2024 Volume 263 Issue 4 Pages 217-226
Helicobacter pylori (H. pylori) plays an important role in chronic atrophic gastritis (CAG). Interestingly, Shaoyao Gancao decoction (SGD), a traditional Chinese analgesic prescription, has the efficacy of relaxing spasms and relieving pain. Here, we aimed to identify whether SGD alleviates CAG and the underlying mechanism. A CAG mouse model was developed using H. pylori colonization and a high-salt diet. Histological staining was used to study the histopathological damage changes, and RT-qPCR assays the production of inflammatory responses in the gastric mucosa of mice. H. pylori and a high-salt diet induced gastric mucosal damage and apoptosis of gastric mucosal epithelial cells in mice, eliciting a significant inflammatory response. Treatment with SGD alleviated CAG-induced gastric mucosal damage, reduced apoptosis of gastric mucosal epithelial cells, and inhibited the inflammatory response. Bioinformatics was then used to construct the pharmacological network of SGD to explore its potential targets. SGD inhibited inflammatory response in mice with CAG by suppressing the expression of MAOB. Overexpression of MAOB impaired the therapeutic effect of SGD on inflammation in mice with CAG. Collectively, our findings indicated that SGD has the potential to alleviate CAG via downregulating MAOB.
Atrophic gastritis refers to the loss of gastric glands in the context of chronic inflammation principally caused by Helicobacter pylori (H. pylori) infection or autoimmunity (Shah et al. 2021). Chronic gastritis is composed of atrophic or non‐atrophic gastritis, and the complicated immune response induced by H. pylori is the major factor in its inflammation (Yao et al. 2023). Chronic atrophic gastritis (CAG) contributes to a great chance of gastric cancer, predominantly when there is extensive intestinal metaplasia, and H. pylori infection (active or past) can be found in approximately two-thirds of CAG patients (Lahner et al. 2018). Better diagnosis and management of patients with CAG are imperative to improve the quality of life and to relieve the social and economic burden caused by CAG (Lahner et al. 2019). TCM has exceptional advantages in the management of gastrointestinal disorders (Teschke et al. 2015; Wang et al. 2021).
TCM is characterized by a constant curative effect, high safety, less toxicity, and low price, which has great advantages in improving symptoms and can remarkably promote H. pylori eradication and reduce adverse reactions (Li et al. 2021c). Shaoyao Gancao decoction (SGD), derived from the “Typhoid Theory” proposed by Zhang Zhongjing, is composed of Paeonia lactiflora Pall and Glycyrrhiza uralensis Fisch, having the potential to relax spasms and relieve pain (Bi et al. 2014). Seven major ingredients of SGD involve paeoniflorin, albiflorin, liquiritin, isoliquiritin, liquiritin apioside, isoliquiritin apioside, as well as glycyrrhizic acid (Liu et al. 2019). SGD relieves visceral hyperalgesia in post-inflammatory irritable bowel syndrome (Shao et al. 2020), indicating its effects on gastrointestinal disorders. There are about 23 Paeonia species, 2 subspecies, and 7 varieties, and Radix Paeoniae Rubra (Chinese name Chishao) has been used to treat gastritis (Li et al. 2021a). The Glycyrrhiza radix (licorice) represents the most traditionally applied medicinal plant in Asia to treat many disorders, such as cough, asthma, respiratory tract infections, as well as gastritis (Kim et al. 2021). It has been reported that special licorice extracts can attenuate H. pylori-induced gastritis and tumorigenesis due to their antioxidative and anti-inflammatory effects (Park et al. 2014). However, there are no clues about the efficacy of SGD on H. pylori-induced CAG. As a consequence, the main aim of this research was to dissect whether the administration of SGD could alleviate the CAG in mice infected with H. pylori. The network pharmacology study enabled us to dissect the potential targets of TCM, including SGD, thereby providing a basis for further drug development research (Li et al. 2020; Chen et al. 2022; Zhou et al. 2023). Therefore, we also explored the underlying mechanism of SGD using a pharmacological network and KEGG enrichment analysis. This study is beneficial to better understanding the possible mechanisms of SGD in the management of CAG.
The expression profiles associated with chronic superficial gastritis and CAG were downloaded from the GSE163416 dataset to analyze the expression of genes. The HERB 2.0 database (http://herb.ac.cn/) was used to download the predicted targets of peony and licorice, the main components of SGD. In detail, the terms “peony” and “licorice” were used as the keywords, and the related gene targets in the search result were downloaded. The KEGG pathway analysis on the pharmacological targets of peony and licorice was conducted using Sangerbox Tools. The pharmacological networks of peony and licorice were established using Cytoscape 3.7.2 software.
H. pylori cultureH. pylori Sydney strain (SS1, Bio-091269, BioBW, Beijing, China) was cultured in tryptic soy agar medium spiked with 5% goat serum at 37°C under microaerobic conditions for 3 d. The strain was collected into fresh broth medium, centrifuged at 3,000 g for 5 min, and resuspended in the broth at 109 CFU/mL.
SGD preparationSGD was prepared with Paeonia lactiflora Pall and Glycyrrhiza uralensis Fisch as the prescription at 1:1, and the quality of herbs met the Pharmacopoeia of the People’s Republic of China 2020. In brief, Paeonia lactiflora Pall and Glycyrrhiza uralensis Fisch were mixed and powdered, followed by the addition of distilled water (1:10, w/v). After that, the samples were loaded into an ultrasonic-microwave combined reaction system at 600 W and a constant temperature of 80°C for 25 min. After the reaction termination, the supernatant was separated, and the residue was extracted twice by the same method. The supernatant was mixed, and the concentrate was dried at 55°C under reduced pressure to obtain the dried extract powder, which was stored at −20°C.
Animal handingAll animal studies were carried out following the protocols approved by the Animal Care and Use Committee of Heilongjiang University of Chinese Medicine. Thirty-six 5-week-old C57BL/6 male mice (Vital River, Beijing, China) were maintained in an SPF room with a 12 h:12 h light-dark cycle during which the mice were guaranteed food and drinking water ad libitum.
After one week, the mice were allocated to six groups: the control, CAG, CAG + phosphate-buffered saline (PBS), CAG + SGD, overexpression (OE)-negative control (NC), and OE-MAOB groups (n = 6). For CAG induction, the mice were injected intraperitoneally with 20 mg/kg pantoprazole, a proton pump inhibitor to reduce gastric acidity and promote colonization by H. pylori, three times a week. Next, 0.1 mL (108 CFU/mL) of H. pylori suspension was inoculated using a gastric intubation needle four times a week for 24 weeks (Park et al. 2021). The mice in the control group were administrated with equal doses of PBS. During this period, the mice were fed with AIN-76A high-salt diet containing 7.5% NaCl, and the control group was provided with a normal diet.
As regards mice in the CAG + SGD group, gavage treatment (5 g/kg) was started at week 10 once a day until week 24 (Park et al. 2021). MAOB expression was upregulated in mice by tail vein injection of adeno-associated viruses (AAV) harboring MAOB overexpression vectors (Shanghai Genechem Co., Ltd., Shanghai, China) 2 weeks before the start of the experiment as per the manufacturer’s protocol. After 24 weeks, the mice were subjected to an intraperitoneal injection of 150 mg/kg of sodium pentobarbital for euthanasia, and the gastric mucosa was dissected and separated into two parts after washing in saline. One portion was stored at −80℃, and the other portion was fixed with 10% formalin overnight and paraffin-embedded.
Histopathologic injuryParaffin-embedded sections of mouse gastric mucosa were dewaxed in xylene for 5 min. After rehydration using gradient concentration ethanol solution, the sections were stained with hematoxylin staining solution for 10 min, fractionated with 5% acetic acid for 30 s, washed with 95% ethanol, stained with eosin staining solution for 1 min, dehydrated with gradient concentration ethanol, treated with xylene, and sealed with neutral gum. The histopathological changes of mouse gastric mucosa were scored under the microscope by three gastroenterologists who were unaware of the grouping using defined histological damage indices. The histopathologic injury score is the sum of the inflammation score and the erosion score as previously described (Park et al. 2021).
Detection of apoptosis by TUNEL assayApoptosis was detected using the TUNEL Kit (C1091, Beyotime, Shanghai, China). Paraffin-embedded sections of gastric mucosa were dewaxed and dehydrated. After that, the sections were incubated with DNase-free Proteinase K and with 3% hydrogen peroxide solution (both for 20 min at room temperature). After being treated with biotin labeling solution for 1 h at 37°C in darkness, the sections were treated with reaction termination solution for 10 min and with Streptavidin-HRP working solution for 0.5 h (both at room temperature). The nuclei were counter-stained with hematoxylin staining solution after a chromogenic reaction using DAB chromogenic solution. After gradient ethanol dehydration and xylene clearing, the sections were sealed with neutral gum. The epithelial cell apoptosis in gastric mucosal tissues was observed under a microscope. The apoptosis rate was counted.
ImmunohistochemistryAfter deparaffinization and rehydration, paraffin-embedded sections of gastric mucosa were heated with citrate for antigen retrieval, and 3% hydrogen peroxide was used to remove endogenous peroxidase, followed by sealing the sections with 5% goat serum for 1 h. The sections were then incubated with MAOB-specific primary antibody (1:1,000, ab259928, Abcam, Cambridge, UK) overnight at 4°C and with HRP-coupled goat anti-rabbit secondary antibody (1:2,000, ab6721, Abcam) at room temperature for 1 h. The sections were color-developed by DAB, nuclei counter-stained using hematoxylin, cleared with xylene, and dehydrated using ethanol. The sections were sealed with neutral gum and then observed under a microscope, and the percentage of positive cells was calculated.
RT-qPCRTotal RNA was isolated from mouse gastric mucosa stored in a −80°C refrigerator using TRIzol reagent (Invitrogen Inc., Carlsbad, CA, USA). A spectrophotometer was used to examine RNA concentration and purity, and the reverse transcription reaction was performed using EasyScript Reverse Transcriptase [M-MLV, RNaseH-] (AE101-02, Transgene Biotech, Beijing, China). An ABI Prism 7900 using TransStart Tip Green qPCR SuperMix (AQ141-01, Transgene Biotech) was used for fluorescent qPCR reactions. Taking GAPDH as the reference, the relative expression of mRNAs was assessed using the 2−ΔΔCT method. The primer sequences are listed in Table 1.
Primers sequences of real-time PCR analyses for mRNA expression.
IL, interleukin; TNF-α, tumor necrosis factor alpha; MAOB, monoamine oxidase-B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
To assess whether SGD produces antimicrobial effects on H. pylori colonization in the gastric mucosa of mice, we collected RNA from the gastric mucosa and then performed qPCR by using H. pylori 16S rRNA-specific primers. The results represent the mean and error between groups through the relative quantitative calculation of the ΔCt (threshold cycle) value (Jung et al. 2020).
Western blot assaysMouse gastric mucosa stored at a −80°C refrigerator was lysed using RIPA lysis buffer. Tissue homogenate was subjected to centrifugation at 12,000 g for 15 min at 4°C to harvest the supernatant, and the protein concentrations were quantified using Pierce BCA Protein Quantification Kit (23225, Thermo Fisher Scientific Inc., Waltham, MA, USA). After being heated at 100℃ for 3 min, the protein was transferred to a PVDF membrane by SDS-PAGE. The membranes were blocked in 5% skim milk for 30 min and probed overnight at 4°C using specific primary antibodies to MAOB (1:1,000, ab259928, Abcam) and GAPDH (1:2,000, ab9485, Abcam). Goat anti-rabbit secondary antibody (1:2,000, ab6721, Abcam) coupled with HRP was applied for a 1-h incubation at room temperature. Immunoblots were visualized by ECL and quantified using Image J software.
Statistical analysesGraphPad Prism software (GraphPad, San Diego, CA, USA) was applied to visualize the results. Data were presented as mean ± SD. The differences between the group means were calculated by an unpaired t-test or one-way ANOVA. Statistical significance was accepted at p < 0.05.
A CAG mouse model was induced using H. pylori SS1 and a high-salt diet. It was revealed by HE staining that the gastric mucosal epithelial glands of control mice were structurally intact and tightly arranged, and no significant inflammatory infiltration was observed. However, the CAG mice exhibited sparse mucosal epithelium, reduced glands, and inflammatory infiltration, while SGD treatment alleviated the production of gastritis in mice (Fig. 1A). TUNEL showed significant apoptosis in the gastric mucosal epithelial cells of CAG mice, and gavage treatment of SGD reduced the level of apoptosis (Fig. 1B). Measurement of inflammatory factors in gastric mucosa using RT-qPCR demonstrated that the H. pylori SS1 infection led to an inflammatory response in mice. SGD treatment alleviated the increased expression of inflammatory factors, and the mRNA expression of IL-6, IL-1β, and TNF-α was remarkably reduced (Fig. 1C-E). Finally, we analyzed the level of H. pylori colonization in the gastric tissues of mice, and the results showed a significant decrease in the level of H. pylori in the gastric tissues after the treatment of SGD (Fig. 1F).
H. pylori-induced CAG in mice.
(A) Histopathology of gastric mucosal epithelial glands in CAG mice or treated with SGD by HE staining and histopathology score. (B) The apoptosis in epithelial cells of gastric mucosa tissues of CAG mice or treated with SGD using TUNEL assay. (C) Detection of IL-6 expression in gastric mucosal tissues of CAG mice or treated with SGD by RT-qPCR. (D) Detection of IL-1β expression in gastric mucosal tissues of CAG mice or treated with SGD by RT-qPCR. (E) Detection of TNF-α expression in gastric mucosal tissues of CAG mice or treated with SGD by RT-qPCR. Data were shown as mean ± SD. (F) H. pylori levels in gastric mucosal tissues of CAG mice after SGD treatment were examined using qPCR. n = 6. *p < 0.05 versus the control or CAG + PBS group (unpaired t-test or one-way ANOVA).
To probe the detailed targets of SGD on CAG, we predicted the genetic targets of peony and licorice, the main components of SGD, in the HERB 2.0 database. The pharmacological networks were exhibited in Fig. 2A, B. KEGG pathway analysis showed that the pharmacological targets of both peony and licorice were enriched in the TNF signaling (Fig. 2C, D), confirming their effects on the inflammatory response.
Prediction of the targets of SGD.
(A) The pharmacological network of the targets of peony using HERB 2.0. (B) The pharmacological network of the targets of licorice using HERB 2.0. (C) KEGG pathway enrichment analysis of the pathways enriched by peony targets. (D) KEGG pathway enrichment analysis of the pathways enriched by licorice targets.
We analyzed the differentially expressed genes in the gastric mucosa of CAG and chronic superficial gastritis in the GSE163416 database, and 3524 differentially expressed genes were obtained (Fig. 3A). We intersected all the targets of SGD with the differentially expressed genes in CAG, and the results showed 11 intersecting genes (Fig. 3B). Analyzing the expression of intersecting genes in the GSE163416 dataset, we plotted a heatmap in Fig. 3C. We found that MAOB was most significantly differentially expressed in CAG (p-value = 6.40E-09), and MAOB expression was significantly elevated in CAG (LogFC = 2.230795998).
We then evaluated the expression of MAOB in mice with CAG. RT-qPCR and western blot demonstrated that MAOB expression was upregulated in the gastric mucosa of CAG mice (Fig. 3D, E). Moreover, the MAOB expression was remarkably reduced in response to SGD (Fig. 3F, G).
MAOB is a possible target of SGD in CAG.
(A) The volcano map of differentially expressed genes in gastric mucosa of CAG and chronic superficial gastritis in GEO dataset GSE163416. (B) The intersection of differentially expressed genes in the GSE163416 dataset with the targets of SGD in HERB 2.0. (C) Heatmap showing the expression of intersecting genes in gastric mucosal tissue in the GSE163416 dataset. (D) RT-qPCR detection of MAOB expression in gastric mucosal tissues of CAG mice. (E) Western blot detection of MAOB expression in the gastric mucosa of CAG mice. (F) RT-qPCR detection of MAOB expression in gastric mucosal tissues of CAG mice treated with SGD or PBS. (G) Western blot detection of MAOB expression in the gastric mucosa of CAG mice treated with SGD or PBS. Data were shown as mean ± SD. n = 6. *p < 0.05 versus the control or CAG + PBS group (unpaired t test).
To confirm that the alleviating effect of SGD on CAG was elicited through the inhibition of MAOB, we overexpressed MAOB in mice and treated them with SGD. RT-qPCR (Fig. 4A), western blot assays (Fig. 4B), and immunohistochemistry (Fig. 4C) on mouse gastric mucosa confirmed the successful overexpression of MAOB. HE staining showed that MAOB overexpression contributed to increased gastric mucosal injury and inflammatory infiltration in mice (Fig. 4D), and the level of apoptotic cells in the tissue was also increased (Fig. 4E). The expression of IL-6 (Fig. 4F), IL-1β (Fig. 4G), and TNF-α (Fig. 4H) was also remarkably augmented in the gastric mucosal tissues of MABO overexpressing mice.
Overexpression of MAOB reverses the therapeutic effect of SGD on CAG in mice. AAV containing MAOB overexpression vectors were injected into mice, followed by CAG modeling and SGD treatment.
(A) RT-qPCR detection of MAOB expression in gastric mucosal tissues of CAG mice after AAV injection. (B) Western blot detection of MAOB expression in the gastric mucosa of CAG mice after AAV injection. (C) Immunohistochemical detection of MAOB expression in the gastric mucosa of CAG mice after AAV injection. (D) Histopathology of gastric mucosal epithelial glands in mice by HE staining and histopathology score. (E) The apoptosis in epithelial cells of gastric mucosa tissues of mice using TUNEL assay. (F) Detection of IL-6 expression in gastric mucosal tissues of mice by RT-qPCR. (G) Detection of IL-1β expression in gastric mucosal tissues of mice by RT-qPCR. (H) Detection of TNF-α expression in gastric mucosal tissues of mice by RT-qPCR. Data were shown as mean ± SD. n = 6. *p < 0.05 versus the OE-NC group (unpaired t-test).
CAG represents a chronic gastric mucosal inflammation related to the loss of gastric glandular cells, replaced by intestinal-type epithelium and fibrous tissue (Tong et al. 2021). H. pylori has a high rate of infection and antibiotic resistance and poses a threat to humans, and TCM has the potential to mitigate drug resistance and facilitate the eradication of H. pylori (Li et al. 2021b). Even though the function of SGD has been implicated in ethanol-induced gastric ulcers in rats (Jin et al. 2022), its therapeutic effect on H. pylori-related diseases, particularly CAG, remains virtually unexplored. Our findings revealed that SGD can relieve CAG in mice induced by H. pylori, which was at least partly related to the downregulation of MAOB.
Inflammatory cell infiltration and apoptosis of gastric mucosal cells were revealed to be two prominent alterations in CAG (Zhang and Wang 2019; Kim et al. 2020). We observed consistent results in the gastric mucosa of mice induced with H. pylori and a high-salt diet, indicating successful modeling. The anti-inflammatory effects of SGD have been described in a substantial set of diseases, including polycystic ovary syndrome (Chang et al. 2021), cerebral ischemia-reperfusion injury (Zhang et al. 2016; Lu et al. 2022), and Parkinson’s disease (Chen et al. 2021a). Meanwhile, SGD has been reported to reduce the apoptosis rate of the interstitial cells of Cajal induced by hypercholesterolemia (Zhu et al. 2021). Likewise, our in vivo evidence showed the anti-inflammatory (IL-6, IL-1β, and TNF-α levels) and anti-apoptotic effects of SGD on the gastric mucosa. As its name indicates, peony and licorice are the main integrates of SGD. Kwon et al. found that HemoHIM, an herbal preparation consisting of roots of peony, significantly alleviated gastric injury in indomethacin-induced gastric ulcers (Kwon et al. 2019). Moreover, the growth-inhibiting and bactericidal effects of Paeonia lactiflora root have been verified by Ngan et al. (Ngan et al. 2012). Consistently, Wittschier et al. showed that aqueous extracts and polysaccharides from liquorice roots repressed the adhesion of H. pylori to the gastric mucosa (Wittschier et al. 2009). These findings were in line with our observations that SGD treatment repressed the colonization of H. pylori in the gastric mucosa.
Various methods were used to analyze the targets of SGD and to elucidate the underlying mechanism in diseases (Feng et al. 2020). We combined the network pharmacology and KEGG enrichment analysis here. The TNF signaling pathway was found to be enriched by targets of both peony and licorice, which partially corroborated the suppressing effects of SGD on the TNF-α levels. Intriguingly, monoclonal antibodies targeting TNF-α have been investigated to prevent inflammation-related gastrointestinal cancer (Lee et al. 2016). Probiotics remarkably alleviated H. pylori-evoked gastric pathology, including alleviated inflammatory infiltration and fewer precancerous lesions by blocking pro-inflammatory pathways, involving the TNF signaling pathway (He et al. 2022). Tsai et al. also found that H. pylori-induced sensitivity to TNF-related apoptosis-inducing ligand-mediated apoptosis in gastric epithelial cells during gastric mucosal damage (Tsai and Hsu 2017).
To further characterize the mechanism underlying the gastroprotective effects of SGD in CAG, we downloaded the data from the GSE163416 dataset to obtain differentially expressed genes and intersected them with targets of both peony and licorice. A total of 11 intersecting genes were found. MAOB whose upregulation was the most pronounced in CAG was selected. MAO with 2 isoforms, A and B, located at the outer mitochondrial membrane are flavoenzyme membranes, and MAO inhibition has emerged as a pharmacological target to mitigate oxidative stress and chronic inflammation (Sturza et al. 2019; Tripathi and Ayyannan 2019). Interestingly, in the “Paeoniflorin-targets-metabolites” interaction network, MAOB was found as one of the five key targets (Chen et al. 2021b). In the study here, we revealed that the expression of MAOB was enhanced in CAG mice and downregulated by SGD, indicating its involvement in the effects of SGD. Finally, we corroborated that the effects of SGD on CAG were achieved at least partially through the MAOB suppression using rescue experiments where MAOB overexpression overturned the anti-inflammatory properties of SGD.
As for the limitation of this study, first, only one dose of SGD has been administered to mice, and the effects of more doses are worth exploring. In addition, DUOX2 was also identified as a modifier in the preventive effects of MAO-based antidepressants on indomethacin-induced gastric lesions (Cao et al. 2020). Therefore, the deeper mechanism of MAOB in SGD needs to be fully studied. Lastly, the detailed molecular mechanisms of SGD inhibiting MAOB expression should be further explored using molecular docking.
ConclusionIn conclusion, the work here revealed that SGD plays an alleviating role in CAG mice. In addition, its mechanism is related to the manipulation of the TNF pathway and the downregulation of MAOB (Fig. 5). This study has offered fresh insights into the mechanism of SGD in the management of CAG. Nevertheless, further clinical trials are necessary to confirm our conclusions.
Schematic presentation showing Shaoyao Gancao decoction (SGD) mitigated chronic atrophic gastritis (CAG).
Inhibition of MAOB expression by SGD alleviated CAG caused by Helicobacter pylori.
The authors declare no conflict of interest.
