The American Journal of Chinese Medicine, Vol. 42, No. 2, 337–347 © 2014 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X14500220
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Puerarin Attenuates Calcification of Vascular Smooth Muscle Cells Qiong Lu,* Da-Xiong Xiang,* Hai-Yan Yuan,* Yiwen Xiao,* Ling-Qing Yuan† and Hua-Bing Li‡ *Department †Institute
of Pharmacy
of Metabolism and Endocrinology ‡Department
of Radiology The Second Xiangya Hospital, Central South University Changsha, People’s Republic of China
Abstract: Several studies demonstrate that estradiol can prevent arterial calcification. However, little is known regarding the effect of puerarin, a phytoestrogen extracted from Radix Puerariae, on arterial calcification. The aim of the present study was to determine whether puerarin reduced osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs). The CVSMCs were isolated from mice aorta and treated with different concentrations of puerarin. The alkaline phosphatase (ALP) activity, osteocalcin secretion and Runx2 expression were determined. To examine whether estrogen receptors (ERs) PI3K and Akt play a role in this effect, ICI182789, phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-Ooctadecylcarbonate (HIMO) was used. Our results showed puerarin could inhibit ALP activity, osteocalcin secretion and Runx2 expression in CVSMCs. Puerarin could induce the activation of Akt. Furthermore, pretreatment of ICI182780, LY294002, HIMO could abolish the effect of puerarin on ALP activity in CVSMCs. Our experiment demonstrated that puerain could attenuate the osteoblastic differentiation of VSMCs through the ER/PI3K-Akt signal pathway. Keywords: Puerarin; Vascular Smooth Muscle Cells; Estrogen Receptor; Akt; PI3K.
Introduction Cardiovascular disease is the leading cause of mortality and morbidity worldwide. Recently, more attention has been paid to arterial calcification, which results in a decline in the elasticity of blood vessels and is related to hypertension and heart failure (Shao et al.,
Correspondence to: Dr. Hua-Bing Li, Department of Radiology, The Second Xiangya Hospital, Central South University, Changsha, People’s Republic of China. Tel/Fax: (þ86) 731-8529-2007, E-mail: [email protected]
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2010; Fadini et al., 2011; EVOLVE Trial Investigators et al., 2012), especially in patients suffering from diabetes and end-stage renal disease (Hwang et al., 2012). Recently, data from in vitro and in vivo studies on the mechanism regulating arterial calcification demonstrate that arterial calcification is a complicated pathological process, which involves transdifferentiation of vascular smooth muscle cells (VSMCs). The VSMCs transdifferentiate from a fibroblastic to an osteoblastic phenotype by upregulating the osteochondrogenic transcription factor Runx2 and a series of osteoblast-related markers, such ALP and osteoclacin (Abedin et al., 2004; Yuan et al., 2011; Shan et al., 2011; Cui et al., 2012; Liang et al., 2012; Thompson and Towler, 2012). Previous studies have suggested that estrogen inhibited vascular lesion progression (Tiyasatkulkovit et al., 2012) and ameliorated coronary artery calcification (Wu et al., 2007). However, the side effects and the gender limitation obstruct the application of estrogen. Phytoestrogens are polyphenolic, non-steroidal plant compounds with estrogenlike biological activity. The phytoestrogen puerarin, a daidzein-8-C glucoside, is an isoflavone extracted from Radix Puerariae (Tiyasatkulkovit et al., 2012). It has been widely used for various medicinal purposes in traditional Chinese medicines for thousands of years (Wang et al., 2012; Xutian et al., 2012). Recent studies have indicated puerarin may be useful in the management of various disorders, such as coronary artery disease, osteoporosis, atherosclerosis, myocardial infarction, and liver fibrosis (Siow et al., 2007; Wu et al., 2007; Meng et al., 2009; Tang et al., 2012; Li et al., 2013). Several studies have confirmed that puerarin could attenuate VSMC proliferation (Zhu et al., 2010) and stimulate osteoblast differentiation (Zhang et al., 2012; Urasopon et al., 2008; Tiyasatkulkovit et al., 2012), which suggests that puerarin may have an effect on osteoblastic differentiation of VSMCs. In the present study, we used calcifying CVSMCs, a specific subpopulation of VSMCs that could express osteoblastic phenotype gene and spontaneously form a calcification nodule, to investigate the effect of puerarin on osteoblastic differentiation of CVSMCs and the signal pathway involved. Materials and Methods Reagents Puerarin was purchased from Sigma-Aldrich Co. ICI82780. A specific estrogen receptor (ER) inhibitor was purchased from Tocris Bioscience (Brisol, UK). PI3K inhibitor LY294002 and Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-Ooctadecylcarbonate (HIMO) were purchased from Calbiochem (San Diego, CA, USA). Puerarin was dissolved in dimethyl sulfoxide (DMSO). The cells treated with the solvent DMSO alone, without puerarin, served as a control. Cell Culture Primary VSMCs were isolated from the thoracic aortas of mice, as previously described (Liang et al., 2012). Briefly, the mouse aortas were isolated under sterile conditions, and
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the tunica media was separated from the aortas. The tissue was fragmented (1–2 mm 3 Þ, the aortas were minced and digested in 5 ml of digestion solution (0.125 mg/mL elastase, 0.25 mg/ml soybean trypsin inhibitor, 10 mg/mL collagenase I, 2.0 mg/mL crystallized bovine albumin, and 15 mM HEPES) at 37 C for 45 min. The cellular digests were filtered through sterile 100 m nylon mesh, centrifuged at 1,000 rpm for 10 min, and washed twice in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% FBS (Gibico-BRL Corp., NY, USA) CVSMCs were isolated from cultures in which multicellular nodules appeared spontaneously. Cells were cloned by limiting dilution and single cell harvesting from these nodule-forming cultures. Cloned lines were identified as CVSMCs by their positive staining with monoclonal antibody -actin, and by their ability to express high levels of ALP and to form calcified nodules. To determine the downstream intracellular signaling pathways affected by puerarin treatment, we explored the relationship of Akt phosphorylation of CVSMCs and treatment with puerarin. Then, CVSMCs were pretreated with 10 nM ICI182780, 10 M LY294002 (a PI3-K inhibitor) or 10 M HIMO (an Akt inhibitor) for 2 h prior to puerarin treatment. ALP activities were assessed after 48 h. The approval was granted by the Second Xiangya Hospital of Central South University Ethics Review Board, China.
Measurements of Alkaline Phosphatase (ALP) Activity and Osteocalcin Secretion Confluent CVSMCs were washed twice with PBS, and the cells were lysed into solution containing 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.02% NaN3 and 1 g/ml aprotinin. The lysates were centrifuged at 13000 rpm for 10 min at 4 C, and then the supernatants were homogenized and assayed for ALP activity by measuring -nitrophenol release at 37 C using spectrophotometry. Protein expression was normalized to total cellular protein by a Bradford protein assay. Osteocalcin secretion into the culture media was measured with a radioimmunoassay kit (DiaSorin Corp., Stillwater, MN, USA) according to the manufacturer’s instructions. Protein expression was normalized to total cellular protein by a Bradford protein assay.
Western Blotting Western blotting was performed, as previously described (Liang et al., 2012; Wu et al., 2013). At each designated time point, the cells were harvested and lysed with RIPA buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 30 mM sodium pyrophosphate, 5 mM EDTA, 2 mM Na3VO4, 5 mM NaF, 1 mM phenylmethyl-sulfonyl-fluoride (PMSF) and protease inhibitor cocktail). Equal amounts of protein were loaded on 10% Tris-Glycine gels and transferred onto polyvinylidene difluoride (PVDF) membrane antibodies using p-Akt. AKT, Runx2 and β-actin were all purchased from cell signaling. The membrane was reprobed with secondary antibody for 1 h. Blots were processed using an ECL kit (Amersham Biosciences) and exposed to the film.
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Mineral Nodules Formation Assay To determine the effect of puerarin on the matrix calcification, CVSMCs were incubated in 24-well plates for 24 h, and treated with puerarin or vehicle for 12 days. Cultures were fed every 2 days by replacing with fresh medium and reagents. The extent of mineralized matrix was determined by Alizarin Red S staining. Cells were fixed in 70% ethanol for 1 h at room temperature and stained with 0.1% (w/v) Alizarin Red S for 30 min. Next, cell preparations were washed with PBS to eliminate non-specific staining. The stained matrix was photographed using a digital microscope. Alizarin Red S staining was released from the cell matrix by incubation in cetyl-pyridinium chloride for 15 min. The amount of released dye was quantified by spectrophotometry at 540 nm. Results were then normalized to total cellular protein values. Statistical Analyses Results are expressed as mean SD. Comparisons were made using a one-way ANOVA. All experiments were repeated at least three times, and representative experiments are shown. Results Effect of Puerarin on Osteoblastic Differentiation of CVSMCs To determine whether puerarin affected the osteoblastic differentiation of CVSMCs, the ALP activity and osteocalcin secretion, which are early and late markers for the differentiation of an osteoblast, were measured. As showed in Fig. 1A, puerarin inhibited the ALP activity in CVSMCs in a dose dependent manner. After 48 h of culture, 0.1 m/L of puerarin had no effect on the ALP activity, while the ALP activity decreased significantly at a concentration of 1 m/L of puerarin, and the maximum effect of puerarin on ALP activity was reached at 10 m/L. Figure 1B shows the effects of puerarin on osteocalcin secretion in cultured CVSMCs. Treatment with 0.1–100 M puerarin caused a significant decrease in osteocalcin production and the maximal effect of puerarin was reached after the addition of 10 M puerarin. The expression of Runx2, the key transcription factor for osteoblastic differentiation, was determined via western blot. Our result showed that the expression of Runx2 was inhibited significantly after treatment with puerarin for 48 h in CVSMCs (Fig. 1C). The effect of puerarin on mineralization of the matrix, a marker of osteoblast terminal differentiation, was determined using Alizarin Red stain. As shown in Figs. 1D and 1E, in the presence of 10 M puerarin, the calcification nodule and the calcium deposition decreased significantly. Puerarin Activated Akt Signaling Pathway in CVSMCs Recent studies have demonstrated that Akt signaling pathways mediate puerarin action (Hwang et al., 2011; Liu et al., 2012). We examined whether puerarin induced Akt
ALP activities (nmol/min.mgprotein)
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Osteocalcin secreti (ng/mg proteinon)
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(E) Figure 1. Effects of puerarin on osteoblastic differentiation of CVSMCs. (A) Effect of puerarin on ALP activity. The cells were cultured with or without 0.1–100 m/L puerarin for the 48 h. ALP activity was determined and normalized to cell total protein. (B) Effect of puerarin on osteocalcin secretion. The cells were cultured with or without 0.1–100 m/L puerarin for the 48 h. ALP activity was determined and normalized to cell total protein. (C) Effect of puerarin on Runx2 expression. The cells were cultured for 48 h with or without 10 m/L puerarin. The expression of Runx2 was measured by western blotting. (E) Representative microscopic view at a magnification of 100 of the Alizarin Red S staining in 24-well plates for control cells and cells treated with puerarin in 12-day cultures. Left panel showed CVSMCs incubated with vehicle, and the right panel showed CVSMCs incubated with 10 M puerarin for 12 days. (F) Effect of puerarin on calcium deposition. The cells were cultured for 12 days with or without 10 m/L puerarin. The calcium contents of the cell layers were measured by the atomic absorption spectroscopy method. (**p < 0:01 vs. control, *p < 0:05 vs. control, n ¼ 3).
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(B)
Figure 2. Puerarin promoted Akt activation through ER in CVSMCs. (A) Cells were exposed to 10 m/L puerarin for 0–60 min. Cell lysates were subjected to western blotting and incubated with antibodies against p-Akt and Akt. The representative results are shown. (B) Cells were incubated with ICI18278 for 2 h before being treated with 10 M puerarin for 15 min. Cell lysates were subjected to western blotting and incubated with antibodies against p-Akt and Akt. The representative results are shown.
signaling in CVSMCs. As shown in Fig. 2A, puerarin stimulated the activity of a specific phosphorylated Akt (p-Akt) in the CVSMCs after 5 min of incubation, when determined by the increase in the p-Akt levels, the peak activation of p-Akt occurred at 15–30 min.
ALP activities (nmol/min.mg protein)
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(A)
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Figure 3. ER/PI3K-Akt signaling pathway mediated the inhibitory effect of puerarin on osteoblastic differentiation of CVSMCs. Cells were homogenized for the ALP activity assay. Cells were incubated with ICI182780 (10 nM), HIMO (10 M) or LY294002 (10 M) for 2 h before being treated with 10 M puerarin for 48 h (n ¼ 5; **p < 0:01 vs. control).
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Previous studies demonstrated that the ER is expressed in VSMCs. To explore the mechanism where puerarin activates Akt though the ER, we used an ER inhibitor to test the activation of Akt. Our results showed puerarin could activate AKT; however, this effect could be blocked by the ER antagonist ICI182780.
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Puerarin Attenuated Osteoblastic Differentiation of CVSMCs Through the ER/PI3K-Akt Signaling Pathway To investigate whether ER mediated the osteoregulatory actions of puerarin in CVSMCs, CVSMCs were pretreated with high affinity ER antagonist ICI182780. Our results showed that CVSMCs treated with puerarin could significantly decrease the ALP activity, while the presence of ICI182780 could attenuate the inhibition effect of puerarin on ALP activity. To better understand the signal pathway involved in the effect of puerarin on osteoblastic differentiation of CVSMCs, we further examined whether Akt and its upstream signal PI3K involved in the effect of Puerarin on osteoblastic differentiation of CVSMCs. The PI3K inhibitor LY294002 and Akt inhibitor HIMO were employed. Our results showed that the inhibition of PI3K and Akt signal pathways significantly attenuated the decreasing effect of puerarin on ALP activity in CVSMCs (Fig. 3). These results strongly suggested that the inhibitory effect of puerarin on osteoblastic differentiation of CVSMCs was possibly in an ER/PI3K-AKT dependent manner.
Discussion A recent clinical study has demonstrated that the serum estradiol level is negatively correlated with a coronary artery calcium score, which suggested an increased serum estradiol concentration might decrease the calcified-plaque burden of coronary arteries (Bourassa et al., 1996). Moreover, in the controlled Women’s Health Initiative Study, postmenopausal women, aged 50 to 59 years and treated with long-term estrogen therapy, had lower levels of coronary artery calcification than those who received placebo (Manson et al., 2007), and ER expression in coronary arteries was correlated with advanced coronary calcification (Liu et al., 2005; Christian et al., 2006). Meanwhile, data from an in vitro experiment suggested that estrogen inhibits VSMCs calcification (Verhaar et al., 1994; Rzewuska-Lech et al., 2005; Osako et al., 2010). However, whether phytoestrogen has the effect of osteoblastic differentiation of VSMCs is still unknown. Phytoestrogen is a natural compound of plant origin and possesses a structural similarity to estrogen and can bind to ER with minimal cancer risk. Recently, the tuberous root extract of phytoestrogen-puerarin has been suggested to have several functions. Moreover, puerarin was found to promote the differentiation of osteoblast in vitro and to prevent osteoporosis in ovariectomized rats by increasing bone mineral density and bone mineral content (Urasopon et al., 2008). These results demonstrate that puerarin could regulate the osteoblast differentiation. However, to the best of our knowledge, whether or not puerarin can affect the osteoblast differentiation of VSMCs has not been still not reported. Our
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present study investigated the effect of puerarin on osteoblastic differentiation of VSMCs and the mechanism involved. CVSMCs were treated with a different concentration of puerarin to investigate whether puerarin affected osteoblastic differentiation of CVSMCs. The results showed puerarin significantly decreased ALP activities, osteocalcin secretion and mineral nodules formation. Runx2 is a transcription factor involved in the differentiation of an osteoblast, and our result showed puerarin inhibited the expression of Runx2 in CVSMCs. These data suggested that puerarin had an inhibitory effect on osteoblastic differentiation of CVSCMs. The present experiment appeared to contradict a previous study, which showed that puerarin promoted osteoblast differentiation (Urasopon et al., 2008; Sheu et al., 2012; Tiyasatkulkovit et al., 2012). We speculated that this might be a biphasic effect on osteoblastic differentiation of VSMCs and osteoblast differentiation. Estrodial has been suggested to increase osteoblast differentiation while inhibiting osteoblastic differentiation of VSMCs (Verhaar et al., 1994; Robinson et al., 1997; Rzewuska-Lech et al., 2005; Osako et al., 2010), and several cytokines have been identified that have these biphasic effects on the osteogenic differentiation of VSMCs. For example, adiponectin attenuates the osteoblastic differentiation of VSMCs (Luo et al., 2009), while adiponectin stimulates osteoblast differentiation in mesenchymalprogenitor cells (Lee et al., 2009). Moreover, clinical data suggests that arterial calcification is frequently associated with osteoporosis, which is called the “calcification paradox” (Persy and D’Haese, 2009). The contradictory association of enhanced calcium deposition in blood vessels and decreased bone mineralization has attracted many researchers’ attention; the precise mechanisms underlying the complex interplay of bone and vasculature remain to be elucidated. To gain further insight into the mechanism by which puerarin inhibited the osteoblastic differentiation of CVSMCs, we evaluated a signal pathway event. Previous studies showed puerarin performs its effect through ER depend or non-ER depend mechanisms (Cheng et al., 2012; Michihara et al., 2012). Our results demonstrated that the ER antagonist blocked the effect of puerarin on ALP activity of CVSMCs, which suggested puerarin attenuated osteoblastic differentiation of CVSMCs through the ER. Previous studies suggested that the PI3K/AKT pathway is involved in the regulation of a serial of cellular processes including growth, proliferation and differentiation. A series of studies demonstrated that the activation of Akt might attenuate the osteoblastic differentiation of VSMC (Radcliff et al., 2005; Duan et al., 2011; Shan et al., 2011; Xie et al., 2011). Our present study showed that puerarin induced the activation of AKt in VSCMs. Moreover, ER antagonist blunted the effect of puerarin on AKT activation. Treatment with LY294002 (PI3K inhibitor), or HIMO (Akt inhibitor), abolished the effect of puerarin on the inhibitory effect of ALP activity in CVSMCs. Our present finding suggested a crucial role for puerarin in decreasing osteoblastic differentiation of VSMCs, and ER/PI3K-AKT is implicated as being involved in this effect. These results suggest puerarin plays a protective role against arterial calcification.
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Acknowledgments This work was supported by grants from Development and Reform Commission of Hunan (20111318, 20121493), Hunan Provincial Department of Finance (2011-43), Hunan province science and technology project (2012FJ6027) .
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Puerarin Attenuates Calcification of Vascular Smooth Muscle Cells Qiong Lu,* Da-Xiong Xiang,* Hai-Yan Yuan,* Yiwen Xiao,* Ling-Qing Yuan† and Hua-Bing Li‡ *Department †Institute
of Pharmacy
of Metabolism and Endocrinology ‡Department
of Radiology The Second Xiangya Hospital, Central South University Changsha, People’s Republic of China
Abstract: Several studies demonstrate that estradiol can prevent arterial calcification. However, little is known regarding the effect of puerarin, a phytoestrogen extracted from Radix Puerariae, on arterial calcification. The aim of the present study was to determine whether puerarin reduced osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs). The CVSMCs were isolated from mice aorta and treated with different concentrations of puerarin. The alkaline phosphatase (ALP) activity, osteocalcin secretion and Runx2 expression were determined. To examine whether estrogen receptors (ERs) PI3K and Akt play a role in this effect, ICI182789, phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-Ooctadecylcarbonate (HIMO) was used. Our results showed puerarin could inhibit ALP activity, osteocalcin secretion and Runx2 expression in CVSMCs. Puerarin could induce the activation of Akt. Furthermore, pretreatment of ICI182780, LY294002, HIMO could abolish the effect of puerarin on ALP activity in CVSMCs. Our experiment demonstrated that puerain could attenuate the osteoblastic differentiation of VSMCs through the ER/PI3K-Akt signal pathway. Keywords: Puerarin; Vascular Smooth Muscle Cells; Estrogen Receptor; Akt; PI3K.
Introduction Cardiovascular disease is the leading cause of mortality and morbidity worldwide. Recently, more attention has been paid to arterial calcification, which results in a decline in the elasticity of blood vessels and is related to hypertension and heart failure (Shao et al.,
Correspondence to: Dr. Hua-Bing Li, Department of Radiology, The Second Xiangya Hospital, Central South University, Changsha, People’s Republic of China. Tel/Fax: (þ86) 731-8529-2007, E-mail: [email protected]
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2010; Fadini et al., 2011; EVOLVE Trial Investigators et al., 2012), especially in patients suffering from diabetes and end-stage renal disease (Hwang et al., 2012). Recently, data from in vitro and in vivo studies on the mechanism regulating arterial calcification demonstrate that arterial calcification is a complicated pathological process, which involves transdifferentiation of vascular smooth muscle cells (VSMCs). The VSMCs transdifferentiate from a fibroblastic to an osteoblastic phenotype by upregulating the osteochondrogenic transcription factor Runx2 and a series of osteoblast-related markers, such ALP and osteoclacin (Abedin et al., 2004; Yuan et al., 2011; Shan et al., 2011; Cui et al., 2012; Liang et al., 2012; Thompson and Towler, 2012). Previous studies have suggested that estrogen inhibited vascular lesion progression (Tiyasatkulkovit et al., 2012) and ameliorated coronary artery calcification (Wu et al., 2007). However, the side effects and the gender limitation obstruct the application of estrogen. Phytoestrogens are polyphenolic, non-steroidal plant compounds with estrogenlike biological activity. The phytoestrogen puerarin, a daidzein-8-C glucoside, is an isoflavone extracted from Radix Puerariae (Tiyasatkulkovit et al., 2012). It has been widely used for various medicinal purposes in traditional Chinese medicines for thousands of years (Wang et al., 2012; Xutian et al., 2012). Recent studies have indicated puerarin may be useful in the management of various disorders, such as coronary artery disease, osteoporosis, atherosclerosis, myocardial infarction, and liver fibrosis (Siow et al., 2007; Wu et al., 2007; Meng et al., 2009; Tang et al., 2012; Li et al., 2013). Several studies have confirmed that puerarin could attenuate VSMC proliferation (Zhu et al., 2010) and stimulate osteoblast differentiation (Zhang et al., 2012; Urasopon et al., 2008; Tiyasatkulkovit et al., 2012), which suggests that puerarin may have an effect on osteoblastic differentiation of VSMCs. In the present study, we used calcifying CVSMCs, a specific subpopulation of VSMCs that could express osteoblastic phenotype gene and spontaneously form a calcification nodule, to investigate the effect of puerarin on osteoblastic differentiation of CVSMCs and the signal pathway involved. Materials and Methods Reagents Puerarin was purchased from Sigma-Aldrich Co. ICI82780. A specific estrogen receptor (ER) inhibitor was purchased from Tocris Bioscience (Brisol, UK). PI3K inhibitor LY294002 and Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-Ooctadecylcarbonate (HIMO) were purchased from Calbiochem (San Diego, CA, USA). Puerarin was dissolved in dimethyl sulfoxide (DMSO). The cells treated with the solvent DMSO alone, without puerarin, served as a control. Cell Culture Primary VSMCs were isolated from the thoracic aortas of mice, as previously described (Liang et al., 2012). Briefly, the mouse aortas were isolated under sterile conditions, and
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the tunica media was separated from the aortas. The tissue was fragmented (1–2 mm 3 Þ, the aortas were minced and digested in 5 ml of digestion solution (0.125 mg/mL elastase, 0.25 mg/ml soybean trypsin inhibitor, 10 mg/mL collagenase I, 2.0 mg/mL crystallized bovine albumin, and 15 mM HEPES) at 37 C for 45 min. The cellular digests were filtered through sterile 100 m nylon mesh, centrifuged at 1,000 rpm for 10 min, and washed twice in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% FBS (Gibico-BRL Corp., NY, USA) CVSMCs were isolated from cultures in which multicellular nodules appeared spontaneously. Cells were cloned by limiting dilution and single cell harvesting from these nodule-forming cultures. Cloned lines were identified as CVSMCs by their positive staining with monoclonal antibody -actin, and by their ability to express high levels of ALP and to form calcified nodules. To determine the downstream intracellular signaling pathways affected by puerarin treatment, we explored the relationship of Akt phosphorylation of CVSMCs and treatment with puerarin. Then, CVSMCs were pretreated with 10 nM ICI182780, 10 M LY294002 (a PI3-K inhibitor) or 10 M HIMO (an Akt inhibitor) for 2 h prior to puerarin treatment. ALP activities were assessed after 48 h. The approval was granted by the Second Xiangya Hospital of Central South University Ethics Review Board, China.
Measurements of Alkaline Phosphatase (ALP) Activity and Osteocalcin Secretion Confluent CVSMCs were washed twice with PBS, and the cells were lysed into solution containing 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.02% NaN3 and 1 g/ml aprotinin. The lysates were centrifuged at 13000 rpm for 10 min at 4 C, and then the supernatants were homogenized and assayed for ALP activity by measuring -nitrophenol release at 37 C using spectrophotometry. Protein expression was normalized to total cellular protein by a Bradford protein assay. Osteocalcin secretion into the culture media was measured with a radioimmunoassay kit (DiaSorin Corp., Stillwater, MN, USA) according to the manufacturer’s instructions. Protein expression was normalized to total cellular protein by a Bradford protein assay.
Western Blotting Western blotting was performed, as previously described (Liang et al., 2012; Wu et al., 2013). At each designated time point, the cells were harvested and lysed with RIPA buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 30 mM sodium pyrophosphate, 5 mM EDTA, 2 mM Na3VO4, 5 mM NaF, 1 mM phenylmethyl-sulfonyl-fluoride (PMSF) and protease inhibitor cocktail). Equal amounts of protein were loaded on 10% Tris-Glycine gels and transferred onto polyvinylidene difluoride (PVDF) membrane antibodies using p-Akt. AKT, Runx2 and β-actin were all purchased from cell signaling. The membrane was reprobed with secondary antibody for 1 h. Blots were processed using an ECL kit (Amersham Biosciences) and exposed to the film.
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Mineral Nodules Formation Assay To determine the effect of puerarin on the matrix calcification, CVSMCs were incubated in 24-well plates for 24 h, and treated with puerarin or vehicle for 12 days. Cultures were fed every 2 days by replacing with fresh medium and reagents. The extent of mineralized matrix was determined by Alizarin Red S staining. Cells were fixed in 70% ethanol for 1 h at room temperature and stained with 0.1% (w/v) Alizarin Red S for 30 min. Next, cell preparations were washed with PBS to eliminate non-specific staining. The stained matrix was photographed using a digital microscope. Alizarin Red S staining was released from the cell matrix by incubation in cetyl-pyridinium chloride for 15 min. The amount of released dye was quantified by spectrophotometry at 540 nm. Results were then normalized to total cellular protein values. Statistical Analyses Results are expressed as mean SD. Comparisons were made using a one-way ANOVA. All experiments were repeated at least three times, and representative experiments are shown. Results Effect of Puerarin on Osteoblastic Differentiation of CVSMCs To determine whether puerarin affected the osteoblastic differentiation of CVSMCs, the ALP activity and osteocalcin secretion, which are early and late markers for the differentiation of an osteoblast, were measured. As showed in Fig. 1A, puerarin inhibited the ALP activity in CVSMCs in a dose dependent manner. After 48 h of culture, 0.1 m/L of puerarin had no effect on the ALP activity, while the ALP activity decreased significantly at a concentration of 1 m/L of puerarin, and the maximum effect of puerarin on ALP activity was reached at 10 m/L. Figure 1B shows the effects of puerarin on osteocalcin secretion in cultured CVSMCs. Treatment with 0.1–100 M puerarin caused a significant decrease in osteocalcin production and the maximal effect of puerarin was reached after the addition of 10 M puerarin. The expression of Runx2, the key transcription factor for osteoblastic differentiation, was determined via western blot. Our result showed that the expression of Runx2 was inhibited significantly after treatment with puerarin for 48 h in CVSMCs (Fig. 1C). The effect of puerarin on mineralization of the matrix, a marker of osteoblast terminal differentiation, was determined using Alizarin Red stain. As shown in Figs. 1D and 1E, in the presence of 10 M puerarin, the calcification nodule and the calcium deposition decreased significantly. Puerarin Activated Akt Signaling Pathway in CVSMCs Recent studies have demonstrated that Akt signaling pathways mediate puerarin action (Hwang et al., 2011; Liu et al., 2012). We examined whether puerarin induced Akt
ALP activities (nmol/min.mgprotein)
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(E) Figure 1. Effects of puerarin on osteoblastic differentiation of CVSMCs. (A) Effect of puerarin on ALP activity. The cells were cultured with or without 0.1–100 m/L puerarin for the 48 h. ALP activity was determined and normalized to cell total protein. (B) Effect of puerarin on osteocalcin secretion. The cells were cultured with or without 0.1–100 m/L puerarin for the 48 h. ALP activity was determined and normalized to cell total protein. (C) Effect of puerarin on Runx2 expression. The cells were cultured for 48 h with or without 10 m/L puerarin. The expression of Runx2 was measured by western blotting. (E) Representative microscopic view at a magnification of 100 of the Alizarin Red S staining in 24-well plates for control cells and cells treated with puerarin in 12-day cultures. Left panel showed CVSMCs incubated with vehicle, and the right panel showed CVSMCs incubated with 10 M puerarin for 12 days. (F) Effect of puerarin on calcium deposition. The cells were cultured for 12 days with or without 10 m/L puerarin. The calcium contents of the cell layers were measured by the atomic absorption spectroscopy method. (**p < 0:01 vs. control, *p < 0:05 vs. control, n ¼ 3).
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(B)
Figure 2. Puerarin promoted Akt activation through ER in CVSMCs. (A) Cells were exposed to 10 m/L puerarin for 0–60 min. Cell lysates were subjected to western blotting and incubated with antibodies against p-Akt and Akt. The representative results are shown. (B) Cells were incubated with ICI18278 for 2 h before being treated with 10 M puerarin for 15 min. Cell lysates were subjected to western blotting and incubated with antibodies against p-Akt and Akt. The representative results are shown.
signaling in CVSMCs. As shown in Fig. 2A, puerarin stimulated the activity of a specific phosphorylated Akt (p-Akt) in the CVSMCs after 5 min of incubation, when determined by the increase in the p-Akt levels, the peak activation of p-Akt occurred at 15–30 min.
ALP activities (nmol/min.mg protein)
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Figure 3. ER/PI3K-Akt signaling pathway mediated the inhibitory effect of puerarin on osteoblastic differentiation of CVSMCs. Cells were homogenized for the ALP activity assay. Cells were incubated with ICI182780 (10 nM), HIMO (10 M) or LY294002 (10 M) for 2 h before being treated with 10 M puerarin for 48 h (n ¼ 5; **p < 0:01 vs. control).
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Previous studies demonstrated that the ER is expressed in VSMCs. To explore the mechanism where puerarin activates Akt though the ER, we used an ER inhibitor to test the activation of Akt. Our results showed puerarin could activate AKT; however, this effect could be blocked by the ER antagonist ICI182780.
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Puerarin Attenuated Osteoblastic Differentiation of CVSMCs Through the ER/PI3K-Akt Signaling Pathway To investigate whether ER mediated the osteoregulatory actions of puerarin in CVSMCs, CVSMCs were pretreated with high affinity ER antagonist ICI182780. Our results showed that CVSMCs treated with puerarin could significantly decrease the ALP activity, while the presence of ICI182780 could attenuate the inhibition effect of puerarin on ALP activity. To better understand the signal pathway involved in the effect of puerarin on osteoblastic differentiation of CVSMCs, we further examined whether Akt and its upstream signal PI3K involved in the effect of Puerarin on osteoblastic differentiation of CVSMCs. The PI3K inhibitor LY294002 and Akt inhibitor HIMO were employed. Our results showed that the inhibition of PI3K and Akt signal pathways significantly attenuated the decreasing effect of puerarin on ALP activity in CVSMCs (Fig. 3). These results strongly suggested that the inhibitory effect of puerarin on osteoblastic differentiation of CVSMCs was possibly in an ER/PI3K-AKT dependent manner.
Discussion A recent clinical study has demonstrated that the serum estradiol level is negatively correlated with a coronary artery calcium score, which suggested an increased serum estradiol concentration might decrease the calcified-plaque burden of coronary arteries (Bourassa et al., 1996). Moreover, in the controlled Women’s Health Initiative Study, postmenopausal women, aged 50 to 59 years and treated with long-term estrogen therapy, had lower levels of coronary artery calcification than those who received placebo (Manson et al., 2007), and ER expression in coronary arteries was correlated with advanced coronary calcification (Liu et al., 2005; Christian et al., 2006). Meanwhile, data from an in vitro experiment suggested that estrogen inhibits VSMCs calcification (Verhaar et al., 1994; Rzewuska-Lech et al., 2005; Osako et al., 2010). However, whether phytoestrogen has the effect of osteoblastic differentiation of VSMCs is still unknown. Phytoestrogen is a natural compound of plant origin and possesses a structural similarity to estrogen and can bind to ER with minimal cancer risk. Recently, the tuberous root extract of phytoestrogen-puerarin has been suggested to have several functions. Moreover, puerarin was found to promote the differentiation of osteoblast in vitro and to prevent osteoporosis in ovariectomized rats by increasing bone mineral density and bone mineral content (Urasopon et al., 2008). These results demonstrate that puerarin could regulate the osteoblast differentiation. However, to the best of our knowledge, whether or not puerarin can affect the osteoblast differentiation of VSMCs has not been still not reported. Our
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present study investigated the effect of puerarin on osteoblastic differentiation of VSMCs and the mechanism involved. CVSMCs were treated with a different concentration of puerarin to investigate whether puerarin affected osteoblastic differentiation of CVSMCs. The results showed puerarin significantly decreased ALP activities, osteocalcin secretion and mineral nodules formation. Runx2 is a transcription factor involved in the differentiation of an osteoblast, and our result showed puerarin inhibited the expression of Runx2 in CVSMCs. These data suggested that puerarin had an inhibitory effect on osteoblastic differentiation of CVSCMs. The present experiment appeared to contradict a previous study, which showed that puerarin promoted osteoblast differentiation (Urasopon et al., 2008; Sheu et al., 2012; Tiyasatkulkovit et al., 2012). We speculated that this might be a biphasic effect on osteoblastic differentiation of VSMCs and osteoblast differentiation. Estrodial has been suggested to increase osteoblast differentiation while inhibiting osteoblastic differentiation of VSMCs (Verhaar et al., 1994; Robinson et al., 1997; Rzewuska-Lech et al., 2005; Osako et al., 2010), and several cytokines have been identified that have these biphasic effects on the osteogenic differentiation of VSMCs. For example, adiponectin attenuates the osteoblastic differentiation of VSMCs (Luo et al., 2009), while adiponectin stimulates osteoblast differentiation in mesenchymalprogenitor cells (Lee et al., 2009). Moreover, clinical data suggests that arterial calcification is frequently associated with osteoporosis, which is called the “calcification paradox” (Persy and D’Haese, 2009). The contradictory association of enhanced calcium deposition in blood vessels and decreased bone mineralization has attracted many researchers’ attention; the precise mechanisms underlying the complex interplay of bone and vasculature remain to be elucidated. To gain further insight into the mechanism by which puerarin inhibited the osteoblastic differentiation of CVSMCs, we evaluated a signal pathway event. Previous studies showed puerarin performs its effect through ER depend or non-ER depend mechanisms (Cheng et al., 2012; Michihara et al., 2012). Our results demonstrated that the ER antagonist blocked the effect of puerarin on ALP activity of CVSMCs, which suggested puerarin attenuated osteoblastic differentiation of CVSMCs through the ER. Previous studies suggested that the PI3K/AKT pathway is involved in the regulation of a serial of cellular processes including growth, proliferation and differentiation. A series of studies demonstrated that the activation of Akt might attenuate the osteoblastic differentiation of VSMC (Radcliff et al., 2005; Duan et al., 2011; Shan et al., 2011; Xie et al., 2011). Our present study showed that puerarin induced the activation of AKt in VSCMs. Moreover, ER antagonist blunted the effect of puerarin on AKT activation. Treatment with LY294002 (PI3K inhibitor), or HIMO (Akt inhibitor), abolished the effect of puerarin on the inhibitory effect of ALP activity in CVSMCs. Our present finding suggested a crucial role for puerarin in decreasing osteoblastic differentiation of VSMCs, and ER/PI3K-AKT is implicated as being involved in this effect. These results suggest puerarin plays a protective role against arterial calcification.
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Acknowledgments This work was supported by grants from Development and Reform Commission of Hunan (20111318, 20121493), Hunan Provincial Department of Finance (2011-43), Hunan province science and technology project (2012FJ6027) .
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