Short communications

Effects of β-sitosterol oral administration onthe proliferation and differentiation of neuralstem cells

Azadeh Hamedi a, Amir Ghanbari b, Vahid Saeidi c, Razieh Razavipour c,Hassan Azari b,d,*a Medicinal Plants Processing Research Center, Department of Pharmacognosy, School of Pharmacy, ShirazUniversity of Medical Sciences, Shiraz, Iranb Neural Stem Cell and Regenerative Neuroscience Laboratory, Department of Anatomical Sciences, ShirazSchool of Medicine, Shiraz University of Medical Sciences, Shiraz, Iranc Student Research Center Committee, International Branch, Shiraz University of Medical Sciences, Shiraz, Irand Neural Stem Cell and Regenerative Neuroscience Laboratory, Shiraz Stem Cell Institute, Shiraz University ofMedical Sciences, Shiraz, Iran

A B S T R A C T

Considering the presence of β-sitosterol in different functional foods and the potential role

of neural stem cells (NSCs) in treating neurodegenerative diseases, this study was de-

signed to evaluate the effects of oral administration of β-sitosterol on proliferation and dif-

ferentiation of NSCs. Adult mice were divided into three groups. For 2 weeks, the control

group received vehicle and the low and high dose groups received 33 and 99 mg/kg/day of

β-sitosterol, respectively. Harvested NSCs from the subventricular zone (SVZ) were plated

in neurosphere culture and the size and number of resulting neurospheres, and also their

in vitro differentiation, were evaluated. β-Sitosterol at 99 mg/kg/day significantly increased

proliferation of NSCs as evidenced by their neurosphere forming frequency (719.2 ± 53.65)

compared to the control group (457.6 ± 21.35). Mean neurosphere diameter was signifi-

cantly larger in both high and low dose β-sitosterol groups compared to the control group.

β-Sitosterol did not affect in vitro differentiation of NSCs.

© 2014 Elsevier Ltd. All rights reserved.

A R T I C L E I N F O

Article history:

Received 7 January 2014

Received in revised form 26 March

2014

Accepted 28 March 2014

Available online 17 April 2014

Keywords:

β-Sitosterol

Neural stem cell

Subventricular zone

Proliferation

Neurosphere assay

1. Introduction

Phytosterols (including sterols and stanols) are essentialtriterpenoid molecules that stabilize phospholipid bilayers ofplant cell membranes (Moreau, Whitaker, & Hicks, 2002). Whilebeing similar in having a sterol ring structure, they differ in

carbon side chain, as well as in the presence, number and po-sition of double bonds. Moreover, phytosterols can be foundin glycosidic forms in seeds and nuts and in the free form oresterified with fatty/cinnamic acid in seed oils (Lampi, Piironen,& Toivo, 2003) and also as synthetic phytosteryl phenolates,the esters of phytosterols with phenolic acids (Tan & Shahidi,2013). Phytosterols also exhibit a wide range of in vivo biologi-

* Corresponding author. Tel.: +98 7112304372.E-mail address: azarihasan@sums.ac.ir (H. Azari).

http://dx.doi.org/10.1016/j.jff.2014.03.0211756-4646/© 2014 Elsevier Ltd. All rights reserved.

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cal activities such as anti-tumor (Awad & Fink, 2000; Awad,Downie, & Fink, 2000; Bradford & Awad, 2007), antioxidant, anti-diabetic (Gupta, Sharma, Dobhal, Sharma, & Gupta, 2011; Tan& Shahidi, 2013), immunomodulatory (Bouic, 2002; Lee et al.,2007; Paniagua-Pérez et al., 2008), and anti-inflammatory func-tions (Yuk et al., 2007).

β-Sitosterol (24α-ethylcholesterol), campesterol (24α-methylcholesterol), and stigmasterol (Δ22, 24α-ethylcholesterol)are examples of sterols that are predominantly found in higherplants as well as in human food (Kritchevsky & Chen, 2005;Phillips, Ruggio, & Ashraf-Khorassani, 2005). Among these phy-tosterols, β-sitosterol has mostly been the center of attentionby scientists and pharmaceutical industries. For example,β-sitosterol is used as a supplement to treat hypercholester-olemia (Scholle, Baker, Talati, & Coleman, 2009), benign pros-tatic hyperplasia (Berges, Kassen, & Senge, 2000), and also toprotect against cardiovascular diseases (Genser et al., 2012).Moreover, because of its well-known cholesterol-lowering prop-erty (Alhazzaa, Oen, & Sinclair, 2013; Wong, 2014), β-sitosterolalone or in combination with other phytosterols (in free or es-terified form) is used in a variety of enriched commercial foodssuch as spreads, fruits juice, milk, and yoghurt. Safety con-cerns regarding the use of β-sitosterol as an available supple-ment in pharmacies and food industries have been addressedin different in vivo and clinical studies (Clifton et al., 2004;Davidson et al., 2001; Hendriks, Brink, Meijer, Princen, & Ntanios,2003; Katan et al., 2003; Nestel, Cehun, Pomeroy, Abbey, &Weldon, 2001). Lack of any adverse effects in these studies hasled to the safety approval for using phytosterols, phytostanols,and their esterified derivatives by the US Food and Drug Ad-ministration (FDA) (FDA, 2010) and European Union ScientificCommittee (Palou, Serra, & Pico, 2003), except for individualswith sitosterolemia, a rare autosomal recessive disorder (Lee,Lu, & Patel, 2001; Salen et al., 1992).

Neural stem cells (NSCs) reside in special niches along theventricular neuraxis of the central nervous system especiallyin the subventricular zone (SVZ) of the lateral wall of the lateralventricle and the subgranular zone (SGZ) of the hippocam-pus (Golmohammadi et al., 2008). Recruitment of NSCs rep-resents a promising therapeutic strategy for treatment ofneurodegenerative diseases (Rossi & Cattaneo, 2002). In a recentstudy, it was shown that supplementing culture medium witha glucoside derivative of β-sitosterol (daucosterol) promotes theproliferation of hippocampal neural stem cells (Jiang et al., 2014).Considering the presence of β-sitosterol in different func-tional foods and the potential role of NSCs in prevention and/or treatment of neurodegenerative diseases, the aim of thisstudy was to evaluate the effects of oral administration ofβ-sitosterol on proliferation and differentiation of NSCs.

2. Materials and methods

2.1. Materials

β-Sitosterol, sodium carboxymethyl cellulose (CMC), soybeantrypsin inhibitor, epidermal growth factor (EGF), basic fibro-blast growth factor (bFGF), heparin and normal goat serum (NGS)were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, USA).Trypsin–ethylenediaminetetraacetic acid (EDTA) and fetal calf

serum (FCS) were purchased from Gibco (Grand Island, NY, USA).Mouse monoclonal anti-β-III-tubulin was purchased fromPromega (Madison,WI, USA). Rabbit polyclonal anti-glial fibrilaryacidic protein (GFAP) was obtained from DakoCytomation(Hamburg, Germany). Goat anti-mouse Alexa-Fluor 568, goatanti-rabbit Alexa-Fluor 488 and 4’,6-diamidino-2-phenylindole(DAPI) were purchased from Molecular Probes (Eugene, OR, USA).The remaining chemicals and solvents were of analytical gradeand purchased from Merck (Darmstadt, Germany). To preparean aqueous suspension of β-sitosterol, 0.1% (w/v) CMC wasadded to distilled water and the mixture was sonicated for30 min at 40 °C.

2.2. Experimental animals

Adult male BALB/c mice (25–30 g, the Laboratory Animal Centerof Shiraz University of Medical Sciences, Shiraz, Iran) werehoused in groups of two or three during the course of studyunder a 12-hour light/dark cycle at room temperature at25 ± 1 °C with free access to chow diet and water. Animal ex-periments were conducted in accordance with the Institu-tional Guidelines and Animal Ordinance (Ethics Committee ofShiraz University of Medical Sciences, Shiraz, Iran). Fifteenanimals were divided into two test groups (N = 5) that re-ceived oral suspension of β-sitosterol at 33 mg/kg/day (low dosegroup) or at 99 mg/kg/day (high dose group) for 14 days. Thecontrol group was treated with the same volume of the vehicle(0.1 ml of an aqueous suspension of 0.1% CMC) for 14 days.

2.3. Isolation and expansion of neural stem cells

On the day following the last treatment, mice from each groupwere anesthetized using 4% isoflurane and then underwent cer-vical dislocation to harvest the brain (animal protocol #2013/12). Using a dissection microscope, the SVZ of both lateralventricles was microdissected from the rostral part of the brainextending between the olfactory bulb and the crossing of theanterior commissure to harvest neural stem and progenitor cells(Azari, Rahman, Sharififar, & Reynolds, 2010; Siebzehnrubl,Vedam-Mai, Azari, Reynolds, & Deleyrolle, 2011). After mincingwith a razor blade, the harvested tissue was digested in 0.05%trypsin–EDTA (37 °C, 5–7 min). Soybean trypsin inhibitor wasused to quench the enzyme activity. The suspension was spun(110 g, 5 min) to remove the supernatant and the cell pellet wasresuspended in neurosphere medium (500 μl) and mechani-cally dissociated to achieve a single cell suspension. After dis-carding the supernatant, cells harvested from each brain wereresuspended in complete neurosphere medium (5 ml) supple-mented with EGF (20 ng/ml), bFGF (10 ng/ml) and heparin (2 μg/ml). The suspension was plated in a T25 flask and incubatedin a humidified incubator with 5% CO2 for 8 days.

2.4. Neurosphere forming frequency and sizedetermination

After 8 days in culture, the cell culture flasks from each groupwere secured on the stage of an Olympus inverted micro-scope to count the number and to measure the size of theneurospheres generated in different conditions. Starting fromthe top right part and ending at the bottom left part of each

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flask, the entire flask was monitored field by field and allneurospheres bigger than or around 50 μm in diameter werecounted (Golmohammadi et al., 2008) and recorded as theneurosphere forming frequency per brain. The meanneurosphere forming frequency was compared among groups.Simultaneously, applying a systematic random samplingmethod, we imaged every 30th neurosphere throughout theentire flask using an Olympus CKX41 digital camera (Olympus,Center Valley, PA, USA). A total of 20–25 neurospheres per flaskwere analyzed and the diameter of each neurosphere was de-termined using stereological software (Stereolite, SUMS, Shiraz,Iran). The mean neurosphere diameter (μm) was comparedamong groups. All data were presented as mean ± SEM for eachgroup.

2.5. Neural stem cell differentiation assay

The harvested neurospheres from each group were dissoci-ated into single cells, plated in differentiation assay and thedifferentiation capacity of the harvested NSCs was investi-gated as described before (Azari et al., 2011; Azari, Sharififar,Fortin, & Reynolds, 2012). Briefly, cells (2–3 × 105 cells/ml) wereplated in complete neurosphere medium supplemented withEGF (20 ng/ml), bFGF (10 ng/ml) and 5% FCS in 24-well platesthat were coated with poly-L-ornithine. The plates were incu-bated for 3–4 days in a humidified incubator with 5% CO2.Then,the medium of each well was replaced with the same mediumcontaining 5% FCS but without growth factors. After 4 days,the cultures were fixed using cold paraformaldehyde (PFA, 4%,20 min at room temperature).

2.6. Immunofluorescence

PFA fixed cultures were processed for immunofluorescence todetermine the percentage of β-III-tubulin and GFAP-expressingprogeny of differentiating neural stem cells. After washing thefixed samples with phosphate buffered saline (PBS) to removePFA, the primary antibody solution containing mouse mono-clonal anti-β-III-tubulin (1:2000) and rabbit polyclonal anti-GFAP (1:500) in PBST (PBS + 0.1% Triton-X) supplemented with10% NGS was added to each well of the 24-well plates. Theplates were incubated for 1 h at room temperature, the primaryantibody was removed and the wells were washed with PBS.Then, the secondary antibody solution containing goat anti-mouse Alexa-Fluor 568 and goat anti-rabbit Alexa-Fluor 488(1:700) in PBST supplemented with 10% NGS were added to eachwell and incubated for 45 min at room temperature in the dark.To counterstain the nuclei, DAPI (1:1000) was also added to thesecondary antibody solution. A fluorescent microscope(Olympus IX-71) equipped with a Canon EOS digital camera wasused to take representative pictures of each well (10–15 fields/well). Cells were counted after merging images using AdobePhotoshop CS4 and the data were presented as a percentageof total cells counted.

2.7. Statistical analysis

The results were expressed as means ± SEM and analyzed usingGraphPad Prism (Version 6.01, San Diego, CA, USA) software.Analysis of variance (ANOVA) followed by Tukey’s multiple com-

parisons was used to analyze the data. The level of signifi-cance for all comparisons was set at P < 0.05.

3. Results

3.1. Effects of β-sitosterol on neural stem cell proliferation

In both β-sitosterol treated and non-treated animals, har-vested NSCs from the SVZ proliferated and formedneurospheres after 8 days in culture (Figs. 1A–C). Quantita-tive analysis revealed that the sphere-forming frequency was457.6 ± 21.35 in the control group, 475.8 ± 21.47 in the low dosegroup, and 719.2 ± 53.65 in the high dose group (Fig. 1D). Sta-tistical analysis revealed that treatment of animals with 99 mg/kg/day of β-sitosterol resulted in a significant (P < 0.001) increasein the proliferation of neural stem and progenitors in com-parison to the control group. Moreover, treatment of animalswith 99 mg/kg/day of β-sitosterol resulted in a significant(P < 0.05) increase in the proliferation of neural stem and pro-genitors in comparison to the animals treated with 33 mg/kg/day.

3.2. Effects of β-sitosterol on neurosphere (size) diameter

Comparing different groups showed that the mean neurospherediameter was 106 ± 3.5 μm for the control group, while thismeasure was 130.5 ± 4.3 μm for the low dose group and172.5 ± 4.8 μm for the high dose group. Statistical analysisshowed that both of the β-sitosterol treatment doses led to sig-nificantly larger neurosphers as compared to the control group(P < 0.001). Furthermore, treatment of the animals with 99 mg/kg/day of β-sitosterol resulted in significantly largerneurospheres as compared to 33 mg/kg/day (P < 0.001, Fig. 1E).

3.3. Effects of β-sitosterol on differentiation of neuralstem cells

Different immunoreactive (IR) cells were counted in represen-tative immunostained pictures from each group (Figs. 2A–D)and the mean percentages of β-III-tubulin IR and GFAP IR cellswere 6.58 ± 2.23 and 74.88 ± 3.5 in the control group, 3.71 ± 0.75and 85.16 ± 1.88 in the low dose group and 4.79 ± 0.72 and80.80 ± 3.72 in the high dose group, respectively (Figs. 2E–F). Sta-tistical analysis revealed that these measures were not sig-nificantly different, showing that oral administration ofβ-sitosterol did not have any effects on in vitro differentiationof the harvested NSCs at doses evaluated in this study.

4. Discussion

We investigated the effects of oral administration of low andhigh doses (33 and 99 mg/kg/day) of β-sitosterol on neural stemcell proliferation and differentiation. Treatment of mice withthe high dose of β-sitosterol significantly (P < 0.001) increasedproliferation of neural stem and progenitors as evidenced bya higher sphere-forming frequency in the neurosphere culture.To the best of our knowledge, no reports in the literature exist

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regarding NSC proliferative properties of β-sitosterol follow-ing oral administration in animal models. Jiang et al. showedin an in vitro study that adding daucosterol (a glycoside de-rivative of β-sitosterol) at concentrations of 20–40 μM to the hip-pocampal NSC culture significantly increased their proliferation(Jiang et al., 2014). While our results confirm the findings ofthis study on the proliferative effect of β-sitosterol on NSCs,it also provides solid preclinical evidence for recruiting NSCsafter oral administration of β-sitosterol in mice. It has been dem-onstrated that dietary plant sterols could penetrate the blood–brain barrier (Jansen et al., 2006; Vanmierlo et al., 2008, 2012),so it is highly likely for the β-sitosterol used in this study tocross the blood–brain barrier and increase proliferation of NSCsin the SVZ.

Neurosphere size was significantly (P < 0.001) higher inβ-sitosterol treated mice compared to the control. Further-more, this measure was also significantly higher in the highdose group compared to the low dose group (P < 0.001). It has

been shown that the size of neurospheres has a direct corre-lation with the number of cell divisions of the originalneurosphere-forming cell and its progeny (Mori et al., 2006).Therefore, this implies that administration of β-sitosterol evenat the low dose could promote the proliferation rate of the ac-tively dividing NSC population in the SVZ niche, as evi-denced by generating larger neurospheres in comparison to thecontrol. Accordingly, as the dose of β-sitosterol increased, thesize of the resulting neurospheres also increased in the highdose versus the low dose group. It seems that increasing thedose of β-sitosterol also resulted in recruiting more NSCs intothe pool of dividing cells as evidenced by more neurosphereformation in the high dose group. In agreement with this, Jianget al. reported that adding β-sitosterol-glycoside (daucosterol)to a hippocampal neural stem cell culture resulted in the up-regulation of genes that are mostly involved in mitotic cell pro-liferation but, in contrast, down-regulation of genes that aremostly involved in differentiation (Jiang et al., 2014).

Fig. 1 – Neurosphere forming frequency and size analysis. (A–C) Representative neurospheres grown from thesubventricular zone of control (A), low dose (B), and high dose (C) β-sitosterol treated mice (scal bar = 50 μm). (D) Meanneurosphere forming frequency. (E) Mean neurosphere diameter in control (untreated), low dose (33 mg/kg/day) and highdose (99 mg/kg/day) groups after 8 days in culture. (Data are shown as mean ± SEM, ***P < 0.001 and **P < 0.05)

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On the other hand, none of the oral doses of β-sitosterolapplied in this study could affect differentiation of the har-vested NSCs in comparison to the control. This might bedue to in vitro proliferation of the harvested NSCs withoutthe presence of β-sitosterol prior to differentiation.This washingperiod might have reversed the expression of the differentia-tion gene back to its normal status, which warrants furtherinvestigation.

Many growth factors, including but not limited to EGF, b-FGF,insulin like growth factor-1 (IGF-1) and brain derived neuro-trophic factor (BDNF) have been investigated to activate NSCproliferation (Benraiss, Chmielnicki, Lerner, Roh, & Goldman,2001; Mason, 2007; Popken et al., 2004; Wong & Guillaud, 2004).However, therapeutic application of these compounds is hin-dered by their expensive costs and their chemical instability.We believe that recruiting NSCs using natural chemical com-pounds such as β-sitosterol, which can be found in relatively

high amounts in seeds, nuts and seed oils, and currently areused as dietary supplements, would be of great clinical trans-lational value. Nevertheless, further investigation is needed toevaluate the effects of β-sitosterol on proliferation and differ-entiation of neural stem cells in vivo, in order to find possibleapplications in different neurodegenerative disease models, topave the way for future clinical translations.

5. Conclusion

β-Sitosterol is a phytosterol with high occurrence in seeds, nuts,and seed oil and also with well-known cholesterol loweringproperties. Several supplements containing this component aresold in pharmacies, and the FDA allows manufacturers toproduce functional foods containing β-sitosterol. Thus, evalu-

Fig. 2 – Immunoflourescence analysis of differentitated neural stem cell cultures: (A) GFAP staining (green); (B) β-III-tubulinstaining (red); (C) DAPI-stained nuclei (blue); (D) photoshop merged picture of all three stains, after 8 days in differentiationculture; (E) mean pecentage of total β-III-tubulin-IR cells; (D) mean percentage of total GFAP-IR cells in differentiated NSCcultures from control (untreated), low dose (33 mg/kg/day), and high dose (99 mg/kg/day) β-sitosterol-treated mice after8 days of differentiation. (Scale bar = 50 μm. Data are shown as mean ± SEM, IR = immunoreactive.)

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ating the useful biological activities and the possible side effectsof β-sitosterol has interested researchers as well as food andpharmaceutical industries. The results of this study show thatoral administration of β-sitosterol significantly enhances theproliferation of neural stem cells without any change to theirin vitro differentiation.

Conflict of interest

The authors have no competing or conflicts of interest todeclare.

Acknowledgments

This study was part of the Pharm. D. thesis project of VahidSaeidi and was financed by the international branch of ShirazUniversity of Medical Sciences (grant # 12). The authors wouldlike to thank Ms. Sharareh Sharififar and Mr. Jeff M. Fortin fortheir critical review of the final version of this manuscript.

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