Gastroprotective effects of Machilus zuihoensis Hayata bark against acidic ethanol-induced gastric ulcer in mice

Abstract

Background and aim

In traditional medicine, Machilus zuihoensis Hayata bark (MZ) is used in combination with other medicines to treat gastric cancer, gastric ulcer (GU), and liver and cardiovascular diseases. This study aims to evaluate the gastroprotective effects and possible mechanism(s) of MZ powder against acidic ethanol (AE)-induced GU and its toxicity in mice.

Experimental procedure

The gastroprotective effect of MZ powder was analyzed by orally administering MZ for 14 consecutive days before AE-inducing GU. Ulcer index (UI) and protection percentage were calculated, hematoxylin and eosin staining and periodic acid-Schiff staining were performed, and gastric mucus weights were measured. The antioxidative, anti-inflammatory, and anti-apoptotic mechanisms, and possible signaling pathway(s) were studied.

Results and conclusion

Pretreatment with MZ (100 and 200 mg/kg) significantly decreased 10 μL/g AE-induced mucosal hemorrhage, edema, inflammation, and UI, resulted in protection percentages of 88.9% and 93.4%, respectively. MZ pretreatment reduced AE-induced oxidative stress by decreasing malondialdehyde level and restoring superoxide dismutase activity. MZ pretreatment demonstrated anti-inflammatory effects by reducing both serum and gastric tumor necrosis factor-α, interleukin (IL)-6, and IL-1β levels. Furthermore, MZ pretreatment exhibited anti-apoptotic effect by decreasing Bcl-2 associated X protein/B-cell lymphoma 2 ratio. The gastroprotective mechanisms of MZ involved inactivations of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and mitogen activated protein kinase (MAPK) signaling pathways. Otherwise, 200 mg/kg MZ didn't induce liver or kidney toxicity. In conclusion, MZ protects AE-induced GU through mucus secreting, antioxidative, anti-inflammatory, and anti-apoptotic mechanisms, and inhibitions of NF-κB and MAPK signaling pathways.

Graphical abstract

Highlights

• •MZ pretreatment alleviates the AE-induced mucosal hemorrhage.
• •MZ pretreatment increases mucosal glycoprotein accumulation and secretion of mucus.
• •MZ pretreatment reduces AE-induced inflammatory cytokine expressions.
• •MZ pretreatment effectively attenuates AE-induced Bax/Bcl-2 ratios.
• •MZ pretreatment reduces AE-induced NF-κB activation and MAPK signaling.

Abbreviations

AE

acidic ethanol

ALT

alanine aminotransferase

AST

aspartate aminotransferase

Bax

Bcl-2 associated X protein

Bcl-2

B-cell lymphoma 2

BSA

bovine serum albumin

BUN

blood urea nitrogen

CBX

carbenoxolone disodium salt

CREA

creatinine

ECL

enhanced chemiluminescent

ERK

extracellular regulated protein kinases

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GC

gas chromatography

GU

gastric ulcer

H&E

hematoxylin and eosin

HRP

horseradish peroxidase

IL

interleukin

JNK

c-Jun N-terminal kinase

LPS

lipopolysaccharides

MAPK

mitogen activated protein kinase

MDA

malondialdehyde

MS

mass spectrometry

MZ

Machilus zuihoensis Hayata bark

NBF

neutral buffered formalin

NF-κB

nuclear factor kappa-light-chain enhancer of activated B cells

OFR

oxygen free radicals

PAGE

polyacrylamide gel electrophoresis

PAS

periodic acid-Schiff

PBS

phosphate buffered saline

PCR

polymerase chain reaction

PU

peptic ulcer

RO

reverse osmosis

ROS

reactive oxygen species

SDS

sodium dodecyl sulfate

SOD

superoxide dismutase

TBST

Tris-buffered saline with 0.1% Tween 20

TNF-α

tumor necrosis factor-α

Taxonomy (classification by EVISE)

identify the disease/health condition: gastric ulcer.

the experimental approach: in vivo studies.

the methodology: The gastroprotective effect of MZ powder was analyzed by orally administering MZ for 14 consecutive days before AE-inducing GU. Ulcer index and protection percentage were calculated, hematoxylin and eosin staining and periodic acid-Schiff staining were performed, and gastric mucus weights were measured. The antioxidative, anti-inflammatory, and anti-apoptotic mechanisms, and possible signaling pathway(s) were studied.

1. Introduction

Approximately 4 million people suffer from peptic ulcer (PU) each year.¹ It is estimated that 5–10% of people will develop PU during their lifetime and the annual incidence is about 0.1–0.3%.² PU can be classified as gastric ulcer (GU) and duodenal ulcer. GU is caused by rupture of the gastric wall mucosal barrier, which penetrates the muscularis mucosa. The pathogenesis of GU is multifactorial and has not been completely elucidated. Known factors include smoking, hydrochloric acid (HCl), ischemia, non-steroidal anti-inflammatory drugs, hypoxia, alcohol, and Helicobacter pylori infection. Otherwise, gastrointestinal (GI) mucosa can be protected with bicarbonate, prostaglandin (PG), mucus, growth factors, and adequate blood flow.³

Current clinical drugs for treating GU mainly include proton pump inhibitors, antacids, antibiotics, and antihistamines, that usually cause serious side effects such as GI reactions, hepatotoxicity, nephrotoxicity, hypergastrinemia, and nerve damage.⁴ However, there is still no complete effective treatment for GU. Therefore, different GU animal models have been used to screen new therapeutic components for protecting the gastric mucosa.⁵

It has been reported that alcohol can quickly erode gastric mucosa to induce GU. High concentrations of ethanol can cause mucosal membrane damage and increase permeability of gastric acid and infiltration of neutrophils, leading to GU.⁶ The ethanol-induced functional alterations and morphologic changes in animal models, such as abnormal secretion of gastric acid and mucus, cause hemorrhagic and necrotic foci in gastric mucosa, resembling those in human GU patients.⁵ The mechanisms of ethanol-induced gastric lesions include inflammatory response, oxidative stress, and apoptosis.^7,8 Thus, ethanol-induced GU animal models were established for the assessment of potential medicines with gastroprotective effects and reduced side effects. Furthermore, acidic ethanol (AE) was also used to induce GU by increased oxidative stress and corrosive damage to gastric mucus. Traditional medicinal herbs and their ingredients from Korea, Japan, and China have been used to treat GU by way of anti-apoptotic, antioxidative, and anti-inflammatory activities in ethanol- or AE-induced animal models.^5,9 Many studies have shown that the molecular mechanisms of protecting ethanol-induced GU are through MAPK and NF-κB signaling pathways in animal models.10, 11, 12

The genus Machilus includes evergreen species found in Southeast Asia. Machilus zuihoensis Hayata bark (MZ) was originally endemic to Taiwan and spread to Southeast Asia.¹³ MZ contains mucilage and is used as a raw material for making incense and in some ancient folk remedies including incense ash. According to the Complete Essentials of the Materia Medica, MZ powder promotes the secretion of digestive juices and eliminates GI gas.¹⁴ In traditional medicine, MZ has been found to be extremely effective for relieving gastroesophageal reflux, abdominal pain, flatulence, and stomach pain. Furthermore, in traditional Chinese medicine, MZ is used in combination with others to treat gastric cancer, GU, and liver and cardiovascular diseases. The in vitro functions of MZ include the leaf extract showing anti-inflammatory and neutralizing free radical effects on RAW264.7 macrophages¹⁵, the extract from leaf and stem exhibiting cytotoxic effects on cancer cell lines¹⁶, and the root extract observing anti-inflammatory activity.¹⁷ However, there was no scientific report for the in vivo function of MZ in both animal and human. The aim of this study is to elucidate the gastroprotective effect and mechanism(s) of MZ against AE-induced GU in mice.

2. Materials and methods

2.1. Chemicals

Carbenoxolone sodium (CBX) was purchased from UNI-ONWARD Corp. (New Taipei, Taiwan). H&E staining kit was purchased from Rapid Science Co., Ltd. (Taichung, Taiwan). PAS staining kit, HCl, ethanol, acetonitrile, and formic acid were supplied by Sigma-Aldrich (St. Louis, MO, USA). MDA and SOD detection kits were purchased from Elabscience Biotechnology Inc. (Bethesda, MD, USA). TNF-α, IL-1β, and IL-6 ELISA kits were supplied by Invitrogen Inc. (Carlsbad, CA, USA). Primary antibodies to NF-κB p65 (GTX102090), p38 (GTX110520), and phosphor-p38 (GTX486146) were obtained from GeneTex Inc. (Irvine, CA, USA). Primary antibodies to Bcl-2 (#2876), Bax (#2772), ERK1/2 (#9102), phospho-ERK1/2 (#9101), JNK (#9252), phospho-NF-κB p65 (#3033), and GAPDH (#3033) were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Primary antibodies to phosphor-JNK (sc-6254) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Goat anti-mouse conjugated HRP and goat anti-rabbit conjugated HRP antibodies were purchased from Abcam PLC (Cambridge, UK). ECL solution was supplied by Biokit Biotechnology Inc. (Miaoli, Taiwan).

2.2. Source and authentication of MZ

MZ powder was purchased from Indonesia North Sumatra Trading Company (Cat. No. 2021201000) and called Top Nanxiang. This medicinal material was appraised by the Changhua County Chinese Medicine Merchants Association and pharmacists at Yongsheng Chinese Medicine Company and Yueshi Pharmacy.

The MZ powder (1 g) was extracted with 10 mL of ethyl acetate through ultrasonic oscillation at 25 °C for 20 min. Solution was concentrated under reduced pressure, and the concentrated material was resolved in 1 mL of ethyl acetate. The Shimadzu GC/MS analysis system consisted of a Shimadzu GC-2010 gas chromatograph, Shimadzu GCMS-QP2010 mass spectrometer and Shimadzu AOC-20i + s autosampler. A Rtx-5MS (30 m × 0.25 mm ID × 0.25 μm df, Restek) was used as stationary phase and helium was the carrier gas. The total program time was 40 min with stating at 40 °C for 2 min and increasing temperature at 5 ^°C/min. The injection volume was 1 μL and mass range was 45–450 m/z. Results were identified by NIST17/FFNSC1.2/WILEYB Mass Spectral Library with Similarity index (SI) over 90 (Fig. 1). The relative concentration of each component (peak) was calculated by dividing the peak area with the total area multiplied by 100%.

Fig. 1.

(A) Macroscopic gross lesions of the gastric mucosa. PBS treated mice (WT), mice treated with 200 mg/kg MZ only (MZ200), mice treated with AE only (AE), mice pretreated with 100 mg/kg MZ before AE treatment (MZ100+AE), mice pretreated with 200 mg/kg MZ before AE treatment (MZ200+AE), and mice pretreated with 100 mg/kg CBX before AE treatment (CBX + AE). (B) Ulcer indexes (left) and protection percentages (right) were calculated for each group. Mice treated with 100 mg/kg MZ (MZ100), 200 mg/kg MZ (MZ200) and 100 mg/kg CBX (CBX), respectively, then treated with AE were underlined. ∗∗∗ (p < 0.001) denotes significant difference compared with the AE group.

2.3. Preparations of MZ, CBX, and AE

Twenty-five milligrams of MZ powder were completely dissolved in 1 mL PBS using a mortar for grinding. CBX was also dissolved in PBS. AE was prepared in a final concentration of 0.3 M HCl and 50% ethanol.

2.4. Animals and experimental design

Five-week-old male BALB/cByJNarl mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All mice were maintained in the Animal Center of Chung Shan Medical University under 12 h light/dark cycle, at 22–24 °C and 50–60% humidity, and supplied with RO water and rodent diet 5001 ad libitum. All animal protocols were followed according to the guidelines of the institutional animal committee of Chung Shan Medical University with protocol number 2354.

Mice were acclimatized in the Animal Center for more than 1 week. Before the start of experiments, they were fasted for 12 h and weighed ≥25g. Mice were randomly divided into six groups (n = 5 for each group): PBS treatment group (WT), 200 mg/kg MZ toxicity group (MZ200), AE treatment group (AE), low (100 mg/kg, MZ100+AE) and high (200 mg/kg, MZ200+AE) doses of MZ treatment groups, and 100 mg/kg CBX treatment group (CBX + AE). Since CBX is one of clinical drugs to treat GU, the CBX treatment group was used as a positive control group in this experiment.¹⁸ MZ or an equal volume of PBS was administered every day for 14 days. On the 15th day, GU was induced 1 h after PBS, MZ, and CBX treatments, respectively. All treatments were orally.

2.5. AE-induced GU in mice

Mice were fasted for 12 h and water was withheld for 2 h before administration of AE to induce GU. Except for the WT and MZ200 groups, mice were administered 10 μL/g AE to induce GU. Two hours later, all mice were anesthetized and sacrificed by overdose of isoflurane (Baxter, CA, USA). Blood was immediately collected by cardiac puncture. Stomach, the right lobe of liver, and kidneys were isolated and stored at −80 °C for biochemical and Western blot analyses.

2.6. Evaluations of gastric mucosal lesions

After each mouse was sacrificed, the stomach was removed and untied along the greater curvature. Images of the stomach were captured by digital camera. Total gastric mucosal area and ulcer area were calculated by ImageJ software. Ulcer index (UI) and protection percentage were calculated by the following formulas¹⁹:

$$\mathrm{U}\mathrm{I}=\mathrm{u}\mathrm{l}\mathrm{c}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}/\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{g}\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{c}\mathrm{m}\mathrm{u}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}\ast 100\%$$

$$\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}=\left[({\mathrm{U}\mathrm{I}}_{\mathrm{A}\mathrm{E}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}–{\mathrm{U}\mathrm{I}}_{\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}})/{\mathrm{U}\mathrm{I}}_{\mathrm{A}\mathrm{E}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}\right]\ast 100\%$$

2.7. H&E staining of stomach, kidney, and liver sections

The stomach, kidney, and the right lobe of liver were fixed in 10% neutral buffered formalin (NBF) and then embedded in paraffin. Gastric, hepatic, and renal tissues were cut into 3 μm sections and stained with H&E. Results were scanned to create digitalized images with TissueFAXS Plus cytometry and TissueFAXS 4.0 Viewer software (TissueGnostics GmbH, Vienna, Austria).

2.8. PAS staining of stomach sections

To evaluate gastric mucus distribution, mucosal glycoproteins were observed via PAS staining. Stomach was fixed in 10% NBF and embedded in paraffin. Gastric tissue was cut into 5 μm sections then stained with PAS. PAS staining scores were calculated by ImageJ software using the following formula: PAS staining score = brick-red area/total area ∗ 100%

2.9. Gastric mucus weights

Chyme of stomach in each mouse was removed with a clip. The mucosa of stomach was scraped slightly using histological glass slide with the mucus adhering to the slide. Mucus was weighed using electronic balance.²⁰

2.10. Preparations of stomach homogenates and measurements of MDA levels and SOD activities

Stomach homogenates (10% (w/v)) were prepared by homogenization with ice-cold PBS containing 1.15% (w/v) potassium chloride using tissue homogenizer (Polytron, Heidolph RZR 1, Germany), followed by centrifugation at 10,000 g (4 °C) for 10 min with refrigerated centrifuge Rotofix 32 (Hettich Zentrifugen, Germany) to collect supernatants. Protein concentrations of supernatants were measured by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). MDA levels and SOD activities in stomach homogenates were measured by MDA kit and SOD kit, respectively.

2.11. Measurements of inflammatory cytokine levels in both serum and liver

Blood samples were allowed to sit at 37 °C for 30–40 min before centrifugation (3,000 g, at 4 °C for 10 min) to collect sera and then stored at −80 °C until use. Liver homogenates (10% (w/v)) were prepared by homogenization with ice-cold PBS using tissue homogenizer, then centrifuged at 10,000 g (4 °C) for 20 min to collect supernatants. Protein concentrations of supernatants were measured by Bio-Rad Protein Assay. Serum and hepatic TNF-α, IL-1β, and IL-6 levels were measured using TNF-α, IL-1β, and IL-6 ELISA kits, respectively.

2.12. Measurements of gastric mRNA levels of inflammatory cytokines by real-time PCR

Total mRNA was extracted from each stomach by Trizol reagent (Life Technologies Corp., Carlsbad, CA, USA) and then reverse transcribed using Superscript II system (Life Technologies) and oligo-dT primer. Levels of TNF-α, IL-1β, IL-6, and GAPDH mRNAs were detected by real-time PCR assays conducted on a Bio-Rad CFX Real-Time PCR Detection System with labeling by Fast SYBR Green Master Mix (Thermo Fisher Scientific). The primers used are listed in Supplementary Table 1. GAPDH mRNA level was used as an internal control. Threshold cycle (Ct) values were acquired by accompanying software. The relative ratios of mRNA levels were quantified using the comparative 2(−ΔΔCt) method and normalized against GAPDH levels. The relative TNF-α, IL-6, and IL-1β mRNA expression levels in the WT group were all defined as 1.0.

2.13. Measurements of gastric protein levels by Western blotting

Stomach proteins were prepared by homogenization with RIPA buffer (Sigma-Aldrich), then centrifuged (10,000 g, at 4 °C for 20 min) to collect supernatants. Protein concentrations of supernatants were measured by Bio-Rad Protein Assay. Sixty micrograms of proteins were separated by 12% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% BSA in TBST, then incubated with primary antibodies against Bax, Bcl-2, NF-κB p65, phospho-NF-κB p65, JNK, phosphor-JNK, ERK, phosphor-ERK, p38, phosphor-p38 and GAPDH, respectively. After washed with TBST, membranes were incubated with secondary antibodies. After washed with TBST, signals on membranes were visualized with ECL solution and intensities of bands were quantified using AlphaEase FC software 6.0 (BIOZ, Los Altos, CA, USA). The levels of GAPDH were served as loading controls for normalization of band densities.

2.14. Measurements of serum biochemical parameters

Serum samples were sent to the National Laboratory Animal Center for analyses of AST and ALT activities, and BUN and CREA levels.

2.15. Statistical analyses

At least three independent experiments were performed. Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using Microsoft Office Excel 2016 software (Microsoft Crop., Redmond, WA, USA) and Student's t-test for comparing results of MZ200 group with WT group in Fig. 7, and using SPSS for Windows, version 25 (SPSS, Inc., Chicago, IL, USA) and one-way ANOVA followed by Tukey post hoc comparison for other results. A value of P < 0.05 indicated statistical significance.

Fig. 7.

(A) Histological examination of hepatic (upper) and renal (lower) tissues by H&E staining. Scale bars: 200 μm. Serum liver (B) and kidney (C) biochemical markers, and hepatic TNF-α (left) and IL-6 (right) levels (D). PBS treated mice (WT) and mice treated with 200 mg/kg MZ only (MZ200). ∗ (p < 0.05) denotes significant difference compared with the WT group.

3. Results and discussions

3.1. GC/MS fingerprint profile of MZ extract

As shown in Supplementary Figs. 1 and 11 major peaks of the MZ extract were identified by GC/MS. The retention time, compound name and relative concentration were displayed in Supplementary Table 2. The major components were β-caryophyllene epoxide (10.84%), β-caryophyllene (8.67%), β-guaiene (5.64%), and spathulenol (5.14%). These components that were similar to those in leaf oils from M. thunbergii²¹, M. kusanoi²², M. konishii²³ and M. obovatifolia²⁴ strongly supported the powder from MZ bark. Among the major components, β-caryophyllene epoxide, β-caryophyllene and spathulenol are three sesquiterpenes that are known to have anti-inflammatory and gastric protective effects.25, 26, 27 Some essential oils or herbal extracts that have protective effects on the stomach contain a high proportion of β-caryophyllene epoxide and/or spathulenol in their main components.28, 29, 30, 31

3.2. Effects of MZ on AE-induced GU

In ethanol-induced animal models, ethanol rapidly penetrates gastric mucosa causing membrane damage, cell exfoliation, and erosion. Due to increased permeability of the cell membrane by HCl, AE further deepens the necrosis and increases tissue injury by increasing oxidative stress and corrosive damage to gastric mucus. According to the literature, the change in appearance of gastric mucosa in AE model is more obvious than that in absolute ethanol model.⁹ Our results show that AE induces obvious dark hemorrhagic necrosis of mucosa (Fig. 1A, AE) which is consistent with the findings of previous studies.⁵ Gastric mucosa was covered in mucosal blood with extensive hemorrhagic necrosis (Fig. 1, AE). The severities of hemorrhagic necrosis were greatly reduced in MZ (MZ100+AE, MZ200+AE) and CBX (CBX + AE) pretreatment groups in comparison with the AE group. Ulcer indexes were significantly reduced in all MZ and CBX pretreatment groups when compared with the AE group (Fig. 1B, left panel). Pretreatment with MZ100, MZ200, and CBX resulted in protection percentages of 88.9%, 93.4% and 88.5%, respectively (Fig. 1B, right panel). Results of Fig. 1 show that MZ100 and MZ200 pretreatments can effectively reduce the gastric injury induced by AE.

According to previous results of GC/MS fingerprint profiles of MZ extract, the first two main components of MZ extract are β-caryophyllene epoxide (10.84%) and β-caryophyllene (8.67%), accounting for 19.51% of total extract. Among the main components extracted from Copaifera langsdorffii, α-humulene, β-caryophyllene and caryophyllene oxide show the highest capacity to protect against gastric injury induced by AE.³² In the MZ extract, α-humulene shows a much lower amount (1.8%) than β-caryophyllene (8.64%) and β-caryophyllene oxide (10.84%). The β-caryophyllene, which is a major component found in many plants, such as clove, basil and cinnamon, shows anti-inflammatory and anti-bacterial effects.³³ The β-caryophyllene also shows capacity to inhibit Helicobacter pylori infection both in vivo and in vitro.²⁵ Otherwise, spathulenol is the major component (27.1%) of Falcaria vulgaris oil and the F. vulgaris extract, and exhibits gastroprotective effect in ethanol induced GU.³⁴ Furthermore, spathulenol is also the major component (22.5%) of the essential oil of Croton rhamnifolioides, and prevents gastric lesions in absolute ethanol, AE or indomethacin induced mouse GU models.²⁸ From these previous reports, we suggest that β-caryophyllene epoxide (10.84%), β-caryophyllene (8.67%), spathulenol (5.14%) and α-humulene (1.8%) are major components of MZ contributed to gastroprotective capacity.

3.3. Effects of MZ on AE-induced gastric mucosal histopathology

Histopathological examinations were performed to demonstrate protective effects of MZ on gastric mucosa. On H&E staining, mucosal epithelia of WT and MZ200 groups were intact without lesions (Fig. 2, WT, MZ200). Mucosal hemorrhage, submucosal edema accompanied by inflammatory cell infiltration, and severe cell necrosis were observed in the AE group (AE). Although CBX pretreatment reduced the AE-induced gastric mucosal damage, there was still submucosal edema (CBX + AE). Pretreatments with MZ100 and MZ200 (MZ100+AE, MZ200+AE) alleviated the AE-induced mucosal hemorrhage, edema, and inflammation compared with the AE group.

Fig. 2.

Histological examinations of gastric tissues by H&E staining. PBS treated mice (WT), mice treated with AE only (AE), mice pretreated with 100 mg/kg MZ before AE treatment (MZ100+AE), mice pretreated with 200 mg/kg MZ before AE treatment (MZ200+AE), and mice pretreated with 100 mg/kg CBX before AE treatment (CBX + AE). The multifocal degeneration/necrosis (arrow) with hemorrhage (arrow head) were found in the mucosal layer of stomach in indicated groups. Scale bars: 200 μm (upper panel for each group) and 100 μm (lower panel for each group).

3.4. Effects of MZ on AE-induced gastric mucus content and gastric wall mucus secretion

Mucus provides a protective barrier in the lining of the GI tract. PAS staining was used to assess gastric mucosal lesions due to its ability to display glycoproteins in gastric mucosa. On PAS staining, dark red signals were detected in all WT, MZ200, MZ100+AE, MZ200+AE, and CBX + AE groups (Fig. 3A). However, this intense coloration was not observed in the AE group. PAS staining results indicated greater accumulation of glycoproteins in the gastric mucosa in all CBX and MZ pretreated groups when compared with the AE group. PAS scores of the MZ200+AE group were higher than those of the MZ100+AE group, showing that the protective effect of MZ is dose-dependent (data not shown). Furthermore, pretreatments with MZ and CBX significantly increased mucus weights to the level of the WT group and MZ pretreatments also showed a dose-dependent manner (Fig. 3B).

Fig. 3.

(A) Histological examination of gastric tissues by PAS staining. Brick red area represents mucus distribution (arrow). PBS treated mice (WT), mice treated with 200 mg/kg MZ only (MZ200), mice treated with AE only (AE), mice pretreated with 100 mg/kg MZ before AE treatment (MZ100+AE), mice pretreated with 200 mg/kg MZ before AE treatment (MZ200+AE), and mice pretreated with 100 mg/kg CBX before AE treatment (CBX + AE). Scale bars: 300 μm. (B) Mucus weights of each group were calculated. Mice treated with 100 mg/kg MZ (MZ100), 200 mg/kg MZ (MZ200) and 100 mg/kg CBX (CBX), respectively, then treated with AE were underlined. ∗ (p < 0.05) denotes significant difference compared with the AE group.

To summarize, MZ pretreatments increase accumulation of glycoproteins in mucosa and secretions of mucus, then protect the gastric lining against AE-induced GU.

3.5. Effects of MZ on AE-induced lipid peroxidation levels and antioxidative enzyme activities

Ethanol-induced gastric mucosal damage is related to oxidative stress.³⁵ Ethanol metabolism induces oxygen free radicals (OFR), causing gastric mucosal vascular endothelial damage, microcirculation disturbance, and ischemia. OFR tends to react with unsaturated fatty acids in cell membrane to produce MDA, which affects the fluidity and permeability of cell membrane. However, OFR can be scavenged by the antioxidant system, such as SOD. SOD converts harmful superoxide to hydrogen peroxide, resulting in low SOD activity. Therefore, ethanol-induced GU is associated with oxygen-derived radicals and imbalance in antioxidant defense mechanisms. To investigate the correlation of MZ protective effect and oxidative stress, MDA levels and SOD activities in stomach were evaluated. There was high MDA level in the AE group compared with the WT group (Fig. 4A). However, there were restorations of MDA levels in both MZ200 and CBX pretreatment groups equivalent to those in the WT group. Furthermore, SOD activities were greatly reduced in the AE group (Fig. 4B). However, SOD activities were also restored in all MZ and CBX pretreatment groups. These results show that MZ protects AE-induced GU due to its antioxidative capacity.

Fig. 4.

Gastric MDA levels (A) and SOD activities (B). Serum levels (C) and gastric relative transcript levels (D) of TNF-α (left), IL-6 (middle), and IL-1β (right), respectively. PBS treated mice (WT) and mice treated with AE only (AE). Mice treated with 100 mg/kg MZ (MZ100), 200 mg/kg MZ (MZ200) and 100 mg/kg CBX (CBX), respectively, then treated with AE were underlined. ∗ (p < 0.05), ∗∗ (p < 0.01) and ∗∗∗ (p < 0.001) denote significant differences compared with the AE group.

3.6. Effects of MZ on expressions of inflammatory cytokines in AE-induced GU

In addition to the endogenous antioxidant system of gastric mucosa, mucosal damage caused by ethanol-induced ROS can be protected by the immune response.⁷ In generating ROS, AE administration provokes the inflammatory response, which releases a great number of inflammatory cytokines such as TNF-α, IL-6, and IL-1β.^6,20 TNF-α is secreted by macrophages and a potent stimulator of neutrophil infiltration into gastric mucosa during GU induction. An elevated level of IL-6 activates neutrophils at the inflammatory site and triggers the oxidative pathway responsible for local tissue damage in GU.³⁶ Therefore, inhibiting productions of TNF-α and IL-6 can reduce infiltration of neutrophils then reduce ROS production.³⁷

To analyze the AE-induced inflammatory response, pro-inflammatory factors, TNF-α, IL-1β, and IL-6, in sera and gastric tissues were analyzed. AE treatment significantly increased serum levels of TNF-α, IL-6, and IL-1β for 3.6 folds, 9.5 folds, and 5.4 folds, respectively, compared with the WT group (Fig. 4C). MZ100, MZ200, and CBX pretreatments decreased serum TNF-α levels to 44.4%, 44.4%, and 39.2%, respectively, compared with the AE group. MZ100, MZ200, and CBX pretreatments also decreased serum IL-6 levels to 32.4%, 10.4%, and 30.9%, respectively, and serum IL-1β levels to 21.3%, 14.3%, and 42.2%, respectively, when compared with the AE group. Thus, MZ and CBX pretreatments significantly reduced the AE-induced serum levels of pro-inflammatory cytokines.

To further demonstrate whether MZ pretreatment reduces expressions of pro-inflammatory cytokines in AE-induced GU, relative mRNA levels of TNF-α, IL-6, and IL-1β in stomach were detected using real-time PCR. AE-induced mRNA expressions of TNF-α, IL-6, and IL-1β were 4.9 folds, 2.1 folds, and 7.1 folds higher than those in the WT group, respectively (Fig. 4D). MZ200, and CBX pretreatments significantly reduced TNF-α expressions to 47% and 18%, respectively, when compared with the AE group. However, MZ100 and MZ200, but not CBX, pretreatments significantly reduced IL-6 expressions to 27% and 30%, respectively, when compared with the AE group. Compared with the AE group, the reductions of IL-1β expression levels in both MZ100 and MZ200 pretreatment groups did not reach statistical significance.

To summarize, AE-induced TNF-α, IL-6, and IL-1β expressions in both sera and gastric tissues (Fig. 4C and D) were similar to those in a previous study.³⁸ These results indicated that MZ and CBX pretreatments reduce AE-induced inflammatory cytokine expressions in sera and in parts of gastric tissues. It has been shown that the components isolated from MZ leaves inhibit expressions of IL-1β, TNF-α, IL-6, and induced nitric oxide synthase induced by lipopolysaccharides (LPS) in vitro¹⁵. In this study, MZ exhibits anti-inflammatory ability in vivo to protect against AE-induced GU.

3.7. Effects of MZ on AE-induced gastric Bax/Bcl-2 ratios

When ethanol-induced ROS exceeds the capacity of gastric mucosal antioxidant system, the intrinsic pathway of cell apoptosis is initiated.³⁹ In the AE group, pro-apoptotic Bax expressions were significantly increased but anti-apoptotic Bcl-2 expressions were significantly decreased in gastric tissues compared with the WT group (Fig. 5A–C). MZ200 and CBX pretreatments significantly decreased Bax expressions compared with the AE group, but they had no significant effect on Bcl-2 expressions (Fig. 5A–C). Furthermore, Bax/Bcl-2 ratios were significantly increased in the AE group compared with the WT group, and MZ200 or CBX pretreatments restored the AE-induced elevations (Fig. 5D). Therefore, these results suggest that MZ200 pretreatment effectively attenuates AE-induced gastric apoptosis.

Fig. 5.

(A) Expression levels of gastric Bax, Bcl-2, and GAPDH proteins. PBS treated mice (WT), mice treated with AE only (AE), mice pretreated with 100 mg/kg MZ before AE treatment (MZ100+AE), mice pretreated with 200 mg/kg MZ before AE treatment (MZ200+AE), and mice pretreated with 100 mg/kg CBX before AE treatment (CBX + AE). Ratios of Bax to GAPDH (B), Bcl-2 to GAPDH (C), and Bax to Bcl-2 (D) were calculated. Mice treated with 100 mg/kg MZ (MZ100), 200 mg/kg MZ (MZ200) and 100 mg/kg CBX (CBX), respectively, then treated with AE were underlined. ∗ (p < 0.05) and ∗∗ (p < 0.01) denote significant differences compared with the AE group.

3.8. Effects of MZ on AE-induced NF-kB and MAPK levels

Ethanol-induced ROS activates NF-κB and then trigger transcriptions of inflammatory cytokines and chemokines.⁴⁰ TNF-α is the prime contributor to activating the NF-κB pathway. Therefore, ethanol-induced TNF-α activates NF-κB synergistically. More studies have confirmed that blocking NF-κB activation is beneficial to the repair of ulcer damage.^11,41 NF-κB is a transcription factor which is related to the pathogenesis of ethanol-induced GU.¹¹ To decipher the gastroprotective mechanism(s) of MZ against AE-induced GU, expression levels of phospho-NF-κB p65 (p-NF-κB) and GAPDH in gastric tissues were detected. AE significantly increased p-NF-κB/GAPDH ratios compared with the WT group (Fig. 6A and B). However, AE-induced elevations of p-NF-κB/GAPDH ratios were significantly decreased by all MZ and CBX pretreatments. These results indicate that MZ pretreatment effectively reduces AE-induced NF-κB activation. These results are similar to a previous study using MZ leaf extract to show inhibition of LPS-induced IL-1β via NF-κB pathway in vitro¹⁵.

Fig. 6.

Expression levels of gastric p-NF-κB p65, NF-κB p65, and GAPDH proteins (A), and phosphorylated and total levels of MAPKs and GAPDH (C). PBS treated mice (WT), mice treated with AE only (AE), mice pretreated with 100 mg/kg MZ before AE treatment (MZ100+AE), mice pretreated with 200 mg/kg MZ before AE treatment (MZ200+AE), and mice pretreated with 100 mg/kg CBX before AE treatment (CBX + AE). (B) Ratios of p-NF-κB p65 to GAPDH, (D) ratios of p-ERK to ERK (upper), p-JNK to JNK (middle), and p-p38 to p38 (lower) were calculated. Mice treated with 100 mg/kg MZ (MZ100), 200 mg/kg MZ (MZ200) and 100 mg/kg CBX (CBX), respectively, then treated with AE were underlined. ∗ (p < 0.05), ∗∗ (p < 0.01), and ∗∗∗ (p < 0.001) denote significant differences compared with the AE group.

Gastric inflammation induced by ethanol may be achieved through ROS release and then activating the MAPK pathway.⁴¹ In our study, phosphorylations of MAPK family proteins including ERK, JNK, and p38 were significantly increased following AE treatment compared with the WT group (Fig. 6C and D). Compared with the AE group, the ratios of p-ERK/ERK significantly decreased both in MZ and CBX pretreatments, the ratios of p-JNK/JNK significantly decreased only in CBX pretreatment, and the ratios of p-p38/p38 significantly decreased in both MZ100 and CBX pretreatments (Fig. 6D). In summary, MZ pretreatment reduces AE-induced the MAPK signaling.

AE appears to activate the MAPK and NF-κB pathways to induce pro-inflammatory factor expressions and GU. Previously, Camellia japonica mitigating AE-induced inflammation and GU were demonstrated through attenuating MAPK/NF-κB signal pathways.¹¹ In our study, MZ pretreatment also inactivates AE-induced MAPK and NF-κB pathways, resulting in marked alleviations of TNF-α, IL-6, and IL-1β expressions.

3.9. Evaluating potential side effects of oral administration of MZ

Body weight change is one of the reference indicators of drug side effects. Compared with the WT group, there were no significant differences in body weight changes and feed intakes (Table 1), and no mortality or behavior change in mice of all MZ treated group. Furthermore, there were no significant increases in serum markers of liver damage, ALT and AST activities (Fig. 7B), and of kidney damage, BUN and CREA (Fig. 7C) in the MZ200 group compared with the WT group. There were no obvious damages in the histological structures of liver (upper panel) and kidney (lower panel) of mice in both WT and MZ200 groups (Fig. 7A). Moreover, hepatic levels of inflammatory cytokines, TNF-α and IL-6, were both similar in the MZ200 and WT groups (Fig. 7D). In summary, oral treatment of 200 mg/kg MZ for 14 consecutive days does not induce liver or kidney toxicities and any obvious side effect in mice.

Table 1.

The body weight changes and feed and water intakes of mice in different indicated groups during 14 experimental days.

Groups	Body weight changes (g)	Feed intakes per day per mouse (g)	Water intakes per day per mouse (mL)
WT	1.94 ± 0.15	3.68 ± 0.36	3.91 ± 0.30
MZ200	2.02 ± 0.50	4.24 ± 0.50	4.15 ± 0.15
MZ100+AE	1.52 ± 0.20	3.71 ± 0.24	3.06 ± 0.06a
MZ200+AE	1.92 ± 0.76	3.84 ± 0.53	3.33 ± 0.33

^aShows significant difference with the WT group.

3.10. Conclusions

Results of this study demonstrates that MZ possesses a gastroprotective function in AE-induced gastric lesions in mice. This result is consistent with the contents of the ancient Chinese Materia Medica.¹⁴ The protective mechanisms of MZ mainly involve increasing mucus secretion and antioxidative, anti-inflammatory, and anti-apoptotic abilities to reduce AE-induced mucosal damage. Furthermore, MZ regulates the NF-κB and MAPK signaling pathways to achieve its gastroprotective effects. The MZ powder used in this study contains 10.84% β-caryophyllene epoxide, 8.67% β-caryophyllene, 5.14% spathulenol and 1.8% α-humulene. These four substances totally account for 26.45% of the MZ extract, and all of them show gastroprotective effects against GU induced by absolute ethanol, AE, indomethacin, or Helicobacter pylori infection induced GU as a pure substance or the major component of essential oils or plant extracts. Thus, we suggest that these 4 components are main components of MZ for gastroprotective ability.

Authors’ contributions

Conception and design of study: S.-C. Huang and S.-H. Wang; Acquisition of data: X.-Z. Yan, H.-C. Lin, J.-C. Tsai, J.-W. Liao and K.-T. Wang; Analysis and/or interpretation of data: M.-S. Tsai and P.-Y. Lai; Drafting the manuscript: W.-J. Wu and S.-H. Wang; Revising the manuscript critically for important intellectual content: Y.-J. Lee, K.-T. Wang and M.-S. Tsai. All authors have read and agreed to the published version of the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

Supplementary Figure 1.

The GC/MS fingerprint profile of the MZ extract.