Exploring the anti gastric cancer mechanism of triterpenoids from Inonotus obliquus based on network pharmacology and molecular docking
Gastric cancer is a common malignant tumor worldwide, with a relatively poor prognosis and a serious threat to human life and health. More than 70% of new cases of gastric cancer occur in developing countries, and about 50% of cases occur in eastern Asia, mainly in China. The incidence rate and mortality of gastric cancer rank second in malignant tumors in China, and the incidence rate has not yet declined. It is a major disease endangering the health of residents.
Inonotus obliquus (I0) is a medicinal fungus in the family Polyporus, and its resources are mainly distributed in the eastern region of China, mainly in Jilin and Heilongjiang provinces. Clinical pharmacological studies have confirmed that Botrytis cinerea has a wide range of pharmacological effects, including hypoglycemic, lipid-lowering, anti-tumor, antiviral, and hepatoprotective effects. In the 16th century, Botrytis cinerea was already a popular medicinal and edible herb among the people in Northeast China. Research has found that patients with advanced gastric cancer who take Botrytis cinerea have increased appetite, improved mood, and can also reduce their pain. Research has shown that lanosterol type tetracyclic triterpenoid compounds include Botrytis cinerea, lanosterol, and mycotic acid. Due to its significant anti-tumor effect, triterpenoids of Inonotus obliquus (IOT) from Inonotus obliquus can effectively inhibit tumor cell proliferation. Our research group conducted preliminary studies on the extraction and purification process of triterpenoids from Inonotus obliquus, as well as their in vitro anti-tumor effects. The results showed that when the triterpenoid concentrations of 20, 50, and 100 μ g/mL were applied to human gastric cancer cell lines for 24 and 48 hours, the cell growth inhibition rates were 27, 80%, 58.41%, and 42.50%, 52.70%, and 66.14%, respectively. The purified Inonotus obliquus triterpenoids significantly inhibited the growth of human gastric cancer cell lines (P<0.05), but the target and mechanism of action of Inonotus obliquus triterpenoids against gastric cancer are not yet clear.
Network pharmacology is a new method of analyzing the targets and mechanisms of drug intervention in diseases from multiple perspectives based on fundamental interdisciplinary theories. This study used network pharmacology to screen potential targets of triterpenoid active compounds from Inonotus obliquus for gastric cancer, explore their molecular mechanisms, construct a multi-target and multi pathway pharmacological network for their anti gastric cancer effects, and conduct molecular docking verification. This provides a scientific basis for further research on the mechanism of action and drug development of triterpenoid compounds from Inonotus obliquus against gastric cancer.

Gastric cancer is the second malignant tumor in incidence rate, which has become an urgent public health problem. At present, more and more people are paying attention to natural foods and health drugs. Botrytis cinerea is praised as the “Siberian Ganoderma” because it can inhibit the metastasis of cancer cells, reduce the possibility of cancer recurrence, improve the tolerance of cancer patients, and reduce the discomfort caused by radiotherapy and chemotherapy, which is highly favored by researchers. This study is based on the theoretical foundation of network pharmacology and molecular docking, and uses relevant databases and drawing software to investigate the mechanism of action of triterpenoids from Inonotus obliquus on gastric cancer.
The results of network pharmacology indicate that the triterpenoid active ingredients of Inonotus obliquus include 24 components, including Inonotus obliquus alcohol, Betulinic acid, Thrombotic acid, lanosterol, etc. Research has shown that betulinic acid has significant anti-tumor activity on various tumor cells, and can exert anti-tumor effects by activating NF kB without any toxic side effects. Additionally, studies have shown that betulinic acid may induce cell apoptosis by upregulating the expression of Caspase-3 and Cyto-3 proteins. Both lanosterol and mycorrhizal acid can inhibit the G0/G1 cell cycle, reduce cancer cell proliferation, promote cell apoptosis, and exert anti-tumor effects. The anti-tumor effect of thrombotic acid is related to the regulation of P-glycoprotein; Nomura et al. found that inotodiol can activate key hydrolytic enzymes during apoptosis, promote the cleavage of PARP and Caspase-3 proteins, initiate apoptosis signals, and promote cancer cell apoptosis. Betuline can upregulate Bax protein expression and downregulate Bcl-2 protein expression, thereby promoting cell apoptosis.
Five key targets were identified through the “active ingredient target pathway” network and PPI protein interaction network, including MAPK1 ALB、NFKB1、MAPK8、CASP3。 Research has shown that serum albumin (ALB), as one of the indicators currently used to reflect nutritional status, is the most abundant and functionally important component in serum proteins. It is an important nutrient in the human body and has a protective effect on the gastric wall. The decrease in plasma levels indicates poor nutritional status in postoperative patients with gastrointestinal tumors. Chen et al. found in their study comparing the clinical outcomes of nutritional intervention in gastrointestinal malignancies that patients who received nutritional support therapy had significantly higher serum ALB levels compared to those who did not receive nutritional support therapy. Albumin levels can be considered as an independent prognostic factor for gastric cancer after surgery. The NFKB1 gene has functional binding sites in the promoters and enhancers of many genes. It can specifically bind to specific sites such as TNF – α, IL-6, and IL-8, participate in inflammation and immune responses, mediate physiological processes such as cell adhesion, differentiation, proliferation, and apoptosis, and play an important role in the occurrence and development of various immune inflammatory diseases and tumors. Jiang et al. found that the mutant genotype CT of rs4648127 located in the intron region of the NFKB1 gene is significantly correlated with susceptibility to gastric cancer and can significantly reduce the risk of gastric cancer susceptibility. More scholars have found that the increased risk of gastric cancer in elderly people over 65 years old is closely related to the polymorphism of the NFKB1 promoter gene (-94 insertions/deletions of ATTG). Through research, MAPK1 may be a downstream target gene of miR-217 in gastric cancer cells. miR-217 can target MAPK1, reduce its expression level, and control the metastasis and invasion of gastric cancer cells. In this study, it was found that the pharmacological substances in the triterpenoids of Botrytis cinerea can act on MAPK1 MAPK8、MAPK10、MAPK14， And it participates in the MAPK signaling pathway, exerting biological effects; Many of the anti gastric cancer targets of triterpenoid active compounds from Botrytis cinerea involve apoptotic proteins such as CASP3 and CASP7. Studies have shown that the pathogenesis of gastric cancer is related to low expression of CASP3 and inhibition of cell apoptosis in human gastric cancer tissue. KEGG pathway analysis also indicates that the mechanism of action of triterpenoid active ingredients from Inonotus obliquus against gastric cancer is related to cancer signaling pathways. The apoptotic pathway is regulated by proteins such as CASP3, CASP7, and CASP9, which can directly cause tumor cell apoptosis and inhibit its proliferation. The TNF signaling pathway is the most common inflammatory pathway, and the receptor protein TNFR1 on the cell membrane can bind to the extracellular free ligand TNF – α, activating the signaling pathway. A cascade reaction occurs, activating the apoptotic protein Caspase-3, triggering a series of inflammatory reactions, ultimately leading to cell apoptosis. Research has found that the water extract of Botrytis cinerea can reduce the levels of TNF – α and IL-4, and significantly decrease the expression of signal transduction factor STAT1 and transcription activation factor STAT6, leading to tumor cell apoptosis. The Toll like receptor signaling pathway has strong positive expression of TLR4 and TLR9 at both the gene and protein levels in gastric cancer tissue. By binding to ligands, it activates the NF kB pathway and regulates various pro-inflammatory genes such as IL-8, IL-1, and IL-6. Therefore, the Toll like receptor signaling pathway can monitor the metastasis of malignant tumor cells through the defense mechanism of the body’s immune system. It is speculated that the triterpenoid active ingredients of Betula platyphylla can act on multiple targets and pathways, thereby exerting anti gastric cancer effects.
Molecular docking, as a major method of computer-aided drug design, has been widely applied in various aspects of new drug development. Generally, Vina divides the binding energies of docking molecules and targets into three categories: -4.0 kJ/mol<binding energy<0 kJ/mol, indicating that the two have binding activity- 7.0kJ/mol<binding energy<-4.0kJ/mol, indicating good binding activity; The binding energy<-7.0 kJ/mol indicates strong binding activity. According to the docking results, the binding energies of betulinic acid to various protein targets such as CASP3, MAPK1, MAPK8, and NFKB1 are less than -7.0 kJ/mol, indicating strong binding ability. Betulinic acid forms hydrophobic interactions with CASP3 active site residues TYR204 (C), LEU168 (C), and PHE256 (C), and forms a hydrogen bond with MAPK8 active site residue LEU110 (A). It also forms hydrophobic interactions with MET111 (A), ASN114 (A), VAL158 (A), and NFKB1 active site residues PHE310 (A) and LYS275 (P). Has good binding ability and forms hydrophobic interactions with active site residues such as ILE523 (A), GLU529 (A), LYS519 (A), etc. 21,24-cyclopentadiene lanostane-3B, 21,25-trihydroxy-8-ene forms two hydrogen bonds with the MAPK1 active site residue MET108 (A), forming hydrophobic interactions with ALA52 (A), VAL39 (A), TYR36 (A), and others. Analysis of the binding models between key components and various proteins reveals that each key active component enters the active sites of each protein crystal and forms hydrogen bonds or hydrophobic interactions with the active sites. Therefore, it is speculated that the triterpenoid components of Botrytis cinerea may regulate pathways related to gastric cancer through the aforementioned key proteins, thereby improving and treating gastric cancer.
In summary, this article predicted the complex molecular network relationship of triterpenoid active ingredients from Inonotus obliquus against gastric cancer through network pharmacology methods. The results showed that the active ingredients of triterpenoid compounds from Inonotus obliquus against gastric cancer may be: Inonotus obliquus alcohol, betulinic acid, lanosterol, thrombotic acid, etc. The key targets involved are ALB, MAPK1, NFKB1, MAPK8, CASP3, etc. The main signaling pathways involved are TNF signaling pathway, cancer signaling pathway, MAPK signaling pathway, Toll like receptor signaling pathway, etc. Finally, through molecular docking verification, it was found that 5 key target proteins have good binding effects with 24 active ingredients. This article provides ideas and methods for the study of related mechanisms of action, and provides a basis for further exploration of the anti gastric cancer mechanism of triterpenoids from Inonotus obliquus.