Genistein ameliorated experimentally induced gastric ulcer in rats via inhibiting gastric tissues fibrosis by modulating Wnt/β-catenin/TGF-β/PKB pathway

ABSTRACT

Objectives

Gastric ulcer (GU) is a prevalent chronic digestive disease affecting about 10% of the world's population leading to gastrointestinal perforation and bleeding. Genistein is a legume flavonoid with antioxidants, anti-inflammatory and antibacterial activities. Therefore, we aimed to investigate the ability of genistein to reduce experimentally induced GU in rats by affecting gastric tissue fibrosis Wnt/β-catenin/TGF-β/SMAD4 pathway.

Methods

Thirty rats were used. Ten rats served as control, and GU was induced in twenty rats using a single dose of indomethacin (80 mg/kg) orally. Following induction of GU, ten were treated with genistein 25 mg/kg orally. The gastric tissues were isolated to investigate markers of gastric fibrosis, Wnt, β-catenin, transforming growth factor (TGF)-β, SMAD4, and Protein kinase B (PKB). In addition, gastric sections were stained with PAS and anti-TGF-β antibodies.

Results

Investigation GU micro-images revealed degeneration in both surface cells and glandular epithelial cells, which was improved by genistein. In addition, treatment with genistein significantly reduced the expression of Wnt, β-catenin, TGF-β, SMAD4, and PKB.

Conclusion

Besides antioxidant activity, genistein improves experimentally induced GU in rats, at least in part, via reduction of gastric tissue fibrosis as indicated by reduction in expression of Wnt, β-catenin, TGF-β, SMAD4, and PKB.

KEYWORDS:

• β-catenin
• gastric ulcer
• protein kinase B (PKB)
• SMAD4
• transforming growth factor (TGF)-β
• Wnt
• malondialdehyde (MDA)
• hydrogen peroxide
• catalase
• superoxide dismutase (SOD)

Introduction

Gastric ulcer (GU) is an inflammatory disorder in the gastric mucosa that results from either immune-mediated or infectious pathogens affecting the stomach and causes upper abdominal pain and nausea, most located on the lesser curvature and extending beyond the mucosa [1]. The body deals with GU through a complex healing procedure that consists of cell migration, proliferation, angiogenesis, and extracellular matrix deposition. All these processes eventually lead to the rebuilding of tissue structure and architecture inside the area of the ulcer scar. Many factors controlled this process, including cytokines, growth factors, and hormones [2]. Understanding the mechanisms underlying the mucosal defense allows for suppressing the pathogenesis of GU disease and contributes to the development of new anti-ulcer therapies.

Wnt/β-catenin is a pathway that controls a wide range of biological processes throughout the whole life of mammals. Aberration within this pathway results in many ailments such as cancer, inflammatory and immune, and metabolic diseases [3]. Extracellular Wnt activates the β-catenin-dependent, canonical signaling pathway or the β-catenin-independent, non-canonical pathway via several receptors [4]. Transforming growth factor (TGF)-β is a key profibrotic mediator in fibrotic diseases [5]. TGF-β is a multifunctional cytokine regulating many biological operations such as proliferation, differentiation, apoptosis, adhesion, migration, and wound repair [6].

Genistein is a natural isoflavone (4′, 5,7-trihydroxy isoflavone) found in soybeans and its products. It was previously documented that the antioxidant nature of isoflavones has a vital role in neutralizing free radicals and reducing the inflammatory reactions leading to the prevention of chronic diseases, namely cardiovascular diseases, osteoporosis, and hormone-related cancers [7]. Genistein is a phytoestrogen that has been reported to promote β-catenin degradation by enhancing its phosphorylation prompting suppression of the nuclear concentration of β-catenin [8]. Moreover, genistein might have potential regulatory activity on TGF signaling using SMAD as molecular targets [9]. Finally, genistein promotes dose-dependent upregulation of WIF1, an inhibitor of Wnt [10]. Therefore, we conducted this study to investigate the anti-ulcerative potential of genistein against indomethacin-induced GU by affecting gastric tissue fibrosis Wnt/β-catenin/TGF-β/SMAD4 pathway.

Methods

Animals and treatment outlines

Thirty Sprague Dawley rats, weighed 180–200 g, were used. They were kept in standard temperature conditions with a regular 12 h light/12 h dark cycle. The research protocol was approved by the Research Ethics Committee of the Faculty of Pharmacy, Delta University for Science and Technology (FPDU-REC), and approval No. (FPDU19/202) was granted. Rats were divided into three groups:

Control group. Ten rats were starved for 24 h with full access to water. Rats were given 0.5% CMC with oral gavage and left during the whole experiment without any treatment.

GU group. Twenty rats were starved for 24 h with full access to water then they were administered 80 mg/kg indomethacin orally for induction of GU. Out of these rats, ten rats were treated with the vehicle.

GU treated with genistein. After induction of GU, ten rats were treated with 25 mg/kg genistein (Sigma Aldrich Chemicals Co., St Louise, MO, U.S.A.) by oral gavage on a daily basis for seven days.

Genistein was used previously twice to attenuate indomethacin-induced gastric lesions in rats using oral 10 mg/kg [11] and oral 100 mg/kg [12]. Therefore, preliminary studies tested four different concentrations of genistein 25, 50, 75, and 100 mg/kg. The dose of 25 mg/kg was selected as it was found to produce the best results.

Sample collection

After animal sacrifice, the whole stomach was removed, measured, and weighed. Then, a piece of the stomach was fixed in 10% buffered formalin and utilized for the morphologic and Immunohistochemistry examination. Another part was homogenized in sodium-potassium phosphate buffer (pH 7.4). The supernatant was stored at –80 °C.

Morphologic analysis and immunohistochemistry

A piece of the stomach was cut into sections of five-micrometer each. Then some sections were stained with periodic acid–Schiff stain (PAS). Other sections were immune stained with monoclonal anti-transforming growth factor (TGF)-β (Sigma Aldrich Chemicals Co., St Louise, MO, U.S.A.) at 4°C as described previously by our group [13-15]. The investigations used a digital camera-aided computer system (Nikon Digital Camera, Japan).

Evaluation of oxidative stress and antioxidant activities

Gastric tissue levels of malondialdehyde (MDA), hydrogen peroxide, superoxide dismutase (SOD), and catalase were quantified using commercially available kits purchased from BioDiagnostic Co. (Giza, Egypt) using BioTek spectrophotometer, Highland, VT, U.S.A.

Enzyme-linked immunosorbent (ELISA) assay

Commercially available ELISA kits were used for β-catenin, TGF-β, SMAD4, and Protein kinase B (PKB) (MyBioSource, Inc., San Diego, CA, US) according to manufacturer’s instructions using Spectro UV-VIS Double Beam PC Scanning Spectrophotometer (Labomed Inc., Los Angeles, CA, US).

Quantitative real-time polymerase chain reaction (RT–PCR)

The gene expression of Wnt, β-catenin, TGF-β, SMAD4, and Protein kinase B (PKB) mRNA levels in rat gastric lysate performed as described previously by our group [16-18]. GAPDH was used as a housekeeping gene and internal reference control. The work was done using Applied Biosystem StepOne Plus, Foster City, CA, U.S.A. The gene-specific PCR primers used were summarized in Table 1.

Table 1. The primers set used for detection of gene expression in rats.

Name	Sequence	Reference sequence
GAPDH	Forward	5′-CCATCAACGACCCCTTCATT-3`	NM_017008.4
	Reverse	5′-CACGACATACTCAGCACCAGC-3`
Wnt	Forward	5′-CTGACCTGATGCAGACGCAAG-3`	NM_003391
	Reverse	5′-AGGAGCCACCTGTAGCTCTCATGTA-3`
β-catenin	Forward	5′-TCCGTCGCCGGTCCACACCC-3`	NM_031144.3
	Reverse	5′-TCACCAACTGGGACGATATG-3`
AKT	Forward	5′-TAGGCATCCCTTCCTTACAG-3′	NM033230
	Reverse	5′-GCCCGAAGTCCGTTATCT-3′
TGF-β1	Forward	5′-GCTAATGGTGGACCGCAACAAC-3`	NM021578
	Reverse	5′-CAGCAGCCGGTTACCAAG-3`
SMAD4	Forward	5′-GGATGAAGTCCTGCACACCA-3`	NM_019275.3
	Reverse	5′-GTTGAAGCACTGCCACCTTG-3`

Statistical analysis

Presentation of quantitative variables is done using mean ± standard error. The sample distribution normality is evaluated using Kolmogorov–Smirnov (K–S). A one-way variance analysis (ANOVA) compared the significance between the studied groups. If significance was present, the post hoc Bonferroni correction test was used. All the statistical analyses in the study were performed by SPSS version 20 (Chicago, IL, U.S.A.). Statistical significance was predefined as P < 0.05.

Results

Effect of genistein on GU

Indomethacin triggered GU in rats as indicated morphologically by the presence of mucosal hemorrhagic lesions compared to the control group (Figure 1(a)). In addition, indomethacin prompted significant reduction of gastric solution pH in rats (Figure 1(b)) and dramatically 10-fold elevated mucous production (Figure 1(c)) with a concomitant increase in stomach/body weight ratio (Figure 1(d)) as compared to control rats. However, genistein treatment ameliorated the mucosal hemorrhagic lesions and reduced the mucous production by 63% beside improving the stomach/body weight ratio as compared to GU group rats.

Figure 1. Effect of gastric ulcer (GU) and 25 mg/kg genistein on stomach morphology (a), showing normal appearance of the stomach from a control rat. The stomach of the GU rat showed mucosal hemorrhagic lesions and areas of ulceration. The stomach from the genistein group showed great reduction in hemorrhage and ulcer areas. (b) represented gastric solution pH, (c) represented mucus production and (d) represented stomach/body weight ratio (d). * Significant difference as compared with control group at p < 0.05. # Significant difference as compared with GU group at p < 0.05.

Effect of genistein on gastric tissue structure

Microsections from GU group stained with PAS exhibited marked decrease in mucosal layer (yellow arrows) as compared to the control group. In addition, genistein administration partially restored the architecture of the mucosal layer (yellow arrows) in animals treated with indomethacin indicating its potential protective effect (Figure 2).

Figure 2. Gastric sections stained with PAS showing normal pink stained mucous content in mucosal layer in control group (a), glandular gastric sections from gastric ulcer group with marked deficiency in mucosal layer as indicated by the yellow arrows (b) and partial restoration of mucosal layer in gastric ulcer group treated with genistein (c). Scale bar 100 µm.

Effect of genistein treatment on GU-induced oxidative stress

GU rats displayed a 5.56 and 4.96-fold increases in gastric MDA and hydrogen peroxide levels, respectively as compared with the control group. In addition, GU rates showed 63% and 74% reduction in gastric catalase and SOD levels, respectively, compared with the control group. However, genistein administration significantly ameliorated markers of oxidative stress (hydrogen peroxide and MDA) and restored the decrease in the antioxidant enzymes catalase and SOD levels (Figure 3).

Figure 3. Effect of gastric ulcer (GU) and 25 mg/kg genistein on gastric oxidative stress and antioxidant markers. (a) Malondialdehyde (MDA), (b) hydrogen peroxide, (c) catalase and (d) superoxide dismutase (SOD) levels. * Significant difference as compared with control group at p < 0.05. # Significant difference as compared with GU group at p < 0.05.

Effect of genistein treatment on GU-induced alteration in Wnt and β-catenin expression

As Wnt is the chief regulator of β-catenin, the gene expression of both Wnt and β-catenin have been determined to evaluate the effect of genistein in rats with GU. Figures 4(a) revealed that Wnt gene expression was significantly up regulated in GU group compared with the control group. This result was accompanied by a dramatic increase in gene expression and protein content of β-catenin (Figure 4(b,c)). Moreover, the genistein administration significantly alleviated the increases in Wnt and β-catenin expression levels compared to the corresponding GU group.

Figure 4. Effect of gastric ulcer (GU) and 25 mg/kg genistein on gene expression of Wnt (a) and β-catenin (b) as well as the gastric level of β-catenin (c). * Significant difference as compared with control group at p < 0.05. # Significant difference as compared with GU group at p < 0.05.

Effect of genistein on GU-induced expression of TGF-β1

Assessment of gastric tissues revealed significant elevations in TGF- β1 genes expression and gastric TGF-β1 levels in GU group as compared to control group and these effects were significantly reduced with genistein treatment (Figure 5(a,b)). These data were further verified by assessing TGF-β1 immune staining (Figure 5(c–e)). Immunostaining images clearly indicated elevation in gastric TGF- β1 expression in rats with GU, and genistein treatment also reduced gastric TGF- β1 immune staining (Figure 5(f)).

Figure 5. Effect of gastric ulcer (GU) and 25 mg/kg genistein on gene expression of transforming growth factor (TGF)-β (a) and its protein level in gastric tissues (b). Gastric sections stained with anti-TGF-β in control group (c), GU group (d) and GU treated with genistein (e), as well as immunohistochemistry score of positive staining (f). Blue arrows indicated areas of immunostaining. Scale bar 100 μm. * Significant difference as compared with control group at p < 0.05. # Significant difference as compared with GU group at p < 0.05.

Effect of genistein on GU-induced expression of SMAD4

Similarly, there were two to three folds significant elevations in SMAD4 expression and gastric levels in GU rats compared to control group and genistein significantly reduced these changes when compared with GU rats (Figure 6).

Figure 6. Effect of gastric ulcer (GU) and 25 mg/kg genistein on gene expression of suppressor of Mothers against Decapentaplegic (SMAD)4 (a) and its gastric protein level (b). * Significant difference as compared with control group at p < 0.05. # Significant difference as compared with GU group at p < 0.05.

Effect of genistein on GU-induced expression of PKB

As previous results, there were two to three folds significant elevations in PKB expression and gastric levels in GU rats compared to control group and genistein significantly reduced these changes when compared with GU rats (Figure 7).

Figure 7. Effect of gastric ulcer (GU) and 25 mg/kg genistein on gene expression of protein kinase B (PKB) (a) and its gastric protein level (b). * Significant difference as compared with control group at p < 0.05. # Significant difference as compared with GU group at p < 0.05.

Discussion

GU is a recognized disease with various complications, such as bleeding, perforation, and obstruction. It is mainly caused by Helicobacter pylori infection and non-steroidal anti-inflammatory drugs (NSAIDs). The incidence of GU is dramatically elevated worldwide due to using NSAIDs [19]. In parallel, new therapeutic agents for GU are expensive and may cause intense side effects. Therefore, we planned to investigate the potential therapeutic effects of garcinol against GU.

There are many animal models for induction of GU in rats. We used oral single dose of indomethacin in fasting rats, which was previously proved to cause inflammation, ulceration, and hemorrhage in the stomach [20]. We found that indomethacin resulted in morphological alteration in the stomach by presence of hemorrhage and widely distributed ulcerative areas associated with significant reduction of gastric solution pH, increased mucous production and elevation of stomach/body weight ratio. The ratio of the weight of stomach to body indicated increased weight of stomach due to increased amount of inflammation and hemorrhage in the stomach associated with a reduction in body weight due to reduced food intake and wasting because of pain and burning sensation. In addition, examination micro-sections from GU rats stained with PAS exhibited marked decrease in mucosal layer.

Most signs and symptoms of UC resulted from activation of oxidative and inflammatory pathways. Therefore, we used genistein as a therapeutic agent for treating GU as it has been reported previously to produce anti-inflammatory and antioxidant activities [7]. We found that genistein produced therapeutic effects against GU induced in rats as it produced remarkable improvement in the morphology of rats’ stomach associated with elevation in gastric solution pH and reduction in mucous production and stomach/body ratio as compared with GU rats. Therefore, we moved to the next step by trying to discover the mechanism of action of genistein in GU. Genistein was used previously to attenuate indomethacin induced GU via antioxidant, anti-inflammatory and antiapoptotic activities [11] or via enhancing the expression of iNOS [12]. Therefore, we conducted this study to investigate the protective effects of genistein via ameliorating gastric tissues fibrosis through investigation of Wnt/β-catenin/TGF-β/SMAD4 pathway.

The first pathway to be explored is Wnt/β-catenin pathway, which was proved previously to regulate many biological functions inside the body. β-Catenin acts both as a transcriptional coregulator and an adaptor protein for intracellular adhesion inside plasma membrane. Wnt is the executive controller of β-catenin. It is present in the endoplasmic reticulum then upon stimulation, it passes through the Golgi to plasma membrane and eventually secreted into extracellular space [21]. Activation of Wnt results in induction of β-catenin, which is pivotal in inflammation by its migration inside the nucleus to produce genetic effects. After translocation into the nucleus, β-catenin associates with T cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors and promotes the transcription of its target genes [22]. However, we found significant up regulation of both Wnt and β-catenin compared to control group. Although this was reversed by the administration of genistein which demonstrate improvement in ulceration through significant suppression of Wnt and β-catenin expression. In absence of activation of Wnt, β-catenin present in cytoplasm is mainly disintegrated by adenoma polyposis coli [23]. Therefore, therapeutics targeting β-catenin cellular distribution is a promising potential strategy and helps elucidating the underlying pathophysiological mechanisms for inflammation-implicated diseases and for developing more specific and effective therapeutic options against GU. Genistein was proved previously to inhibit Wnt/β-catenin pathway in bone damage induced by glucocorticoids in rats [24], tendinopathy risk increases with menopause in rats [25], cancer stem cells [26], invasion and migration of colon cancer cells in HT29 cells [10] and adipogenic differentiation in pluripotent mesenchymal stem cells [27]. However, no previous study illustrated the ability of genistein to inhibit Wnt/β-catenin pathway in GU.

We next investigate TGF-β in GU. It is a multifunctional regulatory cytokine that works as profibrotic mediator in fibrotic diseases. TGF-β/Smad pathway is a major player in fibrosis process in many animal models such as thioacetamide induce hepatocellular carcinoma in rats [28], sodium nitrite induce brain tissue damage in rats [29], and Ehrlich ascitis carinoma in mice [30]. Activation of TGF-β1 results in induction of fibrosis by enhancing both canonical or non-canonical TGF-β/Smad signaling pathways resulting in activation of myofibroblasts, production of extracellular matrix (ECM) components and inhibition of ECM destruction [31]. After activation of the signaling pathway of TGF-β/SMAD, SMAD proteins produce complex biological actions ranging from pro-fibrosis to antifibrosis. Furthermore, there are complex interactions between TGF-β/Smad and other signaling pathways [32]. Therefore, targeted inhibition of TGF-β/Smad signaling pathway can be used as a potential therapeutic goal for ameliorating GU. We evaluated TGF-β1 level and SMAD-4 content of gastric tissues and found significant overexpression of both in GU rats as compared with the control rats. However, treatment with genistein significantly suppressed TGF-β1 and SMAD4 expression compared to GU group. Genistein was reported previously to inhibit the expression of TGF-β1 in ovarian oxidative stress damage in rats [33] and d-galactosamine induced liver fibrosis in rats [34]. However, no previous study illustrated the ability of genistein to down regulate TGF-β1 in GU.

Finally, we examined AKT/protein kinase B (PKB) in GU. It consists of three isoforms, PKBα (AKT1), PKBβ (AKT2), and PKBγ (AKT3) [35]. It controls a wide range of cellular responses including cell cycle, cell proliferation, autophagy, and apoptosis. It has a role in GU by regulation of autophagy and apoptosis crosstalk mechanism. Activation of the PI3K-Akt signaling pathway is essential for cell proliferation and migration. It has been reported previously that there is an autophagic flux failure and apoptosis by activating PKB signaling pathway [36]. We found elevated expression of PKB in GU, which was ameliorated by treatment with genistein. Genistein was previously reported to regulate PKB in amyloid-β-induced toxicity [37], type 2 diabetes mellitus [38], and in human tumor cells [39]. However, no previous study illustrated the ability of genistein to regulate PKB in GU.

Conclusion and future directions

Genistein produced therapeutic effects against GU experimentally induced in rats as it restored normal gastric pH, reduced mucous production, decreased stomach/body ratio, and reduced mucosal hemorrhagic lesions. In addition, restored the architecture of the mucosal layer in rats. The protective effects of genistein could be explained by its ability to inhibit gastric tissues fibrosis via antioxidant activities and inhibition of Wnt/β-catenin/TGF-β/SMAD4 pathway. We believe that our results can be readily translated to clinical use for several reasons. Genistein is a safe natural product as illustrated by many previous studies that genistein had a low order of toxicity with a tolerated dose of up to 500 mg/kg/day [40] compared with 25 mg/kg used in our study. Genistein was given orally to rats in our research which could resemble the ability to use it for protection by eating soybeans and other legumes. Among legumes, soybeans contain the highest amount of genistein (26.8–102.5 mg/100 g dry weight), followed by arrowroot (12.6 mg/100 g dry weight) [41]. Therefore, the dose used in this research can be obtained by eating about 100–200 g of soyabeans.

Ethical approval

The research protocol was approved by the Research Ethics Committee of the Faculty of Pharmacy, Delta University for Science and Technology (FPDU-REC), and approval No. (FPDU19/202) was granted.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability

Data are available from the corresponding author upon a suitable request.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.