Abstract 

Objective: To explore the potential mechanism of Cistanche total glycosides on inflammatory bowel disease (IBD) through network pharmacology and animal experimental research. Methods: The Pubchem database and literature were used to collect the three-dimensional structures of the seven main active ingredients in Cistanche total glycosides. Obtain information on active ingredient-related targets and IBD-related gene targets through databases such as PharmMapper, UniProt, and GeneCards. Then draw a Venn diagram between the active ingredient targets of Cistanche and the targets of IBD to obtain the intersection targets, and upload the intersection targets to the String database for protein-protein interaction (PPI) screening analysis. Using the DAVID database to compare the active ingredients of Cistanche total glycosides in IBD

Targets that exert protective effects were analyzed by Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Cytoscape3.8.0 software was used to construct an "active ingredient-target-pathway network" to predict the total number of Cistanche. Targets and pathways of glycosides in treating IBD, and an IBD mouse model was constructed for further verification. Results: There are 254 targets of the total active ingredients of Cistanche that have a protective effect on IBD, and 30 core targets were screened out by PPI analysis. GO functional enrichment and KEGG pathway enrichment found that Cistanche total glycosides may mainly regulate cancer pathways, mTOR pathways, TGF-β pathways, JAK-STAT pathways, AMPK pathways, etc., thereby affecting cell signaling, proliferation, differentiation, and apoptosis. Play a role. Animal experiment results show that Cistanche total glycosides can effectively alleviate weight loss and fecal bleeding in IBD mice, reduce disease activity index, and effectively inhibit the expression of two target proteins in the spleen, mTOR and TGF-β. By sequencing the 16S rDNA amplicon of mouse feces for functional annotation of the PICRUSt2 gene, the analysis found that Cistanche total glycosides regulate IBD disease and cell wall/cell membrane/envelope biogenesis, lipid metabolism, sugar metabolism processes and related defense signal transduction in mice. There is a close relationship with the guidance mechanism, which is consistent with the network pharmacology results. Conclusion: Cistanche total glycosides can pass through multiple components and multiple targets

Points and multiple pathways can effectively prevent IBD, which provides new methods and new ideas for elucidating the clinical application of Cistanche in the treatment of IBD. Its specific mechanism and material basis need to be verified by more in-depth experimental research.

Keywords:Cistanche total glycosides; network pharmacology; inflammation;

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Inflammatory bowel disease (IBD) is a special chronic intestinal inflammatory disease with unknown cause, including two subtypes: Crohn's Disease (CD) and ulcerative colitis (Ulcerative Colitis, UC) [1]. As of 2017, 6.8 million people worldwide have been diagnosed with IBD, and the global incidence and prevalence are increasing year by year. The prevalence of IBD is higher in North America and Western Europe [2-3]. From 1990 to 2019, the incidence rate of IBD in China increased from 1.45/100,000 to 3.62/100,000, with an overall increase of 149.66%; the mortality rate decreased from 0.47/100,000 to 0.33/100,000, with an overall decrease of 29.79%[4] . As China's population ages, a small increase in the high incidence of elderly patients will place a huge burden on the medical and health care system.

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Studies have proven that dietary regulation can help relieve symptoms such as abdominal pain or diarrhea in IBD patients and reduce its complications; it can also regulate the composition and function of intestinal microorganisms, affect intestinal homeostasis, and maintain normal intestinal functions [5]. Cistanche , as a food and medicine plant with a long history, is widely used in the treatment of intestinal diseases in traditional Chinese medicine. It is also considered to have a variety of health effects, so it has attracted much attention in the field of health food [6-7 ]. In 2020, Cistanche (desert) was listed as a food and drug homologous substance, and pilot production and operation work was launched [6-7]. As a food, Cistanche can prevent IBD in daily life; as a medicinal material, it can alleviate symptoms during the treatment of IBD and has certain economic and medicinal value.

Some scholars have found that desert Cistanche aqueous extract can stimulate the immune system, regulate immune activity, and prevent inflammatory bowel disease [8]. Jia et al. [9] found that Echinacoside (ECH) extracted from Cistanche can improve DSS-induced colitis in mice; ECH can prevent cell death and improve mucosa by stimulating intestinal epithelial cell proliferation and upregulating TGF-β. Tissue repair effect[10]. ECH can also alleviate LPS-induced apoptosis and inflammation in rat intestinal epithelial cells by inhibiting the expression level of the mTOR/STAT3 pathway [11]. In addition, verbascoside can act as a free radical scavenger in the body, slowing down the progression of 2,4-Dinitrobenzene sulfonic acid (DNBS)-induced colitis in rats and reducing tissue damage [12].

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Network pharmacology is an interdisciplinary subject that integrates bioinformatics, biochemistry, pharmacology and computational science. It uses big data, bioinformatics and computing tools to analyze and simulate complex drug metabolism in organisms. The prediction of mechanism of action and drug efficacy provides important support for the development of new drugs and the optimization of existing drugs [13-14]. The core of network pharmacology is to establish a network model between drugs and molecules in organisms to reveal the interrelationships between drugs and target proteins, metabolic pathways, cell signaling pathways, etc. [15]. With the continuous advancement of science and technology, network pharmacology will continue to play an important role in the field of medicine and contribute to improving patients' quality of life and health.

Studies have found that Cistanche total glycosides can effectively inhibit the inflammatory response of BV2 cells induced by LPS [16], inhibit the progression of liver cancer [17], protect the liver [18], improve memory and cognition [19-20], and resist aging [ 21] and other functions. At present, there are few research reports on the effect of Cistanche total glycosides on inflammatory bowel disease. This study explores the mechanism of action of Cistanche total glycosides on inflammatory bowel disease based on network pharmacology and animal experiments, and provides new methods and ideas for its clinical application in the treatment of IBD.

1 Materials and instruments

1.1 Reagents

Dextran sodium sulfate (DSS, relative molecular weight 36 000 ~ 50 000), American MP Company; urine and fecal occult blood test kit, Nanjing Jiancheng Bioengineering Institute; absolute ethanol, Sinopharm Chemical Reagent Co., Ltd.; xylene, Shanghai Ling Feng Chemical Reagent Co., Ltd.; PBS buffer, RNA extraction solution, first-strand reverse transcription kit, 2× real-time quantitative PCR amplification premix solution, RTQPCR primers, Wuhan Sevier Biotechnology Co., Ltd.; sterile and enzyme-free water,

HyClone USA.

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1.2 Main instruments and equipment

KZ-III-FP low-temperature grinder, Wuhan Sevier Biotechnology Co., Ltd.; CT15RE high-speed centrifuge, HITACHI Company of Japan; CFX fluorescence quantitative PCR instrument, Bio-rad Company of the United States; SW-CJ-1FD ultra-clean workbench , Suzhou Antai Air Technology Co., Ltd.; NanoDrop2000 ultra-trace spectrophotometer, Thermo Company, Germany; FBZ2001-up-p standard reagent type pure water meter, Qingdao Fulham Technology Co., Ltd.; INFINITE MNANO multi-function microplate reader, TECAN, Switzerland Company, UV1600 UV spectrophotometer, Shanghai Miputa Instrument Co., Ltd.

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