpnI/BglII sites of pGL3-basic vector (Promega). The promoter activity of the human CRF upstream region was analyzed using Dual-Luciferase Reporter Assay System (Promega); 0.25 μg of pGL3/hCRF plasmid and 1 ng of Renilla pRL-SV40 internal control plasmid were transiently transfected into N2A cells using Lipofectamine 2000 transfection reagent (Invitrogen). Twelve hours after transfection, N2A cells were treated with or without forskolin trigger for another 24 h, N2A cells were then lysed with 20 μl of PLB buffer. A 10-μl aliquot of the lysate was mixed with 10 μl of lysis buffer and 10 μl of Stop & Glo reagent in sequence. Firefly luciferase activity and Renilla luciferase activity were measured with a luminometer. As
We aimed to study the effect of the five furocoumarins from P. corylifolia (Fig. 1) on human CRF promoter activity. The regulatory region of human CRF gene was cloned into pGL3-basic plasmid and transfected into N2A cells. The cells were subsequently treated with the furocoumarins psoralenoside, isopsoralenside, psoralidin, psoralen, and isopsoralen, followed by triggering using 15 μM forskolin. As shown in Fig. 2, forskolin incubation increased promoter activity approximately 16 folds compared with untriggered cells. As shown in
Fig. 2. Induction of CRF promoter by forskolin. N2A cells were transfected with PGL3/ hCRFP and pRL-SV40 as internal control. Luciferase activity was measured after treatment with 15 μM forskolin.
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Fig. 3. Effect of furocoumarins from Psoralea corylifolia on human CRF promoter activity induced by forskolin. Luciferase activity was measured after treatment with drugs of indicated concentration for 48 h. The cells were co-transfected with plasmids pGL3/hCRFP and pRL-SV40 as internal control. The graph plots the mean ± SD (n = 3) of fold of difference in promoter activity. ⁎: P b 0.05; ⁎⁎: P b 0.01.
Fig. 3, incubation with psoralen did not result in a significant difference on CRF promoter activity compared with control. However, at higher concentrations, psoralen caused the activity to further increase. Isopsoralen showed no effect on CRF promoter. Psoralidin decreased CRF promoter activity in a dose-dependent manner. The observation that psoralidin can inhibit CRF promoter suggested that by decreasing CRF synthesis, it is a potential candidate for antidepression. Interestingly, the combining treatment of psoralen and psoralidin at the same concentration of 40 μM null each other's effects on CRF promoter activity. Psoralenoside and isopsoralenside treatment exerted no significant inhibitory effect on CRF promoter. Fluoxetine was used as a positive control since it was reported to inhibit CRF promoter activity. Inhibition of forskolin-induced CRF gene transcription by furocoumarins from P. corylifolia We next studied the effects of furocoumarins from P. corylifolia on CRF gene transcription at mRNA level by QRT-PCR. Psoralen, isopsoralen, psoralenoside and isopsoralenside exerted no effect on
Fig. 4. Inhibition of forskolin-induced CRF gene transcription in N2A cells by psoralidin. N2A cells incubated with forskolin were treated with psoralidin and vehicle control CRF mRNA levels were quantified by QRT-PCR and normalized to GAPDH mRNA levels as described in Materials and methods. The QRT-PCR assays were performed in triplicate.
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Fig. 5. Cytotoxic effect of five furocoumarins from Psoralea corylifolia and antidepressant fluoxetine on N2A cells.
forskolin-induced CRF gene transcription, consistent with results of the luciferase reporter assay. Psoralidin significantly inhibited forskolin-triggered CRF mRNA synthesis in a dose-dependent manner as shown in Fig. 4. Furthermore, the dose-dependent trend to inhibit gene transcription at lower concentration from 10 to 40 μM is obvious (Fig. 4). Effect of furocoumarins from P. corylifolia on cell viability To test the cytotoxicity of furocoumarins from P. corylifolia, N2A cells were incubated with 5 to 100 μM of the drugs with a final DMSO concentration of 0.5% for 48 h. MTT assay was performed to examine survival of the cells after drug treatment. Psoralidin demonstrated no significant inhibitory effect on N2A cell proliferation at concentrations lower than 50 μM (Fig. 5). It exerted significant repressive effect on CRF transcription at the concentration of 40 μM. Furthermore, the dose-dependent trend to inhibit CRF gene transcription of psoralidin is obvious at lower concentration as shown in Fig. 4, suggesting that its inhibitory effect on CRF gene transcription was not due to its cytotoxicity. None of the other four components used in the present study evoked a significant toxic effect, when examined by the MTT assay in N2A cells. Discussion Long-term administration of some antidepressant drugs has been shown to decrease CRF mRNA level in the rat hypothalamus (Aubry et al., 1999; Fadda et al., 1995). More recent studies demonstrated that some clinically effective antidepressant drugs, such as imipramine and fluoxetine, inhibited hCRF promoter activity in Neuro-2A cells (Budziszewska et al., 2004). Inhibition of CRF gene promoter activity by antidepressant drugs has been suggested to be a molecular mechanism by which these drugs inhibit the activity of HPA axis (Budziszewska et al., 2004; Kunugi et al., 2006). CRF has been suggested to be a promising drug target for treating depression. Our recent studies also showed that psoralidin from P. corylifolia exerted effective antidepressant activity in animal models as well as ameliorated the swim stress-induced elevations in serum CRF concentration without toxic effects (Yi et al., 2008). We firstly examined the effect of five furocoumarins from P. corylifolia on CRF gene promoter activity in Neuro-2A cells. The main finding of these experiments is the demonstration that psoralidin, one of the compounds under study, was able to inhibit hCRF gene promoter activity in Neuro-2A cells. The inhibitory effect was dose-dependent. Psoralidin exerted a significant inhibitory effect on hCRF promoter activity at a concentration without evoking evident cytotoxicity in Neuro-2A cells. Based on the comparison of the structures of these five
furocoumarins, it seems that the aromatic cycle attached to C-3 rather than the isoprenyl at C-6 is responsible to the significant inhibitory effect on CRF promoter activity of psoralidin. To study the effect of psoralidin on CRF gene transcription, we used quantitative RT-PCR to measure CRF mRNA level. Endogenous CRF mRNA level in Neuro-2A cells was low, but was upregulated by forskolin treatment. We further confirmed the inhibitory effect of psoralidin on CRF gene transcription. Cyclic adenosine 3′,5′-cyclic monophosphate (cAMP)/cAMP-dependent protein kinase (PKA) signal transduction is the main pathway that stimulates CRF gene promoter activity. Forskolin increases cellular cAMP levels and CRF gene transcription induced by forskolin appears to be regulated via cAMP regulatory element (CRE). Besides PKA, recent studies also suggest a possible involvement of Ca2+/calmodulindependent kinase and PI-3K mediated pathway in the intracellular mechanism of antidepressant effects on CRF gene promoter activity (Basta-Kaim et al., 2005). Whether psoralidin exerts its effects on CRF gene at the level of cAMP synthesis and/or involves downstream signaling molecules needs further confirmation. P. corylifolia has long been used for treatment of the symptoms of aging (Jiangsu College of New Medicine, 1977). Our studies showed that extracts from P. corylifolia exhibited in vitro inhibitory actions on monoamine oxidase (MAO) activity (Kong et al., 2001). Our studies also suggested that furocoumarin extracts from P. corylifolia possessed strong antidepressant activity in an animal model (Chen et al., 2005). It is interesting to find opposite effect of psoralen and psoralidin on CRF gene transcriptional activity. We are currently testing their effects in animal behavioral models (Yi et al., 2008). It is reported that some antipsychotic drugs e.g. chlorpromazine or clozapine also potently inhibited CRF gene promoter activity in Neuro-2A cells (Basta-Kaim et al., 2006). The ability of furocoumarins to inhibit the forskolin-induced activity of CRF gene transcription may be a molecular mechanism involved in their inhibitory effect on the HPA axis activity and their antidepressant properties. Our data also support the concept that CRF gene may be an attractive drug target for screening effective antidepressant components from traditional Chinese medicine. Conclusion We found that psoralidin isolated from the seeds of P. corylifolia strongly inhibited forskolin-induced CRF promoter activity and suppressed CRF gene transcription. Recently, we have also shown the antidepressant-like effects of psoralidin in the forced swimming test (FST) in ICR strain of male mice. Furthermore, psoralidin also ameliorated the elevations in serum CRF induced by swimming stress in mice. Hence down-regulation of CRF gene transcription by psoralidin may be involved in the molecular mechanism underlying its potent antidepressant effect. Acknowledgement This project was supported by Hong Kong Jockey Club Institute of Chinese Medicine (JCICM-39-02 to HFK). References Aubry, J.M., Pozzzoli, G., Vale, W.W., 1999. Chronic treatment with the antidepressant amitriptyline decreases CRF-R1 receptor mRNA levels in the rat amygdale. Neuroscience Letters 266, 197–200. Basta-Kaim, A., Budziszewska, B., Jaworska-Feil, L., Tetich, M., Kubera, M., Leskiewicz, M., Lason, W., 2005. Inhibitory effect of imipramine on the human corticotropinreleasing-hormone gene promoter activity operates through a PI3-K/AKT mediated pathway. Neuropharmacology 49, 156–164. Basta-Kaim, A., Budziszewska, B., Jaworska-Feil, L., Tetich, M., Kubera, M., Leskiewicz, M., Otczyk, M., Lason, W., 2006. Antipsychotic drugs inhibit the human corticotropin-releasing-hormone gene promoter activity in neuro-2A cells— an involvement of protein kinases. Neuropsychopharmacology 31, 53–65. Budziszewska, B., Jaworska-Feil, L., Tetich, M., Basta-Kaim, A., Kubera, M., Leskiewicz, M., Lason, W., 2004. Regulation of the human corticotropin-releasing-hormone
Y. Chen et al. / Life Sciences 82 (2008) 1117–1121 gene promoter activity by antidepressant drugs in Neuro-2A and AtT-20 cells. Neuropsychopharmacology 29, 785–794. Chen, Y., Kong, L.D., Xia, X., Kung, H.F., Zhang, L., 2005. Behavioral and biochemical studies of total furocoumarins from seeds of Psoralea corylifolia in the forced swimming test in mice. Journal of Ethnopharmacology 96, 451–459. Erhardt, A., Ising, M., Unschuld, P.G., Kern, N., Lucae, S., Putz, B., Uhr, M., Binder, E.B., Holsboer, F., Keck, M.E., 2006. Regulation of the hypothalamic–pituitary–adrenocortical system in patients with panic disorder. Neuropsychopharmacology 31, 2515–2522. Fadda, P., Pani, L., Porcella, A., Fratta, W., 1995. Chronic imipramine, L-sulpiride and mianserine decrease corticotropin releasing factor levels in the rat brain. Neuroscience Letters 192, 121–123. He, J.Q., Tang, X.W., Chen, H.X., Xiang, Z., 2004. Study on treatment of climacteric depression with bushen tiaogan qingxin recipe. Zhongguo Zhong Xi Yi Jie He Za Zhi 24, 889–892. Holsboer, F., Gerken, A., Stalla, G.K., Muller, O.A., 1985. ACTH, cortisol and corticosterone output after ovine corticotrophin-releasing factor challenge during depression and after recovery. Biological Psychiatry 20, 276–286. Jiangsu College of New Medicine, 1977. The Dictionary of the Traditional Chinese Medicine. Shanghai Press of Science & Technology, Shanghai, pp. 1177–1179. Kong, L.D., Tan, R.X., Woo, A.Y., Cheng, C.H.K., 2001. Inhibition of rat brain monoamine oxidase activities by psoralen and isopsoralen: implications for the treatment of affective disorders. Pharmacology and Toxicology 88, 75–80. Kunugi, H., Ida, I., Owashi, T., Kimura, M., Inoue, Y., Nakagawa, S., Yabana, T., Urushibara, T., Kanai, R., Aihara, M., Yuuki, N., Otsubo, T., Oshima, A., Kudo, K., Inoue, T., Kitaichi, Y., Shirakawa, O., Isogawa, K., Nagayama, H., Kamijima, K., Nanko, S., Kanba, S.,
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Higuchi, T., Mikuni, M., 2006. Assessment of the dexamethasone/CRH test as a state-dependent marker for hypothalamic–pituitary–adrenal (HPA) axis abnormalities in major depressive episode: a multicenter study. Neuropsychopharmacology 31, 212–220. Li, Z., Kang, S.S., Lee, S., Rivier, C., 2005. Effect of ethanol on the regulation of corticotropin-releasing factor (CRF) gene expression. Molecular and Cellular Neuroscience 29, 345–354. Linkowski, P., Mendlewicz, J., LeClerc, R., Brasseur, M., Hubain, P., Goldstein, J., Copinschi, G., Van Cauter, E., 1985. The 24-hour profile of ACTH and cortisol in major depressive illness. Journal of Clinical Endocrinology and Metabolism 61, 429–438. Luo, H.C., Qian, R.Q., Zhao, X.Y., Bi, J., Xin, H., Jiang, X., Xu, K., Yan, S., 2006. Clinical observation on effect of danzhi xiaoyao powder in treating depression. Zhongguo Zhong Xi Yi Jie He Za Zhi 26, 212–214. Mantle, F., 2002. The role of alternative medicine in treating postnatal depression. Complementary Therapies in Nursing and Midwifery 8, 197–203. Qiao, C.F., Han, Q.B., Mo, S.F., Song, J.Z., Xu, L.J., Chen, S.L., Yang, D.J., Kong, L.D., Kung, H.F., Xu, H.X., 2006. Psoralenoside and isopsoralenoside, two new benzofuran glycosides from Psoralea corylifolia. Chemical and Pharmaceutical Bulletin 54, 714–716. Wu, J., An, H., Li, Y., Duan, L., 1999. TCM treatment with the modified wendan tang in 40 cases of melancholia. Journal of Traditional Chinese Medicine 19, 296–297. Yi, L.T., Li, Y.C., Pan, Y., Li, J.M., Xu, Q., Mo, S.F., Qiao, C.F., Jiang, F.X., Xu, H.X., Lu, X.B., Kong, L.D., Kung, H.F., 2008. Antidepressant-like effects of psoralidin isolated from the seeds of Psoralea corylifolia in the forced swimming test in mice. Progress in Neuropsychopharmacology and Biological Psychiatry 32, 510–519.
Contents lists available at ScienceDirect
Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Transcriptional regulation of corticotrophin releasing factor gene by furocoumarins isolated from seeds of Psoralea corylifolia Yangchao Chen a, Yuen-Ting Cheung b, Ling-dong Kong c,⁎, Tzi Bun Ng d, Chunfeng Qiao e, Shi-fu Mo e, Hong-xi Xu e, Hsiang-fu Kung b,⁎ a
Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, China Stanley Ho Center for Emerging Infectious Diseases, The Chinese University of Hong Kong, Hong Kong, China State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, China d Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong, China e Hong Kong Jockey Club Institute of Chinese Medicine, Hong Kong, China b c
A R T I C L E
I N F O
Article history: Received 4 December 2007 Accepted 19 March 2008 Keywords: Depression Antidepressant drug Corticotrophin releasing factor Traditional Chinese medicine
A B S T R A C T Dysregulation of the hypothalamic–pituitary–adrenocortical (HPA) system plays a causal role in the development and course of depression. Clinically effective antidepressant drugs normalize the disturbed activity of the HPA axis by inhibition of corticotrophin releasing factor gene promoter activity. Furocoumarins from Psoralea corylifolia have been demonstrated to possess potent antidepressant properties. In order to ascertain whether these coumarin components directly regulate corticotrophin releasing factor (CRF) gene transcription, we studied their effect on CRF promoter activity using the luciferase reporter assay in Neuro-2A cells. CRF promoter was cloned into firefly luciferase reporter vector and co-transfected into Neuro-2A cells with Renilla luciferase plasmid as internal control. CRF promoter transcription activity was induced by forskolin. We found that one of the components of P. corylifolia, psoralidin, strongly inhibited forskolin-induced CRF promoter activity. We further confirmed that psoralidin suppressed CRF gene transcription by quantitative reverse transcription polymerase chain reaction. Hence, down-regulation of CRF gene transcription by psoralidin may be involved in the molecular mechanism underlying its potent antidepressant effect. © 2008 Elsevier Inc. All rights reserved.
Introduction Major depression is frequently associated with hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis (Holsboer et al., 1985; Linkowski et al., 1985; Erhardt et al., 2006), whose proximal part is mediated by secretion of corticotrophin releasing factor (CRF) from the paraventricular nucleus of the hypothalamus. Antidepressant drugs as clinically effective therapeutics normalize the dysregulated HPA axis partly by decreasing CRF synthesis and secretion (Budziszewska et al., 2004). Many studies attempted to identify herbal medicines and their extracts and evaluate their effectiveness in treating depression (He et al., 2004; Li et al., 2005; Luo et al., 2006; Mantle, 2002; Wu et al., 1999). Our preliminary data suggest that furocoumarins from Psoralea corylifolia possess potent antidepressant properties in an animal model (Kong et al., 2001; Chen et al., 2005; Qiao et al., 2006). Recently, we investigated the antidepressant-like effects of psoralidin isolated
from the seeds of P. corylifolia in the forced swimming test (FST) in mice. Psoralidin significantly decreased immobility time and increased swimming behavior without altering climbing behavior in the mouse FST after oral administration for 3 consecutive days at dosage of 60 mg/kg (Yi et al., 2008). Furthermore, no toxicity was observed in mice after psoralidin treatment, suggesting it an effective yet safe antidepressant drug. Psoralidin also ameliorated the elevations in serum CRF induced by swimming stress in mice. However, the exact molecular mechanism has not been elucidated. In the present investigation, we aim to test the effect of five furocoumarins isolated from P. corylifolia, including psoralen, isopsoralen, psoralidin, psoralenoside and isopsoralenside on transcriptional regulation of CRF gene. The effects of the compounds from P. corylifolia on CRF promoter activity and its mRNA level were investigated to explore the molecular mechanism underlying the antidepressant activity of the medicinal herb. Materials and methods
⁎ Corresponding authors. H. Kung is to be contacted at Stanley Ho Center for Emerging Infectious Diseases, 511A Basic Medical Sciences Building, The Chinese University of Hong Kong, Shatin, Hong Kong, China. Tel.: +852 26037743; fax: +852 29944988. Kong, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, China. E-mail addresses: [email protected] (L. Kong), [email protected] (H. Kung). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.03.014
Chemicals Five furocoumarins (1 psoralenoside, 2 isopsoralenside, 3 psoralidin 4 psoralen, 5 isopsoralen) were isolated from P. corylifolia and purified as described in our previous reports (Yi et al., 2008). The purity
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regarding for drug treatment, N2A cells were transfected with pGL3/ hCRF plasmid, 12 h after transfection, N2A cells were exposed to the five furocoumarins at the indicated concentration for 48 h. Afterwards, N2A cells were incubated with 15 μM forskolin or 0.5% DMSO for another 24 h and luciferase activity was measured. Quantitative real-time RT-PCR (QRT-PCR) The QRT-PCR was carried out using SYBR green QRT-PCR Master Mix, 2-Step kit from ABI. Briefly, total RNA was extracted using Trizol reagent as described by the manufacturer (Invitrogen). The cDNA was generated using oligo dT primer mix and total RNA. The cDNA was PCR amplified using primers specific for mouse GAPDH and CRF. The PCR amplication was carried out in an ABI 7500 machine. The Ct (threshold cycle) value of CRF amplification was normalized to that of GAPDH control. The primers for QRT-PCR were— CRF forward: 5-GCT AAC TTT TTC CGC GTG TTG CTG-3; CRF reverse: 5-GGT GGA AGG TGA GAT CCA GAG AG-3; GAPDH forward: 5-AGG TGA CCG CAT CTT CTT GT-3; GAPDH reverse: 5CTT GCC GTG GGT AGA GTC AT-3. Fig. 1. Chemical structure of five furocoumarins from Psoralea corylifolia.
Determination of cell viability
of the chemicals exceeded 98% as judged by HPLC analysis. Their chemical structures are shown in Fig. 1. Forskolin, fluoxetine hydrochloride and dimethylsulfoxide (DMSO) were purchased from Sigma. All drugs were dissolved in DMSO.
Cell viability was determined using the methylthiazoletetrazolium (MTT) assay. MTT (Sigma) was dissolved in PBS and added to the culture to a final concentration of 0.5 mg/ml and incubated for 3 h. The formazan product was extracted with DMSO and detected with a UV spectrophotometer at 595 nm.
Cell culture and drug treatment
Statistical analysis
The mouse neuroblastoma cell line Neuro-2A (N2A) was purchased from American Type Culture Corporation (ATCC). N2A cells were cultured in Minimum Essential Medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and maintained at 37 °C in a humidified atmosphere of 5% CO2. N2A cells (8 × 104 per well) were seeded in a 96-well culture plate 72 h prior to drug treatment. The five furocoumarins were incubated with the cells for 48 h at the indicated concentration. Afterwards, N2A cells were incubated with 15 μM forskolin or 0.5% DMSO for another 24 h.
The data are presented as means ± SEM of three independent experiments in triplicate wells, and the significance of differences between the means was evaluated using one-way analysis of variance (ANOVA). A value of P b 0.05 was considered to be statistically significant in all cases. Results Effect of furocoumarins from P. corylifolia on forskolin-induced CRF gene promoter activity
Constructs and CRF promoter activity assay Human DNA was extracted from the immortalized hepatocyte cell line LO2 by Wizard SV Genomic DNA Purification System (Promega). The regulatory region of human CRF gene (−663 to +124) was amplified by PCR using as forward primer CRF KpnI 5′ CGC GGT ACC GAG AGA CGT CTC CGG GGG C 3′ (28 bp, KpnI recognition site underlined) and as reverse primer CRF BglII 5′ GCG AGA TCT GGC TCA TAA CTC CTT TAT GTG CTT GC 3′ (35 bp, BglII recognition site underlined). The PCR reaction mixture consisted of 50 ng of human genomic DNA, 2 μl (10 μM) of each primer, 5 μl (10 mM) of dNTPs, 5 μl of 10 × PCR buffer, 2 U of Taq polymerase. The fragment was amplified by initial denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 58 °C for 1 min and 72 °C for 1 min, and final extension at 72 °C for 10 min. Purified PCR fragment was sequenced, digested and then ligated into KpnI/BglII sites of pGL3-basic vector (Promega). The promoter activity of the human CRF upstream region was analyzed using Dual-Luciferase Reporter Assay System (Promega); 0.25 μg of pGL3/hCRF plasmid and 1 ng of Renilla pRL-SV40 internal control plasmid were transiently transfected into N2A cells using Lipofectamine 2000 transfection reagent (Invitrogen). Twelve hours after transfection, N2A cells were treated with or without forskolin trigger for another 24 h, N2A cells were then lysed with 20 μl of PLB buffer. A 10-μl aliquot of the lysate was mixed with 10 μl of lysis buffer and 10 μl of Stop & Glo reagent in sequence. Firefly luciferase activity and Renilla luciferase activity were measured with a luminometer. As
We aimed to study the effect of the five furocoumarins from P. corylifolia (Fig. 1) on human CRF promoter activity. The regulatory region of human CRF gene was cloned into pGL3-basic plasmid and transfected into N2A cells. The cells were subsequently treated with the furocoumarins psoralenoside, isopsoralenside, psoralidin, psoralen, and isopsoralen, followed by triggering using 15 μM forskolin. As shown in Fig. 2, forskolin incubation increased promoter activity approximately 16 folds compared with untriggered cells. As shown in
Fig. 2. Induction of CRF promoter by forskolin. N2A cells were transfected with PGL3/ hCRFP and pRL-SV40 as internal control. Luciferase activity was measured after treatment with 15 μM forskolin.
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Fig. 3. Effect of furocoumarins from Psoralea corylifolia on human CRF promoter activity induced by forskolin. Luciferase activity was measured after treatment with drugs of indicated concentration for 48 h. The cells were co-transfected with plasmids pGL3/hCRFP and pRL-SV40 as internal control. The graph plots the mean ± SD (n = 3) of fold of difference in promoter activity. ⁎: P b 0.05; ⁎⁎: P b 0.01.
Fig. 3, incubation with psoralen did not result in a significant difference on CRF promoter activity compared with control. However, at higher concentrations, psoralen caused the activity to further increase. Isopsoralen showed no effect on CRF promoter. Psoralidin decreased CRF promoter activity in a dose-dependent manner. The observation that psoralidin can inhibit CRF promoter suggested that by decreasing CRF synthesis, it is a potential candidate for antidepression. Interestingly, the combining treatment of psoralen and psoralidin at the same concentration of 40 μM null each other's effects on CRF promoter activity. Psoralenoside and isopsoralenside treatment exerted no significant inhibitory effect on CRF promoter. Fluoxetine was used as a positive control since it was reported to inhibit CRF promoter activity. Inhibition of forskolin-induced CRF gene transcription by furocoumarins from P. corylifolia We next studied the effects of furocoumarins from P. corylifolia on CRF gene transcription at mRNA level by QRT-PCR. Psoralen, isopsoralen, psoralenoside and isopsoralenside exerted no effect on
Fig. 4. Inhibition of forskolin-induced CRF gene transcription in N2A cells by psoralidin. N2A cells incubated with forskolin were treated with psoralidin and vehicle control CRF mRNA levels were quantified by QRT-PCR and normalized to GAPDH mRNA levels as described in Materials and methods. The QRT-PCR assays were performed in triplicate.
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Fig. 5. Cytotoxic effect of five furocoumarins from Psoralea corylifolia and antidepressant fluoxetine on N2A cells.
forskolin-induced CRF gene transcription, consistent with results of the luciferase reporter assay. Psoralidin significantly inhibited forskolin-triggered CRF mRNA synthesis in a dose-dependent manner as shown in Fig. 4. Furthermore, the dose-dependent trend to inhibit gene transcription at lower concentration from 10 to 40 μM is obvious (Fig. 4). Effect of furocoumarins from P. corylifolia on cell viability To test the cytotoxicity of furocoumarins from P. corylifolia, N2A cells were incubated with 5 to 100 μM of the drugs with a final DMSO concentration of 0.5% for 48 h. MTT assay was performed to examine survival of the cells after drug treatment. Psoralidin demonstrated no significant inhibitory effect on N2A cell proliferation at concentrations lower than 50 μM (Fig. 5). It exerted significant repressive effect on CRF transcription at the concentration of 40 μM. Furthermore, the dose-dependent trend to inhibit CRF gene transcription of psoralidin is obvious at lower concentration as shown in Fig. 4, suggesting that its inhibitory effect on CRF gene transcription was not due to its cytotoxicity. None of the other four components used in the present study evoked a significant toxic effect, when examined by the MTT assay in N2A cells. Discussion Long-term administration of some antidepressant drugs has been shown to decrease CRF mRNA level in the rat hypothalamus (Aubry et al., 1999; Fadda et al., 1995). More recent studies demonstrated that some clinically effective antidepressant drugs, such as imipramine and fluoxetine, inhibited hCRF promoter activity in Neuro-2A cells (Budziszewska et al., 2004). Inhibition of CRF gene promoter activity by antidepressant drugs has been suggested to be a molecular mechanism by which these drugs inhibit the activity of HPA axis (Budziszewska et al., 2004; Kunugi et al., 2006). CRF has been suggested to be a promising drug target for treating depression. Our recent studies also showed that psoralidin from P. corylifolia exerted effective antidepressant activity in animal models as well as ameliorated the swim stress-induced elevations in serum CRF concentration without toxic effects (Yi et al., 2008). We firstly examined the effect of five furocoumarins from P. corylifolia on CRF gene promoter activity in Neuro-2A cells. The main finding of these experiments is the demonstration that psoralidin, one of the compounds under study, was able to inhibit hCRF gene promoter activity in Neuro-2A cells. The inhibitory effect was dose-dependent. Psoralidin exerted a significant inhibitory effect on hCRF promoter activity at a concentration without evoking evident cytotoxicity in Neuro-2A cells. Based on the comparison of the structures of these five
furocoumarins, it seems that the aromatic cycle attached to C-3 rather than the isoprenyl at C-6 is responsible to the significant inhibitory effect on CRF promoter activity of psoralidin. To study the effect of psoralidin on CRF gene transcription, we used quantitative RT-PCR to measure CRF mRNA level. Endogenous CRF mRNA level in Neuro-2A cells was low, but was upregulated by forskolin treatment. We further confirmed the inhibitory effect of psoralidin on CRF gene transcription. Cyclic adenosine 3′,5′-cyclic monophosphate (cAMP)/cAMP-dependent protein kinase (PKA) signal transduction is the main pathway that stimulates CRF gene promoter activity. Forskolin increases cellular cAMP levels and CRF gene transcription induced by forskolin appears to be regulated via cAMP regulatory element (CRE). Besides PKA, recent studies also suggest a possible involvement of Ca2+/calmodulindependent kinase and PI-3K mediated pathway in the intracellular mechanism of antidepressant effects on CRF gene promoter activity (Basta-Kaim et al., 2005). Whether psoralidin exerts its effects on CRF gene at the level of cAMP synthesis and/or involves downstream signaling molecules needs further confirmation. P. corylifolia has long been used for treatment of the symptoms of aging (Jiangsu College of New Medicine, 1977). Our studies showed that extracts from P. corylifolia exhibited in vitro inhibitory actions on monoamine oxidase (MAO) activity (Kong et al., 2001). Our studies also suggested that furocoumarin extracts from P. corylifolia possessed strong antidepressant activity in an animal model (Chen et al., 2005). It is interesting to find opposite effect of psoralen and psoralidin on CRF gene transcriptional activity. We are currently testing their effects in animal behavioral models (Yi et al., 2008). It is reported that some antipsychotic drugs e.g. chlorpromazine or clozapine also potently inhibited CRF gene promoter activity in Neuro-2A cells (Basta-Kaim et al., 2006). The ability of furocoumarins to inhibit the forskolin-induced activity of CRF gene transcription may be a molecular mechanism involved in their inhibitory effect on the HPA axis activity and their antidepressant properties. Our data also support the concept that CRF gene may be an attractive drug target for screening effective antidepressant components from traditional Chinese medicine. Conclusion We found that psoralidin isolated from the seeds of P. corylifolia strongly inhibited forskolin-induced CRF promoter activity and suppressed CRF gene transcription. Recently, we have also shown the antidepressant-like effects of psoralidin in the forced swimming test (FST) in ICR strain of male mice. Furthermore, psoralidin also ameliorated the elevations in serum CRF induced by swimming stress in mice. Hence down-regulation of CRF gene transcription by psoralidin may be involved in the molecular mechanism underlying its potent antidepressant effect. Acknowledgement This project was supported by Hong Kong Jockey Club Institute of Chinese Medicine (JCICM-39-02 to HFK). References Aubry, J.M., Pozzzoli, G., Vale, W.W., 1999. Chronic treatment with the antidepressant amitriptyline decreases CRF-R1 receptor mRNA levels in the rat amygdale. Neuroscience Letters 266, 197–200. Basta-Kaim, A., Budziszewska, B., Jaworska-Feil, L., Tetich, M., Kubera, M., Leskiewicz, M., Lason, W., 2005. Inhibitory effect of imipramine on the human corticotropinreleasing-hormone gene promoter activity operates through a PI3-K/AKT mediated pathway. Neuropharmacology 49, 156–164. Basta-Kaim, A., Budziszewska, B., Jaworska-Feil, L., Tetich, M., Kubera, M., Leskiewicz, M., Otczyk, M., Lason, W., 2006. Antipsychotic drugs inhibit the human corticotropin-releasing-hormone gene promoter activity in neuro-2A cells— an involvement of protein kinases. Neuropsychopharmacology 31, 53–65. Budziszewska, B., Jaworska-Feil, L., Tetich, M., Basta-Kaim, A., Kubera, M., Leskiewicz, M., Lason, W., 2004. Regulation of the human corticotropin-releasing-hormone
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