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Osthole, a coumadin analog from Cnidium monnieri (L.) Cusson, stimulates corticosterone secretion by increasing steroidogenic enzyme expression in mouse Y1 adrenocortical tumor cells
Journal of Ethnopharmacology 175 (2015) 456–462
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Osthole, a coumadin analog from Cnidium monnieri (L.) Cusson, stimulates corticosterone secretion by increasing steroidogenic enzyme expression in mouse Y1 adrenocortical tumor cells Zhiqiang Pan n, Zhaoqin Fang, Wenli Lu, Xiaomei Liu, Yuanyuan Zhang Basic Medical School of Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 March 2015 Received in revised form 30 August 2015 Accepted 2 October 2015 Available online 9 October 2015
Ethnopharmacological relevance: Osthole is an O-methylated coumadin, which was isolated and purified from the seeds of Cnidium monnieri (L.) Cusson. Osthole is a commonly used traditional Chinese medicine to treat patients with Kidney-Yang deficiency patients, who exhibit clinical signs similar to those of glucocorticoid withdrawal. However, the mechanism of action of osthole is not fully understood. Objective: This study was designed to reveal the effects of osthole on corticosterone production in mouse Y1 cell. Materials and methods: Mouse Y1 adrenocortical cells were used to evaluate corticosterone production, which was quantified by enzyme-linked immunosorbent assay (ELISA) kits. Cell viability was tested using the MTT assay, and the mRNA and protein expression of genes encoding steroidogenic enzymes and transcription factors was monitored by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. Results: Osthole stimulated corticosterone secretion from mouse Y1 cells in a dose- and time-dependent manner, and osthole enhanced the effect of dibutyryl-cAMP (Bu2cAMP) on corticosterone production. Further, osthole also increased StAR and CYP11B1 mRNA expression in a dose-dependent manner and enhanced the expression of transcription factors such as HSD3B1, FDX1, POR and RXRα as well as immediate early genes such as NR4A1. Moreover, osthole significantly increased SCARB1(SRB1) mRNA and StAR protein expression in the presence or absence of Bu2cAMP; these proteins are an important for the transport of the corticosteroid precursor cholesterol transport into mitochondria. Conclusions: Our results show that the promotion of corticosterone biosynthesis and secretion is a novel effect of osthole, suggesting that this agent can be utilized for the prevention and treatment of KidneyYang deficiency syndrome. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Osthole Corticosterone StAR CYP11B1 Y1 adrenocortical cell
1. Introduction Cnidium monnieri (L.) Cusson is a commonly used traditional Chinese medicine to treat Kidney-Yang deficiency patients, who exhibit symptoms suggestive of glucocorticoid deficiency (Zhao et al., 2013). Kidney-Yang deficiency syndrome is a typical condition in Chinese medicine, shares similar clinical signs of the glucocorticoid withdrawal syndrome. However, Kidney-Qi deficiency degree is less than Kidney-Yang deficiency. Additionally, Qi function is weak in Kidney-Qi deficiency syndrome, whereas both Qi function and Yang function are weak or poor in Kidney-Yang deficiency syndrome. Osthole is identified as the main bioactive component isolated from the seeds of C. monnieri (L.) Cusson, that n
Corresponding author. E-mail address: [email protected] (Z. Pan).
http://dx.doi.org/10.1016/j.jep.2015.10.009 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
is known to ameliorate Kindey-Yang deficiency syndrome. Osthole has been shown to have a wide array of biological effects, including anti-inflammatory (Liu et al., 2005), hepatoprotective (Zhang et al., 2011), anti-proliferative (Jarzab et al., 2014), antitumor (Kao et al., 2012), anti-allergic (Chen et al., 1988), anti-osteoporotic (Zhang et al., 2007a) and estrogen-like activities (Hsieh et al., 2004). Additionally, a pharmacological report showed that osthole improved the function of the pituitary–adrenocortical axis in a Kidney-Yang deficiency rat model induced by continuous administration of hydrocortisone acetate (Qin et al., 1997). However, effects of osthole on steroid-producing cells remain unclear. Latter-day traditional Chinese medicine theory holds that KidneyYang deficiency syndrome is related to a hypofunctioning of the hypothalamus–pituitary–adrenal axis (Shen et al., 2007). Corticosterone, the major glucocorticoid secreted by adrenocortical cells, is important for the regulation of glucose, lipid and protein biosynthesis and metabolism. Corticosterone production is
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under the control of circulating adrenocorticotropic hormone (ACTH) secreted by the pituitary, which also plays an important role in stress. It has been reported that components of Chinese herbs exhibit steroid hormone effects. Therefore, it is necessary to discover the effect of osthole on adrenocortical cells. In this study we demonstrate that osthole increased corticosterone secretion from Y1 adrenocortical cells, suggesting that osthole plays an important role in steroidogenesis.
2. Materials and methods 2.1. Materials Osthole (with the chemical structure illustrated in Fig. 1) was obtained from the Shanghai Institute for Food and Drug Control (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1% in all osthole groups and had no effect on cell viability. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium-F12 (DMEM-F12) were purchased from Hyclone Thermo Fisher Scientific (Waltham, MA). 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), Bu2cAMP and β-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). StAR, CYP11A1 and SRB1 antibodies were purchased from Abcam (Cambridge, MA, USA). Mouse Corticosterone EIA kits were obtained from Cayman Chemical Company (Ann Arbor, Michigan, USA). 2.2. Cell culture and treatment The mouse adrenocortical tumor Y1 cells were grown in DMEM-F12 medium containing 1% penicillin–streptomycin and 10% FBS. For analysis of responses to Bu2cAMP and/or osthole, cells were sub-cultured in 6-well plates to approximately 80% confluence. One day before the experiment, the medium was replaced with a low-serum experimental medium (DMEM-F12 medium supplemented with 1% FBS). The next morning, cells were treated with 0.1% DMSO or 25 μM osthole in the presence and absence of 1 mM Bu2cAMP in fresh low-serum experimental medium. For the dose response experiments, after Y1 cells treated for 24 h with 10 μM, 25 μM, 50 μM osthole, the cell culture supernatants were collected and used for hormone analysis, and cells were harvested for genes or proteins expression assay. The vehicle DMSO (0.1%) treated group was used as the control group in all experiments. For the time course experiments, the cell supernatants were collected and cells were harvested after treatment with 10 μM osthole for 15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 12 h as indicated.
Fig. 1. Chemical structure of osthole.
corticosterone was assayed using the mouse corticosterone EIA kit according to the manufacturer’s instructions. 2.5. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted using TRIzol (Invitrogen) according to protocols from the manufacturers. The purity and integrity of the RNA were checked spectroscopically using a NanoDrop 2000/c spectrophotometer (Thermo). Then, for each sample, 2 μg RNA was reverse transcribed to obtain the cDNA template using PrimeScriptsRT reagent Kit (TaKaRa). Each cDNA sample was diluted 5 times for qRT-PCR amplification; qRT-PCR was performed using the fluorescent dye SYBRs Premix Ex Taq™(Tli RNaseH Plus)II (TaKaRa) with a 7500 Fast Real-Time PCR System. Amplification was performed with the following fast time course: 95 °C 30 s, 95 °C 5 s, 65 °C for 30 s for 40 cycles. Relative mRNA expression ΔΔ values were determined by the 2 Ct method using mouse cyclophilin (PPIA) and β-actin as the normalization control (Livak and Schmittgen, 2001). 2.6. Western blotting
Cell viability was tested using the MTT assay according to the supplier’s instructions. Y1 cells were seeded into 96-well plates at a density of 1 104 per well for 24 h. Following treatment with osthole for 24–72 h, medium was removed and cells were washed with PBS. MTT (0.5 mg/mL) was then added to each well and the mixture was incubated for 4 h at 37 °C. MTT reagent was then replaced with DMSO (150 μL per well) to dissolve formazan crystals. After the mixture was shaken at room temperature for 10 min, absorbance was determined at 570 nm using a microplate reader (Bio-Tek, Winooski, VT, USA).
Y1 cells were cultured in 6-well plates and were lysed with RIPA lysis buffer (Beyotime, Haimen, China) supplemented with PMSF. The cell lysates were centrifuged and the supernatants were mixed with 5 Sample buffer (Beyotime, Haimen, China). The proteins were separated through 10% SDS-PAGE gels and electrophoretically transferred to PVDF membranes. The membranes were then blocked by incubating in the blocking buffer (5% nonfat dry milk, 150 mM NaCl, 10 mM Tris–HCl, pH 7.5) for 1 h and then incubated with blocking buffer containing CYP11A1 antibody (1:1000), StAR antibody (1:1000), SRB1 antibody (1:2000) or βactin antibody (1:20,000) overnight at 4 °C. Following extensive washing in Tris-buffered saline containing 0.1% Tween-20 (TBST) buffer, the transfer membranes were further incubated for 1.5 h in 5% nonfat dry milk blotting buffer that contained secondary antibodies (anti-rabbit IgG HRP-linked antibody or anti-mouse IgG HRP-linked antibody). Then, the membranes were washed three times with TBST and were finally developed using an alpha fluorchem E system detection kit (Protein-Simple, USA).
2.4. Measurement of steroid hormone production
2.7. Statistical analysis
Y1 cells were incubated for 16–20 h in low-serum DMEM-F12 medium containing 1% FBS. The cells were then treated with and without Bu2cAMP and/or osthole for the times indicated. The supernatants were collected and stored frozen at 20 °C until
Data were expressed as the means 7SD. Significant differences were accepted at the 0.05 level of probability and were statistically determined with ANOVA followed by a Newman–Keuls post-hoc test using GraphPad Prism4 (San Diego, CA).
2.3. MTT assay
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mRNA expression, and osthole enhanced the effect of Bu2cAMP on CYP11B1 mRNA expression compared to the Bu2cAMP alone group (Fig. 3C). 3.4. Osthole increased StAR and CYP11B1 expression in a dose-dependent manner
Fig. 2. Cell viability assay using MTT tests. Cell viability showed that 1–100 μM osthole had no cell toxicity in Y1 cells treated for 24 h, 48 h or 72 h.
3. Results 3.1. Chemical structure of osthole Osthole is also known as 7-methoxy-8-(3-methyl-2-butenyl) coumarin (Fig. 1); it was originally isolated and purified from the seeds of C. monnieri (L.) Cusson. Osthole, obtained as white crystals, thoroughly and easily dissolved in DMSO. 3.2. Y1 cells viability assay Mouse Y1 cells were treated with 1–100 μM osthole for 24 h, 48 h and 72 h. Cell viability assays showed that concentrations less than or equal to 100 μM exerted no toxicity on Y1 cells (Fig. 2). Thence, 1–100 μM osthole can be used to treat Y1 cells for the mechanism studies. 3.3. Osthole alone or in combination with Bu2cAMP stimulated corticosterone production We assayed corticosterone production after 24 h and 48 h treatment with 10 μM, 25 μM and 50 μM osthole. The results showed that 25 μM and 50 μM osthole significantly increased corticosterone release into the medium after 48 h treatment (Fig. 3A). Furthermore, corticosterone production was measured after a 24 h treatment with or without osthole in the presence and absence of Bu2cAMP. The data indicate that osthole significantly increased corticosterone levels, as did Bu2cAMP alone, and the combination enhanced corticosterone synthesis further (Fig. 3B). Additionally, similar effects were observed on CYP11B1 mRNA expression; that is, 25 μM osthole significantly increased CYP11B1
We studied the dose dependence of the effects of osthole on the expression of genes encoding key proteins involved in corticosterone synthesis. Using doses ranging from 10 mM to 50 mM, we found that osthole stimulated the expression of StAR and CYP11B1 in a dose-dependent manner, with a significant increase observed in response to a 25 μM concentration (Fig. 4A and B). However, these different doses of osthole did not affect the mRNA or protein expression of CYP11A1, which is the initiating enzyme in the synthesis of all steroid hormones; however, Bu2cAMP increased CYP11A1 mRNA and protein levels (Fig. 4C and D). 3.5. Changes in the expression of proteins involved in corticosterone synthesis and transcription factors in response to osthole We detected increased gene expression of several genes encoding proteins involved in corticosterone synthesis and metabolism in response to osthole alone and in the presence of Bu2cAMP following a 24 h treatment. Osthole and Bu2cAMP significantly up-regulated HSD3B1 gene expression, which converts the pregnenolone formed by CYP11A1 to progesterone (Fig. 5A). Osthole alone and in the presence of Bu2cAMP significantly increased the expression of FDX1 and POR, proteins involved in providing the electrons required by CYP11A1 (Fig. 5D and F), although there was no effect of osthole on the FDXR gene (Fig. 5E). Interestingly, Bu2cAMP significantly decreased the expression of HSD11B1,which converts inactive 11-dehydrocorticosterone to corticosterone, and osthole returned expression to a value not significantly different from the control, while having no effect itself (Fig. 5B). Neither Bu2cAMP nor osthole affected the expression of HSD11B2, which reduces active corticosterone to inactive 11dehydrocorticosterone (Fig. 5C). It is noteworthy that osthole increased RXRα gene expression in the presence or absence of Bu2cAMP (Fig. 5G). Bu2cAMP significantly increased the expression of PPARα, which when is activated by pioglitazone increases CYP11B1 expression in human adrenocortical carcinoma cells, as we have previously shown (Pan et al., 2014). Bu2cAMP also upregulated MC2R (the receptor for ACTH) gene expression, but osthole had no effect (Fig. 5H and I).
Fig. 3. Osthole enhanced Bu2cAMP-induced corticosterone production. (A) Mouse Y1 cells were treated with 10 μM, 25 μM or 50 μM osthole (OST) or 0.1% DMSO as the vehicle control (Con) for 24 h and 48 h, and the cell culture supernatants collected and assayed for corticosterone; **Po 0.01 vs control, ***Po 0.001 vs control. (B) Y1 cells were treated with 0.1% DMSO or 25 μM osthole (OST) for 24 h in the presence and absence of 1 mM Bu2cAMP. The cell culture supernatants were collected and corticosterone was assayed by EIA; *Po 0.05 vs control, **P o0.01 vs control, ***P o0.001 vs control, #Po 0.05 vs Bu2cAMP. (C) Y1 cells were treated with or without 25 μM osthole (OST) in the presence or absence of 1 mM Bu2cAMP for 24 h; 0.1% DMSO (Con) was used as the vehicle. CYP11B1 mRNA expression was detected by qRT-PCR; **P o0.01 vs control, ***Po 0.001 vs control, #Po 0.05 vs Bu2cAMP.
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Fig. 4. Mouse Y1 cells responded in a dose-dependent manner to osthole (10 μM, 25 μM and 50 μM OST) after a 24 h treatment, with 0.1% DMSO used as the vehicle control (Con) and 1 mM Bu2cAMP as a positive control. Gene expression was detected by qRT-PCR, and protein expression was assayed by western blot. (A) 25 μM and 50 μM osthole significantly increased StAR mRNA expression; *P o0.05 vs control. (B) 10 μM, 25 μM and 50 μM osthole significantly increased CYP11B1 mRNA expression; *Po 0.05 vs control. (C) CYP11A1 mRNA expression was not affected by osthole, but was up-regulated by 1 mM Bu2cAMP; *Po 0.05 vs control. (D) CYP11A1 protein expression showed no response to osthole, but 1 mM Bu2cAMP enhanced CYP11A1 protein expression.
3.6. The expression of immediate early genes was induced by osthole treatment We also monitored the expression of immediate early genes such as NR4A1 and NR4A2, as well as NR5A1, between 15 min and 12 h of stimulation with 10 μM osthole. The results showed that NR4A1 was significantly increased by osthole at 30 min and 1 h of treatment (Fig. 6A), and osthole also enhanced NR4A2 and NR5A1 gene expression between 30 min and 1 h (Fig. 6B and C). 3.7. Osthole increased SRB1 and StAR gene and protein expression in Y1 cells Finally, we determined SRB1 and StAR mRNA and protein after a 24 h treatment with 25 μM osthole in the presence or absence of Bu2cAMP. The data showed that osthole alone significantly increased SRB1 and StAR mRNA expression, and significantly enhanced the stimulatory effect of Bu2cAMP (Fig. 7A and B). Additionally, StAR protein was also increased by osthole with or without added Bu2cAMP (Fig. 7D). The results suggested that osthole promoted cholesterol uptake and transport to the inner mitochondrial membrane for the synthesis of steroid hormones.
4. Discussion In this report, we found first that osthole stimulated corticosterone production in mouse Y1 adrenocortical cells by inducing the expression of genes encoding steroid hormone synthetic enzymes and proteins involved in corticosterone synthesis. These include enzymes involved in early steps of biosynthesis such as SRB1, StAR, FDX1, POR, and HSD3B1 as well as those involved in promoting corticosterone synthesis, like CYP11B1 and HSD11B1. In traditional Chinese medicine osthole originates from C. monnieri (L.) Cusson, used to treat Kidney-Yang deficiency syndrome, which appears to be related to adrenocortical function (Shen, 2005). Whether or not osthole has direct effects on adrenal steroid
hormone secretion was unknown. We used the Y1 cell line because these tumor cells produce corticosterone as one of the major steroid products, and the regulation of steroid synthesis in Y1 cells is similar to the regulation observed in cells cultured from normal adrenal glands with regard to ACTH (Rainey et al., 2004). Moreover, in traditional Chinese medicine we often study the effect of Chinese herbs effect on mice. Therefore, we selected the Y1 cell as the experimental model. According to reports on the genome-wide effects of ACTH on transcript accumulation in mouse adrenal Y1 cells, ACTH upregulated transcripts include several genes with sterol biosynthesis functions such as StAR, CYP11A1, MC2R, NR5A1, SRB1, LDLR, HMGCR, HSD3B1, NR4A1, NR5A1, POR, FDXR and so on (Schimmer et al., 2006, 2007). Our study showed that osthole also increased CYP11B1, StAR, SRB1, HSD3B1, FDX1, POR, NR4A1 and NR5A1 expression, therefore, osthole showed partial ACTH-like effects. Indeedly, StAR play major roles in intracellular cholesterol transport, which is responsible for the rapid movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane. Moreover, StAR and related proteins containing StARrelated lipid transfer domains such as StarD4 and StarD5 play synchronously major roles in intracellular cholesterol transport (Miller, 2007). Additionally, rodent cells use cholesterol from circulating high-density lipoproteins (HDL) taken up by via scavenger receptor B1 (SRB1) as their principal source of cholesterol for steroidogenesis (Miller and Bose, 2011). In this study, osthole increased StAR mRNA expression in a dose-dependent manner, and also enhanced StAR and SRB1 gene and protein expression with or without Bu2cAMP. The result suggests that osthole play a key role in intracellular cholesterol trafficking. Furthermore, CYP11A1 catalyzes the first step in steroidogenesis in adrenal cells, and conversion of cholesterol to pregnenolone in mitochondria is ratelimiting step in the synthesis of the steroid hormones, therefore, transcription of the CYP11A1 gene encoding P450scc is the factor that determines the net steroidogenic capacity of a cell (Miller and Auchus, 2011). However, in this paper, the data suggests that CYP11A1 gene and protein expression show no response to
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Fig. 5. Gene involved in corticosterone synthesis and metabolism was detected by qRT-PCR after Y1 cells were treated with 25 μM osthole (OST) in the presence or absence of Bu2cAMP for 24 h. (A) Osthole and Bu2cAMP significantly increased HSD3B1 mRNA expression; *Po 0.05 vs control, ***P o 0.001 vs control. (B) Bu2cAMP significantly decreased HSD11B1 mRNA expression and osthole returned expression to a value not significantly different from the control; *P o0.05 vs control. (C) HSD11B2 mRNA expression was not altered by osthole or Bu2cAMP. (D) Osthole and Bu2cAMP significantly increased FDX1 mRNA expression; *Po 0.05 vs control, ***Po 0.001 vs control. (E) FDXR mRNA expression was unchanged by osthole or Bu2cAMP. (F) Osthole and Bu2cAMP significantly increased POR mRNA expression; *P o0.05 vs control, **P o 0.01 vs control. (G) Osthole in the presence of absence of Bu2cAMP significantly increased RXRα mRNA expression; *Po 0.05 vs control. (H) Bu2cAMP in the presence or absence of osthole significantly increased PPARα mRNA expression; *Po 0.05 vs control. (I) Only Bu2cAMP significantly increased MC2R mRNA expression; **Po 0.01 vs control.
different doses of osthole, but Bu2cAMP significantly enhanced CYP11A1 gene and protein expression. The results indicated that osthole treatment for 24 h in Y1 cells did not affect rate-limiting step in steroidogenesis. It is important that CYP11B1 gene expression was increased by osthole in a dose-dependent manner and corticosterone production level was heightened significantly by osthole treatment for 24 h and 48 h. The results suggests that osthole promotes corticosterone biosynthesis and secretion is a novel pharmacological finding.
Acting as a cAMP analog that can permeate the plasma membrane, Bu2cAMP activates cAMP-dependent PKA (Schimmer and Schulz, 1985; Reyland, 1993). Further, we determined whether there were changes in the mRNA or protein expression of members of various pathways, such as PKA, CAMK1, and CREB1; we observed no effect of osthole on these parameters (data not shown). However, since the activity of these molecules is often regulated post-translationally, it is still possible that osthole can affect the cAMP-, protein kinase C- and Ca2 þ -dependent signaling
Fig. 6. The mRNA expression of immediate early genes, such as NR4A1 and NR4A2, as well as NR5A1, were detected by qRT-PCR after Y1 cells were treated for various times as indicated with 10 μM osthole or 0.1% DMSO as the vehicle control (Con). (A) Osthole significantly increased NR4A1 mRNA expression after treatment for 30 min, reaching a peak at 1 h; ***Po 0.001 vs control. (B) Osthole increased NR4A2 mRNA expression after treatment for 30 min and 1 h; *Po 0.05 vs control. (C) Osthole increased NR5A1 mRNA expression after treatment for 30 min and 1 h; *Po 0.05 vs control.
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Fig. 7. Osthole enhanced SRB1 and StAR mRNA and protein expression upon Y1 cell treatment with 25 μM osthole (OST) in the presence or absence of 1 mM Bu2cAMP for 24 h. (A) Osthole and/or Bu2cAMP significantly increased SCARB1 (SRB1) mRNA expression; *Po 0.05 vs control, **P o0.01 vs control, ***Po 0.001 vs control, #Po 0.05 vs Bu2cAMP. (B) Osthole and Bu2cAMP significantly increased StAR mRNA expression, and the combination showed a further enhancement; *P o 0.05 vs control, ***Po 0.001 vs control, ##P o0.01 vs Bu2cAMP. (C) Bu2cAMP with and without osthole significantly increased SRB1 protein expression. (D) Osthole and Bu2cAMP increased StAR protein expression.
pathways in mouse Y1 adrenocortical cells. On the other hand, osthole-mediated facilitation of glutamate release in rat hippocampal nerve endings involves the activation of the cGMP/PKGdependent pathway (Wang et al., 2008; Lin et al., 2012). We hypothesize that osthole promotes corticosterone production by a transcriptional mechanism. In addition, osthole increased the expression of nuclear hormone receptors such as RXRα and PPARα in Y1 cells. It has been reported that PPARα is also regulated by osthole in hepatocytes and cardiac fibroblasts; for example, osthole increases PPARα mRNA expression in mouse liver and cultured hepatocytes in a dose-dependent manner, and decreased its target genes including DGAT and HMG-CoA reductase (Zhang et al., 2007b; Sun et al., 2010). Osthole can also inhibit NF-κB and TGF-β1 expression by activating PPARα/γ, and subsequently enhancing MMP-2/9 expression in cultured mouse cardiac fibroblasts (Chen et al., 2013). It is noteworthy that the orphan nuclear receptors (NR4 and NR5) are described as early response genes; in vitro studies have shown that they take part in regulation of adrenal function. For example, the NR5A1 gene encodes for steroidogenic factor 1, which regulate many aspects of adrenal and reproductive development and function (Achermann et al., 2002; Buaas et al., 2012), and NR5A1 has also been shown to regulate the expression of enzymes involved in steroid production in vitro (Buaas et al., 2012), NR5A1 mutations have also been associated with primary adrenal insufficiency, whereas NR5A1 overexpression dramatically inhibited CYP11B2 expression and decreased aldosterone production in H295R/TR/SF-1 cell line (Ye et al., 2009). Our data showed that NR5A1 induction in response to osthole is weak. The result suggested that NR5A1 may not have an important role on ostholeinduced steroidogenesis. Indeed, NR4 family is essential for adrenal production of mineralocorticoids and glucocorticoids. For example, NR4A1 plays a critical part in the regulation of 3beta-hydroxysteroid dehydrogenase (HSD3B2) transcription (Bassett et al., 2004a). Additionally, in human H295R adrenocortical cells, NR4A1 and NR4A2 are key regulators of aldosterone synthase (CYP11B2) expression, suggesting possible roles of these transcriptional factors in the regulation of adrenocortical steroidogenesis (Bassett et al., 2004b). In this study, NR4A1 was increased significantly
after osthole treatment for 30 min and 1 h suggesting that NR4A1 may have an important role in osthole-induced steroidogenesis.
5. Conclusion In summary, osthole increased corticosterone biosynthesis by increasing the expression of genes encoding steroid hormone synthetic enzymes and proteins and other proteins that can regulate adrenocortical function. Thus, our results provide biological evidence for the mechanisms contributing to the efficacy of osthole, and the traditional Chinese medicine in which it is the active ingredient, for the treatment of Kidney-Yang deficiency syndrome.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81102533) and the Shanghai Municipal Commission of Health and Family Planning Project (No. ZY3-CCCX3-3010). We are thankful to Dr. Wendy B. Bollag, Ph.D. (Professor, Department of Physiology, Georgia Regents University, Augusta, Georgia, USA) for providing her valuable inputs for English and scientific contents of the manuscript.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2015.10.009.
References Achermann, J.C., Ozisik, G., Ito, M., Orun, U.A., Harmanci, K., Gurakan, B., Jameson, J. L., 2002. Gonadal determination and adrenal development are regulated by the orphan nuclear receptor steroidogenic factor-1, in a dose-dependent manner. J. Clin. Endocrinol. Metab. 87, 1829–1833. Bassett, M.H., Suzuki, T., Sasano, H., De Vries, C.J., Jimenez, P.T., Carr, B.R., Rainey, W. E., 2004a. The orphan nuclear receptor NGFIB regulates transcription of 3beta-
462
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hydroxysteroid dehydrogenase: implications for the control of adrenal functional zonation. J. Biol. Chem. 279, 37622–37630. Bassett, M.H., Suzuki, T., Sasano, H., White, P.C., Rainey, W.E., 2004b. The orphan nuclear receptors NURR1 and NGFIB regulate adrenal aldosterone production. Mol. Endocrinol. 18, 279–290. Buaas, F.W., Gardiner, J.R., Clayton, S., Val, P., Swain, A., 2012. In vivo evidence for the crucial role of SF1 in steroid-producing cells of the testis, ovary and adrenal gland. Development 139, 4561–4570. Chen, R., Xue, J., Xie, M., 2013. Osthole regulates TGF-beta1 and MMP-2/9 expressions via activation of PPARalpha/gamma in cultured mouse cardiac fibroblasts stimulated with angiotensin II. J. Pharm. Pharm. Sci. 16, 732–741. Chen, Z.C., Duan, X.B., Liu, K.R., 1988. The anti-allergic activity of osthol extracted from the fruits of Cnidium monnieri (L.) Cusson. Yao Xue Xue Bao 23, 96–99. Hsieh, M.T., Hsieh, C.L., Wang, W.H., Chen, C.S., Lin, C.J., Wu, C.R., 2004. Osthole improves aspects of spatial performance in ovariectomized rats. Am. J. Chin. Med. 32, 11–20. Jarzab, A., Grabarska, A., Kielbus, M., Jeleniewicz, W., Dmoszynska-Graniczka, M., Skalicka-Wozniak, K., Sieniawska, E., Polberg, K., Stepulak, A., 2014. Osthole induces apoptosis, suppresses cell-cycle progression and proliferation of cancer cells. Anticancer Res. 34, 6473–6480. Kao, S.J., Su, J.L., Chen, C.K., Yu, M.C., Bai, K.J., Chang, J.H., Bien, M.Y., Yang, S.F., Chien, M.H., 2012. Osthole inhibits the invasive ability of human lung adenocarcinoma cells via suppression of NF-kappaB-mediated matrix metalloproteinase-9 expression. Toxicol. Appl. Pharmacol. 261, 105–115. Lin, T.Y., Lu, C.W., Huang, W.J., Wang, S.J., 2012. Involvement of the cGMP pathway in the osthole-facilitated glutamate release in rat hippocampal nerve endings. Synapse 66, 232–239. Liu, J., Zhang, W., Zhou, L., Wang, X., Lian, Q., 2005. Anti-inflammatory effect and mechanism of osthole in rats. Zhong Yao Cai 28, 1002–1006. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. Miller, W.L., 2007. StAR search – what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import. Mol. Endocrinol. 21, 589–601. Miller, W.L., Auchus, R.J., 2011. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr. Rev. 32, 81–151. Miller, W.L., Bose, H.S., 2011. Early steps in steroidogenesis: intracellular cholesterol trafficking. J. Lipid Res. 52, 2111–2135. Pan, Z.Q., Xie, D., Choudhary, V., Seremwe, M., Tsai, Y.Y., Olala, L., Chen, X., Bollag, W. B., 2014. The effect of pioglitazone on aldosterone and cortisol production in HAC15 human adrenocortical carcinoma cells. Mol. Cell. Endocrinol. 394, 119–128. Qin, L.P., Zhang, J.Q., Shi, H.P., 1997. Effects of coumarins from Cnidium monnieri on
the function of pituitary–adrenocortical axis in kidney yang deficiency rats. Zhongguo Zhong Xi Yi Jie He Za Zhi 17, 227–229. Rainey, W.E., Saner, K., Schimmer, B.P., 2004. Adrenocortical cell lines. Mol. Cell. Endocrinol. 228, 23–38. Reyland, M.E., 1993. Protein kinase C is a tonic negative regulator of steroidogenesis and steroid hydroxylase gene expression in Y1 adrenal cells and functions independently of protein kinase A. Mol. Endocrinol. 7, 1021–1030. Schimmer, B.P., Cordova, M., Cheng, H., Tsao, A., Goryachev, A.B., Schimmer, A.D., Morris, Q., 2006. Global profiles of gene expression induced by adrenocorticotropin in Y1 mouse adrenal cells. Endocrinology 147, 2357–2367. Schimmer, B.P., Cordova, M., Cheng, H., Tsao, A., Morris, Q., 2007. A genome-wide assessment of adrenocorticotropin action in the Y1 mouse adrenal tumor cell line. Mol. Cell. Endocrinol. 265–266, 102–107. Schimmer, B.P., Schulz, P., 1985. The roles of cAMP and cAMP-dependent protein kinase in forskolin’s actions on Y1 adrenocortical tumor cells. Endocr. Res. 11, 199–209. Shen, Z.Y., 2005. Studies of gene networks and singal transduction of kidney deficiency syndrome by syndrome differentiation through drug effects. Zhongguo Zhong Xi Yi Jie He Za Zhi 25, 1125–1128. Shen, Z.Y., Dong, J.C., Cai, D.F., 2007. Important action of improving adrenocortical function for certain diseases recovery. Zhongguo Zhong Xi Yi Jie He Za Zhi 27, 364–367. Sun, F., Xie, M.L., Xue, J., Wang, H.B., 2010. Osthol regulates hepatic PPAR alphamediated lipogenic gene expression in alcoholic fatty liver murine. Phytomedicine 17, 669–673. Wang, S.J., Lin, T.Y., Lu, C.W., Huang, W.J., 2008. Osthole and imperatorin, the active constituents of Cnidium monnieri (L.) Cusson, facilitate glutamate release from rat hippocampal nerve terminals. Neurochem. Int. 53, 416–423. Ye, P., Nakamura, Y., Lalli, E., Rainey, W.E., 2009. Differential effects of high and low steroidogenic factor-1 expression on CYP11B2 expression and aldosterone production in adrenocortical cells. Endocrinology 150, 1303–1309. Zhang, J., Xue, J., Wang, H., Zhang, Y., Xie, M., 2011. Osthole improves alcohol-induced fatty liver in mice by reduction of hepatic oxidative stress. Phytother. Res. 25, 638–643. Zhang, Q., Qin, L., He, W., Van Puyvelde, L., Maes, D., Adams, A., Zheng, H., De Kimpe, N., 2007a. Coumarins from Cnidium monnieri and their antiosteoporotic activity. Planta Med. 73, 13–19. Zhang, Y., Xie, M., Xue, J., Gu, Z., 2007b. Osthole improves fat milk-induced fatty liver in rats: modulation of hepatic PPAR-alpha/gamma-mediated lipogenic gene expression. Planta Med. 73, 718–724. Zhao, L., Wu, H., Qiu, M., Sun, W., Wei, R., Zheng, X., Yang, Y., Xin, X., Zou, H., Chen, T., Liu, J., Lu, L., Su, J., Ma, C., Zhao, A., Jia, W., 2013. Metabolic signatures of kidney yang deficiency syndrome and protective effects of two herbal Extracts in rats using GC/TOF MS. Evid. Based Complement. Altern. Med. 2013, 540957.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Osthole, a coumadin analog from Cnidium monnieri (L.) Cusson, stimulates corticosterone secretion by increasing steroidogenic enzyme expression in mouse Y1 adrenocortical tumor cells Zhiqiang Pan n, Zhaoqin Fang, Wenli Lu, Xiaomei Liu, Yuanyuan Zhang Basic Medical School of Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 March 2015 Received in revised form 30 August 2015 Accepted 2 October 2015 Available online 9 October 2015
Ethnopharmacological relevance: Osthole is an O-methylated coumadin, which was isolated and purified from the seeds of Cnidium monnieri (L.) Cusson. Osthole is a commonly used traditional Chinese medicine to treat patients with Kidney-Yang deficiency patients, who exhibit clinical signs similar to those of glucocorticoid withdrawal. However, the mechanism of action of osthole is not fully understood. Objective: This study was designed to reveal the effects of osthole on corticosterone production in mouse Y1 cell. Materials and methods: Mouse Y1 adrenocortical cells were used to evaluate corticosterone production, which was quantified by enzyme-linked immunosorbent assay (ELISA) kits. Cell viability was tested using the MTT assay, and the mRNA and protein expression of genes encoding steroidogenic enzymes and transcription factors was monitored by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. Results: Osthole stimulated corticosterone secretion from mouse Y1 cells in a dose- and time-dependent manner, and osthole enhanced the effect of dibutyryl-cAMP (Bu2cAMP) on corticosterone production. Further, osthole also increased StAR and CYP11B1 mRNA expression in a dose-dependent manner and enhanced the expression of transcription factors such as HSD3B1, FDX1, POR and RXRα as well as immediate early genes such as NR4A1. Moreover, osthole significantly increased SCARB1(SRB1) mRNA and StAR protein expression in the presence or absence of Bu2cAMP; these proteins are an important for the transport of the corticosteroid precursor cholesterol transport into mitochondria. Conclusions: Our results show that the promotion of corticosterone biosynthesis and secretion is a novel effect of osthole, suggesting that this agent can be utilized for the prevention and treatment of KidneyYang deficiency syndrome. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Osthole Corticosterone StAR CYP11B1 Y1 adrenocortical cell
1. Introduction Cnidium monnieri (L.) Cusson is a commonly used traditional Chinese medicine to treat Kidney-Yang deficiency patients, who exhibit symptoms suggestive of glucocorticoid deficiency (Zhao et al., 2013). Kidney-Yang deficiency syndrome is a typical condition in Chinese medicine, shares similar clinical signs of the glucocorticoid withdrawal syndrome. However, Kidney-Qi deficiency degree is less than Kidney-Yang deficiency. Additionally, Qi function is weak in Kidney-Qi deficiency syndrome, whereas both Qi function and Yang function are weak or poor in Kidney-Yang deficiency syndrome. Osthole is identified as the main bioactive component isolated from the seeds of C. monnieri (L.) Cusson, that n
Corresponding author. E-mail address: [email protected] (Z. Pan).
http://dx.doi.org/10.1016/j.jep.2015.10.009 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
is known to ameliorate Kindey-Yang deficiency syndrome. Osthole has been shown to have a wide array of biological effects, including anti-inflammatory (Liu et al., 2005), hepatoprotective (Zhang et al., 2011), anti-proliferative (Jarzab et al., 2014), antitumor (Kao et al., 2012), anti-allergic (Chen et al., 1988), anti-osteoporotic (Zhang et al., 2007a) and estrogen-like activities (Hsieh et al., 2004). Additionally, a pharmacological report showed that osthole improved the function of the pituitary–adrenocortical axis in a Kidney-Yang deficiency rat model induced by continuous administration of hydrocortisone acetate (Qin et al., 1997). However, effects of osthole on steroid-producing cells remain unclear. Latter-day traditional Chinese medicine theory holds that KidneyYang deficiency syndrome is related to a hypofunctioning of the hypothalamus–pituitary–adrenal axis (Shen et al., 2007). Corticosterone, the major glucocorticoid secreted by adrenocortical cells, is important for the regulation of glucose, lipid and protein biosynthesis and metabolism. Corticosterone production is
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under the control of circulating adrenocorticotropic hormone (ACTH) secreted by the pituitary, which also plays an important role in stress. It has been reported that components of Chinese herbs exhibit steroid hormone effects. Therefore, it is necessary to discover the effect of osthole on adrenocortical cells. In this study we demonstrate that osthole increased corticosterone secretion from Y1 adrenocortical cells, suggesting that osthole plays an important role in steroidogenesis.
2. Materials and methods 2.1. Materials Osthole (with the chemical structure illustrated in Fig. 1) was obtained from the Shanghai Institute for Food and Drug Control (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1% in all osthole groups and had no effect on cell viability. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium-F12 (DMEM-F12) were purchased from Hyclone Thermo Fisher Scientific (Waltham, MA). 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), Bu2cAMP and β-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). StAR, CYP11A1 and SRB1 antibodies were purchased from Abcam (Cambridge, MA, USA). Mouse Corticosterone EIA kits were obtained from Cayman Chemical Company (Ann Arbor, Michigan, USA). 2.2. Cell culture and treatment The mouse adrenocortical tumor Y1 cells were grown in DMEM-F12 medium containing 1% penicillin–streptomycin and 10% FBS. For analysis of responses to Bu2cAMP and/or osthole, cells were sub-cultured in 6-well plates to approximately 80% confluence. One day before the experiment, the medium was replaced with a low-serum experimental medium (DMEM-F12 medium supplemented with 1% FBS). The next morning, cells were treated with 0.1% DMSO or 25 μM osthole in the presence and absence of 1 mM Bu2cAMP in fresh low-serum experimental medium. For the dose response experiments, after Y1 cells treated for 24 h with 10 μM, 25 μM, 50 μM osthole, the cell culture supernatants were collected and used for hormone analysis, and cells were harvested for genes or proteins expression assay. The vehicle DMSO (0.1%) treated group was used as the control group in all experiments. For the time course experiments, the cell supernatants were collected and cells were harvested after treatment with 10 μM osthole for 15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 12 h as indicated.
Fig. 1. Chemical structure of osthole.
corticosterone was assayed using the mouse corticosterone EIA kit according to the manufacturer’s instructions. 2.5. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted using TRIzol (Invitrogen) according to protocols from the manufacturers. The purity and integrity of the RNA were checked spectroscopically using a NanoDrop 2000/c spectrophotometer (Thermo). Then, for each sample, 2 μg RNA was reverse transcribed to obtain the cDNA template using PrimeScriptsRT reagent Kit (TaKaRa). Each cDNA sample was diluted 5 times for qRT-PCR amplification; qRT-PCR was performed using the fluorescent dye SYBRs Premix Ex Taq™(Tli RNaseH Plus)II (TaKaRa) with a 7500 Fast Real-Time PCR System. Amplification was performed with the following fast time course: 95 °C 30 s, 95 °C 5 s, 65 °C for 30 s for 40 cycles. Relative mRNA expression ΔΔ values were determined by the 2 Ct method using mouse cyclophilin (PPIA) and β-actin as the normalization control (Livak and Schmittgen, 2001). 2.6. Western blotting
Cell viability was tested using the MTT assay according to the supplier’s instructions. Y1 cells were seeded into 96-well plates at a density of 1 104 per well for 24 h. Following treatment with osthole for 24–72 h, medium was removed and cells were washed with PBS. MTT (0.5 mg/mL) was then added to each well and the mixture was incubated for 4 h at 37 °C. MTT reagent was then replaced with DMSO (150 μL per well) to dissolve formazan crystals. After the mixture was shaken at room temperature for 10 min, absorbance was determined at 570 nm using a microplate reader (Bio-Tek, Winooski, VT, USA).
Y1 cells were cultured in 6-well plates and were lysed with RIPA lysis buffer (Beyotime, Haimen, China) supplemented with PMSF. The cell lysates were centrifuged and the supernatants were mixed with 5 Sample buffer (Beyotime, Haimen, China). The proteins were separated through 10% SDS-PAGE gels and electrophoretically transferred to PVDF membranes. The membranes were then blocked by incubating in the blocking buffer (5% nonfat dry milk, 150 mM NaCl, 10 mM Tris–HCl, pH 7.5) for 1 h and then incubated with blocking buffer containing CYP11A1 antibody (1:1000), StAR antibody (1:1000), SRB1 antibody (1:2000) or βactin antibody (1:20,000) overnight at 4 °C. Following extensive washing in Tris-buffered saline containing 0.1% Tween-20 (TBST) buffer, the transfer membranes were further incubated for 1.5 h in 5% nonfat dry milk blotting buffer that contained secondary antibodies (anti-rabbit IgG HRP-linked antibody or anti-mouse IgG HRP-linked antibody). Then, the membranes were washed three times with TBST and were finally developed using an alpha fluorchem E system detection kit (Protein-Simple, USA).
2.4. Measurement of steroid hormone production
2.7. Statistical analysis
Y1 cells were incubated for 16–20 h in low-serum DMEM-F12 medium containing 1% FBS. The cells were then treated with and without Bu2cAMP and/or osthole for the times indicated. The supernatants were collected and stored frozen at 20 °C until
Data were expressed as the means 7SD. Significant differences were accepted at the 0.05 level of probability and were statistically determined with ANOVA followed by a Newman–Keuls post-hoc test using GraphPad Prism4 (San Diego, CA).
2.3. MTT assay
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mRNA expression, and osthole enhanced the effect of Bu2cAMP on CYP11B1 mRNA expression compared to the Bu2cAMP alone group (Fig. 3C). 3.4. Osthole increased StAR and CYP11B1 expression in a dose-dependent manner
Fig. 2. Cell viability assay using MTT tests. Cell viability showed that 1–100 μM osthole had no cell toxicity in Y1 cells treated for 24 h, 48 h or 72 h.
3. Results 3.1. Chemical structure of osthole Osthole is also known as 7-methoxy-8-(3-methyl-2-butenyl) coumarin (Fig. 1); it was originally isolated and purified from the seeds of C. monnieri (L.) Cusson. Osthole, obtained as white crystals, thoroughly and easily dissolved in DMSO. 3.2. Y1 cells viability assay Mouse Y1 cells were treated with 1–100 μM osthole for 24 h, 48 h and 72 h. Cell viability assays showed that concentrations less than or equal to 100 μM exerted no toxicity on Y1 cells (Fig. 2). Thence, 1–100 μM osthole can be used to treat Y1 cells for the mechanism studies. 3.3. Osthole alone or in combination with Bu2cAMP stimulated corticosterone production We assayed corticosterone production after 24 h and 48 h treatment with 10 μM, 25 μM and 50 μM osthole. The results showed that 25 μM and 50 μM osthole significantly increased corticosterone release into the medium after 48 h treatment (Fig. 3A). Furthermore, corticosterone production was measured after a 24 h treatment with or without osthole in the presence and absence of Bu2cAMP. The data indicate that osthole significantly increased corticosterone levels, as did Bu2cAMP alone, and the combination enhanced corticosterone synthesis further (Fig. 3B). Additionally, similar effects were observed on CYP11B1 mRNA expression; that is, 25 μM osthole significantly increased CYP11B1
We studied the dose dependence of the effects of osthole on the expression of genes encoding key proteins involved in corticosterone synthesis. Using doses ranging from 10 mM to 50 mM, we found that osthole stimulated the expression of StAR and CYP11B1 in a dose-dependent manner, with a significant increase observed in response to a 25 μM concentration (Fig. 4A and B). However, these different doses of osthole did not affect the mRNA or protein expression of CYP11A1, which is the initiating enzyme in the synthesis of all steroid hormones; however, Bu2cAMP increased CYP11A1 mRNA and protein levels (Fig. 4C and D). 3.5. Changes in the expression of proteins involved in corticosterone synthesis and transcription factors in response to osthole We detected increased gene expression of several genes encoding proteins involved in corticosterone synthesis and metabolism in response to osthole alone and in the presence of Bu2cAMP following a 24 h treatment. Osthole and Bu2cAMP significantly up-regulated HSD3B1 gene expression, which converts the pregnenolone formed by CYP11A1 to progesterone (Fig. 5A). Osthole alone and in the presence of Bu2cAMP significantly increased the expression of FDX1 and POR, proteins involved in providing the electrons required by CYP11A1 (Fig. 5D and F), although there was no effect of osthole on the FDXR gene (Fig. 5E). Interestingly, Bu2cAMP significantly decreased the expression of HSD11B1,which converts inactive 11-dehydrocorticosterone to corticosterone, and osthole returned expression to a value not significantly different from the control, while having no effect itself (Fig. 5B). Neither Bu2cAMP nor osthole affected the expression of HSD11B2, which reduces active corticosterone to inactive 11dehydrocorticosterone (Fig. 5C). It is noteworthy that osthole increased RXRα gene expression in the presence or absence of Bu2cAMP (Fig. 5G). Bu2cAMP significantly increased the expression of PPARα, which when is activated by pioglitazone increases CYP11B1 expression in human adrenocortical carcinoma cells, as we have previously shown (Pan et al., 2014). Bu2cAMP also upregulated MC2R (the receptor for ACTH) gene expression, but osthole had no effect (Fig. 5H and I).
Fig. 3. Osthole enhanced Bu2cAMP-induced corticosterone production. (A) Mouse Y1 cells were treated with 10 μM, 25 μM or 50 μM osthole (OST) or 0.1% DMSO as the vehicle control (Con) for 24 h and 48 h, and the cell culture supernatants collected and assayed for corticosterone; **Po 0.01 vs control, ***Po 0.001 vs control. (B) Y1 cells were treated with 0.1% DMSO or 25 μM osthole (OST) for 24 h in the presence and absence of 1 mM Bu2cAMP. The cell culture supernatants were collected and corticosterone was assayed by EIA; *Po 0.05 vs control, **P o0.01 vs control, ***P o0.001 vs control, #Po 0.05 vs Bu2cAMP. (C) Y1 cells were treated with or without 25 μM osthole (OST) in the presence or absence of 1 mM Bu2cAMP for 24 h; 0.1% DMSO (Con) was used as the vehicle. CYP11B1 mRNA expression was detected by qRT-PCR; **P o0.01 vs control, ***Po 0.001 vs control, #Po 0.05 vs Bu2cAMP.
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Fig. 4. Mouse Y1 cells responded in a dose-dependent manner to osthole (10 μM, 25 μM and 50 μM OST) after a 24 h treatment, with 0.1% DMSO used as the vehicle control (Con) and 1 mM Bu2cAMP as a positive control. Gene expression was detected by qRT-PCR, and protein expression was assayed by western blot. (A) 25 μM and 50 μM osthole significantly increased StAR mRNA expression; *P o0.05 vs control. (B) 10 μM, 25 μM and 50 μM osthole significantly increased CYP11B1 mRNA expression; *Po 0.05 vs control. (C) CYP11A1 mRNA expression was not affected by osthole, but was up-regulated by 1 mM Bu2cAMP; *Po 0.05 vs control. (D) CYP11A1 protein expression showed no response to osthole, but 1 mM Bu2cAMP enhanced CYP11A1 protein expression.
3.6. The expression of immediate early genes was induced by osthole treatment We also monitored the expression of immediate early genes such as NR4A1 and NR4A2, as well as NR5A1, between 15 min and 12 h of stimulation with 10 μM osthole. The results showed that NR4A1 was significantly increased by osthole at 30 min and 1 h of treatment (Fig. 6A), and osthole also enhanced NR4A2 and NR5A1 gene expression between 30 min and 1 h (Fig. 6B and C). 3.7. Osthole increased SRB1 and StAR gene and protein expression in Y1 cells Finally, we determined SRB1 and StAR mRNA and protein after a 24 h treatment with 25 μM osthole in the presence or absence of Bu2cAMP. The data showed that osthole alone significantly increased SRB1 and StAR mRNA expression, and significantly enhanced the stimulatory effect of Bu2cAMP (Fig. 7A and B). Additionally, StAR protein was also increased by osthole with or without added Bu2cAMP (Fig. 7D). The results suggested that osthole promoted cholesterol uptake and transport to the inner mitochondrial membrane for the synthesis of steroid hormones.
4. Discussion In this report, we found first that osthole stimulated corticosterone production in mouse Y1 adrenocortical cells by inducing the expression of genes encoding steroid hormone synthetic enzymes and proteins involved in corticosterone synthesis. These include enzymes involved in early steps of biosynthesis such as SRB1, StAR, FDX1, POR, and HSD3B1 as well as those involved in promoting corticosterone synthesis, like CYP11B1 and HSD11B1. In traditional Chinese medicine osthole originates from C. monnieri (L.) Cusson, used to treat Kidney-Yang deficiency syndrome, which appears to be related to adrenocortical function (Shen, 2005). Whether or not osthole has direct effects on adrenal steroid
hormone secretion was unknown. We used the Y1 cell line because these tumor cells produce corticosterone as one of the major steroid products, and the regulation of steroid synthesis in Y1 cells is similar to the regulation observed in cells cultured from normal adrenal glands with regard to ACTH (Rainey et al., 2004). Moreover, in traditional Chinese medicine we often study the effect of Chinese herbs effect on mice. Therefore, we selected the Y1 cell as the experimental model. According to reports on the genome-wide effects of ACTH on transcript accumulation in mouse adrenal Y1 cells, ACTH upregulated transcripts include several genes with sterol biosynthesis functions such as StAR, CYP11A1, MC2R, NR5A1, SRB1, LDLR, HMGCR, HSD3B1, NR4A1, NR5A1, POR, FDXR and so on (Schimmer et al., 2006, 2007). Our study showed that osthole also increased CYP11B1, StAR, SRB1, HSD3B1, FDX1, POR, NR4A1 and NR5A1 expression, therefore, osthole showed partial ACTH-like effects. Indeedly, StAR play major roles in intracellular cholesterol transport, which is responsible for the rapid movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane. Moreover, StAR and related proteins containing StARrelated lipid transfer domains such as StarD4 and StarD5 play synchronously major roles in intracellular cholesterol transport (Miller, 2007). Additionally, rodent cells use cholesterol from circulating high-density lipoproteins (HDL) taken up by via scavenger receptor B1 (SRB1) as their principal source of cholesterol for steroidogenesis (Miller and Bose, 2011). In this study, osthole increased StAR mRNA expression in a dose-dependent manner, and also enhanced StAR and SRB1 gene and protein expression with or without Bu2cAMP. The result suggests that osthole play a key role in intracellular cholesterol trafficking. Furthermore, CYP11A1 catalyzes the first step in steroidogenesis in adrenal cells, and conversion of cholesterol to pregnenolone in mitochondria is ratelimiting step in the synthesis of the steroid hormones, therefore, transcription of the CYP11A1 gene encoding P450scc is the factor that determines the net steroidogenic capacity of a cell (Miller and Auchus, 2011). However, in this paper, the data suggests that CYP11A1 gene and protein expression show no response to
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Fig. 5. Gene involved in corticosterone synthesis and metabolism was detected by qRT-PCR after Y1 cells were treated with 25 μM osthole (OST) in the presence or absence of Bu2cAMP for 24 h. (A) Osthole and Bu2cAMP significantly increased HSD3B1 mRNA expression; *Po 0.05 vs control, ***P o 0.001 vs control. (B) Bu2cAMP significantly decreased HSD11B1 mRNA expression and osthole returned expression to a value not significantly different from the control; *P o0.05 vs control. (C) HSD11B2 mRNA expression was not altered by osthole or Bu2cAMP. (D) Osthole and Bu2cAMP significantly increased FDX1 mRNA expression; *Po 0.05 vs control, ***Po 0.001 vs control. (E) FDXR mRNA expression was unchanged by osthole or Bu2cAMP. (F) Osthole and Bu2cAMP significantly increased POR mRNA expression; *P o0.05 vs control, **P o 0.01 vs control. (G) Osthole in the presence of absence of Bu2cAMP significantly increased RXRα mRNA expression; *Po 0.05 vs control. (H) Bu2cAMP in the presence or absence of osthole significantly increased PPARα mRNA expression; *Po 0.05 vs control. (I) Only Bu2cAMP significantly increased MC2R mRNA expression; **Po 0.01 vs control.
different doses of osthole, but Bu2cAMP significantly enhanced CYP11A1 gene and protein expression. The results indicated that osthole treatment for 24 h in Y1 cells did not affect rate-limiting step in steroidogenesis. It is important that CYP11B1 gene expression was increased by osthole in a dose-dependent manner and corticosterone production level was heightened significantly by osthole treatment for 24 h and 48 h. The results suggests that osthole promotes corticosterone biosynthesis and secretion is a novel pharmacological finding.
Acting as a cAMP analog that can permeate the plasma membrane, Bu2cAMP activates cAMP-dependent PKA (Schimmer and Schulz, 1985; Reyland, 1993). Further, we determined whether there were changes in the mRNA or protein expression of members of various pathways, such as PKA, CAMK1, and CREB1; we observed no effect of osthole on these parameters (data not shown). However, since the activity of these molecules is often regulated post-translationally, it is still possible that osthole can affect the cAMP-, protein kinase C- and Ca2 þ -dependent signaling
Fig. 6. The mRNA expression of immediate early genes, such as NR4A1 and NR4A2, as well as NR5A1, were detected by qRT-PCR after Y1 cells were treated for various times as indicated with 10 μM osthole or 0.1% DMSO as the vehicle control (Con). (A) Osthole significantly increased NR4A1 mRNA expression after treatment for 30 min, reaching a peak at 1 h; ***Po 0.001 vs control. (B) Osthole increased NR4A2 mRNA expression after treatment for 30 min and 1 h; *Po 0.05 vs control. (C) Osthole increased NR5A1 mRNA expression after treatment for 30 min and 1 h; *Po 0.05 vs control.
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Fig. 7. Osthole enhanced SRB1 and StAR mRNA and protein expression upon Y1 cell treatment with 25 μM osthole (OST) in the presence or absence of 1 mM Bu2cAMP for 24 h. (A) Osthole and/or Bu2cAMP significantly increased SCARB1 (SRB1) mRNA expression; *Po 0.05 vs control, **P o0.01 vs control, ***Po 0.001 vs control, #Po 0.05 vs Bu2cAMP. (B) Osthole and Bu2cAMP significantly increased StAR mRNA expression, and the combination showed a further enhancement; *P o 0.05 vs control, ***Po 0.001 vs control, ##P o0.01 vs Bu2cAMP. (C) Bu2cAMP with and without osthole significantly increased SRB1 protein expression. (D) Osthole and Bu2cAMP increased StAR protein expression.
pathways in mouse Y1 adrenocortical cells. On the other hand, osthole-mediated facilitation of glutamate release in rat hippocampal nerve endings involves the activation of the cGMP/PKGdependent pathway (Wang et al., 2008; Lin et al., 2012). We hypothesize that osthole promotes corticosterone production by a transcriptional mechanism. In addition, osthole increased the expression of nuclear hormone receptors such as RXRα and PPARα in Y1 cells. It has been reported that PPARα is also regulated by osthole in hepatocytes and cardiac fibroblasts; for example, osthole increases PPARα mRNA expression in mouse liver and cultured hepatocytes in a dose-dependent manner, and decreased its target genes including DGAT and HMG-CoA reductase (Zhang et al., 2007b; Sun et al., 2010). Osthole can also inhibit NF-κB and TGF-β1 expression by activating PPARα/γ, and subsequently enhancing MMP-2/9 expression in cultured mouse cardiac fibroblasts (Chen et al., 2013). It is noteworthy that the orphan nuclear receptors (NR4 and NR5) are described as early response genes; in vitro studies have shown that they take part in regulation of adrenal function. For example, the NR5A1 gene encodes for steroidogenic factor 1, which regulate many aspects of adrenal and reproductive development and function (Achermann et al., 2002; Buaas et al., 2012), and NR5A1 has also been shown to regulate the expression of enzymes involved in steroid production in vitro (Buaas et al., 2012), NR5A1 mutations have also been associated with primary adrenal insufficiency, whereas NR5A1 overexpression dramatically inhibited CYP11B2 expression and decreased aldosterone production in H295R/TR/SF-1 cell line (Ye et al., 2009). Our data showed that NR5A1 induction in response to osthole is weak. The result suggested that NR5A1 may not have an important role on ostholeinduced steroidogenesis. Indeed, NR4 family is essential for adrenal production of mineralocorticoids and glucocorticoids. For example, NR4A1 plays a critical part in the regulation of 3beta-hydroxysteroid dehydrogenase (HSD3B2) transcription (Bassett et al., 2004a). Additionally, in human H295R adrenocortical cells, NR4A1 and NR4A2 are key regulators of aldosterone synthase (CYP11B2) expression, suggesting possible roles of these transcriptional factors in the regulation of adrenocortical steroidogenesis (Bassett et al., 2004b). In this study, NR4A1 was increased significantly
after osthole treatment for 30 min and 1 h suggesting that NR4A1 may have an important role in osthole-induced steroidogenesis.
5. Conclusion In summary, osthole increased corticosterone biosynthesis by increasing the expression of genes encoding steroid hormone synthetic enzymes and proteins and other proteins that can regulate adrenocortical function. Thus, our results provide biological evidence for the mechanisms contributing to the efficacy of osthole, and the traditional Chinese medicine in which it is the active ingredient, for the treatment of Kidney-Yang deficiency syndrome.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81102533) and the Shanghai Municipal Commission of Health and Family Planning Project (No. ZY3-CCCX3-3010). We are thankful to Dr. Wendy B. Bollag, Ph.D. (Professor, Department of Physiology, Georgia Regents University, Augusta, Georgia, USA) for providing her valuable inputs for English and scientific contents of the manuscript.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2015.10.009.
References Achermann, J.C., Ozisik, G., Ito, M., Orun, U.A., Harmanci, K., Gurakan, B., Jameson, J. L., 2002. Gonadal determination and adrenal development are regulated by the orphan nuclear receptor steroidogenic factor-1, in a dose-dependent manner. J. Clin. Endocrinol. Metab. 87, 1829–1833. Bassett, M.H., Suzuki, T., Sasano, H., De Vries, C.J., Jimenez, P.T., Carr, B.R., Rainey, W. E., 2004a. The orphan nuclear receptor NGFIB regulates transcription of 3beta-
462
Z. Pan et al. / Journal of Ethnopharmacology 175 (2015) 456–462
hydroxysteroid dehydrogenase: implications for the control of adrenal functional zonation. J. Biol. Chem. 279, 37622–37630. Bassett, M.H., Suzuki, T., Sasano, H., White, P.C., Rainey, W.E., 2004b. The orphan nuclear receptors NURR1 and NGFIB regulate adrenal aldosterone production. Mol. Endocrinol. 18, 279–290. Buaas, F.W., Gardiner, J.R., Clayton, S., Val, P., Swain, A., 2012. In vivo evidence for the crucial role of SF1 in steroid-producing cells of the testis, ovary and adrenal gland. Development 139, 4561–4570. Chen, R., Xue, J., Xie, M., 2013. Osthole regulates TGF-beta1 and MMP-2/9 expressions via activation of PPARalpha/gamma in cultured mouse cardiac fibroblasts stimulated with angiotensin II. J. Pharm. Pharm. Sci. 16, 732–741. Chen, Z.C., Duan, X.B., Liu, K.R., 1988. The anti-allergic activity of osthol extracted from the fruits of Cnidium monnieri (L.) Cusson. Yao Xue Xue Bao 23, 96–99. Hsieh, M.T., Hsieh, C.L., Wang, W.H., Chen, C.S., Lin, C.J., Wu, C.R., 2004. Osthole improves aspects of spatial performance in ovariectomized rats. Am. J. Chin. Med. 32, 11–20. Jarzab, A., Grabarska, A., Kielbus, M., Jeleniewicz, W., Dmoszynska-Graniczka, M., Skalicka-Wozniak, K., Sieniawska, E., Polberg, K., Stepulak, A., 2014. Osthole induces apoptosis, suppresses cell-cycle progression and proliferation of cancer cells. Anticancer Res. 34, 6473–6480. Kao, S.J., Su, J.L., Chen, C.K., Yu, M.C., Bai, K.J., Chang, J.H., Bien, M.Y., Yang, S.F., Chien, M.H., 2012. Osthole inhibits the invasive ability of human lung adenocarcinoma cells via suppression of NF-kappaB-mediated matrix metalloproteinase-9 expression. Toxicol. Appl. Pharmacol. 261, 105–115. Lin, T.Y., Lu, C.W., Huang, W.J., Wang, S.J., 2012. Involvement of the cGMP pathway in the osthole-facilitated glutamate release in rat hippocampal nerve endings. Synapse 66, 232–239. Liu, J., Zhang, W., Zhou, L., Wang, X., Lian, Q., 2005. Anti-inflammatory effect and mechanism of osthole in rats. Zhong Yao Cai 28, 1002–1006. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. Miller, W.L., 2007. StAR search – what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import. Mol. Endocrinol. 21, 589–601. Miller, W.L., Auchus, R.J., 2011. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr. Rev. 32, 81–151. Miller, W.L., Bose, H.S., 2011. Early steps in steroidogenesis: intracellular cholesterol trafficking. J. Lipid Res. 52, 2111–2135. Pan, Z.Q., Xie, D., Choudhary, V., Seremwe, M., Tsai, Y.Y., Olala, L., Chen, X., Bollag, W. B., 2014. The effect of pioglitazone on aldosterone and cortisol production in HAC15 human adrenocortical carcinoma cells. Mol. Cell. Endocrinol. 394, 119–128. Qin, L.P., Zhang, J.Q., Shi, H.P., 1997. Effects of coumarins from Cnidium monnieri on
the function of pituitary–adrenocortical axis in kidney yang deficiency rats. Zhongguo Zhong Xi Yi Jie He Za Zhi 17, 227–229. Rainey, W.E., Saner, K., Schimmer, B.P., 2004. Adrenocortical cell lines. Mol. Cell. Endocrinol. 228, 23–38. Reyland, M.E., 1993. Protein kinase C is a tonic negative regulator of steroidogenesis and steroid hydroxylase gene expression in Y1 adrenal cells and functions independently of protein kinase A. Mol. Endocrinol. 7, 1021–1030. Schimmer, B.P., Cordova, M., Cheng, H., Tsao, A., Goryachev, A.B., Schimmer, A.D., Morris, Q., 2006. Global profiles of gene expression induced by adrenocorticotropin in Y1 mouse adrenal cells. Endocrinology 147, 2357–2367. Schimmer, B.P., Cordova, M., Cheng, H., Tsao, A., Morris, Q., 2007. A genome-wide assessment of adrenocorticotropin action in the Y1 mouse adrenal tumor cell line. Mol. Cell. Endocrinol. 265–266, 102–107. Schimmer, B.P., Schulz, P., 1985. The roles of cAMP and cAMP-dependent protein kinase in forskolin’s actions on Y1 adrenocortical tumor cells. Endocr. Res. 11, 199–209. Shen, Z.Y., 2005. Studies of gene networks and singal transduction of kidney deficiency syndrome by syndrome differentiation through drug effects. Zhongguo Zhong Xi Yi Jie He Za Zhi 25, 1125–1128. Shen, Z.Y., Dong, J.C., Cai, D.F., 2007. Important action of improving adrenocortical function for certain diseases recovery. Zhongguo Zhong Xi Yi Jie He Za Zhi 27, 364–367. Sun, F., Xie, M.L., Xue, J., Wang, H.B., 2010. Osthol regulates hepatic PPAR alphamediated lipogenic gene expression in alcoholic fatty liver murine. Phytomedicine 17, 669–673. Wang, S.J., Lin, T.Y., Lu, C.W., Huang, W.J., 2008. Osthole and imperatorin, the active constituents of Cnidium monnieri (L.) Cusson, facilitate glutamate release from rat hippocampal nerve terminals. Neurochem. Int. 53, 416–423. Ye, P., Nakamura, Y., Lalli, E., Rainey, W.E., 2009. Differential effects of high and low steroidogenic factor-1 expression on CYP11B2 expression and aldosterone production in adrenocortical cells. Endocrinology 150, 1303–1309. Zhang, J., Xue, J., Wang, H., Zhang, Y., Xie, M., 2011. Osthole improves alcohol-induced fatty liver in mice by reduction of hepatic oxidative stress. Phytother. Res. 25, 638–643. Zhang, Q., Qin, L., He, W., Van Puyvelde, L., Maes, D., Adams, A., Zheng, H., De Kimpe, N., 2007a. Coumarins from Cnidium monnieri and their antiosteoporotic activity. Planta Med. 73, 13–19. Zhang, Y., Xie, M., Xue, J., Gu, Z., 2007b. Osthole improves fat milk-induced fatty liver in rats: modulation of hepatic PPAR-alpha/gamma-mediated lipogenic gene expression. Planta Med. 73, 718–724. Zhao, L., Wu, H., Qiu, M., Sun, W., Wei, R., Zheng, X., Yang, Y., Xin, X., Zou, H., Chen, T., Liu, J., Lu, L., Su, J., Ma, C., Zhao, A., Jia, W., 2013. Metabolic signatures of kidney yang deficiency syndrome and protective effects of two herbal Extracts in rats using GC/TOF MS. Evid. Based Complement. Altern. Med. 2013, 540957.