Fructooligosaccharide supplementation ameliorates gut associated metabolic dysregulation in estrogen-deprived rats via targeting oxidative stress, inflammation, apoptosis, and gut microbiome

Rats in the control, sham, and FOS Per Se group remain healthy throughout the experimental protocol with no substantial changes in basic physiological parameters, obesity markers, lipid profile, liver function biomarkers, and liver & colon health. Rats in the estrogen-deprived (323.2 ± 1.4; 31.36%) and vehicle-treated estrogen-deprived (322.0 ± 0.7; 30.88%) groups exhibited a considerable upsurge ( p  < 0.001) in BW, with a notable decrease ( p  < 0.001) in uterine horn weight (0.12 ± 0.006; 79.49% and 0.13 ± 0.004; 77.24%, respectively) and serum estradiol (E2) levels (11.1 ± 0.21; 80.19% and 12.0 ± 0.13; 78.74%, resp.) with respect to control rats. FOS (50 & 100 mg/kg) & 17 βE2 (10 µg/kg) considerably ( p  < 0.001) inhibits BW upsurge in estrogen-deprived rats. Additionally, 17 βE2 substantially ( p  < 0.001) recovers uterine horn weight and E2 levels in estrogen-deprived rats, but FOS (50 & 100 mg/kg) had no impact. Compared to 17 βE2, FOS exhibited no substantial variation in BW growth in estrogen-deprived rats. The administration of FOS at 25 mg/kg in estrogen-deprived rats did not showed any effect on BW, uterine horn weight, and E2 levels (Fig.  2 a–d) Fig. 2 Impact of FOS on basic physiological markers in estrogen-deprived rats ( n  = 6). ( a ): BW at 28th day; ( b ): % difference in BW; ( c ): Weight of uerine horn; ( d ): E2 levels; ( e ): Food intake; ( f ): Weight of liver; ( g ): Weight of visceral adipose tissue; ( h ): BMI. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on basic physiological markers in estrogen-deprived rats ( n  = 6). ( a ): BW at 28th day; ( b ): % difference in BW; ( c ): Weight of uerine horn; ( d ): E2 levels; ( e ): Food intake; ( f ): Weight of liver; ( g ): Weight of visceral adipose tissue; ( h ): BMI. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Anthropometric and adiposity markers such as food intake (24.3 ± 0.07; 43.23 & 24.6 ± 0.07; 45.0%, resp.), liver weight (5.71 ± 0.007; 9.72 & 5.74 ± 0.008; 10.33%, resp.), visceral adipose tissue weight (3.33 ± 0.04; 80.0 & 3.29 ± 0.09; 77.92%, resp.), BMI (0.85 ± 0.003; 90.03 & 0.87 ± 0.003; 92.61%, resp.), waist hip ratio (1.27 ± 0.005; 14.41 & 1.26 ± 0.006; 13.81%, resp.), change in abdominal circumference (4.41 ± 0.10; 87.94 & 4.40 ± 0.08; 87.23%, resp.), total cholesterol (132.8 ± 4.24; 73.02 & 130.5 ± 3.74; 71.25%, resp.), LDL cholesterol (54.0 ± 1.06; 67.2 & 55.1 ± 1.35; 68.32%, resp.), TG (127.5 ± 0.76; 64.51 & 126.3 ± 1.64; 63.01%, resp.), and VLDL (25.5 ± 0.15; 64.51 & 25.26 ± 0.32; 63.01%, resp.) were seen substantially ( p  < 0.001) elevated, and HDL cholesterol (12.5 ± 0.76; 71.26 & 12.0 ± 1.39; 72.41%, resp.) levels were seen to drop noticeably ( p  < 0.001) in estrogen-deprived and vehicle-treated estrogen-deprived rats with respect to control rats. Ingestion of both treatments, such as FOS (50 & 100 mg/kg) and 17 βE2, substantially restores ( p  < 0.001) anthropometric and adiposity markers in OVX rats towards normal levels. No effect was seen on anthropometric and adiposity markers in estrogen-deprived rats by the administration of FOS 25 mg/kg (Figs.  2 e-h, & 3 a-g) Liver function biomarkers such as serum bilirubin (1.75 ± 0.07; 80.02 & 1.71 ± 0.17; 79.21%, resp.), serum albumin (7.35 ± 0.07; 61.0 & 7.43 ± 0.07; 61.9%, resp.), SGOT (182.5 ± 0.76; 78.56 & 183.4 ± 0.75; 79.76%, resp.) and SGPT (163.5 ± 0.75; 81.12 & 165.6 ± 0.76; 82.23%, resp.) levels were considerably upsurged ( p  < 0.001) in rats of the estrogen-deprived and vehicle-treated estrogen-deprived group relative to control rats, but ingestion of FOS (50 & 100 mg/kg) and 17 βE2 positively restores ( p  < 0.001) these levels and improves liver functions in OVX rats towards normal. FOS 25 mg/kg showed no effect on liver function biomarkers in estrogen-deprived rats (Fig.  3 h-k) In comparison to 17 βE2-administered rats, FOS-supplemented rats showed substantial differences in liver weight, weight of visceral adipose tissue, HDL, LDL, TG, VLDL, serum bilirubin, serum albumin, SGOT, and SGPT levels in OVX rats. Fig. 3 Impact of FOS on anthropometric & adiposity markers, and liver function biomarkers in OVX rats ( n  = 6). ( a ): Waist hip ratio; ( b ): Change in abdominal circumference; ( c ): Total cholesterol; ( d ): HDL cholesterol; ( e ): LDL cholesterol; ( f ): Triglyceride; ( g ): VLDL; ( h ): Serum bilirubin; ( i ): Serum albumin; ( j ): SGOT; ( k ): SGPT. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on anthropometric & adiposity markers, and liver function biomarkers in OVX rats ( n  = 6). ( a ): Waist hip ratio; ( b ): Change in abdominal circumference; ( c ): Total cholesterol; ( d ): HDL cholesterol; ( e ): LDL cholesterol; ( f ): Triglyceride; ( g ): VLDL; ( h ): Serum bilirubin; ( i ): Serum albumin; ( j ): SGOT; ( k ): SGPT. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Estrogen-deprived and vehicle-treated estrogen-deprived rats displayed a substantial upsurge in oxidative stress ( p  < 0.001), as evidenced by elevated LPO levels (3.58 ± 0.14; 84.87% and 3.50 ± 0.13; 82.43%, resp.) and decreased levels of TT (0.36 ± 0.06; 73.80% and 0.39 ± 0.05; 72.14%, resp.), SOD activity (0.33 ± 0.06; 80.19% and 0.35 ± 0.06; 79.20%, resp.), CAT activity (1.33 ± 0.06; 50.31% and 1.35 ± 0.05; 49.68%, resp.), and GSH levels (1.41 ± 0.13; 85.97% and 1.38 ± 0.12; 86.30%, resp.) with respect to control rats. Supplementation with FOS at 50 & 100 mg/kg, together with 17 βE2 at 10 µg/kg, dramatically ( p  < 0.001) reduces elevated oxidative stress levels to normal in OVX rats, as seen by the restoration of GSH levels, LPO levels, TT levels, SOD activity, and CAT activity. No impact on oxidative stress in the liver was seen in estrogen-deprived rats treated with FOS at a dosage of 25 mg/kg. Nonetheless, FOS-treated rats exhibited a substantial reduction in oxidative stress levels compared to 17 βE2-treated rats (Fig.  4 a-e) Fig. 4 Impact of FOS on estrogen deprivation-induced oxidative stress in the liver ( n  = 6). ( a ): LPO; ( b ): TT; ( c ): SOD; ( d ): CAT; ( e ): GSH. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on estrogen deprivation-induced oxidative stress in the liver ( n  = 6). ( a ): LPO; ( b ): TT; ( c ): SOD; ( d ): CAT; ( e ): GSH. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Rats in the estrogen-deprived and vehicle-treated estrogen-deprived group exhibited substantial ( p  < 0.001) inflammation in hepatocytes as indicated by elevation of TNF-α (963.3 ± 11.1; 84.12 & 970.0 ± 12.9; 84.87%, resp.), CRP (9.85 ± 0.05; 79.11 & 9.74 ± 0.07; 79.51%, resp.), LPS (9.73 ± 0.14; 56.4 & 9.71 ± 0.09; 56.87%, resp.), and NF-kB/p65 (1261.6 ± 131.5; 86.1 & 1270.1 ± 29.3; 85.88%, resp.) levels and upregulation in the expressions of TNF-α (2.83- & 3.09-fold, resp.) and IL-6 (2.70- & 2.91-fold, resp.) genes with respect to control rats. FOS (50 & 100 mg/kg) supplementation and standard drug 17 βE2 substantially ( p  < 0.001) mitigate inflammatory markers in the liver of estrogen-deprived rats, but no result was seen by FOS 25 mg/kg. With this, we also saw a substantial ( p  < 0.001) difference between FOS & 17 βE2-treated OVX rats (Fig.  5 a-e) With this, we also saw a substantial upsurge ( p  < 0.001) in apoptosis as indicated by upregulation of Bax (1.64- & 1.65-fold, resp.) and downregulation of Bcl-2 (0.62- & 0.60-fold change) gene expressions in rats of estrogen-deprived and vehicle-treated estrogen-deprived groups relative to control rats. This rise in apoptosis was substantially ( p  < 0.001) restored by the ingestion of FOS (50 & 100 mg/kg) & 17 βE2 in estrogen-deprived rats, but no result was seen by FOS 25 mg/kg. However, while comparing with 17 βE2, FOS also showed a substantial difference ( p  < 0.001) in apoptosis in estrogen-deprived rats (Fig.  5 g and h) Estrogen deficiency in estrogen-deprived & vehicle-treated estrogen-deprived group rats substantially ( p  < 0.001) reduces estrogen receptor expressions, such as ER-α (0.55- & 0.51-fold, resp.) and ER-β (0.70- & 0.67-fold, resp.) in comparison to control rats, but 17 βE2 in estrogen-deprived rats substantially ( p  < 0.001) restores ER-α (1.13-fold) and ER-β (1.08-fold) gene expressions. No effect was seen on ER-α (0.57-fold) and ER-β (0.63-fold) gene expressions in OVX rats when administered with FOS at doses of 25, 50, & 100 mg/kg (Fig.  5 i and j) Fig. 5 Impact of FOS on inflammation, apoptosis, and estrogen receptors in the liver of OVX rats ( n  = 6). ( a ): TNF-α levels; ( b ): CRP levels; ( c ): LPS levels; ( d ): NF-κB/p65 levels; ( e ): TNF-α gene expressions; ( f ): IL-6 gene expressions; ( g ): Bax gene expressions; ( h ): Bcl-2 gene expressions; ( i ): ER-α gene expressions; and (j): ER-β gene expressions. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on inflammation, apoptosis, and estrogen receptors in the liver of OVX rats ( n  = 6). ( a ): TNF-α levels; ( b ): CRP levels; ( c ): LPS levels; ( d ): NF-κB/p65 levels; ( e ): TNF-α gene expressions; ( f ): IL-6 gene expressions; ( g ): Bax gene expressions; ( h ): Bcl-2 gene expressions; ( i ): ER-α gene expressions; and (j): ER-β gene expressions. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Rats in the estrogen-deprived & vehicle-treated estrogen-deprived group showed the presence of higher dead and inflamed hepatocytes with respect to control rats, but treatment with standard drug-17 βE2 and FOS 50 & 100 mg/kg both restored the liver health as evidenced by reducing inflammation and death of hepatocytes. FOS 25 mg/kg showed no improvement on liver health with respect to estrogen-deprived rats (Fig.  6 a-i & Supplementary Fig. 1). Fig. 6 Impact of FOS on estrogen deficiency-induced changes in liver morphology (Images captured at 100x magnification). ( a ): Control; ( b ): Sham; ( c ): FOS Per Se; ( d ): OVX; ( e ): OVX + Veh; ( f ): OVX + 17 βE2; ( g ): OVX + FOS 25 mg/kg; ( h ): OVX + FOS 50 mg/kg; ( i ): OVX + FOS 100 mg/kg. Black circle indicates the presence of dead hepatocytes. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on estrogen deficiency-induced changes in liver morphology (Images captured at 100x magnification). ( a ): Control; ( b ): Sham; ( c ): FOS Per Se; ( d ): OVX; ( e ): OVX + Veh; ( f ): OVX + 17 βE2; ( g ): OVX + FOS 25 mg/kg; ( h ): OVX + FOS 50 mg/kg; ( i ): OVX + FOS 100 mg/kg. Black circle indicates the presence of dead hepatocytes. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Rats in the estrogen-deprived & vehicle-treated estrogen-deprived group saw a substantial upsurge ( p  < 0.001) in LPO levels (4.41 ± 0.10; 83.9 & 4.65 ± 0.08; 89.4%, resp.), and a substantial decrease in TT levels (0.30 ± 0.05; 83.01 & 0.28 ± 0.06; 83.96%, resp.), SOD activity (0.55 ± 0.06; 69.44 & 0.60 ± 0.07; 66.66%, resp.), CAT activity (0.25 ± 0.04; 89.13 & 0.30 ± 0.05; 86.95%, resp.), and GSH levels (0.93 ± 0.11; 90.58 & 0.98 ± 0.17; 90.08%, resp.), but treatment with FOS (50 & 100 mg/kg) & 17 βE2 both substantially ( p  < 0.001) restore these changes toward normal levels with respect to control rats. No result was seen by the administration of FOS 25 mg/kg on colon oxidative-stress in estrogen-deprived rats. OVX rats, when administered with FOS, also showed a substantial difference in oxidative stress markers with respect to 17 βE2-treated estrogen-deprived rats (Fig.  7 a-e) Fig. 7 Impact of FOS on oxidative stress in the colon of estrogen-deprived rats ( n  = 6). ( a ): LPO levels; ( b ): TT levels; ( c ): SOD activity; ( d ): CAT activity; ( e ): GSH levels. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on oxidative stress in the colon of estrogen-deprived rats ( n  = 6). ( a ): LPO levels; ( b ): TT levels; ( c ): SOD activity; ( d ): CAT activity; ( e ): GSH levels. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Estrogen deficiency in the estrogen-deprived & vehicle-treated estrogen-deprived group of rats substantially ( p  < 0.001) upsurges organ-specific TNF-α levels (933.3 ± 11.1; 87.11 & 940.0 ± 12.9; 86.86%) and TNF-α (3.2- & 3.4-fold, resp.) & IL-6 (3.3- & 3.6-fold, resp.) gene expressions, NF-κB/p65 (1653.3 ± 51.9; 97.10 & 1616.6 ± 105.4; 95.21%, resp.), CRP (10.05 ± 0.05; 61.21 & 9.91 ± 0.07; 60.45%, resp.), and LPS (9.71 ± 0.10; 59.21 & 9.85 ± 0.04; 60.19%, resp.) levels with respect to control rats. Rats fed with FOS 50 & 100 mg/kg and 17 βE2-10ug/kg substantially restored ( p  < 0.001) these inflammatory marker levels towards normal in estrogen-deprived rats, but no effect was seen by FOS at 25 mg/kg. In comparison with 17 βE2, FOS-fed rats also showed a substantial difference ( p  < 0.001) in the mitigation of organ-specific inflammation (Fig.  8 a-f). We also seen substantial upregulation ( p  < 0.001) in the expressions of Bax (1.9- & 1.92-fold, resp.) and marked downregulation in the expressions of the Bcl-2 gene (0.72- & 0.7-fold, resp.) in rats of the estrogen-deprived and vehicle-treated estrogen-deprived group. With this, continuous administration of FOS (50 & 100 mg/kg) and 17 βE2 substantially ( p  < 0.001) restores the apoptosis towards normal levels in estrogen-deprived rats, but no effect was seen by FOS 25 mg/kg. FOS also showed a substantial difference ( p  < 0.001) in the expressions of the Bax gene relative to 17 βE2, but no alteration was seen in the Bcl-2 gene expressions (Fig.  8 g and h). Fig. 8 Impact of FOS on inflammation in the colon of estrogen-deprived rats ( n  = 6). ( a ): TNF-α levels; ( b ): NF-κB/p65 levels; ( c ): CRP levels; ( d ): LPS levels; ( e ): TNF-α gene expressions; ( f ): IL-6 gene expressions; ( g ): Bax gene expressions; and ( h ): Bcl-2 gene expressions. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on inflammation in the colon of estrogen-deprived rats ( n  = 6). ( a ): TNF-α levels; ( b ): NF-κB/p65 levels; ( c ): CRP levels; ( d ): LPS levels; ( e ): TNF-α gene expressions; ( f ): IL-6 gene expressions; ( g ): Bax gene expressions; and ( h ): Bcl-2 gene expressions. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. There was a substantial downregulation ( p  < 0.001) in the expressions of MUC-2 (0.47- and 0.40-fold, respectively), Lipocalin-2 (0.63- and 0.68-fold, respectively), and claudin (0.62- and 0.60-fold, respectively) genes, alongside a substantial upregulation ( p  < 0.001) in the expressions of the TLR-4 gene (2.01- and 2.17-fold change) in OVX and OVX + Veh group rats compared to control rats. Rats administered FOS (50 & 100 mg/kg) and 17 βE2 for 28 days substantially restores the ( p  < 0.001) MUC-2 and tight junction genes (TJG) in estrogen-deprived rats; however, no positive impact was seen with FOS at 25 mg/kg. Rats administered FOS exhibited a notable disparity in the recovery of the mucosal layer and tight junctions in estrogen-deprived rats compared to those receiving 17 βE2 (Fig.  9 a-d). Fig. 9 Impact of FOS on the mucosal layer and tight junctions in the colon of estrogen-deprived rats ( n  = 6). ( a ): MUC-2 ; ( b ): Lipocalin-2 ; ( c ): TLR-4 ; ( d ): Claudin . (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on the mucosal layer and tight junctions in the colon of estrogen-deprived rats ( n  = 6). ( a ): MUC-2 ; ( b ): Lipocalin-2 ; ( c ): TLR-4 ; ( d ): Claudin . (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Estrogen-deprivation rats in the estrogen-deprived & vehicle-treated estrogen-deprived group substantially downregulate ( p  < 0.001) the expressions of beneficial bacteria such as BACT (0.53- & 0.61-fold, resp.), ROS (0.37- & 0.40-fold, resp.), BIF (1.15- & 1.08-fold, resp.), PREV (0.73- & 0.78-fold, resp.), AKK (0.36- & 0.4-fold, resp.), LAB (0.76- & 0.8-fold, resp.), & FAE (0.7- & 0.75-fold, resp.) and upregulates the expressions of pathogenic bacteria such as FIRMI (3.4- & 3.6-fold, resp.), ENTB (3.5- & 3.6-fold, resp.) and RUMI (1.8- & 1.9-fold, resp.) with respect to control rats. Supplementation of FOS (50 & 100 mg/kg) & 17 βE2 in rats with estrogen deficiency substantially restores the beneficial vs. pathogenic bacterial abundance. No protective effect was seen by FOS 25 mg/kg on beneficial and pathogenic bacterial abundance in estrogen-deprived rats. In comparison to 17 βE2, rats fed with FOS also substantially restore ( p  < 0.001) the BACT , PREV , AKK , LAB , and FIRMI bacterial genes expressions, but no difference was seen in the expressions of ROS , BIF , FAE , ENTB , and RUMI bacterial genes (Fig.  10 a and j). Fig. 10 Impact of FOS on GM in estrogen-deprived rats ( n  = 6). ( a ): BACT ; ( b ): ROS ; ( c ): BIF ; ( d ): PREV ; ( e ): AKK ; ( f ): LAB ; ( g ): FAE ; ( h ): FIRMI ; ( i ): ENTB ; ( j ): RUMI . (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on GM in estrogen-deprived rats ( n  = 6). ( a ): BACT ; ( b ): ROS ; ( c ): BIF ; ( d ): PREV ; ( e ): AKK ; ( f ): LAB ; ( g ): FAE ; ( h ): FIRMI ; ( i ): ENTB ; ( j ): RUMI . (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Rats in the estrogen-deprived & vehicle-treated estrogen-deprived group showed damaged mucosal layer, goblet cells, villi structure, tight junctions, and inflammation in the colon, which indicates damaged colon microstructure with respect to control rats. 17 βE2 and FOS 50 & 100 mg/kg administration in rats with estrogen deficiency restores colon microanatomy as indicated by restoration of gut mucosa, goblet cells, villi structure, tight junctions, and reducing inflammation. While comparison with estrogen-deprived rats, FOS 25 mg/kg showed no protective effect on gut health (Fig.  11 a-i & Supplementary Fig. 2) Fig. 11 Impact of FOS on estrogen deficiency-induced changes in colon morphology (All images captured at 100x magnification). ( a ): Control; ( b ): Sham; ( c ): FOS Per Se; ( d ): OVX; ( e ): OVX + Veh; ( f ): OVX + 17 βE2; ( g ): OVX + FOS 25 mg/kg; ( h ): OVX + FOS 50 mg/kg; ( i ): OVX + FOS 100 mg/kg. Plus sign in red color indicates the presence of damaged goblet cells. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group. Impact of FOS on estrogen deficiency-induced changes in colon morphology (All images captured at 100x magnification). ( a ): Control; ( b ): Sham; ( c ): FOS Per Se; ( d ): OVX; ( e ): OVX + Veh; ( f ): OVX + 17 βE2; ( g ): OVX + FOS 25 mg/kg; ( h ): OVX + FOS 50 mg/kg; ( i ): OVX + FOS 100 mg/kg. Plus sign in red color indicates the presence of damaged goblet cells. (a) p  < 0.05 vs. control group; (aa) p  < 0.01 vs. control group; (aaa) p  < 0.001 vs. control group; (b) p  < 0.05 vs. OVX group; (bb) p  < 0.01 vs. OVX group; (bbb) p  < 0.001 vs. OVX group; (c) p  < 0.05 vs. OVX + 17 βE2 group; (cc) p  < 0.01 vs. OVX + 17 βE2 group; (ccc) p  < 0.001 vs. OVX + 17 βE2 group.

FOS 50 & 100 mg/kg ameliorates the gut and metabolic impairment in the estrogen-deprived state, commonly known as GAMD. FOS exerts this effect through multiple mechanisms: (1) FOS reduces oxidative stress, inflammation, and apoptosis in both colon and liver; (2) FOS promotes gut mucosa, tight junction proteins within the goblet cells, and promotes beneficial bacteria and diminishes pathogenic bacteria. Through these multiple mechanisms, FOS ameliorates anthropometric and adiposity markers and the lipid profile in estrogen-deprived rats. Thus, FOS could be a safer, and effective for postmenopausal GAMD. The present study did not include a comprehensive metagenomic analysis of the gut microbiome, the absence of SCFA measurements, the limited mechanistic exploration, and the reliance on a single animal model, which represent limitation. In future studies, high-throughput metagenomic sequencing will be employed to obtain a more detailed characterization of microbial diversity and functional potential. Additionally, the current investigation assessed the expression of only ten selected gut microbial targets. Future work will aim to expand this analysis to include a broader range of microbial taxa to provide a more comprehensive understanding of microbiome alterations associated with the studied condition. Future work will also include SCFA measurements, in vitro studies, and adding more than one animal model.

Estrogen deficiency not only dysregulates metabolic health 37 but also responsible for the deterioration of gut health 38 , hence causes GAMD 39 . OVX is the most widely accepted model to induce similar postmenopausal complications in rodents 40 – 42 . In the current study, to induce estrogen-GAMD, first, rats were bilaterally OVX and then various anthropometric and adiposity markers, lipid profile, liver function biomarkers, oxidative stress, inflammatory and apoptotic markers, gut dysbiosis biomarkers, and GM were analyzed to confirm GAMD. As earlier evidenced, estrogen deficiency followed by OVX causes the development of metabolic impairment 43 which can be seen by impairment in anthropometric & adiposity markers 37 , dysbalance in lipid profile 44 & liver function biomarkers 45 , 46 . OVX also causes an enhancement of oxidative stress 47 , development of inflammation and apoptosis 48 , 49 . Estrogen deficiency is responsible for the deterioration of gut health, as indicated by the destruction of mucosa and tight junctions present within the gut 30 , 50 , upsurges inflammation 51 , 52 , apoptosis 53 , and dysregulates the GM 7 , 54 – 56 . Our current study also supports this and reports similar results. Thus, collectively, estrogen deficiency impairs metabolic and gut health, which may further causes GAMD. To date, hormone replacement therapy, most commonly 17 βE2, has been prescribed worldwide to mitigate postmenopausal complications, but it exerts various side effects, such as breast cancer, cervical cancer, and endometriosis, due to its uterotrophic effect 57 , 58 . Thus, our main aim was to find new solutions that may restore gut-microbiome-metabolic axis impairment without producing uterotrophic effects. FOS, which is a prebiotic most commonly available in the market and can be obtained from fruits and vegetables employed in the current study to ameliorate this axis. There is very limited data available, which suggests that FOS can improve metabolic health in an estrogen-deprived state. Estrogen, which is an important gonadal hormone, supports metabolic health by maintaining a balance between ghrelin, leptin, and neuropeptides 59 , 60 . Estrogen is directly involved in the development and deposition of adipose tissues and is involved in the regulation of the lipogenesis process. Estrogen also exerts a uterotrophic effect by maintaining E2 levels and overall uterus health 61 . Earlier studies reported that treatment with 17 βE2 improves metabolic status in OVX rats by reducing further weight gain, feed intake, reduces adiposity index, and overall abdominal obesity 60 , 62 . 17 βE2 also supports uterus health via improving E2 levels and uterus weight. These results were also correlated with human data 60 , 63 . 17 βE2 also improves lipid profiles such as cholesterol, TG, HDL-C, and LDL-C in rats with estrogen deficiency 60 , 64 . Thus, FOS, which is a non-estrogenic molecule, also supports metabolic health in animal models as indicated by a reduction in further weight gain 65 . FOS exerts this effect due to its modulatory effect on GM, preventing inflammation and improving intestinal barrier function 65 . FOS in C57BL/6 J male mice with non-alcoholic fatty liver disease ameliorates obesity markers and lipid profile, such as maintenance of liver weight, fat weight, serum AST, and serum TG levels, which further improves metabolic health 23 . We also observed that administration of FOS (50 & 100 mg/kg) and 17 βE2 in estrogen-deprived rats prevents BW gain, improves anthropometric and adiposity markers, restores lipid profile, and liver function biomarkers. With this, 17 βE2 but not FOS exerts a uterotrophic effect as evidenced by increased E2 levels, uterine weight, and activation of estrogen receptors ( ER-α & ER-β ). Thus, FOS (50 & 100 mg/kg) positively restores anthropometric, adiposity, and obesity related markers. Estrogen maintains oxidative stress, inflammation, and apoptosis. Oxidative stress triggers an inflammatory cascade and activates inflammatory cytokines, which further enhance apoptosis. Collectively, all these factors exacerbate the condition and are responsible for the development of metabolic and gut diseases 66 , 67 . Earlier studies reported that 17 βE2 exerts antioxidant and antiapoptotic effects via reducing lipid peroxidation levels, increasing SOD activity, MDA levels, and catalase activity, and also attenuates apoptosis 68 , 69 . Similarly, other studies also reported that 17 βE2 reduces inflammation by decreasing TNF-α, CRP, and NF-kB/p65 levels and is also involved in the regulation of apoptosis and acts as a potent anti-inflammatory agent 48 , 69 . Collectively, 17 βE2 promotes colon and liver health via maintaining healthy morphological structure 69 , 70 . Estradiol regulates apoptosis via maintaining the balance between Bax and Bcl-2 in OVX rats 71 . FOS intake reduces oxidative stress via reducing malondialdehyde, increasing SOD activity, and suppressing reactive oxygen species production 20 , 72 . FOS also acts as a potent anti-inflammatory agent via reducing pro-inflammatory cytokines such as TNF-α , IL-6 , IL-1β and also reduces CRP and LPS levels 17 , 23 . FOS supplementation also reduces the progression of disease via balancing apoptotic markers such as Bax and Bcl-2 73 . In concurrence with earlier studies, our current study also supported these findings and suggests that treatment with FOS (50 & 100 mg/kg) and 17 βE2 for a total of 28 days attenuates oxidative stress, inflammation, and apoptosis in the liver and colon. With this, FOS (50 & 100 mg/kg) and 17 βE2 both recover the healthy morphological status of the liver and colon in estrogen-deprived conditions. The gut contains numerous beneficial/harmful bacteria involved in various biological processes 74 . Higher proportion of BACT present in the healthy state and higher proportion of FIRMI present in the diseased state 75 , 76 . Imbalance in beneficial vs. harmful bacteria causes gut dysbiosis 74 . Gut dysbiosis further facilitates the release of LPS (endotoxin produced by harmful bacteria), pro-inflammatory cytokines, and increases TLR-4 and degrades tight junctions (claudin, & lipocalin-2) and mucosal layer 74 , 77 , 78 . This further contributes to developing leaky gut. Estrogen maintains this equilibrium in the gut and promotes gut homeostasis 79 . The estrogen in the gut reduces inflammation, epithelial damage, and gut microbes translocation 80 . 17 βE2 also promotes gut health via maintaining gut mucosa, tight junctions, and changed the GM structure 38 , 70 , 81 . FOS maintains the gut mucosa, such as MUC-2 and tight junctions, such as Claudin , ZO-1 , lipocalin-2 , and TLR-4 , present within the goblet cells 82 , 83 . FOS maintains the equilibrium between BACT and FIRMI ; both are the major gut bacteria that cover all gut health and disturbance in balance involved in the development of a diseased state 76 , 84 . FOS administration promotes LAB , Proteobacteria , Parabacteroides , BACT , ROS , & Blautia sp. and changed the GM 26 , 84 . However, our current study supports these studies and reports similar results. In our study, both FOS (50 & 100 mg/kg) and 17 βE2 regulate gut health via promoting the mucosal layer, tight junction, and rebalancing the gut dysbiotic state into a gut eubiotic state via promoting beneficial bacteria and diminishing pathogenic bacteria. FOS are widely recognized for their prebiotic potential, primarily through their fermentation by GM into short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate 85 , which play a pivotal role in regulating host metabolic homeostasis 86 , inflammation, and intestinal barrier function 87 which are non-estrogen receptor-dependent mechanisms. With this FOS may target enteroendocrine cells to release hormones such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) which further regulate metabolic homeostasis 88 . Although the present study demonstrated that FOS supplementation significantly improved gut–metabolic axis dysfunction in estrogen-deprived rats, but SCFA levels were not assessed. This limits the ability to directly link GM modulation with downstream metabolic benefits. Our current study suggests that FOS (a well-known prebiotic) at a dose of 50 & 100 mg/kg promotes gut and metabolic health in an estrogen-deprived state. Collectively, FOS 50 & 100 mg/kg ameliorates GAMD induced by estrogen deficiency.

Fructooligosaccharide was obtained from NOW Foods, U.S. 17 β-estradiol (17 βE2), RNAlater, and TRIZOL were purchased from Sigma-Aldrich, USA. The cDNA synthesis kit was bought from Thermo-Scientific. SYBR Green was purchased from Bio-Rad Lab. Female Sprague Dawley rats weighing between 200 and 250 g and 7–8 weeks of age were acquired from Panjab University, Chandigarh. Rats were kept at the animal facility of M.M. College of Pharmacy in Haryana, India, after being purchased. Rats were housed under regular laboratory settings with a 12-hour light/dark cycle and unrestricted access to food and water ad libitum. The study was approved by the Institutional Animal Ethics Committee (IAEC) of M.M. College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana-133207 vide protocol number: MMCP-IAEC-152, dated March 16, 2024. All animal experiments were conducted in accordance with the guidelines of the Committee for the Control and Supervision of Experiments on Animals (CCSEA) and complied with relevant institutional and national regulations for the care and use of laboratory animals. The study was conducted and reported in accordance with the ARRIVE Guidelines. Rats were separated into six-groups, with each group containing 6 to 8 animals. Group 1: Control group: Rats remained normal and had free access to food & water. Group 2: Sham group: Rats in the sham group underwent similar surgical conditions, except the removal of ovaries, and remained untreated. Group 3: FOS Per Se: Normal rats were administered with FOS 50 mg/kg; Group 4: OVX group: Using surgery, both ovaries were removed. Group 5: OVX + Veh group: Rats in this group were bilaterally OVX, and 0.9% normal saline was administered as a vehicle. Group 6: OVX + 17 βE2 (10 µg/kg) group: Rats in this group were bilaterally OVX, and 17 βE2-10 µg/kg/ s.c was administered for 28 days via dissolving in sesame oil 24 . Group 7: OVX + FOS (25 mg/kg) group: Estrogen-deprived rats were administered with FOS-25 mg/kg/ p.o (Normal saline was used to dissolve the FOS) 25 . Group 8: OVX + FOS (50 mg/kg) group: Rats in this group were bilaterally OVX and were administered with FOS-50 mg/kg/ p.o 25 – 28 . Group 9: OVX + FOS (100 mg/kg) group: Estrogen-deprived rats were given FOS-100 mg/kg/ p.o 25 – 28 . Doses of 25, 50, and 100 mg/kg were selected based on previous studies and our prior findings to cover a low-to-high dose range and evaluate potential dose-dependent effects 25 – 27 . Earlier reports have demonstrated that FOS at 50 mg/kg significantly modulates gut microbiota and improves behavioral outcomes in stress models 26 , while doses of 50 and 100 mg/kg have been shown to exert neuroprotective and microbiota-restorative effects 27 . Consistent with these findings, our previous study also reported significant anti-inflammatory effects at 50 and 100 mg/kg, whereas no appreciable effect was observed at 25 mg/kg 25 . Therefore, the inclusion of 25 mg/kg in the present study was intended to establish a lower threshold of efficacy and to better characterize the dose–response relationship of FOS. To induce postmenopausal GAMD in rats, a bilateral OVX approach was employed, as earlier described 29 . After a week of recovery period, drug treatment was started for a total of 28 days. (Fig.  1 ) Fig. 1 Methodology followed. Methodology followed. At last, different physiological, biochemical, molecular, histopathological, & gene expression parameters were measured. BW was measured at 0-day and 28th day by using a digital weighing balance. Food intake, water intake, & common health were assessed. Various anthropometric & adiposity markers were analysed and recorded. Abdominal circumference was measured using ordinary tape. Lees’s index was calculated using the formula: cube root of BW (g)/naso to anal length (cm) 30 . Body mass index was calculated using the formula BW (g) divided by the square of length (cm²) 30 . The waist-hip ratio (WHR) was determined by dividing the circumference of the waist by that of the hips 30 . Food consumption was measured on the 0th and 28th days of the study by calculating the difference between the initial food weight and the remaining food in each cage from the previous day (g/day). This was expressed as average feed intake (g/day/rat) by dividing the total feed intake by the number of animals per cage 30 . For faecal collection, rats were sited in cages one by one. First, the cage was sanitised properly by using 75% ethanol. Rats were acclimatised to the cage before collecting faecal matter. Then rats were placed one by one, and were allowed to excrete faecal pellets. After excretion, faecal pellets were collected in a sterile urine container using sterile forceps and immediately stored at −80 °C until further use 31 – 33 . Rats were euthanised using higher doses of anaesthesia (ketamine/xylazine), and blood was collected via the retroorbital route and then centrifuged at 1000 × g for 13 min for serum separation. Separated serum was obtained and preserved at −20 °C for molecular analyses. Rats were perfused with cooled phosphate-buffered saline (PBS), then the liver, colon, and adipose tissues were removed and thoroughly rinsed in normal saline to determine weight using a digital balance. Collected organs were then preserved at −20 °C for biochemical and molecular analyses. Colon & liver tissues were stored at room temperature for histopathological studies. The resected tissues were cleaned, weighed, and homogenised by centrifuging at 1000 × g (4 °C). The transparent supernatant was transferred to a separate container and thereafter stored at −80 °C until needed 31 – 33 . Following the procedure described in Erba diagnostic kits (purchased from Erba Diagnostics Pvt. Ltd), serum was used to measure lipid profile biomarkers such as total cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol, as well as liver function biomarkers such as albumin, bilirubin, AST, and ALT. Liver and colon homogenates were used to measure key markers of oxidative stress, such as reduced glutathione (GSH), lipid peroxidation (LPO), total thiols (TT), catalase (CAT) activity, and superoxide dismutase (SOD) activity 34 – 36 . Serum estradiol, liver & colon NF-κB p65, liver & colon LPS (Cusabio-Technology LLC), liver & colon CRP, & liver & colon TNF-α levels were measured using manufacturer instructions available in ELISA kits (ElabScience). RNA was extracted, converted to cDNA, and then quantified as per the procedure mentioned in our earlier published studies, without any modification 31 – 33 . In liver and colon tissues, the genes Bcl-2 , Bax , IL-6 , and TNF-α were quantified. In the colon, measurements were made of the genes MUC-2 , Lipocalin-2 , Claudin , and TLR-4 . Hepatic tissue was used to assess the levels of ER-α and ER-β. The data were analysed using the ∆∆Ct method, and the findings were shown as a relative fold change compared to the control group. β-actin functioned as an internal control. Primers used in the current study were taken from our earlier published studies without any change 31 – 33 and the same has been mentioned in the Supplementary Table 1. DNA was isolated and quantified as per the methodology mentioned in our earlier studies without any change 31 – 33 . The ∆∆Ct method was used for the analysis of data, and the relative fold change was used to express the values. Total bacteria (TB) served as an internal control. Primer sequences for bacterial populations such as Enterobacteriaceae ( ENTB) , Faecalibacterium (FAE) , Lactobacillus ( LAB) , Bacteroides (BACT) , Prevotella (PREV) , Firmicute (FIRMI) , Bifidobacterium (BIF) , Roseburia (ROS) , Ruminococcus (RUMI) , and Akkermensia (AKK) were sourced from our earlier studies without any alteration 31 – 33 and same has been mentioned in the Supplementary Table 2. Tissues from the liver and colon were stored at room temperature in 10% buffered formalin. Samples were sectioned using a rotary microtome at a thickness of 5–6 μm after undergoing further processing for paraffin embedding. Floating sections were attached to a glass slide and left to air-dry for the whole night. Haematoxylin and eosin (H & E) was used to stain air-dried slides. Slides were examined with the help of a light microscope. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., CA, USA). Data are presented as mean ± SEM. Group comparisons were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant.

Estrogen, a key gonadal steroid hormone, is best known for its role in regulating the female reproductive system 1 . However, its influence extends far beyond the reproductive tract, exerting widespread effects on the brain, liver, cardiovascular system, kidneys, skin, bones, and most notably, the gastrointestinal tract 1 – 3 . Estrogen is crucial for maintaining homeostasis, modulating immune responses, preserving metabolic balance, and promoting antioxidant defense mechanisms via interacting with its receptors, particularly ER-α & ER-β 4 – 6 . Estrogen regulates glucose and lipid metabolism, influences mitochondrial function, and protects against insulin resistance, further improving metabolic health 7 , 8 . Within the gut, estrogen contributes to maintaining intestinal barrier integrity, modulating microbial diversity, and suppressing pro-inflammatory signalling pathways 9 . These effects are crucial for sustaining a healthy gut associated metabolic dysregulation (GAMD) 7 , 10 . Estrogen deficiency, which occurs during menopause or following ovariectomy (OVX), disrupts the gut metabolic axis 7 , 10 . The decline in circulating estrogen levels during the menopausal or postmenopausal state is associated with increased oxidative stress, heightened inflammatory cytokine production, dysregulated apoptosis, and substantial alterations in gut microbiome (GM) composition 7 , 11 . These alterations may result in heightened intestinal permeability (leaky gut), systemic inflammation, and hepatic impairment, collectively known as hallmarks of gut-microbiome-metabolic axis disruption 7 , 12 . Consequently, estrogen deficiency is strongly linked to the onset of metabolic impairment, including non-alcoholic fatty liver disease (NAFLD), insulin resistance, obesity, inflammatory bowel conditions, and gut dysbiosis 13 , 14 . Although hormone replacement therapy (HRT) remains the conventional approach to mitigating the adverse effects of estrogen loss, long-term HRT use has been associated with several risks, including breast cancer, thromboembolic events, and cardiovascular complications 15 . This has driven interest in safer, non-hormonal therapeutic alternatives that can target the underlying pathophysiological mechanisms without associated higher risks. Fructooligosaccharide (FOS), a class of non-digestible prebiotic fibers, has garnered interest for its potential in restoring gut and metabolic health 16 , 17 . FOS modulates GM composition, enhances short-chain fatty acid (SCFA) production, strengthens intestinal barrier function, and exerts antioxidant and anti-inflammatory effects 18 , 19 . FOS also affects visceral and central adiposity, prevents obesity, atherosclerosis, and non-alcoholic fatty liver disease 20 – 23 . However, to date, the gap remains persistent that FOS may be beneficial in protecting the gut and associated metabolic impairment in estrogen-deprived rats without producing uterotrophic effects. Thus, we aimed to examine the therapeutic potential of FOS in ameliorating GAMD in estrogen-deprived rats via targeting apoptosis, inflammation, oxidative stress, & GM. Understanding the mechanistic role of FOS in this context could offer valuable insights into the development of safer alternatives to HRT for managing menopause-associated metabolic and gastrointestinal complications.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1