Estrogen receptor modulatory effects of germinated brown rice bioactives in the uterus of rats through the regulation of estrogen-induced genes

Figures & data

Figure 1 Flow chart of the study design, showing the control and the treatment groups.

Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GBR, germinated brown rice; GABA, γ-amino butyric acid; ORZ, γ-oryzanol; OVX, ovariectomized; REM, Remifemin.

Table 1 Gene description, accession number, primer sequence, and the product size of the selected genes

Gene description	Accession number	Primer sequence	Product size	Comment
Peptidylprolyl isomerase A	NM_017101	F: 5′-AGGTGACACTATAGAATATTCTGTAGCTCAGGAGAGCA-3′ R: 5′-GTACGACTCACTATAGGGATTGAAGGGGAATGAGGAAAA-3′	145	House-keeping gene
Estrogen receptor-β	NM_012754	F: 5′-AGGTGACACTATAGAATATGGGTATCATTACGGCGTTT-3′ R: 5′-GTACGACTCACTATAGGGAATAACACTTGCGAAGTCGGC-3′	197	Gene related to uterine proliferation
Calcium binding protein	X16635	F: 5′-AGGTGACACTATAGAATA-3′ R: 5′-TTCAGGAGGCTGGAGAAGTTGGA-3′	257	Gene related to uterine proliferation
Complement protein	NM_016994	F: 5′-AGGTGACACTATAGAATACTGTTGCATAACCCAGCCTT-3′ R: 5′-GTACGACTCACTATAGGGAACGGACAGCCACAGTTTTGT-3′	277	Gene related to uterine proliferation
Heat shock 70kD protein 1A	NM_031971	F: 5′-AGGTGACACTATAGAATATCTTGTCTGCCTCCGATTTC-3′ R: 5′-GTACGACTCACTATAGGGACGAAGGCGTAGAGATTCCAG-3′	297
Interleukin-4 receptor	NM_133380	F: 5′-AGG TGA CAC TAT AGA ATA TCC GCA CTT CTA CGT GTG AG-3′ R: 5′-GTA CGA CTC ACT ATA GGG ATG GAG TGT GAG GTT GTC TGG-3′	317	Inflammation
Kanr				Internal control

Figure 2 Relative messenger RNA expression of ER-β gene in the uterus of OVX rats treated with GBR bioactives, EST, or REM, orally for 8 weeks.

Notes: Levels not connected by the same letters are significantly different (P<0.05); group treated with EST 0.2 mg/kg showed an upregulation of ER-β gene when compared to other treatment groups (P<0.001); group treated with GBR 200 mg/kg also showed an upregulation of ER-β gene compared to other groups (P<0.05).
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GBR, germinated brown rice; GABA, γ-amino butyric acid; mRNA, messenger ribonucleic acid; ORZ, γ-oryzanol; OVX, ovariectomized; REM, Remifemin; ER-β, estrogen receptor-β.

Figure 3 Relative mRNA expression of the CaBP9k gene in the uterus of OVX rats treated with GBR bioactives, EST, or REM, orally for 8 weeks.

Notes: Levels not connected by the same letters are significantly different (P<0.05); groups treated with ASG 200 mg/kg, EST 0.2 mg/kg, and ORZ 200 mg/kg showed an upregulation of the CaBP9k gene compared to other treated groups (P<0.05).
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GBR, germinated brown rice; GABA, γ-amino butyric acid; mRNA, messenger ribonucleic acid; ORZ, γ-oryzanol; OVX, ovariectomized; REM, Remifemin; CaBP9k, calcium-binding protein.

Figure 4 Relative mRNA expression of the C3 gene in the uterus of OVX rats treated with GBR bioactives, EST, or REM, orally for 8 weeks.

Notes: Levels not connected by the same letters are significantly different (P<0.05); groups treated with EST 0.2 mg/kg showed an upregulation of the C3 gene compared to other treated groups (P<0.05).
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GBR, germinated brown rice; GABA, γ-amino butyric acid; mRNA, messenger ribonucleic acid; ORZ, γ-oryzanol; OVX, ovariectomized; REM, Remifemin; C3, complement protein.

Figure 5 Relative mRNA expression of the HSP70 gene in the uterus of OVX rats treated with GBR bioactives, EST, or REM, orally for 8 weeks.

Notes: Levels with different letters are significantly different (P<0.05); group treated with EST 0.2 mg/kg showed higher expression of the HSP70 gene compared to other groups (P<0.05); groups treated with GBR and GABA 100 mg/kg also showed higher expression of the HSP70 gene compared to other groups (P<0.05).
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GBR, germinated brown rice; GABA, γ-amino butyric acid; mRNA, messenger ribonucleic acid; ORZ, γ-oryzanol; OVX, ovariectomized; REM, Remifemin; HSP70, heat shock protein 70 kDa.

Figure 6 Relative mRNA expression of the IL-4 receptor gene in the uterus of OVX rats treated with GBR bioactives, EST, or REM, orally for 8 weeks.

Notes: Levels with different letters are significantly different (P<0.05); group treated with EST 0.2 mg/kg showed higher expression of the IL-4 receptor gene compared to all other groups (P<0.05); group treated with ASG100 mg/kg also induces a high upregulation when compared to other treated groups (P<0.05); group treated with GBR 200 mg/kg induces an upregulation of the IL-4 receptor gene similar to the expression exhibits by sham non-treated group.
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GBR, germinated brown rice; GABA, γ-amino butyric acid; IL, interleukin; mRNA, messenger ribonucleic acid; ORZ, γ-oryzanol; OVX, ovariectomized; REM, Remifemin.

Table 2 Serum sex steroid hormone concentration in OVX rats after 8 weeks of intervention with GBR phenolics, ASG, GABA, ORZ, EST, and REM at various doses administered orally

Groups	Estrogen (pg/mL)			Progesterone (ng/mL)			FSH (mlU/mL)			LH (mlU/mL)
	2nd week	4th week	8th week	2nd week	4th week	8th week	2nd week	4th week	8th week	2nd week	4th week	8th week
SHAM	38.23±1.43^a	34.87±1.76^a	35.32±1.06^a	0.76±0.14	0.64±0.03	0.72±0.13	1.96±0.04^a	2.64±0.05^a	4.21±0.05^b	0.13±0.03	0.13±0.01	0.14±0.03
OVX	17.83±0.43^c	15.31±1.06^c	16.74±0.47^c	0.64±0.21	0.51±0.03	0.54±0.06	2.08±0.07^a	2.10±0.09^a	2.21±0.03^a	0.13±0.03	0.13±0.04	0.13±0.03
EST0.2	32.64±0.37^b	30.39±1.35^b	30.29±0.68^b	0.57±0.17	0.65±0.03	0.63±0.07	2.10±0.02^a	2.88±0.04^a	8.74±0.03^b	0.14±0.03	0.13±0.03	0.14±0.03
REM10	21.52±1.08^c	21.32±0.92^c	22.78±0.54^c	0.57±0.21	0.61±0.06	0.53±0.04	1.85±0.03^a	3.24±0.06^b	3.12±0.11^b	0.13±0.03	0.13±0.03	0.14±0.04
REM20	22.63±0.97^c	21.78±1.04^c	23.29±1.57^c	0.67±0.03	0.57±0.12	0.65±0.07	1.78±0.02^a	2.01±0.02^a	4.22±0.07^b	0.13±0.04	0.13±0.03	0.14±0.02
GBR100	22.75±0.22^c	23.94±1.62^b	21.86±1.24^c	0.56±0.23	0.64±0.13	0.65±0.03	2.19±0.09^a	3.61±0.06^b	4.12±0.11^b	0.13±0.04	0.14±0.02	0.14±0.01
GBR200	23.56±0.57^c	24.11±1.43^b	20.94±0.98^c	0.61±0.25	0.58±0.11	0.55±0.14	2.75±0.0^a	2.61±0.01^a	4.31±0.09^b	0.14±0.03	0.13±0.03	0.14±0.03
ASG100	22.17±0.31^c	24.42±1.35^b	21.08±1.22^c	0.65±0.15	0.63±0.10	0.74±0.02	1.87±0.04^a	2.79±0.06^b	3.79±0.01^c	0.13±0.04	0.14±0.04	0.13±0.02
ASG200	24.90±0.26^b	24.87±0.72^b	24.11±1.51^b	0.67±0.23	0.59±0.04	0.66±0.06	1.83±0.10^a	3.00±0.10^b	4.99±0.02^c	0.13±0.03	0.13±0.01	0.14±0.03
GABA100	21.09±0.47^c	21.40±1.26^c	20.78±0.54^c	0.55±0.26	0.57±0.14	0.60±0.09	1.95±0.03^a	2.27±0.02^a	5.11±0.08^b	0.14±0.03	0.13±0.03	0.13±0.04
GABA200	23.89±0.73^c	21.54±0.93^c	23.25±1.07^c	0.63±0.11	0.48±0.17	0.64±0.11	1.66±0.13^a	2.30±0.03^a	6.03±0.01^c	0.13±0.02	0.13±0.04	0.13±0.03
ORZ100	20.14±0.41^c	21.43±0.81^c	20.78±0.71^c	0.58±0.04	0.60±0.12	0.681±0.30	2.30±0.04^a	3.00±0.08^b	3.98±0.06^b	0.13±0.03	0.13±0.03	0.14±0.02
ORZ200	26.32±0.74^b	27.02±0.76^b	26.85±1.35^b	0.60±0.14	0.57±0.14	0.64±0.12	2.61±0.05^a	2.14±0.04^a	4.76±0.13^b	0.13±0.02	0.14±0.02	0.14±0.03

Notes: Values are expressed as mean ± SD for three readings; levels not denoted by the same superscript letter on the same row are significantly different.

Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GBR, germinated brown rice; GABA, γ-amino butyric acid; ORZ, γ-oryzanol; OVX, ovariectomized; REM, Remifemin; SD, standard deviation.

Figure 7 Slides showing immunohistochemical staining of ER-β antigen on uterine tissue of OVX rats. (A) Sham non-treated group; (B) OVX non-treated; (C) EST 0.2 mg/kg; (D) GBR phenolics 200 mg/kg; (E) ASG 200 mg/kg; (F) REM 20 mg/kg; (G) GABA 200 mg/kg; and (H) ORZ 200 mg/kg.

Notes: Red arrows show areas of immunohistochemical staining. The sham (non-OVX group) showed light reactivity on the glandular epithelium, while the group treated with GBR phenolics showed an ER-β reactivity at the glandular and some part of the stroma and the group treated with EST showed an immunoreactivity both at the glandular, luminal, and deep in the stromal cells. Other treated groups showed mild reactivity mostly in the stroma.
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GBR, germinated brown rice; GABA, γ-amino butyric acid; ORZ, γ-oryzanol; OVX, ovariectomized; REM, Remifemin; ER-β, estrogen receptor-β.