Protective effect of trillin against ethanol-induced acute gastric lesions in an animal model

Abstract

The purpose of the present study was to evaluate gastroprotective effect of trillin on mucosal lesions induced by ethanol. Mice were orally administered with trillin (25 mg kg⁻¹, 50 mg kg⁻¹, 100 mg kg⁻¹) or OME (20 mg kg⁻¹). 1 hour later, the mice were intragastrically given ethanol (0.2 ml kg⁻¹) and were sacrificed at 4 h after ethanol administration. Compared with the ethanol group, trillin treatment significantly showed increased levels of superoxide dismutase (SOD) in serum, decreased malonaldehyde (MDA) content in serum and decreased activity of myeloperoxidase (MPO) in stomach tissues, which suggested that trillin could prevent oxidative damage. Meanwhile, the serum levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) declined after pretreatment with trillin. In HE staining sections, ameliorative pathological changes of gastric lesions were markedly observed in the trillin group compared with those in the ethanol group. In the mechanistic study, the data showed that pretreatment with the trillin also effectively down-regulated protein expressions of p-NF-κBp65, p-IκBα and COX-2 in stomachs compared with those in the model group. Taken together, it could be concluded that trillin represented a potential therapeutic option to reduce the risk of gastric ulceration.

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1. Introduction

Peptic ulcer is one of the most common gastrointestinal diseases with 4–5% prevalence in human society.¹ The gastric ulcer is characterized by necrosis, infiltration of neutrophils, reduction in blood flow and secretion of inflammatory mediators.^2,3 Major etiologic factors such as Helicobacter pylori infection, excessive use of non-steroidal anti-inflammatory drugs (NSAIDs), alcohol consumption, smoking, psychological and physiological stress can lead to gastric ulcers.⁴ In particular, NSAIDs and alcohol consumption increase the risk for upper gastrointestinal bleeding.⁵ Alcohol is one of the most commonly abused drugs, therefore, alcohol-induced disorders of the gastrointestinal tract are very common. Ethanol-induced gastric injury is a commonly known method applied for the evaluation of therapeutic potencies against gastric ulcers.⁶ Among the causes of gastric lesions, one major causative factor (60% of gastric ulcers) is chronic inflammation due to Helicobacter pylori colonizing the antral mucosa.

It has been shown that infection of Helicobacter pylori could apparently increase the level of gastrin, a peptide hormone which stimulates secretion of gastric acid by the parietal cells.⁷ The main constituent of gastric acid is highly corrosive hydrochloric acid. Therefore, the increase in gastric acid stimulated by gastrin can contribute to the erosion of the mucosa leading to ulcer formation. The pH of gastric acid is usually 1.5 to 3.5 in the stomach lumen. Accordingly, the evaluation of gastric juice acid content (pH) in the stomach can reflect the development of gastric ulcer. In addition, it was suggested that inflammatory cytokines including TNF-α, IL-6 and IL-1β play pivotal roles in the acute phase inflammation as well as in maintenance and regulation of the severity of gastric ulcers.⁸ Overexpression and translocation of the NF-κB subunits (p65 and p50) to the nucleus promote the upregulation of pro-inflammatory mediators such as TNF-α, IL-1β and IL-6.⁹ Increasing evidence revealed that the inhibition of NF-κB activity might result in alleviating the severity of inflammatory diseases.¹⁰

Alternative and complementary medicine has gained global attention because of their widespread applications of Chinese traditional medicine in the field of medicine for treating many diseases.^11–13 The rhizome of Dioscorea nipponica Makino (Dioscoreae nipponicae Rhizoma or Japan Yam Rhizome) has been consumed as a herbal medicine for more than four thousand years in China. In clinical practice, Dioscoreae nipponicae Rhizoma is used for relieving cough and asthma, eliminating rheumatic aches, alleviating pain and improving blood circulation. The rhizome of Dioscorea nipponica is the major part of medicine and the identified active components are steroidal saponins including furostanol saponins and isospirostanol saponins. Saponin of Dioscorea nipponica possesses various pharmacological activities. Among them, trillin (Fig. 1), a steroidal saponin identified from Dioscorea nipponica, has been demonstrated to exert anti-inflammtory and anti-oxidative effects on LPS-induced acute lung injury.¹⁴ However, the report on other biological effects of trillin remains limited. In this study, our objective was to elucidate the effect and the molecular mechanism of trillin on ethanol-induced gastric ulcer.

Fig. 1 The chemical structure of trillin and the effect of trillin on gastric juice acid content (pH). Mice were orally administered with trillin (25 mg kg⁻¹, 50 mg kg⁻¹, 100 mg kg⁻¹) or OME (20 mg kg⁻¹). 1 hour later, the mice were intragastrically given ethanol (0.2 ml kg⁻¹) and were sacrificed at 4 h after ethanol challenge. All values given are the means ± SDs. ^##P < 0.01 vs. control group. *P < 0.05 and **P < 0.01 vs. model group.

2. Materials and methodology

2.1 Reagents

Trillin (purity > 95%) was purchased from Nanjing Ze Lang Pharmaceutical Technology Development CO., LTD (Nanjing, China). Omeprazole (OME) was supplied by Shanghai Xinyijiahua Pharmaceutical Company Limited (Shannxi, China). SOD and MPO were purchased from the Institute of Jiancheng Bioengineering (Nanjing, China). The enzyme-linked immunosorbent assay (ELISA) kits for determinations of IL-6, IL-1β and TNF-α were produced by R&D (Minneapolis, MN, USA). The anti-p-NF-κBp65, NF-κBp65, p-IκBα, IκBα, COX-2 were supplied by Cell Signaling Technology.

2.2 Animal

60 Kunming mice (18–22 g) were purchased from Jiangning Qinglongshan Animal Cultivation Farm (Nanjing, China). The animals were maintained in a temperature controlled room at 20.1–23.11 °C and 40–50% humidity, a 12 h light/dark cycle. Mice were provided with water and standard chow ad libitum. All animal procedures were performed according to protocols approved by the Animal Ethics Committee of China Pharmaceutical University (no. CPU-TCM-2013012) and animal handling followed the dictates of the National Animal Welfare Law of China.

2.3 Experimental protocol for gastric ulcer model

Mice were assigned into five groups randomly (n = 10): groups 1 and 2 were given distilled water orally; group 3 was administered omeprazole (OME) 20 mg kg⁻¹; group 4, 5 and 6 were given trillin respectively (25 mg kg⁻¹, 50 mg kg⁻¹, 100 mg kg⁻¹). An additional hour later, the mice in groups 2–6 were challenged with ethanol (0.2 ml, orally) while those in group 1 were given the same volume of distilled water orally instead. 4 h after inducement, mice were sacrificed and their serum and stomachs were harvested for further studies. Blood samples were collected for the latter estimation including SOD, MDA and pro-inflammatory cytokines. The stomach tissues were fixed in normal 10% neutral-buffered formalin for histopathological examination or stored at −70 °C for further biochemical analysis consisting of MPO and western blots. The gross evaluation of acute gastric lesion was shown in Fig. 2.

Fig. 2 Macroscopic evaluation of the effect of trillin on ethanol-induced gastric ulcer. Mice were orally administered with trillin (25 mg kg⁻¹, 50 mg kg⁻¹, 100 mg kg⁻¹) or OME (20 mg kg⁻¹). 1 hour later, the mice were intragastrically given ethanol (0.2 ml kg⁻¹) and were sacrificed at 4 h after ethanol challenge. These are representative photos from animals in group (A): control; (B): model; (C): OME (20 mg kg⁻¹); (D): trillin (25 mg kg⁻¹); (E): trillin (50 mg kg⁻¹); (F): trillin (100 mg kg⁻¹).

2.4 Measurement of gastric juice acid content (pH)

The stomachs were removed from each mouse and the gastric contents were collected and centrifuged at 4000 rpm for 10 min. The pH of the supernatant for each sample was measured using 0.1 N NaOH as titrant with a pH meter.

2.5 MPO, MDA and SOD levels

At the end of the experiment, the blood was centrifuged at 3000 rpm for 8 min and then the serum samples were stored at −80 °C for pending tests. Stomach tissues were homogenized with cold normal saline and centrifuged at 12 000 rpm for 10 min at 4 °C, the supernatant of the homogenate was collected and stored at −80 °C. The protein content of stomach sample was determined using a BCA protein assay kit. MPO activity in stomachs, MDA and SOD levels in serum were determined using test kits purchased from Nanjing Jiancheng Bioengineering Institute (China, Nanjing). All procedures were carried out according to the manual.

2.6 Cytokine measurements

The blood was centrifuged at 3000 rpm for 8 min and then the serum samples were stored at −80 °C for pending tests. Serum levels of IL-6, TNF-α and IL-1β were measured by ELISA according to the manufacturer's instructions (R&D, Minneapolis, MN, USA). Dilutions of protein standards and samples were added to 96-well ELISA plates, which was followed by the addition of a biotin-conjugated anti-cytokine antibody according to the manufacturer's instructions. The color reaction was accomplished with the substrate solution and then was terminated using the stopping solution. The optical density (OD) of each well was red at 450 nm with a microplate spectrophotometer. All measurements were performed in triplicate.

2.7 Histological procedure and assessment

For histological assessment, the stomachs were fixed in 10% neutral buffered formalin solution for 24 h, sectioned and then embedded in paraffin. The samples were deparaffinized, stained with hematoxylin–eosin (H&E) and examined under a light microscope. After that the specimens were assessed according to the criteria of Laine and Weinstein. Briefly, a 1 cm segment of each histological section was assessed for epithelial cell loss (score: 0–3), edema in the upper mucosa (score: 0–4), hemorrhagic damage (score: 0–4), and the presence of inflammatory cells (score: 0–3), yielding a maximum total score of 14. Afterward, the sections were assessed by an experienced pathologist who was blinded to the treatment.

2.8 Western blot

Stomach tissue was chopped into small pieces and homogenized in ice-cold RIPA buffer containing 0.1% phenylmethylsulfonyl fluoride. The dissolved proteins were collected from the supernatant after centrifugation at 12 000g for 20 min. Protein contents were determined BCA assay kit (Beyotime, Nanjing, China). Protein extracts were separated by a SDS-polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. The membranes were blocked with 5% skim milk in Tris buffer saline and then incubated at 4 °C overnight with respective primary antibodies for anti-NF-κBp65, p-NF-κBp65, p-IκBα, IκBα, and COX-2. After washing with tris-buffered saline–tween 20 (TBST), the membranes were incubated with a horseradish peroxidase conjugated secondary antibodies (1 : 12 000) for 1.5 h at room temperature. The antibody-reactive bands were visualized by using enhanced chemiluminescence detection reagents and a gel imaging system (Tanon Science & Technology Co., Ltd., China).

2.9 Statistical analysis

All data were normally distributed and are presented as means ± SDs. Data comparison was carried out by one-way ANOVA with Tukey multiple comparison. p values less than 0.05 were regarded to reflect a significant difference.

3. Results

3.1 Effect of trillin on gastric juice acid content (pH)

In order to evaluate the effects of change of trillin on gastric acidity in stomach, the gastric juice acid content was assessed. As illustrated in Fig. 1, the pH in the model model mice (1.30 ± 0.25) was only 40.00% of values in the control mice (3.25 ± 0.34), suggesting the induction of gastric lesion. On the contrary, the results (1.62 ± 0.17, 1.97 ± 0.26, 2.47 ± 0.19) after pretreatment with trillin (25, 50, 100 mg kg⁻¹) showed the restoration in different degree with the elevation of 1.25-fold, 1.51-fold, 1.9-fold than that of model group in a dose-dependent manner. Meantime, as expected, we got desirable effects of OME (2.83 ± 0.24) which presented the more apparent inhibition than trillin against gastric ulcer.

3.2 Effects of trillin on MPO, MDA and SOD levels

MDA and SOD are critical indexes of lipid peroxidation. MPO is a key marker of neutrophil infiltration into the gastric mucosa. Notably, the levels of MDA (52.94 ± 5.94) and MPO (4.41 ± 0.41) in the ethanol-induced model group were 4.97-fold, 3.29-fold stronger than those of control group (MDA 10.65 ± 2.42, MPO 1.34 ± 0.22), whereas its SOD activity (14.34 ± 3.30) declined to 32.65% of SOD (43.92 ± 4.62) than that of the control mice. By contrast, treatment with trillin presented dose-dependent response. Among three doses, administration of trillin at 100 mg kg⁻¹ lowered the MDA content by 36.83% (from 52.94 ± 5.94 to 33.44 ± 4.80), decreased MPO level by 41.26% (from 4.41 ± 0.41 to 2.59 ± 0.31), and improved SOD activity by 41.26% (from 14.34 ± 3.30 to 29.44 ± 3.26), respectively. However, treatment with OME was slightly more potent than the effect of trillin at the highest dose (Fig. 3).

Fig. 3 Effects of trillin on MPO, MDA and SOD levels. Mice were orally administered with trillin (25 mg kg⁻¹, 50 mg kg⁻¹, 100 mg kg⁻¹) or OME (20 mg kg⁻¹). 1 hour later, the mice were intragastrically given ethanol (0.2 ml kg⁻¹) and were sacrificed at 4 h after ethanol challenge. All values given are the means ± SDs. ^##P < 0.01 vs. control group. *P < 0.05 and **P < 0.01 vs. model group.

3.3 Effects of trillin on inflammatory cytokines in serum

We measured the levels of important inflammatory factors TNF-α, IL-6 and IL-1β in the gastric tissues to evaluate the anti-inflammatory activity of trillin (25, 50, 100 mg kg⁻¹). The levels of the these cytokines in serum were dramatically increased in model. The serum TNF-α level of model group (139.53 ± 6.79) has displayed near 2-fold higher levels than that of control group (74.01 ± 7.29) after challenge of ethanol, with much more marked elevation in IL-6 (from 31.35 ± 4.15 to 90.72 ± 5.06) and IL-1β (from 18.41 ± 3.37 to 50.80 ± 4.69) levels. Intriguingly, trillin (100 mg kg⁻¹) effectively downregulated the 35.66% of TNF-α, 30.42% of IL-6 and 38.10% of IL-1β levels of model group respectively, which has been considered to be the most significant among three doses (Fig. 4).

Fig. 4 Effects of trillin on inflammatory cytokine IL-1β, IL-6 and TNF-α in serum. Mice were orally administered with trillin (25 mg kg⁻¹, 50 mg kg⁻¹, 100 mg kg⁻¹) or OME (20 mg kg⁻¹). 1 hour later, the mice were intragastrically given ethanol (0.2 ml kg⁻¹) and were sacrificed at 4 h after ethanol challenge. All values given are the means ± SDs. ^##P < 0.01 vs. control group. *P < 0.05 and **P < 0.01 vs. model group.

3.4 Histology of gastric lesions

Gastric ulcer examination by H & E staining showed clear gastric mucosal damage. The stomach from ethanol-induced gastric ulcer more extensive damage to the gastric mucosa, edema and leucocyte infiltration compared with those of control mice, which was suggested by epithelial cell loss (from 0 to 2.67) as well as inflammatory cells score (from 0 to 2.33). However, pretreated with trillin exhibited remarkable protective activity via the reduction of the ulcer area, leucocyte infiltration and submucosal edema in dose-dependent manners. Compared with model group, pretreatment with trillin (25, 50, 100 mg kg⁻¹) showed the ameliorative effects on the epithelial cell loss (2, 1.67, 1.33) and inflammatory cells score (2, 1.67, 1.33) (Fig. 5).

Fig. 5 Effects of trillin on ethanol-stimulated histopathologic changes in stomach tissues. Mice were orally administered with trillin (25 mg kg⁻¹, 50 mg kg⁻¹, 100 mg kg⁻¹) or OME (20 mg kg⁻¹). 1 hour later, the mice were intragastrically given ethanol (0.2 ml kg⁻¹) and were sacrificed at 4 h after ethanol challenge. These are representative photos from animals in group (A): control; (B): model; (C): OME (20 mg kg⁻¹); (D): trillin (25 mg kg⁻¹); (E): trillin (50 mg kg⁻¹); (F): trillin (100 mg kg⁻¹). All values given are the means ± SDs. ^##P < 0.01 vs. control group. *P < 0.05 and **P < 0.01 vs. model group. Effects of trillin on hemorrhagic damage, edema, epithelial cell loss and inflammatory cells were presented.

3.5 Effects of trillin on the protein expressions of NF-κBp65, p-NF-κBp65, p-IκBα, IκBα and COX-2

To explore the potential mechanism of protective effect of trillin on ethanol-induced gastric mucosal damage, we assessed the phosphorylations of NF-κB, IκBα and the expression of COX-2. Pretreatment with trillin at different doses presented dose-dependent response in the regulation of signaling events, and treatment with trillin (100 mg kg⁻¹) exhibited the most potent inhibitory effect. In model group, the levels of the p-NF-κB, p-IκBα and COX-2 in stomachs were dramatically increased to 1.33-fold, 1.34-fold, 1.36-fold of those levels in control, respectively, while the pretreatment with trillin (100 mg kg⁻¹) significantly downregulated the ratio of phosphorylations of NF-κB (from 1.33 to 1.18), IκBα (from 1.34 to 1.16) and the expression of COX-2 (from 1.33 to 1.17) (Fig. 6).

Fig. 6 Effects of trillin on the expressions of p-IκBα, IκBα, p-NF-κB, NF-κB and COX-2. Mice were orally administered with trillin (25 mg kg⁻¹, 50 mg kg⁻¹, 100 mg kg⁻¹) or OME (20 mg kg⁻¹). 1 hour later, the mice were intragastrically given ethanol (0.2 ml kg⁻¹) and were sacrificed at 4 h after ethanol challenge. (A): control; (B): model; (C): OME (20 mg kg⁻¹); (D): trillin (25 mg kg⁻¹); (E): trillin (50 mg kg⁻¹); (F): trillin (100 mg kg⁻¹). All values given are the means ± SDs. ^##P < 0.01 vs. control group. *P < 0.05 and **P < 0.01 vs. model group.

4. Discussion

Trillin, a steroidal saponin identified from Dioscorea nipponica, has been demonstrated to induce multi-nucleation for mitotic arresting in human myeloblast leukemia HL-60 cells, human chronic myelogenous leukemia K562 cells and human promyelocytic leukemia NB₄ cells.¹⁵ However, seldom evidence indicated the trillin-induced multi-nucleation in gastric cell lines. Furthermore, no report focused on the pharmacological effects of trillin on peptic ulcer has been published.¹⁶ In this study, we investigated the gastroprotective activity of trillin in a model of ethanol-induced experimental gastric injury. Ethanol is associated with gastric mucosal damage and intragastrical administration of ethanol is a widely used model of peptic ulcers in mice.¹⁷ The analytical results revealed that oral administration of trillin effectively reversed ethanol-induced gastric injury. The protective activity of trillin was ascertained by comparing the gastric damage the mice in the model group with those in the treatment groups.

As the key cells in gastric tissues, stomach parietal cells are part of epithelial fundic glands of the gastric mucosa and contain an extensive secretory network where gastric acid could release to the lumen of the stomach.¹⁸ Previous research demonstrated the critical role of gastric acid in the progression of gastric ulcers.¹⁹ Thus, the suppression of gastric acid provided an important therapeutic strategy for treatment of peptic ulcers. The experimental results of the present study revealed that the treatment with trillin remarkably ameliorated that gastric acid content facilitating the erosion of the mucosa, which confirmed the protective effects of trillin in peptic ulcer.

The potential antiulcer drugs can exert their protective effect against mucosal lesions through the inhibition of neutrophil infiltration in ulcerated gastric tissue.²⁰ Furthermore, neutrophil accumulation in a gastric mucosa has been shown to induce microcirculatory abnormalities. The neutrophil infiltration into the gastric mucosal tissues is observed by the measurement of MPO activity.²¹ In the present study, we found a significant increase in MPO activity in the stomach following ethanol administration, which confirmed the activation and infiltration of neutrophils in the gastric mucosa caused by ethanol. On the other hand, our data also revealed a key role of oxidative stress in the pathogenesis of gastric ulcer stimulated by ethanol. SOD exhibits obvious physiological activities including anti-inflammatory and anti-oxidant effects in general.²² Additionally, as the end-product of polyunsaturated fatty acid, MDA is often applied as an index to evaluate the lipid peroxidation in experimental design.²³ On the contrary, pretreatment with trillin caused the significant increases in the activity of SOD, as well as the decreases in MDA and MPO levels. Thus, our study demonstrated that trillin exerted gastroprotective physiological activities alleviating neutrophil infiltration and lipid peroxidation induced by growing ROS level through an anti-oxidative system, as evidenced by the attenuation of histological conditions.

One of the pathogenesis features of peptic ulcers is the imbalances between offensive factors, such as gastric acid and protective factors including inflammatory cytokines. The inflammatory cytokine network is implicated in various ulcerogenic factors, which lead to the enhancement of inflammatory responses and the development of gastric mucosal injury.²⁴ Besides, TNF-α is involved in the induction of inflammatory lesion and carcinogenesis in a variety of tissues including the gastric mucosa.²⁵ Hasgul et al. found that TNF-α could suppress the gastric microcirculation around the ulcer and maintain the inflammatory cascade.²⁶ Moreover, overproduction of IL-6 activates neutrophils at the inflammatory site, which initiates oxidative stress and lysosomal enzymes responsible for tissue damage in peptic ulcer disorders. In addition, IL-1β contributes to the development of ethanol-induced gastric mucosal injury and the recruitment of other cytokines such as TNF-α and prostaglandins.^27,28 Meantime, accumulating evidence demonstrated that COX-2 contributed to gastric mucosal defense and the overexpression of COX-2 resulted in a delay of ulcer healing.²⁹ Above all, the possible alterations of pro-inflammatory cytokines and COX-2 expression were investigated. Our experimental results indicated that the serum pro-inflammatory cytokines levels and the gastric COX-2 expression were significantly upregulated due to the necrotizing effects of ethanol. However, the inflammatory status had been favorably reduced in the animals pretreated with trillin, which suggested that the effect of trillin was involved the inhibition in the anti-inflammatory properties. Thus, the results of present study demonstrated that trillin could inhibit the levels of inflammatory cytokines (TNF-α, IL-1β and IL-6) and the expression of COX-2, which showed its anti-inflammatory effect in ethanol-induced gastric ulcer.

It has been demonstrated that NF-κB is considered an ideal target for the molecular therapy of gastric ulcer. Protein kinase C (PKC) have been shown to play a dual role in activation of NF-κB in a number of model systems. Notably, PKCs are able to phosphorylate and activate IκB,³⁰ thus indicating its direct implication in the NF-κB activation process by participating in IκB degradation. Another mechanism by which PKCs regulate NF-κB is the phosphorylation of the p65 subunit.^31,32 In the present study, we firstly discovered that anti-inflammatory effect of trillin might be linked with the down-regulation of NF-κB p65 activity in ethanol induced gastric ulcer in mice. Western blot results displayed that the phosphorylation of NF-κB p65 significantly upregulated in ethanol-induced gastric ulcer in mice and obviously downregulated in groups pretreated with trillin. On the other hand, our experiment suggested that the levels of TNF-α, IL-1β and IL-6 mediated by NF-κB pathway the in gastric ulcer group were distinctly reduced. We presumed that trillin might inhibit early stage of inflammation and modulated upregulation of pro-inflammatory genes through the suppression of NF-κB activation. Moreover, over one hundred target genes for NF-κB have been identified during the past few years. One of them is the COX-2 gene. COX-2 mRNA expression was demonstrated at the ulcer edge during healing of chronic gastric ulcer.³³ In this study, trillin inhibited inflammation and modulated upregulation of multiple pro-inflammatory genes through the suppression of COX-2 activation.

In conclusion, the results of the present report demonstrated that trillin pre-treatment exerted beneficial effects in ethanol-induced gastric ulcer. Further researches are warranted to explore the clinical application of trillin.

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the National Twelve Five Major Drug Discovery Project (no. 2011ZX09102-0 02-01) and Natural Science Foundation of China (81302839).

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Footnote

† Tong Chen and Wenjiao Jiang contributed equally to this work.

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