Immunobiology 220 (2015) 798–806
Contents lists available at ScienceDirect
Immunobiology journal homepage: www.elsevier.com/locate/imbio
Protective effect of ␦-amyrone against ethanol-induced gastric ulcer in mice Weifeng Li, Huan Yao, Xiaofeng Niu ∗ , Yu Wang, Hailin Zhang, Huani Li, Qingli Mu School of Pharmacy, Xi’an Jiaotong University, Xi’an 710061, PR China
a r t i c l e
i n f o
Article history: Received 4 November 2014 Received in revised form 10 December 2014 Accepted 22 December 2014 Available online 30 December 2014 Keywords: ␦-Amyrone Gastric ulcer Ethanol NF-B Inflammatory mediators
a b s t r a c t The purpose of this study is to examine the protective effect of ␦-amyrone on ethanol-induced gastric ulcer in mice. The mice intragastric administration 75% (0.5 mL/100 g) ethanol was pretreated with ␦amyrone (4 and 8 mg/kg) and cimetidine (100 mg/kg) or vehicles in different experimental groups for a continuous three-day, and animals were euthanized 3 h after ethanol ingestion. The gastric lesions were significantly attenuated by ␦-amyrone (4 and 8 mg/kg) as compared to the ulcer control group. Pretreatment with ␦-amyrone prevented the myeloperoxidase (MPO) activity, production of nitric oxide (NO) in serum, expression of inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-B) p65 protein expression. Analysis of cytokines in gastric tissue and serum of ethanol-induced mice showed the levels of tumor necrosis factor-alpha (TNF-␣) and interleukin-6 (IL-6) were decreased by ␦-amyrone in response to NF-B p65. These results suggested that ␦-amyrone exerts its protective effect on experimental gastric ulcer by inhibiting NF-B signaling pathways, which subsequently reduces overproduction of the inducible enzymes iNOS and suppresses the release of the inflammatory factors TNF-␣, IL-6 and NO. Thus, ␦-amyrone shows promise as a therapeutic agent in experimental gastric ulcer. © 2014 Elsevier GmbH. All rights reserved.
Introduction Gastric ulcer, which is a very common global problem today, occurs due to an imbalance between the offensive (gastric acid secretion) and defensive (gastric mucosal integrity) factors (Laine et al., 2008; Shaker et al., 2010). Gastric ulcer is a disease with many etiologies, such as free oxygen radicals, ethanol, gastric hydrochloric acid, and among them alcohol ingestion is a significant contributor to gastric ulceration (Ham and Kaunitz, 2007). There are many different experimental models of gastric ulcer induction, such as ethanol and acetic acid (Shaker et al., 2010). Ethanol-induced gastric ulcer is used as to screen the compound has the potential effect on ulcer. It is well known that ethanol is considered as a cause of gastric damage by altering protective factors, including decreasing mucus production and blood circulation within the mucosa (Choi et al., 2009). The mechanisms underlying the ethanol-induced gastric injury have not been fully clarified, but it is well known that proinflammatory mediators such as cytokines, reactive oxygen
∗ Corresponding author at: School of Pharmacy, Xi’an Jiaotong University, No. 76 Western Yanta Road, Xi’an, Shaanxi 710061, PR China. Tel.: +86 29 82655139; fax: +86 29 82655138. E-mail address: [email protected] (X. Niu). http://dx.doi.org/10.1016/j.imbio.2014.12.014 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
mediators, neutrophil infiltration are major factors which play key roles in the development of ulcer (Jainu and Devi, 2006). It has been shown neutrophil infiltration into the gastric mucosa is a critical process in the pathogenesis of various gastric ulcers and ethanolinduced neutrophil infiltration in the gastric mucosa is related to the genesis of lesions, which can be indicated by determining myeloperoxidase (MPO) activity (La Casa et al., 2000). Likewise, cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-␣) play key roles in maintenance and regulation of gastric ulcer (Liu et al., 2012). Nitric oxide (NO) pathway is a major defensive system in the gastric mucosa. NO produced by inducible NO synthase (iNOS), and previous study indicated that a decrease in iNOS activity in the gastric mucosa occur with the development of gastric mucosal lesions (Nishida et al., 1998). Nuclear factor kappa B (NF-B) is a transcription factor and binds to the B motifs in the promoters of target genes, and thus, induces the transcription of iNOS, pro-inflammatory cytokines IL-6, IL-1, TNF-␣ (Dambrova et al., 2010). Because NF-B plays a critical role in the process of inflammatory response, the effective inhibition of the pathway of NF-B regulated cytokines expression may be beneficial for the treatment of gastric ulcer. ␦-Amyrone (13(18)-oleanen-3-one) (Fig. 1), which is an active constituent extracted and separated from Sedum lineare Thunb., exhibits anti-inflammatory effects in vitro and in vivo of mice (Niu et al., 2011). Our previous study clearly suggested that ␦-amyrone
W. Li et al. / Immunobiology 220 (2015) 798–806
Fig. 1. Structure of ␦-amyrone.
is a potent inhibitor of cyclooxygenase-2 (COX-2), which may relevant to the inhibitor of the production of prostaglandin E2 (PGE2 ), and can inhibit the expression of pro-inflammatory mediators (TNF-␣, IL-6, NO) in mice models or cells (Niu et al., 2014). Although ␦-amyrone is effective in inhibiting inflammation, there has been no information on whether ␦-amyrone is therapeutic on gastric ulcer. Therefore, the purpose of this study is to investigated the potential protective effect of ␦-amyrone on gastric ulcer induced by ethanol and clarify the possible mechanism of ␦-amyrone so that can provide therapeutic alternatives for protecting gastric lesions. Materials and methods Drugs ␦-Amyrone was isolated from Sedum lineare Thunb. As a positive reagent, cimetidine (CMD) was supplied by Xi’an Lijun Pharmaceutical Limited (Shaanxi, China). The kit for biochemical analysis of MPO was purchased from Jiancheng Bioengineering Institute (Nanjing, China). The enzyme-linked immunosorbent assay (ELISA) kit for mouse IL-6 and TNF-␣ were purchased from R&D Systems (Minneapolis, MN, USA). Griess reagent was purchased from Sigma (St. Louis, MO, USA). NF-B p65 and iNOS polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyvinylidene fluoride (PVDF) membranes were purchased from Pall Gelman Laboratory (Ann Arbor, MI, USA). All other reagents used in this study were of analytical grade. Animals Male Kunming mice (22–25 g) were purchased from Experimental Animal Center, Xi’an Jiaotong University (Xi’an, China). They were housed and cared for under standard conditions with a 12 h light/dark circle and were fed with standard pallet diet and water ad libitum. All the animals were acclimatized to the laboratory conditions for a week before the experiments. All experimental procedures utilizing mice were in accordance with National Institute of Health guidelines. Ethanol-induced gastric ulcer in mice This is an extensively used model which seems to cause gastric ulcer, independently from the acid secretion. Gastric ulcerations were induced by intragastric administration of ethanol according
799
to a previously described method (Li et al., 2013). All mice were randomly divided into five groups, each consisting of twelve animals. The groups 1 (normal group) and 2 (ulcer control group) obtained the 1% carboxymethyl cellulose (CMC) (intragastric). Groups 3, 4 were administrated ␦-amyrone (4 and 8 mg/kg, dissolved in 1% CMC, intragastric), respectively. The last group received cimetidine (CMD, 100 mg/kg, dissolved in 1% CMC, intragastric). All drugs were administrated once daily for a period of 3 days. After fasting for 24 h before the experiment, mice were intragastric administration with 75% ethanol (0.5 mL/100 g body weight) to induce the acute ulcer, while the normal group obtained water only. Three hours after induction, animals were sacrificed by cervical dislocation after collecting blood through the retro-orbital plexus. Blood samples were separated by centrifuging for 10 min at 3500 rpm to obtain clear sera and frozen at −20 ◦ C before biochemical estimation. After the mice were euthanized, the stomachs were rapidly removed and opened along the greater curvature, and rinsed gently in icecold saline to remove the gastric contents. Then the stomach was stretched on a piece of blank paper with the mucosal surface facing up and was photographed with a digital camera. Thereafter, each stomach was dichotomized, with one moiety of stomach stored at −20 ◦ C for the assay of MPO or other inflammatory cytokines as described below, and the other moiety was used for histopathologic evaluation and immunohistochemical study. Determination of gastric ulcer index The degree of gastric mucosal damage was examined under a simple microscope and assessed in accordance with a modified scoring system as previously described (Salga et al., 2012). The lesion scores were made as follows: 0: no lesions; 0.5: slight hyperemia or ≤5 petechiae; 1: ≤5 erosions ≤5 mm in length; 1.5: ≤5 erosions ≤5 mm in length and many petechiae; 2: 6–10 erosions ≤5 mm in length; 2.5: 1–5 erosions >5 mm in length; 3: 5–10 erosions >5 mm in length; 3.5: >10 erosions >5 mm in length; 4: 1–3 erosions ≤5 mm in length and 0.5–1 mm in width; 4.5: 4–5 erosions ≤5 mm in length and 0.5–1 mm in width; 5: 1–3 erosions >5 mm in length and 0.5–1 mm in width; 6: 4 or 5 grade 5 lesions; 7: ≥6 grade 5 lesions; 8: complete lesion of the mucosa with hemorrhage. The sum of the total scores was divided by the numbers of animals to obtain the mean ulcer index for each group. Analysis of gastric juice and mucus production The stomach of each animal was cut along the greater curvature. The contents were carefully transferred into a clean container, then centrifuged at 4 ◦ C for 10 min and analyzed for hydrogen ion concentration using digital pH meter. The gastric mucosa of each mice was gently scraped using a glass slide, and the mucus weight measured using electronic weighing balance (Salga et al., 2012). Myeloperoxodase (MPO) assay in gastric tissue Myeloperoxodase is an enzyme found primarily in neutrophil azurophilic granules. MPO has been widely used a biochemical marker for neutrophil infiltration in the injured gastric mucosa (Costa et al., 2013) and MPO activity was determined in accordance with the method described previous (Krawisz et al., 1984) using a MPO detection kit (Nanjing Jiancheng Bioengineering Institute). The stomach tissues were accurately weighted and homogenized in 19 volumes of ice-cold physiological saline. Then homogenates were centrifuged at 4000 rpm at 4 ◦ C for 10 min. MPO activity in the supernatant was measured at 460 nm with a spectrophotometer (756pc Shanghai Spectrum Instrument Co. Ltd., China). MPO activity was expressed as units per gram of tissue.
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W. Li et al. / Immunobiology 220 (2015) 798–806
Measurement of cytokines in gastric tissue The cytokine levels of TNF-␣ and IL-6 in gastric tissues were determined by commercial ELISA kits according to the manufacturer’s instructions. The stomachs (100 mg) were homogenized in 1 mL ice-cold physiological saline and homogenates were centrifuged at 3500 rpm for 10 min at 4 ◦ C. Supernatant of homogenates or standards were added to the coated wells with a biotin-conjugated polyclonal antibody preparation specific for TNF-␣ and IL-6, and incubated for 40 min at 37 ◦ C. After washing, biotinylated antibody was added to every well and incubated at 37 ◦ C for 20 min. Then enzyme conjugated was loaded into each well and incubated at 37 ◦ C for 10 min. After washing, 100 L TMB (3,3 5,5 -tetramethylbenzidine) substrate solution was added. The reaction was stopped with H2 SO4 after incubation at 37 ◦ C for 15 min and plates were read at a wavelength of 450 nm. Experiments were performed three times. Measurement of cytokines in serum The levels of cytokines (TNF-␣ and IL-6) in serum were measured using ELISA kits for mice according to the manufacturer’s specifications. All procedures were performed as the protocol instructions strictly. The samples were analyzed at least three times. NO level in serum The NO level accumulated in serum was measured by reaction with Griess reagent (0.1% N-(1-naphthyl)ethylenediamide dihydrochloride, 1% sulfanilamide in 5% phosphoric acid) to 100 L of incubating at room temperature for 10 min, in accordance with the NO kit instructions. The absorbance at 550 nm was measured using a SpectraMax 250 microplate reader. All experiments were carried out in triplicate. A standard curve was generated using NaNO2 . Histopathological examination of gastric tissue The stomach tissues were fixed in Bouin’s fluid, dehydrated with increasing concentrations of ethanol, embedded in paraffin and sectioned. Sections (5 m thick) were mounted on slides, dewaxed, hydrated and stained with hematoxylin and eosin (H & E). All tissue sections were assessed under a light microscope. The specimens were assessed in accordance with a previous study (Li et al., 2014). 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 (score: 0–4), and the presence of inflammatory cells (score: 0–3). Immunohistochemistry for NF-B p65 The stomach tissues were fixed in Bouin’s fluid, dehydrated with increasing concentrations of ethanol, embedded in paraffin and sectioned. Sections (5 m thick) were mounted on slides. After dewaxing and rehydration in xylol and ethanol, the sections were immersed in 0.01 M citrate buffer in microwave for antigen retrieval, then cooled to room temperature. 3% H2 O2 for 10 min at 37 ◦ C was used to quench endogenous peroxidase activity. After rinsing three times with phosphate-buffered saline (PBS), sections were incubated with polyclonal antibody of NF-B p65 (dilution 1:100) at 4 ◦ C overnight. Then sections were washed with PBS and incubated with secondary antibodies for 30 min at 37 ◦ C. Whereafter, sections were rewashed with PBS and stained in 3,3 -diaminobenzidine (DAB, Sigma, St Louis, MO, USA) until a brown reaction product could be observed, followed by counterstaining with hematoxylin. Finally, the sections were dehydrated
with increasing concentration of ethanol and mounted with neutral gum. The slides were visualized under light microscope and the extent of cell immunopositivity was assessed. Western blotting analysis of gastric tissue Gastric tissues were minced, hand-homogenized in lysis buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 20 mM NaF; 0.5% NP-40; and 1% TritonX-100) containing a protease inhibitor and phosphatase inhibitor cocktail. The homogenates were centrifuged at 10,000 rpm for 10 min at 4 ◦ C, and the supernatant were collected for western blot analysis. The protein contents were quantified by a BCA assay. Prior to electrophoresis, bromophenol blue and dithiothreitol (DTT, final concentration c = 10 mM) 30 L protein from each sample were electrophoretically separated on 10% sodium dodecylsulfate-polyacylamide gel and were transferred onto PVDF membranes. The membranes were blocked for 2 h with 5% skim milk in TBST buffer (20 mM Tris, 500 mM NaCl, pH 7.5 and 1% Tween 20) at room temperature and then incubated with primary antibodies against GAPDH (diluted 1:10,000 dilutions), iNOS/NF-B p65 (diluted 1:100 in TBST) overnight at 4 ◦ C. After washing three times with TBST, the membranes were incubated with a secondary antibody (1:40,000 dilutions) conjugated with horseradish peroxidase for 1 h at room temperature and washed four times with TBST. Immunoreactive bands were developed using an enhanced chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). All western blots were repeated three times from three different experiments. Statistical analysis All statistical analyses were conducted using the GraphPad Software (California, USA). Experimental data were presented as mean ± S.E.M (standard error of mean). Statistical analysis was determined using one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. P-values of
Contents lists available at ScienceDirect
Immunobiology journal homepage: www.elsevier.com/locate/imbio
Protective effect of ␦-amyrone against ethanol-induced gastric ulcer in mice Weifeng Li, Huan Yao, Xiaofeng Niu ∗ , Yu Wang, Hailin Zhang, Huani Li, Qingli Mu School of Pharmacy, Xi’an Jiaotong University, Xi’an 710061, PR China
a r t i c l e
i n f o
Article history: Received 4 November 2014 Received in revised form 10 December 2014 Accepted 22 December 2014 Available online 30 December 2014 Keywords: ␦-Amyrone Gastric ulcer Ethanol NF-B Inflammatory mediators
a b s t r a c t The purpose of this study is to examine the protective effect of ␦-amyrone on ethanol-induced gastric ulcer in mice. The mice intragastric administration 75% (0.5 mL/100 g) ethanol was pretreated with ␦amyrone (4 and 8 mg/kg) and cimetidine (100 mg/kg) or vehicles in different experimental groups for a continuous three-day, and animals were euthanized 3 h after ethanol ingestion. The gastric lesions were significantly attenuated by ␦-amyrone (4 and 8 mg/kg) as compared to the ulcer control group. Pretreatment with ␦-amyrone prevented the myeloperoxidase (MPO) activity, production of nitric oxide (NO) in serum, expression of inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-B) p65 protein expression. Analysis of cytokines in gastric tissue and serum of ethanol-induced mice showed the levels of tumor necrosis factor-alpha (TNF-␣) and interleukin-6 (IL-6) were decreased by ␦-amyrone in response to NF-B p65. These results suggested that ␦-amyrone exerts its protective effect on experimental gastric ulcer by inhibiting NF-B signaling pathways, which subsequently reduces overproduction of the inducible enzymes iNOS and suppresses the release of the inflammatory factors TNF-␣, IL-6 and NO. Thus, ␦-amyrone shows promise as a therapeutic agent in experimental gastric ulcer. © 2014 Elsevier GmbH. All rights reserved.
Introduction Gastric ulcer, which is a very common global problem today, occurs due to an imbalance between the offensive (gastric acid secretion) and defensive (gastric mucosal integrity) factors (Laine et al., 2008; Shaker et al., 2010). Gastric ulcer is a disease with many etiologies, such as free oxygen radicals, ethanol, gastric hydrochloric acid, and among them alcohol ingestion is a significant contributor to gastric ulceration (Ham and Kaunitz, 2007). There are many different experimental models of gastric ulcer induction, such as ethanol and acetic acid (Shaker et al., 2010). Ethanol-induced gastric ulcer is used as to screen the compound has the potential effect on ulcer. It is well known that ethanol is considered as a cause of gastric damage by altering protective factors, including decreasing mucus production and blood circulation within the mucosa (Choi et al., 2009). The mechanisms underlying the ethanol-induced gastric injury have not been fully clarified, but it is well known that proinflammatory mediators such as cytokines, reactive oxygen
∗ Corresponding author at: School of Pharmacy, Xi’an Jiaotong University, No. 76 Western Yanta Road, Xi’an, Shaanxi 710061, PR China. Tel.: +86 29 82655139; fax: +86 29 82655138. E-mail address: [email protected] (X. Niu). http://dx.doi.org/10.1016/j.imbio.2014.12.014 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
mediators, neutrophil infiltration are major factors which play key roles in the development of ulcer (Jainu and Devi, 2006). It has been shown neutrophil infiltration into the gastric mucosa is a critical process in the pathogenesis of various gastric ulcers and ethanolinduced neutrophil infiltration in the gastric mucosa is related to the genesis of lesions, which can be indicated by determining myeloperoxidase (MPO) activity (La Casa et al., 2000). Likewise, cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-␣) play key roles in maintenance and regulation of gastric ulcer (Liu et al., 2012). Nitric oxide (NO) pathway is a major defensive system in the gastric mucosa. NO produced by inducible NO synthase (iNOS), and previous study indicated that a decrease in iNOS activity in the gastric mucosa occur with the development of gastric mucosal lesions (Nishida et al., 1998). Nuclear factor kappa B (NF-B) is a transcription factor and binds to the B motifs in the promoters of target genes, and thus, induces the transcription of iNOS, pro-inflammatory cytokines IL-6, IL-1, TNF-␣ (Dambrova et al., 2010). Because NF-B plays a critical role in the process of inflammatory response, the effective inhibition of the pathway of NF-B regulated cytokines expression may be beneficial for the treatment of gastric ulcer. ␦-Amyrone (13(18)-oleanen-3-one) (Fig. 1), which is an active constituent extracted and separated from Sedum lineare Thunb., exhibits anti-inflammatory effects in vitro and in vivo of mice (Niu et al., 2011). Our previous study clearly suggested that ␦-amyrone
W. Li et al. / Immunobiology 220 (2015) 798–806
Fig. 1. Structure of ␦-amyrone.
is a potent inhibitor of cyclooxygenase-2 (COX-2), which may relevant to the inhibitor of the production of prostaglandin E2 (PGE2 ), and can inhibit the expression of pro-inflammatory mediators (TNF-␣, IL-6, NO) in mice models or cells (Niu et al., 2014). Although ␦-amyrone is effective in inhibiting inflammation, there has been no information on whether ␦-amyrone is therapeutic on gastric ulcer. Therefore, the purpose of this study is to investigated the potential protective effect of ␦-amyrone on gastric ulcer induced by ethanol and clarify the possible mechanism of ␦-amyrone so that can provide therapeutic alternatives for protecting gastric lesions. Materials and methods Drugs ␦-Amyrone was isolated from Sedum lineare Thunb. As a positive reagent, cimetidine (CMD) was supplied by Xi’an Lijun Pharmaceutical Limited (Shaanxi, China). The kit for biochemical analysis of MPO was purchased from Jiancheng Bioengineering Institute (Nanjing, China). The enzyme-linked immunosorbent assay (ELISA) kit for mouse IL-6 and TNF-␣ were purchased from R&D Systems (Minneapolis, MN, USA). Griess reagent was purchased from Sigma (St. Louis, MO, USA). NF-B p65 and iNOS polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyvinylidene fluoride (PVDF) membranes were purchased from Pall Gelman Laboratory (Ann Arbor, MI, USA). All other reagents used in this study were of analytical grade. Animals Male Kunming mice (22–25 g) were purchased from Experimental Animal Center, Xi’an Jiaotong University (Xi’an, China). They were housed and cared for under standard conditions with a 12 h light/dark circle and were fed with standard pallet diet and water ad libitum. All the animals were acclimatized to the laboratory conditions for a week before the experiments. All experimental procedures utilizing mice were in accordance with National Institute of Health guidelines. Ethanol-induced gastric ulcer in mice This is an extensively used model which seems to cause gastric ulcer, independently from the acid secretion. Gastric ulcerations were induced by intragastric administration of ethanol according
799
to a previously described method (Li et al., 2013). All mice were randomly divided into five groups, each consisting of twelve animals. The groups 1 (normal group) and 2 (ulcer control group) obtained the 1% carboxymethyl cellulose (CMC) (intragastric). Groups 3, 4 were administrated ␦-amyrone (4 and 8 mg/kg, dissolved in 1% CMC, intragastric), respectively. The last group received cimetidine (CMD, 100 mg/kg, dissolved in 1% CMC, intragastric). All drugs were administrated once daily for a period of 3 days. After fasting for 24 h before the experiment, mice were intragastric administration with 75% ethanol (0.5 mL/100 g body weight) to induce the acute ulcer, while the normal group obtained water only. Three hours after induction, animals were sacrificed by cervical dislocation after collecting blood through the retro-orbital plexus. Blood samples were separated by centrifuging for 10 min at 3500 rpm to obtain clear sera and frozen at −20 ◦ C before biochemical estimation. After the mice were euthanized, the stomachs were rapidly removed and opened along the greater curvature, and rinsed gently in icecold saline to remove the gastric contents. Then the stomach was stretched on a piece of blank paper with the mucosal surface facing up and was photographed with a digital camera. Thereafter, each stomach was dichotomized, with one moiety of stomach stored at −20 ◦ C for the assay of MPO or other inflammatory cytokines as described below, and the other moiety was used for histopathologic evaluation and immunohistochemical study. Determination of gastric ulcer index The degree of gastric mucosal damage was examined under a simple microscope and assessed in accordance with a modified scoring system as previously described (Salga et al., 2012). The lesion scores were made as follows: 0: no lesions; 0.5: slight hyperemia or ≤5 petechiae; 1: ≤5 erosions ≤5 mm in length; 1.5: ≤5 erosions ≤5 mm in length and many petechiae; 2: 6–10 erosions ≤5 mm in length; 2.5: 1–5 erosions >5 mm in length; 3: 5–10 erosions >5 mm in length; 3.5: >10 erosions >5 mm in length; 4: 1–3 erosions ≤5 mm in length and 0.5–1 mm in width; 4.5: 4–5 erosions ≤5 mm in length and 0.5–1 mm in width; 5: 1–3 erosions >5 mm in length and 0.5–1 mm in width; 6: 4 or 5 grade 5 lesions; 7: ≥6 grade 5 lesions; 8: complete lesion of the mucosa with hemorrhage. The sum of the total scores was divided by the numbers of animals to obtain the mean ulcer index for each group. Analysis of gastric juice and mucus production The stomach of each animal was cut along the greater curvature. The contents were carefully transferred into a clean container, then centrifuged at 4 ◦ C for 10 min and analyzed for hydrogen ion concentration using digital pH meter. The gastric mucosa of each mice was gently scraped using a glass slide, and the mucus weight measured using electronic weighing balance (Salga et al., 2012). Myeloperoxodase (MPO) assay in gastric tissue Myeloperoxodase is an enzyme found primarily in neutrophil azurophilic granules. MPO has been widely used a biochemical marker for neutrophil infiltration in the injured gastric mucosa (Costa et al., 2013) and MPO activity was determined in accordance with the method described previous (Krawisz et al., 1984) using a MPO detection kit (Nanjing Jiancheng Bioengineering Institute). The stomach tissues were accurately weighted and homogenized in 19 volumes of ice-cold physiological saline. Then homogenates were centrifuged at 4000 rpm at 4 ◦ C for 10 min. MPO activity in the supernatant was measured at 460 nm with a spectrophotometer (756pc Shanghai Spectrum Instrument Co. Ltd., China). MPO activity was expressed as units per gram of tissue.
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W. Li et al. / Immunobiology 220 (2015) 798–806
Measurement of cytokines in gastric tissue The cytokine levels of TNF-␣ and IL-6 in gastric tissues were determined by commercial ELISA kits according to the manufacturer’s instructions. The stomachs (100 mg) were homogenized in 1 mL ice-cold physiological saline and homogenates were centrifuged at 3500 rpm for 10 min at 4 ◦ C. Supernatant of homogenates or standards were added to the coated wells with a biotin-conjugated polyclonal antibody preparation specific for TNF-␣ and IL-6, and incubated for 40 min at 37 ◦ C. After washing, biotinylated antibody was added to every well and incubated at 37 ◦ C for 20 min. Then enzyme conjugated was loaded into each well and incubated at 37 ◦ C for 10 min. After washing, 100 L TMB (3,3 5,5 -tetramethylbenzidine) substrate solution was added. The reaction was stopped with H2 SO4 after incubation at 37 ◦ C for 15 min and plates were read at a wavelength of 450 nm. Experiments were performed three times. Measurement of cytokines in serum The levels of cytokines (TNF-␣ and IL-6) in serum were measured using ELISA kits for mice according to the manufacturer’s specifications. All procedures were performed as the protocol instructions strictly. The samples were analyzed at least three times. NO level in serum The NO level accumulated in serum was measured by reaction with Griess reagent (0.1% N-(1-naphthyl)ethylenediamide dihydrochloride, 1% sulfanilamide in 5% phosphoric acid) to 100 L of incubating at room temperature for 10 min, in accordance with the NO kit instructions. The absorbance at 550 nm was measured using a SpectraMax 250 microplate reader. All experiments were carried out in triplicate. A standard curve was generated using NaNO2 . Histopathological examination of gastric tissue The stomach tissues were fixed in Bouin’s fluid, dehydrated with increasing concentrations of ethanol, embedded in paraffin and sectioned. Sections (5 m thick) were mounted on slides, dewaxed, hydrated and stained with hematoxylin and eosin (H & E). All tissue sections were assessed under a light microscope. The specimens were assessed in accordance with a previous study (Li et al., 2014). 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 (score: 0–4), and the presence of inflammatory cells (score: 0–3). Immunohistochemistry for NF-B p65 The stomach tissues were fixed in Bouin’s fluid, dehydrated with increasing concentrations of ethanol, embedded in paraffin and sectioned. Sections (5 m thick) were mounted on slides. After dewaxing and rehydration in xylol and ethanol, the sections were immersed in 0.01 M citrate buffer in microwave for antigen retrieval, then cooled to room temperature. 3% H2 O2 for 10 min at 37 ◦ C was used to quench endogenous peroxidase activity. After rinsing three times with phosphate-buffered saline (PBS), sections were incubated with polyclonal antibody of NF-B p65 (dilution 1:100) at 4 ◦ C overnight. Then sections were washed with PBS and incubated with secondary antibodies for 30 min at 37 ◦ C. Whereafter, sections were rewashed with PBS and stained in 3,3 -diaminobenzidine (DAB, Sigma, St Louis, MO, USA) until a brown reaction product could be observed, followed by counterstaining with hematoxylin. Finally, the sections were dehydrated
with increasing concentration of ethanol and mounted with neutral gum. The slides were visualized under light microscope and the extent of cell immunopositivity was assessed. Western blotting analysis of gastric tissue Gastric tissues were minced, hand-homogenized in lysis buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 20 mM NaF; 0.5% NP-40; and 1% TritonX-100) containing a protease inhibitor and phosphatase inhibitor cocktail. The homogenates were centrifuged at 10,000 rpm for 10 min at 4 ◦ C, and the supernatant were collected for western blot analysis. The protein contents were quantified by a BCA assay. Prior to electrophoresis, bromophenol blue and dithiothreitol (DTT, final concentration c = 10 mM) 30 L protein from each sample were electrophoretically separated on 10% sodium dodecylsulfate-polyacylamide gel and were transferred onto PVDF membranes. The membranes were blocked for 2 h with 5% skim milk in TBST buffer (20 mM Tris, 500 mM NaCl, pH 7.5 and 1% Tween 20) at room temperature and then incubated with primary antibodies against GAPDH (diluted 1:10,000 dilutions), iNOS/NF-B p65 (diluted 1:100 in TBST) overnight at 4 ◦ C. After washing three times with TBST, the membranes were incubated with a secondary antibody (1:40,000 dilutions) conjugated with horseradish peroxidase for 1 h at room temperature and washed four times with TBST. Immunoreactive bands were developed using an enhanced chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). All western blots were repeated three times from three different experiments. Statistical analysis All statistical analyses were conducted using the GraphPad Software (California, USA). Experimental data were presented as mean ± S.E.M (standard error of mean). Statistical analysis was determined using one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. P-values of
