Effect and mechanism of Acoritataninowii Rhizoma and Curcumae Radix on chronic gastritis: a network pharmacology study


 Background

Chronic gastritis (CG) is an inflammatory disease which is one of the common diseases of the digestive system. To investigate the mechanisms of herbal pair Acoritataninowii Rhizoma(Shichangpu, AR) and Curcumae Radix༈Yujin, CR༉ in treatment of CG based on the network pharmacology.
Methods

The possible active ingredients and targets of AR-CR were obtained by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The UniProt database was used to query the human gene corresponding to each target protein. The genes related to CG were collected from the GeneCards database, the OMIM database, the DisGeNET database and the PharmGKB database. Intersected the target genes of AR-CR and CG, then protein-protein interaction(PPI) network was constructed by STRING website. The overlapped genes were subjected to gene ontology༈GO༉enrichment and Kyoto encyclopedia of genes and genomes༈KEGG༉pathway enrichment analyses by David.
Results

45 intersection genes were obtained, and there were 40 targets in the PPI network for protein interaction, the kernel targets with Degree ≥ 10 included AKT1, TNF, JUN, MAPK3, MAPK8 and MAPK1. The Go enrichment analysis was mainly related to protein binding, enzyme binding, protein homodimerization activity, etc. The KEGG pathway enrichment analyses mainly involved the Pathways in cancer, TNF signaling pathway, Apoptosis, and VEGF signaling pathway.
Conclusion

AR-CR might delayed, blocked or reversed the atrophy, intestinal metaplasia, dysplasia and canceration of gastric mucosa by targeting key proteins and signal pathways,achieved the effect of the treatment of CG.


Results
45 intersection genes were obtained, and there were 40 targets in the PPI network for protein interaction, the kernel targets with Degree ≥ 10 included AKT1, TNF, JUN, MAPK3, MAPK8 and MAPK1. The Go enrichment analysis was mainly related to protein binding, enzyme binding, protein homodimerization activity, etc. The KEGG pathway enrichment analyses mainly involved the Pathways in cancer, TNF signaling pathway, Apoptosis, and VEGF signaling pathway.
Conclusion AR-CR might delayed, blocked or reversed the atrophy, intestinal metaplasia, dysplasia and canceration of gastric mucosa by targeting key proteins and signal pathways,achieved the effect of the treatment of CG.
Background CG is an in ammatory disease that the epithelium of the gastric mucosa is invaded by various pathogenic factors, and it is one of the common diseases of the digestive system. The recurrence of this disease seriously affects the quality of life of the patient. Chronic atrophic gastritis with intestinal metaplasia and intraepithelial neoplasia increased the risk of gastric cancer, which has attracted more and more attention in clinical [1] . Based on endoscopy and pathological examination, it included chronic non-atrophic gastritis and chronic atrophic gastritis [2] . Dampness-heat syndrome of spleen-stomach is one of the common syndromes of chronic gastritis [1] . Dampness-heat disease is extensive, the pathogenesis is complex, the course of disease is long, and the treatment is di cult.
Herbal pair AR-CR is sourced from Wen Bing Quan Shu. The nature and avor of AR is pungent-warm which could dissipate cold and dispel dampness and strong spleen yang to accelerate transportation and transformation, the qi and avor of AR is aromatous which could resolve dampness-turbidity. AR is an indispensable herb to treat patients who are dampness-heat of spleen-stomach with yellow-slimy fur, or patients who are cold-dampness encumbering the spleen with white-slimy fur [3] . CR acts on qi aspect to move qi and relieve depression, and blood aspect to cool the blood and dissipate stasis. AR combination with CR could move qi and relieve depression, clear the heart and open the ori ces,dissipate stasis and resolve phlegm. Herbal pair AR-CR have good clinical effect on patients with spleen-stomach dampnessheat syndrome of CG with the state of anxious or depressed. Professor Lao's Qingzhuo Anzhong Decoction is mainly composed of AR and CR, which is quite effective in the treatment of spleen-stomach dampness-heat syndrome of CG [4] .
However, the mechanism of herbal pair AR-CR on CG is not clear. In this study, we applied network pharmacology to analyze the effective target of herbal pair AR-CR in the treatment of CG in an integrated and systematic way. Through the topological network analysis, the effective pathways would be deeply explored,and expounded the mechanism of herbal pair AR-CR in the treatment of CG.

AR-CR Target prediction
All ingredients contained in AR and CR were collected from the TCMSP (http://tcmspw.com/tcmsp.php).
Screen the retrieved ingredients, and the compound Oral bioavailability (OB) screening thresholds were OB ≥ 30%, and the compound Drug likeness (DL) screening thresholds were DL ≥ 0.18. Candidate compound targets were retrieved by searching the TCMSP. The targets were transformed using the UniProt knowledge database (https://www.uniprot.org/), with the selected species as Homo sapiens.
Data were combined to gain gene symbol after removed duplicate items.

PPI network of AR-CR
After the target genes of AR-CR and CG were intersected, the PPI network was constructed using the STRING (https://string-db.org/), which is an update online database, known as revealing target protein interaction analysis, then used Cytoscape3.7.2 for visualization of protein networks.

Gene enrichment analysis
The Database for DAVID (https://david.ncifcrf.gov/) server was employed to perform the GO enrichment and the KEGG pathway analysis for the overlapped targets associated with AR-CR and CG. Then, analyzed the in uence of overlapped target proteins in gene function and signal pathway, and used Cytoscape3.7.2 to construct the target-pathway network. biological process such as response to drug, response to estradiol.

Discussion
Chinese medicine has a history of thousands of years, and herb medicines exert extensive biological and pharmacological effects through a variety of compounds and targets. Network pharmacology is an available tool to identify alternative targets for herbal medicines and to develop multi-target drugs. In this study, we analyzed the PPI of AR-CR on the effective targets of CG, and obtained multiple kernel targets including AKT1, TNF, JUN, MAPK3, MAPK8 and MAPK1. In order to clarify how the AR-CR worked through these targets, the GO enrichment analysis and the KEGG pathway enrichment analysis were further performed in this study. Through the GO enrichment analysis, we thought that the herb pair AR-CR was able to regulate the protein binding, enzyme binding, response to drug, response to estradiol, etc. The enrichment results of the KEGG signaling pathway showed that the main function of AR-CR might be related to the Pathways in cancer, TNF signaling pathway, Apoptosis, and VEGF signaling pathway, etc.
In the PPI network analysis, one of the key genes such as AKT1 is tightly related to gastrointestinal cancers [5] . The activation of AKT1 induces cell proliferation through the stimulation of cell cycle progression and inhibits the intrinsic apoptotic pathway [6] . The amount of AKT1 protein can stimulate the proliferation of neoplastic cells,and AKT1 was overexpressed in 8% of tumors and associated with a trend for a poor overall survival [6] . Rescue experiments demonstrated that decreased proliferation and increased apoptosis of gastric cancer cells induced by miRNA4903p overexpression were partially suppressed by AKT1 overexpression [7] .
TNF is an important regulator of in ammation and immunity, including TNF -α and TNF -β. TNF -α is the main in ammatory cytokine, which maintains homeostasis by regulating in ammation, cell proliferation, differentiation, survival and apoptosis [8] . The increased expression of TNF-α in the sequential evolution of gastric cancer, the gastric precancerous conditions such as chronic gastritis, intestinal metaplasia, dysplasia and gastric adenocarcinoma patients showed the high expression of TNF-α [9] . Xu et al. reported that TNF-α was a major part in in ammatory, infectious and tumor processes, and is pivotal at the early stages of gastric cancer, and TNF-α rs361525 polymorphism is related to the risk of gastric cancer, especially for Asians [10] . In the development from CG to gastric cancer, TNF signaling pathway plays an important component in the in ammatory response and tumor [11] .
Jun is also known as c-Jun, which played a key component in the tumorigenesis of gastric cancer. Miao et al. reported that KIAA1429 contributed to promoting gastric cancer by regulating c-Jun expression in a m6A independent manner [12] . Peng et al. reported that c-Jun could promote the proliferation, metastasis, and invasion of gastric cancer cells in vitro and in vivo by accelerating the expression of FOXK1 at the transcriptional level [13] .
MAPK is a group of serine/threonine protein kinases that can be activated by different extracellular stimuli, such as cytokines, neurotransmitters, hormones, cell stress and cell adhesion, which regulate cell growth, differentiation, stress adaptation to the environment, in ammatory response and other important cellular physiological and pathological processes. MAPK3, MAPK1 and MAPK8 are member of the MAPK family. The cell proliferation and invasion of gastric cancer cells were reported to be propelled by the activation of MAPK signaling pathway [14] . MAPK3 is also known as ERK1, is an important signal transduction molecule in the ERK/MAPK pathway [15] . The ERK/MAPK signal transduction pathway is widely expressed in various tissues and cells, and it can regulate various biological processes such as cell proliferation, cycle, apoptosis, migration, and invasion [16,17] . Enhanced functional activity of MAPK3 contributed to the development and progression of gastric cancer [18][19][20] . Knockdown of MAPK1 expression in gastric cancer cell lines induced cell growth retardation, apoptosis, and suppressed cell migration as well as invasion [21] . Diao et al. [22] reported that lncRNA GAPLINC was increased and positively correlated with MAPK1 expression in gastric cancer tumor tissues, additionally, the enhancement of gastric cancer cell proliferation and cell cycle progression by lncRNA GAPLINC was dependent on MAPK1. MAPK1 is a direct target gene of miR-585 that promotes the proliferation and metastasis of gastric cancer [23] .MAPK8, which is also known as JNK, is characterized as a pronounced marked protein, is an important cellular pathway triggered in response to DNA damage, and it was also proved to be associated with tumor progression, including cell survival, migration and autophagy [24] . Sun et al. [25] reported that MAPK8 could mediate S/G2 phase cycle arrest and apoptosis, and suppress AKT activation in gastric cancer cells, and dysregulated MAPK8 was observed in several cancers, such as glioblastoma, skin cancer, brain tumor and leukemia [26,27] .
Increased apoptosis is associated with the development of gastric carcinoma, and H. pylori infection induces apoptosis in gastric epithelial cells by stimulating the host's in ammatory/immune responses [28] . Stimuli and inhibitors for apoptosis may act at targets on the cell membrane via Fas and TNF receptors either in the cytoplasm or in the nucleus [28] . The ethanol-induced apoptosis of gastric mucosa could be ascribed to increases in TNF-α level, Bax, and caspase-3 activity [29] . A feature of malignancies is an alteration of cell turnover leading to cell hyperproliferation and to deregulation of apoptosis [30] . With deepening researches, a sustained in ammatory reaction and abnormal apoptosis of gastric mucosa are con rmed as important causes in the pathogenesis of chronic atrophic gastritis, which have drawn growing attention [31,32] . An impaired balance between proliferation and apoptosis was detected in gastric cancer and in atrophic gastritis or intestinal metaplasia in gastric mucosa adjacent to cancer [33] .
VEGF contribute to stimulate the growth and differentiation of vascular endothelial cells, is an essential factor in cancer development and progression, its receptors include VEGFR1 and VEGFR2. VEGF could exhibit different levels of expressions in precancerous lesions and gastric cancer, and it is involved in the occurrence and development of gastric cancer [34] . Canonical VEGF signaling through VEGFR1/R2 regulates the activities of several kinases and ultimately guides cell proliferation, migration, survival, and vascular permeability during vasculogenesis and angiogenesis [35] . Inhibiting the VEGF/VEGFR pathway could cause a rapid and sustained antiangiogenic/antitumor response [36] .

Conclusion
From CG to gastric precancerous lesions, to gastric cancer is a process involving multiple genes, multiple pathways and complicated molecular mechanism. Our study aimed to investigate the mechanisms of AR-CR in the treatment of CG, we performed compounds, targets, pathways prediction and network analysis by using a network pharmacology method. In this study, we thought that herbal pair AR-CR might delayed, blocked or reversed the atrophy, intestinal metaplasia, dysplasia and canceration of gastric mucosa by regulating the above key targets or regulating the above signal pathways, achieved the effect of the treatment of CG, which provided important information for the mechanism of AR-CR in the treatment of CG. Target-pathway diagram