Effects of complex extracts of traditional Chinese herbs on gastric mucosal injury in rats and potential underlying mechanism

Five complex extracts (CEs) of seven Chinese herbs ( Astragalus , Poria cocos , Alpinia officinarum Hance , Radix Puerariae , Ginseng , Licorice , Hericium erinaceus ) were prepared by hot water extraction and evaluated for their effect on gastric ulcer in rats. In rats with acetic acid-induced chronic gastric ulcer, gross and microscopic appearance showed that gastric mucosal injury index and lesion inhibition rate were improved after CEs gavage for 21 days. Pretreatment with CEs for 21 days in rats with acute gastric ulcer could also improve the gastric mucosal injury by ethanol. CE1, CE4, and CE5 showed moreobviouseffectintwomodels.ThecellexperimentsresultsshowedthatCE1,CE4, and CE5 effectively inhibited Wnt signaling activity. Thus, they could protect gastric mucosa through inhibiting Wnt signaling pathway. These results indicated that CE1, CE4, and CE5 had significant protective effects on gastric mucosal injury by inhibiting Wnt signalling pathway and could be developed into safe functional products. peptic high It is believed to be caused by an imbal-ance between the offensive factors (such as reactive oxygen species, alcohol consumption, cigarette smoking, Helicobacter pylori , etc.) and defensive factors (such as mucosal barrier, mucus secretion, bicarbon-Abbreviations: of using at

antagonists, and so on. However, these drugs have unacceptably high incidence of adverse effects and linked to ulcer recurrence (Kangwan et al., 2014). Therefore, exploring more effective antigastric ulcer products with little or even without side effects from natural resources has attracted much attention (Awaad et al., 2013;Jain et al., 2010). Now many medicinal resources have been proven to be conducive to improving gastric ulcers in humans and many animal models (Boylan et al., 2014;Gargano et al., 2017).
A variety of traditional Chinese herbs have been proven to be effective on gastric ulcer and gastric cancer. Polysaccharide isolated from the aqueous extract of Hericium erinaceus mycelium culture has been identified as an active ingredient in ethanol-induced gastric ulcer mice and cell experiments . Astragalus saponins deceased the invasion ability of gastric cancer BGC-823 cell and induced its apoptosis (Miraj & Kiani, 2016;. PGP2a, a polysaccharide isolated from Ginseng roots, has an inhibitory effect on the growth of HGC-27 cells in a dose-dependent manner . Long-term history of traditional Chinese herb application and recent studies have shown that complex extract of herbs are more effective than single extracts (Bai et al., 2014;You et al., 2019). It was found that complexes of herbs provide greater immune stimulation effects than the sum of the same concentrations of single components (Hong et al., 2018). SiJunZiTang, a classic Traditional Chinese Formula consisting of four Chinese herbs, Radix Ginseng, Atractylodes macrocephala, Poria cocos and Glycyrrhiza uralensis, has been demonstrated to show protective effects on intestine and gastric injure .
In this study, five complex extracts (CEs) of traditional Chinese herbs (Astragalus, Poria cocos, Alpinia officinarum Hance, Radix Puerariae, Ginseng, Licorice, and Hericium erinaceus) were prepared and evaluated the effect on chronic and acute gastric ulcer in rats. And the effects of CEs on the Wnt/β-catenin signaling pathway were conducted in cell experiments to discuss the mechanism preliminary.

Acetic acid-induced chronic gastric ulcer and treatment
The animals were randomly divided into seven groups with nine rats in each group: control group, model group, and five experimental groups (CE1, CE2, CE3, CE4, and CE5). Chronic gastric ulcer was induced by acetic acid treatment in rats of model group and experimental groups (Almasaudi et al., 2017;Okabe & Pfeiffer, 1972). Animals were fasted for 12 h before the operation and were given free access to water.
Under anesthesia, the stomach was exposed by a longitudinal incision below the xiphoid process and then was injected with 0.05 mL (v/v) of 2.5% acetic acid into the subserosal layer in the glandular part of the anterior wall. After that, the abdomen was closed to allow the rats to feed normally. From the second day after ulcer induction, the animals were treated with water (control group and model group), or CE1, CE2, CE3, CE4, CE5 (experimental groups) by gavage once daily for 21 day (Table 1). At last, with a 12 h fast and free access to water, the animals were sacrificed and the stomachs were removed for examination.

2.3.3
Ethanol-induced acute gastric ulcer and treatment Protective effects of CEs on acute gastric ulcer were evaluated using an ethanol-induced gastric mucosal lesion model (Al-Qarawi et al., rats were fasted and given free access to water for 12 h, and then received 1.0 mL of absolute ethanol by gavage except the rats in the control group. One hour later, rats were sacrificed and the stomachs were removed for examination.

Stomach tissue sample for examination
After experiment, the rats were killed and abdominal cavity was opened immediately. After ligation at the cardia and pylorus, the stomach was removed, opened along the greater curvature and then rinsed with saline. The stomach tissue was spread out for observation and photographed for macroscopic assessment of gastric mucosal injury.
Next, part of the gastric tissue was fixed in 4% paraformaldehyde solution for histopathological analysis.

Macroscopic assessment
Ulcer occurrence in the gastric mucosal layer was carefully observed.
Meanwhile, number of hemorrhagic spots was recorded and the length and width of ulcer stripes were measured with a vernier caliper. Gastric ulcer index was calculated through Guth method (Park et al., 2015), based on the following scoring criteria: Normally 0 points; 1 point for spot smashing; 2 points for smashing length <1 mm; 3 points for 1-2 mm; 4 points for 3-4 mm; 5 points for > 4 mm. The lesion inhibition rate was calculated using Equation (1) (Zhang et al., 2018): Where A0 is the gastric ulcer index of model group, A1 is the gastric ulcer index of experimental groups.

Histological analysis
Hematoxylin and eosin staining method were used for histopathological observation on general microstructure of gastric mucosa. The most serious parts of ulcer region were chosen to produce paraffin wax tissue sections (4 μm). The sections were stained in hematoxylin and eosin stain, and then observed under a light microscopy. Gastric ulcer index was calculated according to Ateufack et al. (2015) as following: No abnormalities 0 points; 1 point for Lesion involvement range <1/3; 2 point for 1/3-2/3; 3 point for 2/3-Mucosal full layer; 4 point for submucosal. The lesion inhibition rate was calculated using Equation (1)

2.4.3
Luciferase reporter assays for Wnt/β-catenin signaling pathway Wnt3a CM was prepared as following: Wnt3a-secreting L cells were cultured in DMEM with 10% FBS for 4 days. The medium was harvested and sterilized using a 0.22 μm filter. After addition of fresh medium, the cells were cultured for another 3 days, and the medium was collected and combined with the previous medium.
RKO-reporter cells contain two reporter plasmids, TOPflash (containing TCF binding site) and FOPflash (containing mutated TCF binding sites). RKO reporter cells were seeded in 96-well plates at a density of 2.0 × 10 4 cells/ well and incubated at 37 • C in 5% CO 2 environment for 12 h. Then, the cells were treated with Wnt activator and 4.5 mg /mL CE1 and CE5 or 4.8 mg /mL CE4 for another 24 h. Luciferase activity was measured with the dual Luciferase Reporter Gene Assay Kit (Promega) according to the manufacturer's instructions. Wnt signaling activity was calculated as fold change (TOP/FOP Luciferase activity) (Shim et al., 2015).

Statistical analysis
All values were represented as mean ± SEM. Statistical analysis was carried out using SPSS 22.0 software. One-way analysis of variance (ANOVA) was adopted to compare the significant differences among all of groups using Tukey's analysis. Differences were considered to be significant at p < 0.05.

Repairing effect of CEs on acetic acid-induced chronic gastric ulcer in rats
Effect of CEs on body weight and histological morphology of liver in acetic acid-induced chronic gastric ulcer rats The body weights of rats all gradually increased during the experimental period. There was no significant difference in weight gain and clinical/behavior signs among all groups during the experiments.
The morphological changes of liver were evaluated by histological examinations to analyze the hepatotoxicity of the CEs. The liver tissue of each group was similar in shape and there was no significant difference. These results suggested that these CEs are relatively safe.

3.1.2
Repairing effect of CEs on acetic acid-induced chronic gastric injury Figure 1 shows the gross appearance and microscopic images of the representative stomach of each group. In the control group, the gastric mucosa was smooth with no gastric glandular structure loss, disorganized glandular structure, hemorrhage, and submucosal edema, whereas these phenomena were observed in the model group, which indicated that the chronic gastric ulcer was successful generated. In experimental groups, these symptoms were alleviated. There were fewer glandular structure losses and disordered glandular structures in the CE2 and CE3 groups. Remarkably, the gastric mucosa of rats in CE1, CE4, and CE5 groups were almost smooth, with no obvious disordered glandular structure.
The gastric ulcer index and lesion inhibition rate by histological morphology were consistent with those by macroscopic observations (Figure 2). Compared with the model group, gastric ulcer index in the experimental groups decreased in different degree, mostly in CE1, CE4, and CE5 groups. There was no significant difference in gastric ulcer index and efficacy among the experimental groups. However, the CE1, CE4, and CE5 appeared apparent decreases in gastric ulcer index and increases in lesion inhibition rate in comparison with the model group.
Lesion inhibition rate of CE1, CE4, and CE5 were the greatest in the experimental groups, although there were no obvious differences.
It was suggested that CEs could accelerate the healing of chronic gastric ulcer of rats and CE1, CE4, and CE5 were more effective in repairing acetic acid-induced gastric mucosal injury.

3.2.1
Effect of CEs pretreatment on the body weight of ethanol-induced acute gastric ulcer rats During the experiment, the body weight of all rats gradually increased.
There was no significant difference in weight gain, clinical/behavioral signs and adverse events among all groups.

Preventive effect of CEs on ethanol-induced acute gastric injury in rats
Compared with normal appearance of gastric mucosal epithelium and folding in the control group, it was found a large area of gastric mucosa hyperemia, ulcers, or perforations in model group (Figure 3). The gastric ulcer index was also highest in the model group (Figure 4), which indicated the most serious lesion. So, ethanol produced serious acute gastric ulcer in rats. In the experimental groups, it was observed fewer glandular structure losses and disordered glandular structures than in the model group. And gastric ulcer index of rats in experimental groups were less than model group. Especially, the gastric mucosa of rats in CE1, CE4, and CE5 groups were almost smooth with no obvious glandular structure disorder. Meanwhile, there was significant difference in gastric ulcer index between model groups and CE1, CE4, or CE5 groups. And CE1, CE4, and CE5 have the greatest lesion inhibition rate, although there was no significant difference in gastric index and lesion inhibition rate among experimental groups.
The results implied that CEs exerted preventive effects against ethanol-induced acute gastric mucosal injury, especially CE1, CE4, and CE5.

Interaction of bioactive ingredients may affect their bioactivity
The effect of complex extract may be due to the interaction of bioactive ingredients. As is shown in

Inhibitory effect of CEs on the Wnt/β-catenin signaling pathway
To investigate the possible mechanisms, a mechanistic study of CE1, CE4 and CE5 on Wnt/β-catenin signaling pathways was conducted.
The cytotoxic effect of CE1, CE4, and CE5 on macrophages was determined by MTT assay. As shown in Figure 5, no significant changes in the cell viability rate were found after treatment with CE1, CE4, or CE5 at the concentration of 0.56-9.60 mg/mL, and the cell morphology was good under the microscope. Therefore, the 0.56-9.60 mg/mL F I G U R E 2 Effect of complex extracts on the gastric ulcer in acetic acid-induced chronic gastric ulcer rats. Values represent the mean ± SEM (n = 9), **p < 0.01 versus model group; ***p < 0.005 versus model group; #p < 0.05 versus CE2 served as safe concentrations were selected for the luciferase reporter assays.
To verify that the effect of CEs on gastric mucosal lesions is related to Wnt pathway, RKO-reporter cells were treated with CEs for 24 h. It was found that Wnt3a levels were greater when cells grew in Wnt3a-CM than in Wnt3a medium. However, the inhibitory effects of CEs on Wnt3a were much better, which indicated that this Wnt3a suppression was more dramatic in cells treated with these CEs. Combined with the results of effect on gastric mucosal injury, these results showed that the greater Wnt inhibitory activity of CEs, the more significant effect on gastric mucosa. It was confirmed that CEs could protect gastric mucosa by inhibiting Wnt signaling pathway.

DISCUSSION
Gastric ulcers produced by acetic acid become chronic within 2-_3 days after ulcer begins, and the model is very similar to human peptic ulcer disease in terms of pathological features and the healing process (Bonamin et al., 2011). Acetic acid-induced gastric ulcers are caused by a variety of factors, including prostaglandins, growth factors, nitric oxide, and cytokine levels, as well as mucus adhesion patterns and microcirculation (Kobayashi et al., 2001). It has been previously reported that acetic acid can induce ulceration by penetrating the gastric mucosa as well as submucous layers and the muscular layer (Najm, 2011;Okabe & Amagase, 2005). Current treatments for gastric ulcers are based on the use of antisecretory drugs, including type 2 histamine receptor antagonists (H2RA) and proton pump inhibitors (PPIs). Although these drugs are effective, they have been used for a long time, especially PPI which has many side effects associated with gastric repair process (Yu et al., 2017). These limitations made people recognize that the treatment of gastric ulcers requires more effective or auxiliary therapeutic agents. In addition, Chinese herbal medicines have a therapeutic effect on gastric ulcer with fewer side effects and lower cost which is only one-sixth of western medicine. Current study was designed to evaluate the effects of different prescriptions on acetic acid-induced gastric ulcer repair. The results obtained from this work showed that CE1, CE3, and CE4 are able to reduce the gastric ulcer index induced by this model. This speculate that the mechanism is to inhibit the Wnt pathway which can repair the damaged gastric mucosa, for which CE1, CE3, and CE4 can achieve it well.
The Wnt/β-catenin signaling pathway is a highly conserved pathway in the evolution of organisms and regulates many biological processes (Wang et al., 2011). In this way, extracellular signals are delivered to the cells by activating the intracellular domain of the cell surface receptor.
Wnt/β-catenin signaling pathway is an important signaling pathway in gastric cancer, and it is often activated abnormally. Luciferase reporter assay is a method of confirming the function of the β-catenin/TCF complexes by using a reporter containing multimeric TCF recognition sequences (TOPFLASH). It is well known that Wnt signaling directly leads to heterogenous expression of related factors of epithelialmesenchymal transition (EMT), such as N-cadherin and E-cadherin.
Epithelial cells express high levels of E-cadherin, whereas mesenchymal cells express high levels of N-cadherin and lose E-cadherin expression. The development of gastric cancer involves the expression or absence of E-cadherin function. Downregulation of E-cadherin reduces the intensity of cell adhesion in tissues, leading to an increase in cellular motility, which in turn allows cancer cells to invade surrounding tissues through the basement membrane Park et al., 2018;Su et al., 2015;Zhang et al., 2019). When acetic acid is used to induce gastric mucosal injury, Wnt signaling pathway is activated in gastric mucosal epithelial cells, resulting in decreased expression of Ecadherin, which leads to the development of gastric ulcer. This study found that CEs can effectively inhibit the Wnt signaling pathway, which increases the expression of E-cadherin, thus accelerating the repair of gastric mucosal injury caused by acetic acid.

CONCLUSION
CEs have repairing effect on chronic gastric mucosal injury induced by acetic acid and preventive effect on acute gastric mucosal injury induced by ethanol in rats. Among them, CE1, CE4, and CE5 have greater effects. They could protect gastric mucosa by inhibiting Wnt signaling pathway. They may be new safe products on the treatment or prevention of gastric ulcer.

DECLARATIONS
Ethics approval and consent to participate All procedures performed in studies involving animals were handled humanely according to the guidelines of the National Institutes of Health guide (NIH Publications No. 8023, revised 1978) for animal care and use were followed, and the animal protocol was approved by the animal ethics committee of Jinan University.

AVAILABILITY OF DATA AND MATERIALS
All data generated or analyzed during this study are included in this published article.

CONFLICT OF INTERESTS
The authors declare that they have no conflict of interests.

AUTHOR CONTRIBUTIONS
RJY contributed to the conception of the study. YED helped perform the analysis with constructive discussions and revise manuscript. LLY contributed to in vitro studies and manuscript preparation. HM performed the animal experiment and wrote the manuscript. CB helped perform the animal experiment. QCL helped build animal models. GN performed assays for Wnt/-catenin signaling pathway. All authors read and approved the final manuscript.