Elucidation of colon-protective efficacy of diosgenin in experimental TNBS-induced colitis: inhibition of NF-κB/IkB-α and Bax/Caspase-1 signaling pathways

Figures & data

Table 1. Effect of diosgenin on TNBS induced alterations in body weight, colon weight, colon weight to length ratio, ulcer area, ulcer index, disease activity index, macroscopic score and stool consistency score in rats.

Treatment	Body Weight (gm)	Colon weight (gm)	Colon weight to length ratio (gm/cm)	Ulcer area (mm^2)	Ulcer index	% inhibition	Disease Activity Index (DAI) Scores	Macroscopic score	Stool Consistency Score
Normal	224.00 ± 2.35	1.14 ± 0.07	0.16 ± 0.01	0.00 ± 0.00	0.00 ± 0.00	– -	0.17 ± 0.17	0.00 ± 0.00	0.00 ± 0.00
TNBS Control	193.50 ± 2.53^#	3.16 ± 0.06^#	0.44 ± 0.02^#	35.95 ± 1.84^#	74.90 ± 2.10^#	– -	3.33 ± 0.21^#	7.50 ± 0.34^#	2.67 ± 0.21^#
5-ASA (500)	211.20 ± 3.41*^,$	1.70 ± 0.09*^,$	0.23 ± 0.01*^,$	3.02 ± 1.61*^,$	10.25 ± 1.60*^,$	86.31	0.50 ± 0.22*^,$	1.00 ± 0.52*^,$	0.50 ± 0.22*^,$
Dio (50)	198.20 ± 2.69	2.88 ± 0.09	0.40 ± 0.02	29.97 ± 1.82	60.35 ± 1.78	19.43	3.00 ± 0.26	6.50 ± 0.34	2.50 ± 0.22
Dio (100)	203.30 ± 2.58*^,$	2.39 ± 0.09*^,$	0.34 ± 0.02*^,$	23.68 ± 1.37*^,$	49.72 ± 1.45*^,$	33.62	2.17 ± 0.31*^,$	4.17 ± 0.40*^,$	1.50 ± 0.22*^,$
Dio (200)	211.20 ± 3.04*^,$	1.99 ± 0.07*^,$	0.30 ± 0.01*^,$	6.36 ± 1.65*^,$	27.43 ± 1.81*^,$	63.38	1.50 ± 0.22*^,$	2.67 ± 0.42*^,$	0.50 ± 0.22*^,$
Per se	220.70 ± 3.20	1.18 ± 0.07	0.16 ± 0.01	0.00 ± 0.00	0.00 ± 0.00	– -	0.17 ± 0.17	0.00 ± 0.00	0.00 ± 0.00

Data are expressed as mean ± S.E.M. (n = 6) and analyzed by one way ANOVA followed by Tukey’s multiple range test. Data of macroscopical score and stool consistency was analyzed using nonparametric Kruskal-Wallis ANOVA followed by Tukey’s multiple range test. *p < 0.05 as compared to TNBS control group, ^#p < 0.05 as compared to normal group and ^$p < 0.05 as compared to one another (diosgenin and 5-ASA). TNBS: 2,4,6-Trinitrobenzenesulfonic acid; 5-ASA: 5-Aminosalicylic acid; Dio: Diosgenin.

Figure 1. Morphological representation of colons from Normal (a), TNBS control (b), 5-ASA (500 mg/kg) (c), Diosgenin (100 mg/kg) (d), Diosgenin (200 mg/kg) (e) and Per Se (f) treated rats.

Table 2. Effect of diosgenin on TNBS induced alterations in colonic SOD, GSH, MDA, NO, hydroxyproline, and MPO in rats.

Treatment	Colonic Total protein (mg/gm)	Colonic SOD (U/mg of protein)	Colonic GSH (µg/mg protein)	Colonic MDA (nM/mg of protein)	Colonic NO (µg/mL)	Colonic HP (µg/mg tissue)	Colonic MPO (U/g of tissue)
Normal	18.81 ± 2.47	4.88 ± 0.28	1.63 ± 0.17	5.71 ± 1.07	536.40 ± 31.73	2.83 ± 0.39	2.09 ± 0.31
TNBS Control	56.22 ± 6.53^#	0.83 ± 0.16^#	0.46 ± 0.05^#	22.13 ± 1.95^#	1136.00 ± 41.54^#	9.83 ± 0.73^#	11.02 ± 0.56^#
5-ASA (500)	29.21 ± 3.04*^,$	4.37 ± 0.19*^,$	1.01 ± 0.06*^,$	9.69 ± 1.02*^,$	702.00 ± 50.60*^,$	3.81 ± 0.26*^,$	6.11 ± 0.62*^,$
Dio (50)	53.39 ± 4.06	1.39 ± 0.25	0.59 ± 0.05	19.67 ± 0.72	1100.00 ± 39.37	9.71 ± 0.92	9.75 ± 0.50
Dio (100)	44.01 ± 4.97*^,$	2.89 ± 0.19*^,$	1.14 ± 0.10*^,$	17.42 ± 1.38*^,$	872.10 ± 30.29*^,$	8.43 ± 0.57*^,$	8.74 ± 0.70*^,$
Dio (200)	32.51 ± 2.13*^,$	3.34 ± 0.33*^,$	1.16 ± 0.15*^,$	13.93 ± 0.40*^,$	766.20 ± 43.10*^,$	6.13 ± 0.75*^,$	5.72 ± 0.86*^,$
Per se	21.15 ± 0.87	4.49 ± 0.27	1.40 ± 0.10	9.12 ± 0.34	535.70 ± 60.70	4.65 ± 0.66	2.42 ± 0.28

Data are expressed as mean ± S.E.M. (n = 6) and analyzed by one way ANOVA followed by Tukey’s multiple range test. *p < 0.05 as compared to TNBS control group, ^#p < 0.05 as compared to normal group and ^$p < 0.05 as compared to one another (diosgenin and 5-ASA). TNBS: 2,4,6-Trinitrobenzenesulfonic acid; 5-ASA: 5-Aminosalicylic acid; Dio: Diosgenin; SOD: Superoxide dismutase; GSH: Glutathione; MDA: Malondialdehyde; NO: Nitric oxide; HP: Hydroxyproline and MPO: Myeloperoxidase.

Table 3. Effect of diosgenin on TNBS induced alterations in colonic TNF-α, IL-1β, IL-6, IL-10, IFN-γ, iNOs, COX-2 and LTB4 mRNA expressions in rats.

Treatment	TNF-α/β-Actin Ratio	IL-1β/β-Actin Ratio	IL-6/β-Actin Ratio	IL-10/β-Actin Ratio	IFN-γ/β-Actin Ratio	iNOs/β-Actin Ratio	COX-2/β-Actin Ratio	LTB4/β-Actin Ratio
Normal	0.58 ± 0.08	0.05 ± 0.01	0.65 ± 0.06	1.54 ± 0.11	0.24 ± 0.04	0.36 ± 0.04	0.37 ± 0.08	0.67 ± 0.06
TNBS Control	2.03 ± 0.08^#	0.19 ± 0.01^#	1.24 ± 0.09^#	0.54 ± 0.11^#	0.89 ± 0.11^#	0.92 ± 0.05^#	1.95 ± 0.07^#	1.53 ± 0.04^#
5-ASA (500)	0.79 ± 0.11*^,$	0.08 ± 0.01*^,$	0.82 ± 0.07*^,$	1.44 ± 0.07*^,$	0.76 ± 0.05	0.51 ± 0.06*^,$	0.74 ± 0.07*^,$	1.44 ± 0.06
Dio (50)	1.86 ± 0.13	0.18 ± 0.01	1.19 ± 0.10	0.62 ± 0.12	0.74 ± 0.04	0.79 ± 0.05	1.68 ± 0.11	1.38 ± 0.07
Dio (100)	1.20 ± 0.07*^,$	0.12 ± 0.01*^,$	1.03 ± 0.06*^,$	0.96 ± 0.11*^,$	0.54 ± 0.03*^,$	0.57 ± 0.05*^,$	1.28 ± 0.09*^,$	1.29 ± 0.08
Dio (200)	0.91 ± 0.07*^,$	0.09 ± 0.01*^,$	0.83 ± 0.08*^,$	1.13 ± 0.09*^,$	0.46 ± 0.03*^,$	0.47 ± 0.06*^,$	0.96 ± 0.07*^,$	1.35 ± 0.07
Per se	0.68 ± 0.09	0.06 ± 0.01	0.76 ± 0.07	1.36 ± 0.10	0.22 ± 0.04	0.30 ± 0.04	0.66 ± 0.13	0.78 ± 0.04

Data are expressed as mean ± S.E.M. (n = 6) and analyzed by one way ANOVA followed by Tukey’s multiple range test. *p < 0.05 as compared to TNBS control group, ^#p < 0.05 as compared to normal group and ^$p < 0.05 as compared to one another (diosgenin and 5-ASA). TNBS: 2,4,6-Trinitrobenzenesulfonic acid; 5-ASA: 5-Aminosalicylic acid; Dio: Diosgenin; iNOs: Inducible nitric oxide synthase, TNF-α: Tumor necrosis factor-alpha, IL’s: Interleukins, IFN-γ: Interferon-gamma, COX-2: Cyclooxygenase-2 and LTB4: leukotriene B4.

Figure 2. Effect of diosgenin on TNBS induced alterations in colonic NF-κB (a), IκBα (b), Bax (c) and Caspase-1 (d) protein expression in rats. Data are expressed as mean ± S.E.M. (n = 6) and analyzed by one way ANOVA followed by Tukey’s multiple range test. *p < 0.05 as compared to TNBS control group, ^#p < 0.05 as compared to normal group and ^$p < 0.05 as compared to one another (diosgenin and 5-ASA). Representative protein expression of Normal (Lane 1), TNBS control (Lane 2), 5-ASA (500 mg/kg) (Lane 3), Diosgenin (50 mg/kg) (Lane 4), Diosgenin (100 mg/kg) (Lane 5), Diosgenin (200 mg/kg) (Lane 6) and Per Se (Lane 7) treated rats. TNBS: 2,4,6-Trinitrobenzenesulfonic acid; 5-ASA: 5-Aminosalicylic acid; Dio: Diosgenin; p-NF-κB: Phospho Nuclear Factor kappa B; p-IκBα: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha, Bax: Bcl-2-associated × protein and GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 3. Effect of diosgenin on TNBS induced alterations in colon histopathology. Photomicrograph of sections of colon tissue from Normal (a), TNBS control (b), 5-ASA (500 mg/kg) (c), Diosgenin (100 mg/kg) (d), Diosgenin (200 mg/kg) (e) and Per Se (f) treated rats stained with H&E stain. The quantitative representation of histological score (g). Data are expressed as mean ± S.E.M. (n = 3) and analyzed by one way ANOVA followed by the Kruskal-Wallis test was applied for post hoc analysis. *p < 0.05 as compared to TNBS control group, ^#p < 0.05 as compared to normal group and ^$p < 0.05 as compared to one another (diosgenin and 5-ASA). TNBS: 2,4,6-Trinitrobenzenesulfonic acid; 5-ASA: 5-Aminosalicylic acid; Dio: Diosgenin. Red arrow indicated inflammatory infiltration, and yellow arrow indicated necrosis. Images (×40 magnification) are typical and are representative of each study group.