Chemico-Biological Interactions 208 (2014) 18–27
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Protective effect of chelerythrine against ethanol-induced gastric ulcer in mice Wei-Feng Li a,1, Ding-Jun Hao b,1, Ting Fan a,b, Hui-Min Huang a, Huan Yao a, Xiao-Feng Niu a,⇑ a b
School of Pharmacy, Xi’an Jiaotong University, Xi’an 710061, China Xi’an Red Cross Hospital, Xi’an 710054, China
a r t i c l e
i n f o
Article history: Received 12 August 2013 Received in revised form 10 October 2013 Accepted 26 November 2013 Available online 1 December 2013 Keywords: Chelerythrine Gastric ulcer Ethanol Nuclear factor-jB
a b s t r a c t The quaternary benzo[c]phenanthridine alkaloid, chelerythrine (CHE), is of great practical and research interest because of its pronounced, widespread physiological effects, primarily antimicrobial and anti-inflammatory, arising from its ability to interact with proteins and DNA. Although CHE was originally shown to possess anti-inflammatory properties, its effects on acute gastric ulcer have not been previously explored. The aim of the present study is to evaluate the protective effect of CHE on ethanol induced gastric ulcer in mice. Administration of CHE at doses of 1, 5 and 10 mg/kg bodyweight prior to ethanol ingestion dose-dependently inhibited gastric ulcer. The gastric mucosal lesion was assessed by ulcer area, gastric juice acidity, myeloperoxidase (MPO) activities, macroscopic and histopathological examinations. CHE significantly reduced the gastric ulcer index, myeloperoxidase activities, macroscopic and histological score in a dose-dependent manner. In addition, CHE also significantly inhibited nitric oxide (NO) concentration, pro-inflammatory interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) level in serum and gastric mucosal in the mice exposed to ethanol induced ulceration in a dose-dependent manner. In addition, immunohistochemical analysis revealed that CHE markedly attenuated the overexpression of nuclear factor-jB in gastric mucosa of mice. It was concluded that CHE represents a potential therapeutic option to reduce the risk of gastric ulceration. In addition, acute toxicity study revealed no abnormal sign to the mice treated with CHE (15 mg/kg). These findings suggest that the gastroprotective activity of CHE might contribute in adjusting the inflammatory cytokine by regulating the NF-jB signalling pathway. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gastric ulcer is a common disease with multiple etiologies, defined as a discontinuity in the gastric mucosa penetration through the muscularis mucosa [1]. It was reported to be associated with the imbalance between the aggressive factors (physical, chemical or psychological) in the lumen and protective mechanisms in the duodenal mucosa represented by mucus and bicarbonate secretions, as well as by prostaglandins, sulphydryl compounds, polyamines, nitric oxide (NO) and dopamine [2], causing chronic inflammation that leads to a defect in the regulation of Abbreviations: TNF-a, tumor necrosis factor-alpha; IL-6, interleukin-6; NF-jB, nuclear factor kappa B; IjB, inhibitor of jB; CHE, chelerythrine; NO, nitric oxide; MAPK, mitogen-activated protein kinase; CMD, cimetidine; ELISA, the enzyme immunosorbent assay; DAI, disease activity index; MPO, myeloperoxidase. ⇑ Corresponding author. Address: School of Pharmacy, Xi’an Jiaotong University, No. 76 Western Yanta Road, Xi’an City, Shaanxi Province 710061, China. Tel.: +86 29 82655139; fax: +86 29 82655138. E-mail address: [email protected] (X.-F. Niu). 1 These authors have equal contribution. 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.11.011
gastrin production [3]. Gastrointestinal problems have now become a global problem, and many studies were conducted towards fixing it. The ability of some ulcer models to suppress secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a), and protect ethanol ulcer by scavenging of ROS and preventing apoptosis has provided a means for intense investigations [4,5]. It was suggested that, cytokines such as TNF-a, IL-6 and IL-10 play important roles in the acute phase inflammation as well as in maintenance and regulation of the severity of gastric ulcers [6]. Over expression and translocation of the NF-jB subunits (p65 and p50) to the nucleus promote the over expression of proinflammatory mediators such as TNF-a and IL-6 [7]. Increasing evidence reveals that the inhibition of NF-jB activity may lead to alleviating the severity of inflammatory diseases [8]. Therefore, understanding the molecular mechanisms involved in this pathway is an essential step towards countering the damaging effects of pro-inflammatory mediators in gastric ulcer. On the other hand, neutrophil infiltration into the gastric mucosa is also a critical process in the pathogenesis of a variety of gastric ulcers [9,10]. It has been shown that ethanol-induced neutrophil infiltration in the gastric mucosa
W.-F. Li et al. / Chemico-Biological Interactions 208 (2014) 18–27
is closely related to the genesis of lesions [11]. The neutrophil infiltration into the gastricmucosal tissues can be reflected by the determination of the activities of myeloperoxidase (MPO) and NO, both of which serve as key indicators of neutrophil infiltration in various experimental gastric injuries [12,13]. Chelerythrine (CHE) (Fig. 1), a quaternary benzo[c]phenanthridine alkaloid, is common in the Papaveraceae and Rutaceae families of plants. This alkaloid shows a broad range of biological activities, primarily antimicrobial and anti-inflammatory, arising from its ability to interact with proteins and DNA [14]. Mechanisms of the anti-inflammatory effect of CHE may include inhibition of 5-lipoxygenase [15], attenuation of the oxidative burst [16], and blocking the P2X7 receptor activity [17]. Besides, CHE affects various signaling pathways via the inhibition of protein kinase C and mitogen-activated protein kinase phosphatase-1 [18]. Our previous studies clearly suggested that CHE is a potent inhibitor of cyclooxygenase-2, which may be relevant to the inhibition of the release/production of exudates and prostaglandin E2 [19], and can inhibit the expression of pro-inflammatory cytokines in cultured cells and experimental models [20], implying that CHE may be developed to a potential strategy in treatment for gastric ulcers. Besides, our previous studies have also demonstrated that CHE exerted its anti-inflammatory effects by inhibiting proinflammatory cytokines (such as TNF-a) production and interfered with mitogen-activated protein kinase (MAPK) signaling pathways [20]. Since gastric ulcer is the inflammatory disease, we supposed that CHE can be used to treat gastric ulcers. Unfortunately, to date, there has been no information on whether CHE is therapeutic for gastric ulcers. Therefore, we hypothesized that CHE could exert its gastroprotective and ulcer healing actions effect on gastric ulcers by inhibiting proinflammatory cytokines production and the activation of NF-jB signaling pathway. Here, we investigated the effect of CHE on a murine model of ethanol-induced gastric ulcer in order to provide experimental evidence that CHE serves as a possible treatment for patients with gastric ulcers.
2. Materials and methods 2.1. Drugs CHEs were purchased from Xi’an Honson Biotechnology Co., Ltd. (Shannxi, China) and identified by the Pharmacognosy Laboratory, School of Medicine, Xi’an Jiaotong University (Xi’an, China). As the positive control, Cimetidine (CMD) was supplied by Shanghai Xinyi Jiahua Pharmaceutical Company Limited (Shannxi, China). The enzyme immunosorbent assay (ELISA) kit for mouse TNF-a and IL-6 was purchased from R&D Systems (Minneapolis, MN, USA). The kits for biochemical analysis of MPO and nitrites were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). NF-jB p65 polyclonal antibody and anti-Histone (H)4
Fig. 1. Structure of CHE.
19
antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Other reagents were of commercially available analytical grade. 2.2. Animals All experimental procedures utilizing mice were in accordance with National Institute of Health guidelines. Male (25–30 g) Kunming mice were obtained from the Experimental Animal Center, Xi’an Jiaotong University (Xi’an, China). They were maintained in our animal house controlled at constant temperature of 23 ± 2 °C with a relative humidity of 55 ± 5% and a 12 h light/dark cycle. The animals were allowed free access to tap water and standard laboratory chow. 2.3. Ethanol-induced gastric mucosal lesions This is a widely used model that seems to cause gastric ulcer, independently from the acid secretion. Acute gastric lesions were induced by intragastric administration of absolute ethanol in accordance with a previously described method [21]. Animals were randomized into six groups, each consisting of 12 animals. Groups 1 (normal control) and 2 (gastric ulcer control) received 0.9% saline at a dose of 50 ml/kg, groups 3, 4 and 5 were given CHE 1, 5, 10 mg/ kg, respectively, and the last group obtained cimetidine (CMD, 100 mg/kg), an antagonist of H2 receptors, was used as the reference drug. All drugs were administered once daily for 4 days. Drugs were given by gastric gavage, once daily and were suspended in saline. On the last day of treatment, 90 min after drugs administration, absolute ethanol (0.2 ml/animal) was administrated orally to all mice expect normal control group. 4 h after ethanol administration, the animals were anaesthetized with ether and blood was collected by retro orbital puncture for biochemical estimation. The animals were sacrificed by cervical dislocation, and the stomach was removed and opened along the greater curvature, and rinsed gently in PBS. The stomach was stretched on a piece of cork with the mucosal surface facing up and was examined in a standard position to assess the degree of gastric mucosal lesions. The hemorrhagic erosions in the stomach were photographed with a Lecia digital camera. The total and injured gastric lesions were measured using an image analyzer (Leica Micro systems Imaging Solutions Ltd, Cambridge, UK) and are expressed in terms of the percent (%) of the gastric area with lesions. After photo-graphing the gastric lesions, the stomach was stored at 70 °C for later biochemical analysis. 2.4. Determination of macroscopic gastric damage Immediately after the animals were killed, their stomachs were removed, cut along the greater curvature, rinsed with ice-cold isotonic saline, and the mucosal lesions were examined macroscopically. Macroscopic scoring of tissue samples was performed by an observer unaware of the treatment groups. The degree of gastric mucosal damage was evaluated from digital pictures, and rated for gross pathology according to the ulcer score scale described by Schiantarelli et al. [22] using the following scale: 0 = normal mucosa; 1 = hyperemic mucosa or up to 3 small patches; 2 = from 4 to 10 small patches; 3 = more than 10 small or up to 3 medium-sized patches; 4 = from 4 to 6 medium-sized patches; 5 = more than 6 medium-sized or up to 3 large patches; 6 = from 4 to 6 large patches; 7 = from 7 to 10 large patches; 8 = more than 10 large patches or extensive necrotic zones. ‘‘Small’’ was defined as up to 2 mm across (max. diameter), ‘‘medium-sized’’ as between 2 and 4 mm across and ‘‘large’’ as more than 4 mm across.
20
W.-F. Li et al. / Chemico-Biological Interactions 208 (2014) 18–27
2.5. Gastroprotective assessments Gastric ulcer appeared as elongated bands of hemorrhagic lesions in which the elongated lesions are the pathological features that related with ethanol induced ulcer [23]. The length (mm) and the width (mm) of each band was measured using planimeter [(10 mm 10 mm = ulcer area) under dissecting microscope (1.8)]. The area of each ulcer lesion was measured by counting the number of small squares, 2 mm 2 mm, covering the length and width of each ulcer band. The sum of the areas of all lesions for each stomach was applied in the calculation of the ulcer area (UA) wherein the sum of small squares 4 1.8 = UA mm2. The inhibition percentage (I%) was calculated by the following formula as reported with slight modifications: The inhibition percentage was calculated:
½ðUAcontrol UAtreatedÞ=UAcontrol 100%: 2.6. Measurement of gastric juice acidity and mucus production The stomach of each mice was cut along the greater curvature. Gastric contents were 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 obtained was weighed using a precision electronic balance [24]. 2.7. Histological procedure and assessment For histological assessment, the glandular stomach was fixed in 10% neutral buffered formalin solution for 24 h, sectioned, and then embedded in paraffin. Sections 5 mm thick were deparaffinized, stained with hematoxylin–eosin (H&E), and examined under a light microscope. The specimens were then assessed according to the criteria of Laine and Weinstein [25]. 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.
cold PBS (10 mM phosphate buffer pH 7.4, 150 mM NaCl), homogenized and centrifuged at 1000g for 5 min at 4 °C. After washing once with ice cold PBS, cell pellets were suspended in 200 ml low salt buffer (10 mM HEPES pH-7.9, 1.5 mM MgCl2 and 10 mM KCl), incubated for 10 min on ice, followed by addition of 20 ml of 10% NP-40 solution and vigorous mixing for 30 s. Nuclei were collected by centrifugation and resuspending in 50 ml high salt buffer (20 mM HEPES pH-7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol). Both buffers were supplemented with protease inhibitors and 0.5 mM DTT before use. Nuclei were incubated for 15 min on ice, vortexed periodically. Nuclear extracts were obtained by centrifugation at 12,500g for 10 min at 4 °C and stored at 80 °C. Tissue extracts (nuclear) were prepared by resuspending cells 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% Triton X-100) containing a protease inhibitor (Roche Diagnostics, Mannheim, Germany). Protein contents were measured using the bicinchoninic acid method (BCA protein determination kit, Pierce, distributed by KFC Chemikalien, Munchen, Germany) according to the manufacturer’s instructions using 0.2–4 mg/ml BSA as the standard (BSA, Sigma) and an optical density of 570 nm. Prior to electrophoresis, bromophenol blue and dithiothreitol (DTT, finalcon-centration n1=4 10 mM) were added to the samples. For Western blot, 20–60 ml protein from each sample was subjected to SDS polyacrylamide gel electrophoresis on a 10% gel under reducing conditions. Proteins were then transferred onto a polyvinylidene fluoride membrane by wetblotting. The membranes were blocked using 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h and then incubated with the primary antibodies (anti-NF-jB p65, 1:500; anti-Histone (H)4, 1:1000) dilutedin 1% bovineserum albumin (BSA, Sigma) in TBST for 2 h. After extensive washing (three times for 10 min each in TBST), proteins were detected using an anti-rabbit horseradish peroxidase conjugated antibody (diluted 1:15,000 in TBST), and washed again three times with TBST. Immunoreactivebands were developed using an enhanced chemiluminescencesystem (GE Healthcare, Little Chalfont, Buck inghamshire, UK). All Western blots were repeated three times from three different experiments. 2.10. Determination of NO level
2.8. Immunohistochemical analysis of gastric tissue Five lm thick sections were prepared from different animal groups and immunohistochemistry was performed as previously described [26]. Sections were deparaffinised, rehydrated, and were pre-treated in citrate buffer (pH 6.0) in microwave, then cooled to room temperature. Endogenous peroxide was blocked with 3% H2O2 for 10 min. Sections were washed with phosphate-buffered saline (PBS) at 0.01 M, blocked with 10% rabbit serum for 15 min to reduce non-specific antibody-binding and then incubated with the primary antibody (nuclear factor-jB p65, at a ratio of 1:100) at 4 °C overnight. Sections were then rinsed with PBS, reacted with secondary antibodies and a polymer helper for 20 min at 37 °C, and followed by PBS and poly-HRP anti-goat IgG for 20 min at 37 °C. Finally, the slides were rewashed with PBS and developed with 3,30 -diaminobenzidine (DAB), followed by counterstaining with hematoxylin. Sections from the control group were stained using the same method except that PBS was substituted for the first antibody. 2.9. Western blotting analysis of gastric tissue Gastric ulcerated tissues were minced, hand-homogenized and centrifuged at 1000g for 5 min at 4 °C. After washing with ice cold PBS, mouse gastric tissues from different groups was minced in ice
The level of NO in the gastric tissue and serum of the experimental mice were evaluated as total nitrate/nitrite using Griess reagent [27]. The blood samples were centrifuged at 3000 rpm for 10 min; the supernatant was transferred into several new tubes, and stored at 80 °C until analysis. The stomach homogenates in 50 mM potassium phosphate buffers (pH 7.8) were centrifuged at 4000 rpm for 10 min at 4 °C. Fifty microliters of the Griess reagent (0.1% N-(1-naphthyl) ethylenediamidedihydrochloride, 1% sulfanilamide in 5% phosphoric acid) was added to 50 ll supernatant of stomach homogenates and serum, and mixed, after 10 min; the absorbance was measured at 550 nm. The standard curves were obtained by using sodium nitrite and the results expressed as micromoles nitrate/nitrite per gram of protein. 2.11. Determination of MPO activity MPO is an enzyme found primarily in neutrophil azurophilic granules. It has been used extensively as a biochemical marker for granulocyte infiltration into various tissues, including the gastrointestinal tract. The extent of neutrophil accumulation in the gastric mucosa was measured by MPO activity evaluation as previously described [28]. Briefly, 150–200 mg of tissue was homogenized in 1 ml of potassium phosphate buffer (50 mM, pH 6.0) with 0.5% of hexadecyltrimethylammonium bromide (HTAB) for
W.-F. Li et al. / Chemico-Biological Interactions 208 (2014) 18–27
each 50 mg of tissue. Then, homogenates were centrifuged at 40,000g for 10 min at 4 °C. MPO activity in the resuspended pellet was assayed by measuring the change in absorbance at 450 nm using o-dianisidine dihydrochloride and 1% hydrogen peroxide. The results were reported as MPO units/mg of tissue. A unit of MPO activity was defined as that converting 1 lmol of H2O2 to water in 1 min at 22 °C.
21
2.12.2. Assessment of TNF-a and IL-6 in gastric tissue The sample of gastric tissue in phosphate buffered saline (PBS) with protease inhibitor cocktail (Sigma, St. Louis, MO) was homogenized, and supernatant was collected. The concentration of TNF-a and IL-6 was determined by using an ELISA. Values are expressed as pictograms of cytokines per milliliter (pg/ml). 2.13. Statistic analysis
2.12. Cytokine measurements 2.12.1. Assessment of TNF-a and IL-6 in serum At the time of mice sacrifice, to obtain serum, blood samples were centrifuged at 4000 rpm for 10 min; the supernatant were transferred into several new tubes, and stored at 80 °C until analysis. Then the levels of cytokines TNF-a and IL-6 in serum were determined using the ELISA according to the manufacturer’s instruction, respectively. In brief, the microtiter plate provided in these kits have been pre-coated with an antibody specific to TNF-a and IL-6. Serum samples and standards were added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for TNF-a and IL-6, and incubated for 2 h at 37 °C. After washing in the assay buffer, the Horseradish Peroxidase (HRP) conjugated anti-TNF-a and antiIL-6 solutions were added to each microplate well and incubated for 1 h at 37 °C. After washing, 100 ll TMB (3,30 5,50 -tetramethylbenzidine) substrate solution was added, and the reaction was stopped after incubation for 15 min at 37 °C. After adding the stop solution, plates were read at an optical density of 450 nm.
All data were expressed as mean ± S.E.M. (standard error of mean). Statistical analysis was performed using SPSS 17.0 statistical software. The statistical significance of any difference in each parameter among the groups was evaluated by one-way analysis of variance (ANOVA) followed by Tukey test. P-values of
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Protective effect of chelerythrine against ethanol-induced gastric ulcer in mice Wei-Feng Li a,1, Ding-Jun Hao b,1, Ting Fan a,b, Hui-Min Huang a, Huan Yao a, Xiao-Feng Niu a,⇑ a b
School of Pharmacy, Xi’an Jiaotong University, Xi’an 710061, China Xi’an Red Cross Hospital, Xi’an 710054, China
a r t i c l e
i n f o
Article history: Received 12 August 2013 Received in revised form 10 October 2013 Accepted 26 November 2013 Available online 1 December 2013 Keywords: Chelerythrine Gastric ulcer Ethanol Nuclear factor-jB
a b s t r a c t The quaternary benzo[c]phenanthridine alkaloid, chelerythrine (CHE), is of great practical and research interest because of its pronounced, widespread physiological effects, primarily antimicrobial and anti-inflammatory, arising from its ability to interact with proteins and DNA. Although CHE was originally shown to possess anti-inflammatory properties, its effects on acute gastric ulcer have not been previously explored. The aim of the present study is to evaluate the protective effect of CHE on ethanol induced gastric ulcer in mice. Administration of CHE at doses of 1, 5 and 10 mg/kg bodyweight prior to ethanol ingestion dose-dependently inhibited gastric ulcer. The gastric mucosal lesion was assessed by ulcer area, gastric juice acidity, myeloperoxidase (MPO) activities, macroscopic and histopathological examinations. CHE significantly reduced the gastric ulcer index, myeloperoxidase activities, macroscopic and histological score in a dose-dependent manner. In addition, CHE also significantly inhibited nitric oxide (NO) concentration, pro-inflammatory interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) level in serum and gastric mucosal in the mice exposed to ethanol induced ulceration in a dose-dependent manner. In addition, immunohistochemical analysis revealed that CHE markedly attenuated the overexpression of nuclear factor-jB in gastric mucosa of mice. It was concluded that CHE represents a potential therapeutic option to reduce the risk of gastric ulceration. In addition, acute toxicity study revealed no abnormal sign to the mice treated with CHE (15 mg/kg). These findings suggest that the gastroprotective activity of CHE might contribute in adjusting the inflammatory cytokine by regulating the NF-jB signalling pathway. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gastric ulcer is a common disease with multiple etiologies, defined as a discontinuity in the gastric mucosa penetration through the muscularis mucosa [1]. It was reported to be associated with the imbalance between the aggressive factors (physical, chemical or psychological) in the lumen and protective mechanisms in the duodenal mucosa represented by mucus and bicarbonate secretions, as well as by prostaglandins, sulphydryl compounds, polyamines, nitric oxide (NO) and dopamine [2], causing chronic inflammation that leads to a defect in the regulation of Abbreviations: TNF-a, tumor necrosis factor-alpha; IL-6, interleukin-6; NF-jB, nuclear factor kappa B; IjB, inhibitor of jB; CHE, chelerythrine; NO, nitric oxide; MAPK, mitogen-activated protein kinase; CMD, cimetidine; ELISA, the enzyme immunosorbent assay; DAI, disease activity index; MPO, myeloperoxidase. ⇑ Corresponding author. Address: School of Pharmacy, Xi’an Jiaotong University, No. 76 Western Yanta Road, Xi’an City, Shaanxi Province 710061, China. Tel.: +86 29 82655139; fax: +86 29 82655138. E-mail address: [email protected] (X.-F. Niu). 1 These authors have equal contribution. 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.11.011
gastrin production [3]. Gastrointestinal problems have now become a global problem, and many studies were conducted towards fixing it. The ability of some ulcer models to suppress secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a), and protect ethanol ulcer by scavenging of ROS and preventing apoptosis has provided a means for intense investigations [4,5]. It was suggested that, cytokines such as TNF-a, IL-6 and IL-10 play important roles in the acute phase inflammation as well as in maintenance and regulation of the severity of gastric ulcers [6]. Over expression and translocation of the NF-jB subunits (p65 and p50) to the nucleus promote the over expression of proinflammatory mediators such as TNF-a and IL-6 [7]. Increasing evidence reveals that the inhibition of NF-jB activity may lead to alleviating the severity of inflammatory diseases [8]. Therefore, understanding the molecular mechanisms involved in this pathway is an essential step towards countering the damaging effects of pro-inflammatory mediators in gastric ulcer. On the other hand, neutrophil infiltration into the gastric mucosa is also a critical process in the pathogenesis of a variety of gastric ulcers [9,10]. It has been shown that ethanol-induced neutrophil infiltration in the gastric mucosa
W.-F. Li et al. / Chemico-Biological Interactions 208 (2014) 18–27
is closely related to the genesis of lesions [11]. The neutrophil infiltration into the gastricmucosal tissues can be reflected by the determination of the activities of myeloperoxidase (MPO) and NO, both of which serve as key indicators of neutrophil infiltration in various experimental gastric injuries [12,13]. Chelerythrine (CHE) (Fig. 1), a quaternary benzo[c]phenanthridine alkaloid, is common in the Papaveraceae and Rutaceae families of plants. This alkaloid shows a broad range of biological activities, primarily antimicrobial and anti-inflammatory, arising from its ability to interact with proteins and DNA [14]. Mechanisms of the anti-inflammatory effect of CHE may include inhibition of 5-lipoxygenase [15], attenuation of the oxidative burst [16], and blocking the P2X7 receptor activity [17]. Besides, CHE affects various signaling pathways via the inhibition of protein kinase C and mitogen-activated protein kinase phosphatase-1 [18]. Our previous studies clearly suggested that CHE is a potent inhibitor of cyclooxygenase-2, which may be relevant to the inhibition of the release/production of exudates and prostaglandin E2 [19], and can inhibit the expression of pro-inflammatory cytokines in cultured cells and experimental models [20], implying that CHE may be developed to a potential strategy in treatment for gastric ulcers. Besides, our previous studies have also demonstrated that CHE exerted its anti-inflammatory effects by inhibiting proinflammatory cytokines (such as TNF-a) production and interfered with mitogen-activated protein kinase (MAPK) signaling pathways [20]. Since gastric ulcer is the inflammatory disease, we supposed that CHE can be used to treat gastric ulcers. Unfortunately, to date, there has been no information on whether CHE is therapeutic for gastric ulcers. Therefore, we hypothesized that CHE could exert its gastroprotective and ulcer healing actions effect on gastric ulcers by inhibiting proinflammatory cytokines production and the activation of NF-jB signaling pathway. Here, we investigated the effect of CHE on a murine model of ethanol-induced gastric ulcer in order to provide experimental evidence that CHE serves as a possible treatment for patients with gastric ulcers.
2. Materials and methods 2.1. Drugs CHEs were purchased from Xi’an Honson Biotechnology Co., Ltd. (Shannxi, China) and identified by the Pharmacognosy Laboratory, School of Medicine, Xi’an Jiaotong University (Xi’an, China). As the positive control, Cimetidine (CMD) was supplied by Shanghai Xinyi Jiahua Pharmaceutical Company Limited (Shannxi, China). The enzyme immunosorbent assay (ELISA) kit for mouse TNF-a and IL-6 was purchased from R&D Systems (Minneapolis, MN, USA). The kits for biochemical analysis of MPO and nitrites were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). NF-jB p65 polyclonal antibody and anti-Histone (H)4
Fig. 1. Structure of CHE.
19
antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Other reagents were of commercially available analytical grade. 2.2. Animals All experimental procedures utilizing mice were in accordance with National Institute of Health guidelines. Male (25–30 g) Kunming mice were obtained from the Experimental Animal Center, Xi’an Jiaotong University (Xi’an, China). They were maintained in our animal house controlled at constant temperature of 23 ± 2 °C with a relative humidity of 55 ± 5% and a 12 h light/dark cycle. The animals were allowed free access to tap water and standard laboratory chow. 2.3. Ethanol-induced gastric mucosal lesions This is a widely used model that seems to cause gastric ulcer, independently from the acid secretion. Acute gastric lesions were induced by intragastric administration of absolute ethanol in accordance with a previously described method [21]. Animals were randomized into six groups, each consisting of 12 animals. Groups 1 (normal control) and 2 (gastric ulcer control) received 0.9% saline at a dose of 50 ml/kg, groups 3, 4 and 5 were given CHE 1, 5, 10 mg/ kg, respectively, and the last group obtained cimetidine (CMD, 100 mg/kg), an antagonist of H2 receptors, was used as the reference drug. All drugs were administered once daily for 4 days. Drugs were given by gastric gavage, once daily and were suspended in saline. On the last day of treatment, 90 min after drugs administration, absolute ethanol (0.2 ml/animal) was administrated orally to all mice expect normal control group. 4 h after ethanol administration, the animals were anaesthetized with ether and blood was collected by retro orbital puncture for biochemical estimation. The animals were sacrificed by cervical dislocation, and the stomach was removed and opened along the greater curvature, and rinsed gently in PBS. The stomach was stretched on a piece of cork with the mucosal surface facing up and was examined in a standard position to assess the degree of gastric mucosal lesions. The hemorrhagic erosions in the stomach were photographed with a Lecia digital camera. The total and injured gastric lesions were measured using an image analyzer (Leica Micro systems Imaging Solutions Ltd, Cambridge, UK) and are expressed in terms of the percent (%) of the gastric area with lesions. After photo-graphing the gastric lesions, the stomach was stored at 70 °C for later biochemical analysis. 2.4. Determination of macroscopic gastric damage Immediately after the animals were killed, their stomachs were removed, cut along the greater curvature, rinsed with ice-cold isotonic saline, and the mucosal lesions were examined macroscopically. Macroscopic scoring of tissue samples was performed by an observer unaware of the treatment groups. The degree of gastric mucosal damage was evaluated from digital pictures, and rated for gross pathology according to the ulcer score scale described by Schiantarelli et al. [22] using the following scale: 0 = normal mucosa; 1 = hyperemic mucosa or up to 3 small patches; 2 = from 4 to 10 small patches; 3 = more than 10 small or up to 3 medium-sized patches; 4 = from 4 to 6 medium-sized patches; 5 = more than 6 medium-sized or up to 3 large patches; 6 = from 4 to 6 large patches; 7 = from 7 to 10 large patches; 8 = more than 10 large patches or extensive necrotic zones. ‘‘Small’’ was defined as up to 2 mm across (max. diameter), ‘‘medium-sized’’ as between 2 and 4 mm across and ‘‘large’’ as more than 4 mm across.
20
W.-F. Li et al. / Chemico-Biological Interactions 208 (2014) 18–27
2.5. Gastroprotective assessments Gastric ulcer appeared as elongated bands of hemorrhagic lesions in which the elongated lesions are the pathological features that related with ethanol induced ulcer [23]. The length (mm) and the width (mm) of each band was measured using planimeter [(10 mm 10 mm = ulcer area) under dissecting microscope (1.8)]. The area of each ulcer lesion was measured by counting the number of small squares, 2 mm 2 mm, covering the length and width of each ulcer band. The sum of the areas of all lesions for each stomach was applied in the calculation of the ulcer area (UA) wherein the sum of small squares 4 1.8 = UA mm2. The inhibition percentage (I%) was calculated by the following formula as reported with slight modifications: The inhibition percentage was calculated:
½ðUAcontrol UAtreatedÞ=UAcontrol 100%: 2.6. Measurement of gastric juice acidity and mucus production The stomach of each mice was cut along the greater curvature. Gastric contents were 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 obtained was weighed using a precision electronic balance [24]. 2.7. Histological procedure and assessment For histological assessment, the glandular stomach was fixed in 10% neutral buffered formalin solution for 24 h, sectioned, and then embedded in paraffin. Sections 5 mm thick were deparaffinized, stained with hematoxylin–eosin (H&E), and examined under a light microscope. The specimens were then assessed according to the criteria of Laine and Weinstein [25]. 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.
cold PBS (10 mM phosphate buffer pH 7.4, 150 mM NaCl), homogenized and centrifuged at 1000g for 5 min at 4 °C. After washing once with ice cold PBS, cell pellets were suspended in 200 ml low salt buffer (10 mM HEPES pH-7.9, 1.5 mM MgCl2 and 10 mM KCl), incubated for 10 min on ice, followed by addition of 20 ml of 10% NP-40 solution and vigorous mixing for 30 s. Nuclei were collected by centrifugation and resuspending in 50 ml high salt buffer (20 mM HEPES pH-7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol). Both buffers were supplemented with protease inhibitors and 0.5 mM DTT before use. Nuclei were incubated for 15 min on ice, vortexed periodically. Nuclear extracts were obtained by centrifugation at 12,500g for 10 min at 4 °C and stored at 80 °C. Tissue extracts (nuclear) were prepared by resuspending cells 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% Triton X-100) containing a protease inhibitor (Roche Diagnostics, Mannheim, Germany). Protein contents were measured using the bicinchoninic acid method (BCA protein determination kit, Pierce, distributed by KFC Chemikalien, Munchen, Germany) according to the manufacturer’s instructions using 0.2–4 mg/ml BSA as the standard (BSA, Sigma) and an optical density of 570 nm. Prior to electrophoresis, bromophenol blue and dithiothreitol (DTT, finalcon-centration n1=4 10 mM) were added to the samples. For Western blot, 20–60 ml protein from each sample was subjected to SDS polyacrylamide gel electrophoresis on a 10% gel under reducing conditions. Proteins were then transferred onto a polyvinylidene fluoride membrane by wetblotting. The membranes were blocked using 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h and then incubated with the primary antibodies (anti-NF-jB p65, 1:500; anti-Histone (H)4, 1:1000) dilutedin 1% bovineserum albumin (BSA, Sigma) in TBST for 2 h. After extensive washing (three times for 10 min each in TBST), proteins were detected using an anti-rabbit horseradish peroxidase conjugated antibody (diluted 1:15,000 in TBST), and washed again three times with TBST. Immunoreactivebands were developed using an enhanced chemiluminescencesystem (GE Healthcare, Little Chalfont, Buck inghamshire, UK). All Western blots were repeated three times from three different experiments. 2.10. Determination of NO level
2.8. Immunohistochemical analysis of gastric tissue Five lm thick sections were prepared from different animal groups and immunohistochemistry was performed as previously described [26]. Sections were deparaffinised, rehydrated, and were pre-treated in citrate buffer (pH 6.0) in microwave, then cooled to room temperature. Endogenous peroxide was blocked with 3% H2O2 for 10 min. Sections were washed with phosphate-buffered saline (PBS) at 0.01 M, blocked with 10% rabbit serum for 15 min to reduce non-specific antibody-binding and then incubated with the primary antibody (nuclear factor-jB p65, at a ratio of 1:100) at 4 °C overnight. Sections were then rinsed with PBS, reacted with secondary antibodies and a polymer helper for 20 min at 37 °C, and followed by PBS and poly-HRP anti-goat IgG for 20 min at 37 °C. Finally, the slides were rewashed with PBS and developed with 3,30 -diaminobenzidine (DAB), followed by counterstaining with hematoxylin. Sections from the control group were stained using the same method except that PBS was substituted for the first antibody. 2.9. Western blotting analysis of gastric tissue Gastric ulcerated tissues were minced, hand-homogenized and centrifuged at 1000g for 5 min at 4 °C. After washing with ice cold PBS, mouse gastric tissues from different groups was minced in ice
The level of NO in the gastric tissue and serum of the experimental mice were evaluated as total nitrate/nitrite using Griess reagent [27]. The blood samples were centrifuged at 3000 rpm for 10 min; the supernatant was transferred into several new tubes, and stored at 80 °C until analysis. The stomach homogenates in 50 mM potassium phosphate buffers (pH 7.8) were centrifuged at 4000 rpm for 10 min at 4 °C. Fifty microliters of the Griess reagent (0.1% N-(1-naphthyl) ethylenediamidedihydrochloride, 1% sulfanilamide in 5% phosphoric acid) was added to 50 ll supernatant of stomach homogenates and serum, and mixed, after 10 min; the absorbance was measured at 550 nm. The standard curves were obtained by using sodium nitrite and the results expressed as micromoles nitrate/nitrite per gram of protein. 2.11. Determination of MPO activity MPO is an enzyme found primarily in neutrophil azurophilic granules. It has been used extensively as a biochemical marker for granulocyte infiltration into various tissues, including the gastrointestinal tract. The extent of neutrophil accumulation in the gastric mucosa was measured by MPO activity evaluation as previously described [28]. Briefly, 150–200 mg of tissue was homogenized in 1 ml of potassium phosphate buffer (50 mM, pH 6.0) with 0.5% of hexadecyltrimethylammonium bromide (HTAB) for
W.-F. Li et al. / Chemico-Biological Interactions 208 (2014) 18–27
each 50 mg of tissue. Then, homogenates were centrifuged at 40,000g for 10 min at 4 °C. MPO activity in the resuspended pellet was assayed by measuring the change in absorbance at 450 nm using o-dianisidine dihydrochloride and 1% hydrogen peroxide. The results were reported as MPO units/mg of tissue. A unit of MPO activity was defined as that converting 1 lmol of H2O2 to water in 1 min at 22 °C.
21
2.12.2. Assessment of TNF-a and IL-6 in gastric tissue The sample of gastric tissue in phosphate buffered saline (PBS) with protease inhibitor cocktail (Sigma, St. Louis, MO) was homogenized, and supernatant was collected. The concentration of TNF-a and IL-6 was determined by using an ELISA. Values are expressed as pictograms of cytokines per milliliter (pg/ml). 2.13. Statistic analysis
2.12. Cytokine measurements 2.12.1. Assessment of TNF-a and IL-6 in serum At the time of mice sacrifice, to obtain serum, blood samples were centrifuged at 4000 rpm for 10 min; the supernatant were transferred into several new tubes, and stored at 80 °C until analysis. Then the levels of cytokines TNF-a and IL-6 in serum were determined using the ELISA according to the manufacturer’s instruction, respectively. In brief, the microtiter plate provided in these kits have been pre-coated with an antibody specific to TNF-a and IL-6. Serum samples and standards were added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for TNF-a and IL-6, and incubated for 2 h at 37 °C. After washing in the assay buffer, the Horseradish Peroxidase (HRP) conjugated anti-TNF-a and antiIL-6 solutions were added to each microplate well and incubated for 1 h at 37 °C. After washing, 100 ll TMB (3,30 5,50 -tetramethylbenzidine) substrate solution was added, and the reaction was stopped after incubation for 15 min at 37 °C. After adding the stop solution, plates were read at an optical density of 450 nm.
All data were expressed as mean ± S.E.M. (standard error of mean). Statistical analysis was performed using SPSS 17.0 statistical software. The statistical significance of any difference in each parameter among the groups was evaluated by one-way analysis of variance (ANOVA) followed by Tukey test. P-values of
