Discussion on the network pharmacology mechanism and molecular targets of Shenqin Baizhu San in the treatment of colorectal cancer
Colorectal cancer (CRC) is currently the third most common malignant tumor in the world. Its incidence rate and mortality continue to increase, and the five-year survival rate has been unsatisfactory. From the perspective of epigenetic changes, the occurrence and development of CRC takes 10-15 years, but risk factors such as dietary preference, excessive consumption of red meat, aging, inflammatory bowel disease, and imbalanced gut microbiota can rapidly promote its development. At present, surgery, radiotherapy, chemotherapy, and targeted therapy are the main treatment methods for CRC. However, the toxic side effects of these treatment methods cannot be ignored, and they have a great impact on the quality of life of patients, seriously restricting their efficacy.

Traditional Chinese medicine (TCM), as an alternative drug for treating tumor diseases, has significant advantages in the treatment of CRC, and its combination with radiotherapy and chemotherapy can significantly improve the survival rate of patients for 1-3 years. SLBZP comes from the “Tai Ping Hui Min He Ji Ju Fang” and mainly consists of ginseng (Ginseng Radix et Rhizoma), Poria (Poria), Atractylodis Macrocepha lae (Rhizoma Atractylodis Macrocepha lae), Lablab Semen Album, Nelumbi nis Semen (Lotus Seed), Dioscorea Hizoma (Yam), Amo mi Fructous (Amo mi Fruitus), Coici Semen (Coix seed), Platycodo nis Radix (Platycodo nis Radix), and Glycyrrhiza (Glycyrrhiza Radix et Rhizoma). Composition of flavored drugs. SLBZP contains a large amount of saponins, flavonoids, steroids, polysaccharides, triterpenoids, and other compounds. In clinical practice, SLBZP combined with radiotherapy and chemotherapy can effectively improve the quality of life of CRC patients, enhance their immunity, and reduce the toxic side effects of radiotherapy and chemotherapy. Wang et al. conducted a randomized controlled clinical study to analyze the effects of SLBZP combined with chemotherapy on postoperative immune function and quality of life in CRC patients. They found that after 12 weeks of treatment, compared with the chemotherapy group, traditional Chinese medicine combined with chemotherapy significantly inhibited the expression of CD3+, CD4+, CD4+/CD8+, increased CD8+expression, improved KPS score, increased disease control rate (88.10%), improved toxic side effects after chemotherapy, and improved patients’ quality of life. Zhang et al. used the combination of SLBZP and mFOLFOX6 regimen to treat postoperative CRC patients, significantly enhancing the body’s immunity and improving the toxic side effects after chemotherapy. Ginseng and Poria cocos, as the main herbs in SLBZP, mainly nourish the five organs of the human body and regulate the spleen and stomach qi. Modern research has shown that the main component of ginseng, ginsenoside Rb2, not only acts on the TGF – β 1/Smad signaling pathway to inhibit the growth, adhesion, EMT, and metastasis of human CRC cells, but also activates the P53 pathway, increases the level of pro apoptotic factor Bax, and promotes apoptosis of HCT-116 and SW620 cells; Poria cocos polysaccharide (PCP), as the most abundant active ingredient in Poria cocos (accounting for about 84% of the total components), has a wide range of biological activities such as anti-tumor, immune regulation, and anti-inflammatory. Animal experiments have also shown that SLBZP can effectively reduce the infiltration of MDSCs and improve the immunosuppressive tumor microenvironment; And by downregulating the levels of TCF – β 1 and N-cadherin, upregulating E-cadherin reduces EMT and β – catenin activation, and mitigates the process of carcinogenesis.

Traditional Chinese medicine is a national treasure of China, and has undergone continuous testing, development, and improvement by generations of medical practitioners. Traditional Chinese medicine emphasizes the importance of the ruler, subject, and assistant. Clinical prescriptions are often composed of a combination of multiple drugs. These complex drugs often have the characteristics of multiple active ingredients, multiple targets of action, and multiple pathway regulation, making them ideal choices for treating complex diseases. However, how to clearly and objectively explore and analyze the molecular mechanisms and interactions of the active ingredients of each drug has become a huge problem that currently limits the development of medicine in China. Therefore, in order to solve the problems faced in the development of traditional Chinese medicine, the method of analyzing the efficacy of traditional Chinese medicine through network pharmacology has emerged. As a new method combining systematic network analysis and pharmacology, it can be used to elucidate the synergistic effects of active ingredients in drugs through compound target disease networks, predict potential mechanisms for treating diseases through multi-target and multi pathway pathways at the molecular level, understand the interaction relationships between genes, proteins, and diseases, and provide new possibilities for pharmacological research of complex traditional Chinese medicine formulas.
In this study, network pharmacology methods were used to analyze the molecular targets and mechanisms of SLBZP’s active ingredients in treating CRC. And the differentially expressed genes between CRC patients and healthy individuals were obtained and screened through the GEO database to obtain CRC related targets. The potential mechanism of SLBZP in treating CRC was analyzed through GO and KEGG.

The unique concept of traditional Chinese medicine has undergone thousands of years of development and inheritance, and is commonly used for the prevention and treatment of various diseases. Traditional Chinese medicine formulas are usually composed of multiple herbs that are used in combination according to the principles of Jun, Chen, Zuo, and Shi to achieve synergistic effects, deliver the medicine to the disease site, and promote the recovery of qi and eliminate evil. CRC belongs to the categories of traditional Chinese medicine, such as “visceral toxicity”, “intestinal wind”, “intestinal mushroom”, “locked anal hemorrhoids”, “dian huang”, “accumulation”, etc. It is often caused by the accumulation of cold, phlegm, dampness, toxin, and blood stasis, which condense into carbuncle and accumulate in the large intestine. The treatment often focuses on strengthening the body and dispelling evil, strengthening the spleen and qi, clearing heat and dampness, achieving the goal of dissipating the accumulation of toxins and blood stasis, enhancing the body’s righteous qi, improving disease symptoms, and improving the quality of life. SLBZP, as a classic formula for strengthening the spleen, promoting dampness, and strengthening the body, is commonly used in the treatment of ulcerative colitis (UC) in clinical practice. It can be combined with radiotherapy and chemotherapy to significantly enhance the immune function of CRC patients, alleviate intestinal inflammatory reactions, optimize intestinal microbiota structure, and so on. Traditional Chinese medicine emphasizes a holistic approach to treating diseases, focusing on the restoration of overall bodily functions and the elimination of underlying causes. Once the concept of network pharmacology was proposed, it was quickly combined with traditional Chinese medicine formulas for research, which can comprehensively elucidate the overall concept of traditional Chinese medicine in treating diseases and analyze the complex mechanisms of action of traditional Chinese medicine formulas. Therefore, with the support of multiple database platforms, bioinformatics methods are used to analyze the complex medicinal components and mechanisms of action of traditional Chinese medicine formulas, making the specific efficacy of the drugs clear and understandable. In this study, 190 compounds and 255 target genes of SLBZP were screened based on OB and DL, and the intersection was obtained by merging 611 differentially expressed genes between CRC patients and healthy individuals. Based on FDR<0.05 and logFC>1, 31 genes related to SLBZP and CRC were ultimately identified. We constructed a SLBZP compound CRC targeting network. The results showed that most compounds of SLBZP can regulate multiple targets, such as quercetin, kaempferol, and coumarin, which act on 21, 5, and 4 targets respectively. They occupy important positions in the network, so they are likely to be key active compounds of SLBZP. Although the number of predicted targets for each individual drug varies, there are many repeated targets of action. It is not difficult to see that multiple active ingredients of SLBZP have synergistic effects on regulating targets. As one of the most widely studied flavonoids, exfoliation has been proved to have strong antioxidant stress and anti-inflammatory activity, and is considered to play an important role in the treatment and prevention of diabetes, cancer, neurodegenerative diseases and cardiovascular diseases. In terms of its effect on tumors, berberine can regulate various biological pathways, induce tumor cell apoptosis, inhibit tumor angiogenesis, and prevent tumor cell proliferation. Research has shown that quercetin, as an effective inhibitor of β – catenin, can induce downregulation of Wnt/β – catenin in CRC SW480 cells, preventing the progression of CRC. Hu et al. studied the impact of Xiexin nanoparticle preparation (35nm) on the tumor microenvironment of bladder cancer by preparing Xiexin nanoparticle preparation. They found that Xiexin granule preparation can increase the permeability of drugs to the tumor barrier, significantly reduce the growth of active fibroblasts and the amount of collagen in the tumor, and normalize the content of collagen in the tumor tissue to achieve the purpose of anti-tumor. Shannai phenol is a natural brassinol found in different plant species. Numerous in vitro experiments have shown that Shannai phenol has significant anti-inflammatory effects and can inhibit tumor development by blocking the tumor cell cycle, preventing tumor angiogenesis, promoting tumor cell apoptosis, and preventing tumor cell migration. However, due to the influence of factors such as transport protein permeability, lipophilicity, mesenteric structure, and intestinal epithelial cell transport on the utilization of drugs in the body, salvianolic acid has a low oral availability, which limits its use as an anticancer agent. However, according to reports, the combination of salvianolic acid and quercetin can significantly enhance the anticancer effect of quercetin by blocking its efflux, and the combination with cisplatin can significantly increase its toxicity to cells. As a candidate anti-cancer drug, sitosterol has also shown significant anti-cancer effects. It can inhibit the proliferation, migration, and formation of capillary networks of tumor endothelial cells by disrupting the TNF – α – VEGFR-2 axis, effectively downregulating the production of inflammatory cytokines, macrophage recruitment, and tumor angiogenesis, and achieving the goal of inhibiting tumor growth. The composition of traditional Chinese medicine is complex, and although network pharmacology can comprehensively analyze the chemical composition and mechanism of action of traditional Chinese medicine, the total effective ingredients and synergistic effects after interactions among all parties cannot be solved. Drug therapy for diseases is the result of the combined action of multiple components. In this study, quercetin, kaempferol, and stigmasterol regulated most of the relevant targets of CRC, and all have anti-inflammatory and anti-tumor properties, making them representative compounds of SLBZP.
The SLBZP predicted targets and CRC related targets were merged and structured using a PPI network. In order to more accurately analyze the interaction relationship between SLBZP and CRC common targets and related proteins, two parameters including DC and BC were set to screen nodes and construct a new network. Through PPI network analysis, it was found that SLBZP has a strong interaction with other proteins in the treatment of CRC, and its therapeutic effect on CRC may synergistically regulate the interaction with other proteins.

Perform GO enrichment analysis on the enrichment of SLBZP targeting CRC targets in BP, CC, and MF. The results indicate that SLBZP regulates certain biological processes, such as extracellular matrix breakdown, collagen breakdown metabolism, and alcohol dehydrogenase activity. Epithelial to mesenchymal transition (EMT) plays a crucial role in both embryonic development and carcinogenesis. In terms of tumors, EMT is related to the invasion, metastasis, drug resistance, and poor prognosis of cancer cells. EMT is usually induced by cytokines such as TCF – β, which activates several signaling pathways, downregulates the expression of E-cadherin and phosphorylates β – catenin protein, reduces the concentration of E-cadherin – β – catenin (E β) complex, and disrupts intercellular adhesion. The extracellular matrix (ECM) undergoes rapid changes in composition and structure during the development of cancer. The tumor ECM is rich in collagen I and fibronectin, and through LOX mediated collagen cross-linking, it reorganizes from a random network to an aligned network, thereby increasing the density of ECM, promoting the survival and proliferation of tumor cells, increasing their motility, promoting tumor cell invasion, and often leading to tumor resistance to radiation therapy. This remodeling of ECM can directly affect the progression of cancer. The deposition and cross-linking of fibrous ECM proteins, including collagen and fibronectin, increase, while the reduction of intercellular adhesion caused by EMT leads to an imbalance in cell matrix and intercellular adhesion, which is also the basis for cancer progression. Approximately 3.6% of cancers worldwide are caused by alcohol consumption, which can be closely associated with gastrointestinal malignancies including colorectal cancer (CRC). Therefore, the International Agency for Research on Cancer (IARC) classifies alcohol as a Group 1 carcinogen in humans. The carcinogenic mechanism of alcohol is: interference with DNA synthesis and repair; Promote gene mutations within cells; Alter metabolic pathways; Excessive consumption of retinoic acid leads to low tissue differentiation, high degeneration, and a pre malignant state. In CRC patients, the activity of alcohol dehydrogenase (ADH) in serum is significantly increased compared to healthy mucosa, while the activity of aldehyde dehydrogenase (ALDH) is significantly reduced, resulting in an abnormal imbalance between the two activities and the production of a large amount of toxic acetaldehyde. SLBZP can prevent the occurrence and development of tumors by intervening in these physiological and pathological processes.

Traditional Chinese medicine has the advantages of multiple components, targets, and pathways in treating diseases. Compound SLBZP also has the same characteristics. Treat CRC with a multi-component, multi-target, and multi pathway approach. In this study, 20 KEGG signaling pathways, including those related to chemical carcinogenesis and Salmonella infection, were significantly enriched. Due to dietary habits and occupational hazards, the incidence of CRC caused by chemical carcinogenesis has been increasing. Food cooked at high temperatures (>130 ° C) or contaminated with air often produces carcinogens such as polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines (HCAs), N-nitroso compounds (NOCs), mycotoxins (aflatoxins), and acrylamide, which urgently need to be recognized as potential risk factors for colorectal cancer (CRC). Most epidemiological studies have observed a positive correlation between HCA exposure and CRC, with a 30% increase in the risk index associated with CRC induced mutations. Nitrates, as effective preservatives added to food products, not only prevent the growth of Clostridium botulinum, but also enhance the color and flavor of processed meat. But increasing evidence suggests that it can lead to endogenous NOC formation and exert a strong carcinogenic effect. The main carcinogenic mechanism of NOC is to increase the production of abnormal crypt foci (ACF) and mucous depletion foci (MDF) in the intestine, causing DNA mutations in intestinal epithelial cells and promoting the occurrence of colorectal cancer (CRC). With the development of high-throughput sequencing, the relationship between gut microbiota and CRC is being revealed. Numerous studies suggest that the structure of gut microbiota is disrupted. Pathogens can alter cellular biology by triggering chronic intestinal inflammation, becoming an important trigger for the development of CRC. Approximately 90 million people are infected with Salmonella each year, and there are over 2500 different serotypes of Salmonella subspecies present in the gut. Salmonella affects multiple signaling pathways associated with tumors, activating the AKT and ERK pathways through effector proteins secreted in cells, which are typically also activated in many cancers. In the Wnt signaling pathway, Salmonella effector non-toxic protein A (AvrA) can be transferred to host cells through T3SS. Under the action of AvrA’s deubiquitinase activity, it prevents the degradation of β – catenin and promotes its massive recruitment in the nucleus. By binding with TCF/LEF, it downregulates the expression of oncogenes MYC and CCND1. At the same time, AvrA can activate AKT to phosphorylate β – catenin on serine, further promoting nuclear β – catenin signaling. In summary, factors such as chemical carcinogenesis, intestinal inflammation, and an increase in the number of pathogenic bacteria are all important factors in the development of CRC. SLBZP, as a classic formula for strengthening the spleen and nourishing qi, can effectively increase the expression of anti-inflammatory factors in the body, enhance immune response, regulate intestinal microbiota, protect intestinal epithelial cells from damage, and prevent the development of CRC. In this study, the above pathways were significantly enriched and closely related to CRC signaling pathways, indicating that the regulation of pathways associated with CRC occurrence may be one of the mechanisms of SLBZP treatment for CRC. In addition, SLBZP may act by regulating other pathways, including the TNF pathway, Toll like receptor pathway, IL-17 pathway, etc.

This study also constructed a gene pathway network to investigate the key target genes of SLBZP for CRC. The results indicate that FOS and IL-1B have the highest BC and may be the core targets. The other top 9 genes (MMP9, VEGFA, ADHIC, STAT3, ADH1A, CCNDI, CXCL2, CLALI, IL-1A) were selected as key target genes. FOS, as a key driving factor for CRC metastasis, is located on the human chromosome 14q21-31 and encodes the nuclear protein c-Fos. The standalone c-Fos protein has no physiological function and must bind with c-Jun to form a heterodimer (AP-1) with transcriptional activation activity AP-1 is closely related to cell proliferation and differentiation. The expression of c-Fos and c-Jun is low in normal tissues and high in many malignant tumors and cancerous processes. Chen et al. analyzed the expression of c-Fos protein in 16 CRC patients’ cancer tissues and adjacent normal tissues to clarify the susceptibility of CRC under the influence of FOS. They found that the expression level of c-Fos protein in CRC tissues was significantly increased, and the survival rate was significantly reduced. Inflammation, as an influencing factor in various stages of carcinogenesis, is increasingly receiving attention. Many studies have shown that regular use of nonsteroidal anti-inflammatory drugs can significantly reduce the risk of developing CRC. This indirectly proves that the inflammatory response promotes the occurrence of CRC. IL-1B (IL-1 β), as a pro-inflammatory “alarm” factor, is activated in the early stages of inflammation and cancer. The expression level of IL-1 β is significantly increased in CRC patients and is related to the severity and prognosis of the disease. Like most other tumor diseases, CRC is a tumor with high angiogenesis, and the efficient generation of blood vessels is an important factor in improving the survival, proliferation, and metastasis of CRC cells. The IL-1 β/IL-6 pathway can promote angiogenesis and tumor cell growth by stimulating the expression of the potential angiogenic factor COX2, and the expression of the three factors is significantly correlated. Overexpression of COX-2 is regulated by the significant increase of IL-1 β and IL6.

This study used network pharmacology methods to explore the mechanism and molecular targets of SLBZP on CRC. Quercetin (21 targets), kaempferol (5 targets), β – carotene (5 targets), stigmasterol (4 targets), isoflavones (4 targets), and diosgenin (3 targets) act on multiple targets because they occupy important positions in the network and may be key active compounds of SLBZP. Therefore, SLBZP may act through the key compounds mentioned above, further affecting specific biological processes, regulating tumor cell proliferation and migration, and exerting therapeutic effects in inhibiting inflammatory responses, preventing chemical carcinogenesis, optimizing gut microbiota structure, and other pathways. FOS, IL-1B, MMP9, VEGFA, ADH1C, STAT1, ADH1A, CCND1, CXCL2, CLAL1, and IL-1A are key genes for SLBZP treatment of CRC, which further confirms the advantages of traditional Chinese medicine formulas in treating complex diseases with multiple components, targets, and pathways.