Upregulation of genes related to bone formation by γ-amino butyric acid and γ-oryzanol in germinated brown rice is via the activation of GABA_B-receptors and reduction of serum IL-6 in rats

Figures & data

Table 1 Serum IL-6 concentration (pg/mL) in OVX rats treated with GBR-phenolic compounds, ASG, GABA, ORZ, EST, or REM over a period of 8 weeks

Treatment groups (mg/kg)	IL-6 concentration (pg/mL)
	Week 2	Week 4	Week 8
ASG, 100	112.81 ± 2.60^d,e	156.67 ± 5.15^b	141.51 ± 17.37^a,b
ASG, 200	107.54 ± 10.36	130.02 ± 2.94^b	142.52 ± 11.17^a,b
EST	99.64 ± 0.59^e	84.91 ± 3.93^b	85.15 ± 3.36^b
GABA, 100	140.74 ± 2.02^c	150.19 ± 4.00^b	121.23 ± 55.02^a,b
GABA, 200	67.52 ± 8.34^g	89.63 ± 2.74^b	95.02 ± 8.12^b
GBR, 100	172.55 ± 5.60^b	158.31 ± 3.05^b	135.77 ± 21.85^a,b
GBR, 200	162.60 ± 8.44	123.16 ± 2.21^b	133.12 ± 22.12^a,b
ORZ, 100	95.46 ± 4.65^e,f	102.91 ± 3.01^b	95.67 ± 4.09^b
ORZ, 200	75.76 ± 2.11^f,g	88.90 ± 1.71^b	84.32 ± 4.10^b
OVX	298.93 ± 6.09^a	374.09 ± 74.77^a	201.69 ± 4.15^a
REM, 100	178.39 ± 2.72^b	155.57 ± 3.59^b	142.05 ± 5.92^a,b
REM, 200	121.67 ± 2.31^c,d	116.70 ± 4.96^b	129.60 ± 28.66^a,b
Sham	75.85 ± 2.08^f,g	79.67 ± 11.34^b	80.36 ± 1.22^b

Notes: Values in the table represent the mean ± standard deviation for three readings (n = 3). Levels not connected by the same superscript letters within the same week are significantly different (P < 0.05).

Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; IL-6, interleukin-6; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin.

Table 2 Body weight (g) in OVX rats treated with GBR-phenolic compounds, ASG, GABA, ORZ, EST, or REM over a period of 8 weeks

Treatment groups (mg/kg)	Body weight(g)
	Week 2	Week 4	Week 8
ASG, 100	264.66 ± 1.63^a,b,c	282.50 ± 3.39^a,b,c	322.50 ± 3.72^b,c
ASG, 200	259.33 ± 6.05^c,d	273.00 ± 4.56^e,f	315.50 ± 5.16^c
OVX + EST	261.83 ± 2.85^a,b,c,d	273.83 ± 4.57^d,e,f	290.16 ± 4.53^d
GABA, 100	265.66 ± 2.25^a,b,c	282.83 ± 3.97^a,b,c	323.16 ± 3.71^b
GABA, 200	261.33 ± 5.12^a,b,c,d	274.83 ± 4.53^c,d,e,f	319.66 ± 3.14^b,c
GBR, 100	264.50 ± 2.58^a,b,c,d	282.83 ± 2.48^a,b,c	322.66 ± 3.44^b,c
GBR, 200	258.83 ± 5.94^c,d	274.16 ± 3.65^d,e,f	316.00 ± 3.03^b,c
ORZ, 100	264.00 ± 1.78^a,b,c,d	278.00 ± 6.81^c,d,e	321.83 ± 2.13^b,c
ORZ, 200	260.00 ± 6.16^b,c,d	276.50 ± 3.61^c,d,e	317.50 ± 3.93^b,c
OVX	264.00 ± 3.74^a,b,c,d	288.83 ± 2.48^a	336.66 ± 4.54^a
REM, 10	260.83 ± 1.72^a,b,c,d	278.33 ± 1.86^b,c,d,e	320.00 ± 3.57^b,c
REM, 20	259.00 ± 3.89^c,d	274.16 ± 3.65^d,e,f	316.00 ± 3.03^b,c
Sham	256.33 ± 5.85^d	267.66 ± 4.13^f	284.66 ± 3.88^d

Notes: Values in the table represent the mean ± standard deviation (n = 6). Levels not connected by the same superscript letters within the same week are significantly different are significantly different (P < 0.05).

Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin.

Table 3 Serum osteocalcin concentration in OVX rats treated with GBR-phenolic compounds, ASG, GABA, ORZ, EST, or REM over a period of 8 weeks

Treatment groups (mg/kg)	Osteocalcin concentration (ng/mL)
	Week 2	Week 4	Week 8
ASG, 100	161.65 ± 12.22^e	176.21 ± 4.25^c,d,e	154.62 ± 2.40^b,c
ASG, 200	201.84 ± 4.19^a,b,c	184.23 ± 7.89^a,b,c,d	172.03 ± 3.66^a,b
EST	204.04 ± 4.60^a,b	189.62 ± 11.66^a,b,c	184.09 ± 4.07^a,b
GABA, 100	190.37 ± 1.54^b,c,d,e	180.56 ± 2.15^b,c,d	131.68 ± 7.83^c
GABA, 200	200.06 ± 6.53^a,b,c	203.45 ± 3.82^a,b	163.96 ± 5.67^a,b,c
GBR, 100	184.21 ± 2.20^b,c,d	160.12 ± 12.24^d,e	153.50 ± 7.74^b,c
GBR, 200	199.89 ± 0.33^a,b,c	193.29 ± 6.44^a,b,c	160.65 ± 1.24^b,c
ORZ, 100	191.56 ± 8.62^b,c,d	170.24 ± 1.49^c,d,e	147.34 ± 3.02^b,c
ORZ, 200	221.24 ± 4.50^a	209.66 ± 3.35^a	203.18 ± 29.64^a
OVX	184.17 ± 8.23^b,c,d,e	173.57 ± 5.80^c,d,e	169.92 ± 13.54^a,b,c
REM, 10	178.39 ± 4.33^c,d,e	182.66 ± 3.24^b,c,d	162.09 ± 3.66^b,c
REM, 20	174.72 ± 7.28^d,e	191.10 ± 2.34^a,b,c	178.93 ± 0.92^a,b
Sham	218.84 ± 3.96^a	151.55 ± 7.42^e	179.91 ± 5.04^a,b

Notes: Values in the table represent the mean ± standard deviation (n = 3). Levels not connected by the same superscript letters within the same week are significantly different (P < 0.05).

Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin.

Table 4 Gene description, accession number, primer sequence, and the product size of the selected genes

Gene description	Accession no	Primer sequence	Product size	Comment
ATP synthase	NM_138883	F: 5′-AGGTGACACTATAGAATACTCTCTGAGTTAAAGACAGTGCTGA-3′(br/)R: 5′-GTACGACTCACTATAGGGAACAATCATCCCACCCATGAT-3′	138	Housekeeping gene
Peptidylprolyl isomerase A (cyclophilin A)	NM_017101	F: 5′-AGGTGACACTATAGAATATTCTGTAGCTCAGGAGAGCA-3′(br/)R: 5′-GTACGACTCACTATAGGGATTGAAGGGGAATGAGGAAAA-3′	145	Housekeeping gene
CGRP	NM_001034015	F: 5′-AGGTGACACTATAGAATAACCTGGTGCGACGGG-3′(br/)R: 5′-GTACGACTCACTATAGGGATGACGATCAGAGTGTGAAGATAGA-3′	157	Gene related to osteoblastic activity
BMP-2	NM 017178	F: 5′-TGA ACA CAG CTG GTC TCAGG-3′(br/)R: 5′-TTA AGA CGC TTC CGC TGTTT-3′	167	Gene related to osteoblastic activity
SPARC	NM_012656	F: 5′-AGGTGACACTATAGAATAGACCTTCGACTCTTCCTGCC-3′(br/)R: 5′-GTACGACTCACTATAGGGAGGAATTCGGTCAGCTCAGAA-3′	177	Gene related to osteoblastic activity
RUNX-2	NM 053470	F: 5′-GCC GGG AAT GAT GAG AACTA-3′(br/)R: 5′-GGA CCG TCC ACT GTC ACTTT-3′	187	Gene related to osteoblastic activity
OSX	NM 181374	F: 5′-GCT GCC TAC TTA CCC GTCTG-3′(br/)R: 5′-GTT GCC CAC TAT TGC CAACT-3′	207	Gene related to osteoblastic activity
Postn	NM_001108550	F: 5′-AGGTGACACTATAGAATAGCCACGACCACTCAACACTA-3′(br/)R: 5′-GTACGACTCACTATAGGGACTGGGGTGTTGACAGGTTG-3′	217	Gene related to osteoblastic activity
Col1&2	NM_053356	F: 5′-AGGTGACACTATAGAATACGAGACCCTTCTCACTCCTG-3′(br/)R: 5′-GTACGACTCACTATAGGGACTGGGGTGTTGACAGGTTG-3′	237	Gene related to osteoblastic activity
GAPDH	NM 017008	F: 5′-GTG GAC CTC ATG GCC TACAT-3′(br/)R: 5′-TGT GAG GGA GAT GCT CAGTG-3′	271	Housekeeping gene
Actb	NM_031144	F: 5′-AGGTGACACTATAGAATAGGCATCCTGACCCTGAAGTA-3′(br/)R: 5′-GTACGACTCACTATAGGGAAGACGCAGGATGGCATGAG-3′	384	Housekeeping gene
KANr				Internal control

Abbreviations:Actb, actin beta; ATP, adenosine triphosphate; BMP-2, bone morphogenic protein-2; Col1&2, collagen 1&2; CGRP, calcitonin receptor; SPARC, secreted protein acidic and rich in cysteine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Runx-2, runt-related transcription factor 2; OSX, osterix; Postn, periostin, osteoblast specific factor.

Figure 1 Relative mRNA expression of CGRP gene in OVX rats treated with EST, REM, GBR, GABA and ORZ in different doses compared to sham and OVX non-treated group.

Notes: Treatment groups were expressed in (mg/kg) for three readings, levels not connected by same letter are significantly different (P < 0.05). ^aGroup treated with GABA at the dose of 100 mg/kg shows the highest significant upregulation of CGRP gene compared to sham, OVX-non treated and all other treatment groups (P < 0.001); ^bgroup treated with REM 20 mg/kg shows a significant upregulation compared to sham, OVX non-treated, EST, ORZ, GBR, and REM 10 mg/kg treated groups (P < 0.001); ^cgroups treated with REM 10 mg/kg, GBR and ASG 200 mg/kg showed an upregulation equal to that of sham (non-OVX group) and greater than that of OVX non-treated group (P < 0.05); ^dgroup treated with GABA 200 mg/kg shows a significant upregulation compared to OVX non-treated group groups treated with ORZ 200 mg/kg, ASG 100 mg/kg, and EST 0.2 mg/kg treated groups (P < 0.05).
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin; mRNA, messenger ribonucleic acid; CGRP, calcitonin receptor.

Figure 2 Relative mRNA expression of BMP-2 gene in OVX rats treated with EST, REM, GBR, GABA and ORZ in different doses compared to sham and OVX non-treated groups.

Notes: Treatment groups were expressed in (mg/kg) for three readings, levels not connected by same letter are significantly different (P < 0.05). ^aGABA 200 mg/kg treated group had the highest expression of BMP-2 gene compared to OVX non-treated, sham non-OVX and all the other treated groups (P < 0.05); ^bgroup treated with REM 10 mg/kg, GABA and ORZ 100 mg/kg showed a significant upregulation compared to sham, OVX non-treated, EST 0.2 mg/kg, ORZ 200, GBR and ASG 100 and 200 mg/kg and REM 20 mg/kg treated groups (P < 0.001).
Abbreviations: ASG, acylated steryl glucosides; BMP, bone morphogenic protein; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin; mRNA, messenger ribonucleic acid.

Figure 3 Relative mRNA expression of SPARC gene in OVX rats treated with EST, REM, GBR, GABA and ORZ in different doses compared to sham and OVX non-treated group.

Notes: Treatment groups were expressed in (mg/kg) for three readings, levels not connected by same letter are significantly different (P < 0.05). ^aGroup treated with GABA 200 mg/kg gave the highest in term of the expression of SPARC gene compared to OVX non-treated, sham non-OVX and all the other treated groups (P < 0.0001); ^bgroup treated with GBR 200 mg/kg gives a significant upregulation of SPARC gene difference compared to OVX non-treated, sham non-OVX and groups treated with EST 0.2 mg/kg, REM 10 and 20 mg/kg, GBR and ASG 100 mg/kg and ORZ 200 mg/kg (P < 0.05); ^cno significant difference (P > 0.05) between the sham non OVX group and groups treated with ASG 200 mg/kg.
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin; mRNA, messenger ribonucleic acid; SPARC, secreted protein acidic and rich in cysteine.

Figure 4 Relative mRNA expression of RUNX-2 gene in OVX rats treated with EST, REM, GBR, GABA and ORZ in different doses compared to sham and OVX non-treated group.

Notes: Treatment groups were expressed in (mg/kg) for three readings, levels not connected by same letter are significantly different (P < 0.05). ^aGroup treated with ORZ 200 mg/kg gave the highest and significant expression of RUNX-2 gene compared to the other treatment groups (P < 0.001); ^bgroups treated with EST 0.2 mg/kg, ASG 100 mg/kg gave almost the same expression in term of RUNX-2 gene compared to sham (P > 0.05) and significantly higher compared to other groups (P < 0.05).
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin; mRNA, messenger ribonucleic acid; RUNX-2, runt-related transcription factor 2.

Figure 5 Relative mRNA expression of Postn gene in OVX rats treated with EST, REM, GBR, GABA and ORZ in different doses compared to sham and OVX non-treated group.

Notes: Treatment groups were expressed in (mg/kg) for three readings, levels not connected by same letter are significantly different (P < 0.05). ^aSham non-OVX group gave the highest expression of Postn gene compared to all treatment groups (P < 0.001); ^bgroup treated with ORZ at 200 mg/kg gives the highest significant upregulation of Postn gene compared to all other treatment groups (P < 0.001); ^cno significant difference was observed when comparing the treatments in these groups (P > 0.05); But significantly higher in terms of expression of Postn compared to OVX non-treated group (P < 0.05).
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin; mRNA, messenger RNA; Postn, periostin, osteoblast specific factor.

Figure 6 Relative mRNA expression of OSX gene in OVX rats treated with EST, REM, GBR, GABA and ORZ in different doses compared to sham and OVX non-treated group.

Notes: Treatment groups were expressed in (mg/kg) for three readings, levels not connected by same letter are significantly different (P < 0.05). ^aGroup treated with ORZ 200 mg/kg gave the highest expression of OSX gene compared to sham, OVX non-treated, and all other treatment groups (P < 0.001); ^bGABA 200 mg/kg treated group gave a significant high expression of OSX gene compared to other treatment groups (P < 0.05); ^cno significant difference (P > 0.05) between the sham non-OVX group compared to groups treated with REM 20 mg/kg, GBR and GABA 100 mg/kg.
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin; mRNA, messenger ribonucleic acid; OSX, osterix.

Figure 7 Relative mRNA expression of col1&2 gene in OVX rats treated with EST, REM, GBR, GABA and ORZ in different doses compared to sham and OVX non-treated group.

Notes: Treatment groups were expressed in (mg/kg) for three readings, levels not connected by same letter are significantly different (P < 0.05). ^aGroup treated with ORZ 200 mg/kg gave the highest significant expression of col1&2 gene compared to other treatment groups (P < 0.001); ^bgroup treated with ORZ 200 mg/kg gave the highest significant expression of col1&2 gene compared to OVX non-treated, sham, and all the other treatment groups (P < 0.0001); ^bno significant difference (P > 0.05) between the sham non-OVX group compared to groups treated with EST 0.2 mg/kg, ASG 100 mg/kg and GABA 200 mg/kg.
Abbreviations: ASG, acylated steryl glucosides; EST, estrogen; GABA, gamma-amino butyric acid; GBR, phenolic extracts from germinated brown rice; ORZ, gamma-oryzanol; OVX, ovariectomized; REM, remifemin; mRNA, messenger ribonucleic acid; col1&2, collagen 1&2.