Anti-Tumor Immunometabolic Molecular Mechanism Of Icariside Ⅰ

Herbal Epimedium is an important Chinese herbal medicine,which has the effects of enhancing body immune function,anti-aging,anti-osteoporosis and anti-tumor.Its main active components are flavonoids.Previous studies have shown that icariin derivatives could effectively kill tumor cells by enhancing the body’s immune system,and are a class of potential natural small-molecule anticancer drugs.At present,the anti-tumor immune drugs widely used in clinical practice are mainly biological macromolecules such as antibodies,and there are few reports of small-molecule immune anti-tumor drugs.Recently,icariin of icariin derivatives has been approved as a domestic class 1 new drug for the treatment of advanced liver cancer.However,icariside Ⅰ,as a trace active component from Herbal Epimedium,due to its very low content(one out of one hundred thousand),it is difficult to obtain only by chemical separation extraction method,so the research and development is rarely reported.This paper based on the study of the overall immunoregulatory function,using immunology,metabolomics and microbiomics technology systematically evaluated the safety and biological activity and anti-tumor immunometabolic molecular mechanism of icariside Ⅰ,the main research contents are as follows:First,this paper conducted in vivo and in vitro safety evaluation for icariside Ⅰ.In vitro cell experiments showed that icariside Ⅰ has some cytotoxicity,with the threshold range of multiple cancer cells ranging from 20 to 35 μM.In vivo experiments explored the possible toxic side effects and immune function effects of different doses of icariside Ⅰ(5,20,80,160,320,640 mg/kg body weight)on mice.Results found that even at the high dose of 640mg/kg body weight,icariside Ⅰ did not have significant effects or damage on the function of major organs such as liver and kidney,indicating that its toxic side effects and its safe drug range were relatively wide.Flow cytometry experiments found that icariside Ⅰ could significantly increase the proportion of multiple immune cell subsets in the peripheral blood of normal mice,indicating that icariside Ⅰ has some systemic immune enhancement effect.Based on the safety evaluation results of icariside Ⅰ,we selected two lower doses(5 and 20mg/kg body weight)of icariside Ⅰ,melanoma mice and melanoma cell models as research subjects to deeply study the anti-tumor immunometabolic molecular mechanism of icariside Ⅰ.It was found that icariside Ⅰ could significantly enhance CD8+ T cells and the level of IFN-γ in the tumor microenvironment(tumor tissue),and interact with protein indindlamine 2,3-dioxygenase 1(IDO1),inhibit the transformation of tryptophan to kynurenine,convert the "immune escape" pathway of tumor cells from IDO1-Kyn-AhR cascade to JAK1-STAT1 pathway,successfully block IFN-γ-induced tumor "immune escape" and activate STAT1 phosphorylation to produce a large number of apoptotic proteins,thus inducing tumor cell apoptosis.These findings suggest that icariside Ⅰ may act as a natural small-molecule anticancer drug targeting the Kyn-AhR pathway for tumor immunotherapy.As the largest immune organ of the body,it is obvious that the regulation of gut microbiota provides an opportunity for tumor immunotherapy.We combine microbiome and metabolomics to further investigate the molecular mechanisms of icariside Ⅰ in assisting tumor immunotherapy by regulating the gut microbiota of tumor-bearing mice.The results found that different doses of icariside Ⅰ(5,20 and 80 mg/kg body weight)could significantly improve the intestinal barrier,immunologic function,the diversity and microbial composition of tumor-bearing mice,especially the abundance of two probiotics(Lactobacillus and Bifidobacteria),and the level of microbial metabolites such as short-chain fatty acids and indole metabolites,reducing the level of systemic inflammation,thus significantly inhibiting the tumor growth in mice.These results showed that icariside Ⅰ could assist in cancer immunotherapy through the regulation of the gut microbiota.In conclusion,this paper uses immunology,targeted metabolomics and microbiomics combined with molecular biology techniques to study in detail the safety and anti-tumor immunometabolic molecular mechanism of trace active ingredient icariside Ⅰ in Herbal Epimedium.It provides detailed basic data and new ideas for the basic research of the Chinese herbal medicine Herbal Epimedium and other authentic herbal medicine.