Gastroprotective effects of diosgenin against HCl/ethanol-induced gastric mucosal injury through suppression of NF-κβ and myeloperoxidase activities

2.1 Chemicals

Diosgenin, omeprazole, HCl, and ethanol (98%) were procured from Santa Cruz Biotechnology Inc., USA (Figure 1). Chemicals for biochemical analysis, antigens, and antibodies for the ELISA assay were obtained from Sigma, St. Louis, USA, and Abcam, USA. Diosgenin was dissolved in 0.1% tween 80 for experimental administration.

Figure 1

Chemical structure of diosgenin.

2.2 Animals

Male Sprague-Dawley (6–7 weeks) rats (180–200 g) were randomly housed in wire bottom cages for the prevention of coprophagy, in a group of ten (n = 10) at 24 ± 2°C temperature, 60% humidity, 12 h light/dark cycle, supplied with standard rodent feed and water.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals and was approved by the ethical committee of Xi’an Third Hospital (Ethical No.: XATH20200827).

2.3 Experimental design and HCl/ethanol-induced gastric injury

All animals were fasted for 24 h and allowed free access to water alone before the initiation of the experiment. The experimental protocol was performed following the methods of Yang et al. [21]. Group I (normal control) was treated with intragastric administration of 0.1% tween 80. Group II served as a gastric model group was administered with HCl/ethanol (0.15 M HCl in 98% ethanol) intragastrically. Group III served as an experimental group treated with diosgenin (20 mg/kg b.w.) by intragastric administration. Group IV served as a positive control treated with omeprazole (20 mg/kg b.w.) by intragastric administration. Groups III and IV received intragastric administration of HCl/ethanol (0.15 M HCl in 98% ethanol), 1 h after the drug administration. The dose of diosgenin was selected based on preliminary studies and past research on the animal model study of diosgenin [18] that was able to prevent oxidative stress; hence, the dosage was modified according to the study of treating gastric mucosal damage in rats [22]. One hour after the administration of HCl/ethanol, the rats were sacrificed through cervical dislocation under deep isoflurane anesthesia, the stomach was dissected longitudinally, and it was washed with ice-cold saline. Consequently, gastric mucosal tissues were subjected to histopathological and biochemical analyses.

2.4 Biochemical analysis

The gastric tissues excised from rats were homogenized in cold phosphate-buffered saline (pH 7.4) and centrifuged at 2,000 ×g for 10 min at 4°C, and the supernatants obtained were subjected to biochemical assays for reduced glutathione (GSH), catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO) levels, and myeloperoxidase (MPO) activity. The remaining aliquots were used for the ELISA assay (Sigma, St. Louis, USA) for interleukin 1-beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α) following the manufacturer’s guidelines. Commercial colorimetric assay kits (Sigma, St. Louis, USA) were used for the determination of CAT, GSH, GPx, SOD, MDA, and NO levels and MPO activity. Protein determination in aliquot was done by Bradford’s assay using bovine serum albumin (BSA) as a standard. Briefly, the decomposition rate of H₂O₂ was observed at 240 nm for CAT determination, formation of 5-thiol-2-nitrobenzoic acid at 412 nm was observed for GSH determination, the oxidation rate of NADPH to NADP⁺ was observed at 340 nm for GPx determination, inhibition rate of pyrogallol auto-oxidation was observed at 470 nm for SOD determination, formation of thiobarbituric acid (TBA) conjugates was observed at 535 nm for MDA determination, production of stable NO metabolite with Griess reagent was observed at 540 nm for NO determination, and degradation rate of peroxide was observed at 412 nm for MPO determination.

2.5 Histopathological analysis

A portion of the gastric tissue was fixed in formaldehyde (4%) for 24 h. The tissues were then dehydrated and paraffin embedded. Embedded tissues were sliced at 5 µm thickness, deparaffinized, and stained with hematoxylin and eosin (H&E). The samples were imaged under the light microscope. Histopathological scoring was performed according to the study by Li et al. [23] by a pathologist who was not aware of experimental groups, mucosal edema was given a score range of 0–4, loss of epithelial cells was given a score range of 1–3, infiltration of inflammatory cells was given a score range of 1–3, and hemorrhage was given a score range of 1–4.

2.6 Western blot

Western blot analysis was performed to evaluate the involvement of the NF-κβ pathway in the gastric mucosal injury induced by HCl/ethanol. Gastric tissues were homogenized using lysis buffer with protease and phosphatase inhibitor cocktail and centrifuged at 4°C for 10 min at 16,000 ×g, and the aliquot was used for western blot analysis. Aliquots with equal amounts of protein were separated through electrophoresis on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk at room temperature for 2 h in Tris-buffered saline with 0.1% Tween 20. Then, the membranes were incubated overnight with primary antibodies against Ikβα (1:1,000 dilutions; ab32518, Abcam, USA), p-Ikβα (1:1,000 dilutions; ab133462, Abcam, USA), p65 NF-κβ (ab16502, Abcam, USA; 1:1,000 dilutions), p-p65 NF-κβ (1:1,000 dilutions; ab86299, Abcam, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:500 dilutions; ab181602, Abcam, USA). The membranes were washed with TBST and further incubated with anti-rabbit horseradish peroxidase–conjugated antibody (1:5,000 dilutions; ab97051, Abcam, USA) for 1 h. Immunoreactive protein expressions were detected and quantified with the enhanced chemiluminescence system (Bio-Rad Lab version 6.0, USA).

2.7 Statistical analysis

All values are displayed as mean ± SEM for 10 rats. Statistical significance was verified with SPSS version 19.0 by performing ANOVA followed by Tukey’s post hoc analysis. All p < 0.05 were regarded as significant.

3.1 Effects of diosgenin on GSH, GPx, SOD, and CAT activities

Antioxidant enzymes and nonenzymatic GSH in the normal and experimental rats are shown in Figure 2. GSH levels were reduced significantly (approximately 59%) in the gastric model group (p < 0.05) from the control group. Similarly, levels of antioxidant enzymes were decreased (p < 0.05) approximately 54% for GPx, 42.5% for SOD, and 48% for CAT by HCl/ethanol in the gastric model group compared to normal. Compared to the model group, GSH, GPx, SOD, and CAT levels were elevated by the intragastric administration of diosgenin (p < 0.05) and by positive control omeprazole.

Figure 2

Diosgenin prevents oxidative stress in gastric ulcer-induced rats. Values are expressed as mean ± SEM of antioxidant and antioxidant enzymes in rats (n = 10). p(a) < 0.05 compared to the normal control. p(b) < 0.05 compared to the gastric ulcer model. GSH = reduced glutathione; GPx = glutathione peroxidase; SOD = superoxide dismutase; CAT = catalase.

3.2 Effects of diosgenin on MDA and NO levels

Lipid peroxidation indicator MDA and NO levels in the normal and experimental rats are shown in Figure 3. HCl/ethanol-induced gastric model rats showed elevated MDA for about fourfolds from the normal group and suppressed NO by 66% (p < 0.05). Instead, intragastric administration of diosgenin decreased MDA and elevated NO (p < 0.05) against the model group, similar to the positive control group.

Figure 3

Diosgenin prevents lipid peroxidation and nitric oxide in gastric ulcer-induced rats. Values are expressed as mean ± SEM of lipid peroxidation and nitric oxide levels in rats (n = 10). p(a) < 0.05 compared to the normal control. p(b) < 0.05 compared to the gastric ulcer model. MDA = malondialdehyde; NO = nitric oxide.

3.3 Effects of diosgenin on IL-1β, IL-6, and TNF-α levels

The levels of pro-inflammatory cytokines in normal and experimental rats are shown in Figure 4. Compared to the normal group, IL-1β, IL-6, and TNF-α were significantly elevated due to HCl/ethanol in the model group (p < 0.05). Remarkably, IL-1β, IL-6, and TNF-α were reduced (p < 0.05) due to intragastric administration of diosgenin from the HCl/ethanol-induced model group. Omeprazole administration exerted similar results as diosgenin.

Figure 4

Diosgenin prevents pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in gastric ulcer-induced rats. Values are expressed as mean ± SEM of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in rats (n = 10). p(a) < 0.05 compared to the normal control. p(b) < 0.05 compared to the gastric ulcer model.

3.4 Effects of diosgenin on MPO activities

MPO activities of the normal and experimental rats are shown in Figure 5. The activities of MPO in HCl/ethanol-induced model rats were high (p < 0.05) up to 40% from the normal group. Compared to the model group, intragastric administration of diosgenin significantly reduced MPO activities (p < 0.05) similar to the positive control group.

Figure 5

Effects of diosgenin on MPO activities in rat gastric tissue. Values are expressed as mean ± SEM of MPO activities in rats (n = 10). p(a) < 0.05 compared to the normal control. p(b) < 0.05 compared to the gastric ulcer model.

3.5 Effects of diosgenin on histopathological changes

Histopathological observations of the normal and experimental rats are shown in Figure 6. The histopathology of the HCl/ethanol-induced gastric injury model showed signs of epithelial cell loss, submucosal edema, hemorrhage, and infiltration of inflammatory cells, indicating mucosal injury compared to the normal rats, which showed normal arrangements and glandular structure of the gastric mucosa. The histopathological scores indicate the mucosal damage caused by HCl/ethanol and the protective measure by diosgenin. Diosgenin administration prevented the changes in rat gastric mucosa by reducing the damage caused by HCl/ethanol, which was comparable to the results of the omeprazole-treated group.

Figure 6

Histopathological changes due to HCl/ethanol-induced gastric mucosal injury and the protective effects of diosgenin. (a) Normal control; (b) gastric model with prominent mucosal injury; (c) diosgenin 20 mg/kg treated gastric tissue with mild mucosal injury; and (d) omeprazole 20 mg/kg treated gastric tissue with very mild mucosal injury. Magnification at 100×. (e) Histopathological scoring of gastric mucosal injury. p(a) < 0.05 compared to the normal control. p(b) < 0.05 compared to the gastric ulcer model.

3.6 Effects of diosgenin on NF-κβ activation

Western blot analysis on the HCl/ethanol-induced gastric mucosal injury-related NF-κβ pathway provided evidence that NF-κβ was activated in the gastric model group by phosphorylation of the subunits as demonstrated by the ratio of phosphorylation in Figure 7. HCl/ethanol administration induced the expression of phosphorylated p65 NF-κβ and Iκβα in the gastric model group compared to the normal control group. Diosgenin significantly suppressed the expressions of phosphorylated Iκβα and p65 NF-κβ compared to the gastric model group.

Figure 7

Western blot analysis on NF-κβ in HCl/ethanol-induced gastric mucosal injury and the protective effects of diosgenin. Phosphorylation ratio of p65 NF-κβ and IκBα was quantified and displayed as bar graphs. The bar graphs are the demonstration of three independent results of the western blot analysis, expressed as mean ± SEM. p(a) < 0.05 compared to the normal control. p(b) < 0.05 compared to the gastric ulcer model. GAPDH was used as the internal standard.