Study On Anti-tumor Activity And Mechanism Of Solid Fermentation Products Of Leucocalocybe Mongolica On H₂₂ Tumor-bearing Mice

As a traditional edible and medicinal fungus,Leucocalocybe mongolica has a long history of application in China and has a variety of pharmacological activities.Owing to increased predation and desertification,wild L.mongolica populations are declining and becoming increasingly rare,which has severely restricted its further application and product development.Microbial fermentation is emerging as a suitable alternative to obtain bioactive compounds from this mushroom.However,there is no data to support whether the solid fermentation products of L.mongolica can replace the wild L.mongolica for drug development.This experiment intended to systematically evaluate the anti-tumor activity and mechanism of solid fermentation products of L.mongolica,and provide data support for the further development and utilization of solid fermentation products of L.mongolica.In this paper,the anti-tumor activity of water extracts,polysaccharides,and petroleum ether extracts of L.mongolica were studied first,and then the method of multi-omics(proteome,metabolome,intestinal microbes)combined analysis was used to analyze the anti-tumor mechanism of the solid fermented petroleum ether extract.The anti-tumor activity results of different extracts isolated from the solid-state fermentation of L.mongolica showed that the extract in each experimental group had no obvious toxic and side effects on mice at a dose of 300 mg/kg/d.This study revealed that the extract isolated from the solid-state fermentation of L.mongolica markedly suppressed the tumor growth in vivo at least partly by enhancing the immune response,inducing apoptosis and inhibiting angiogenesis.The pro-apoptotic effect of the extract is achieved by adjusting the ratio of Bax/Bcl-2 and promoting the expression of P53 protein.The treatment effect of each group showed a dose-dependent trend.The anti-tumor effect of petroleum ether extract was better than polysaccharide and water extract.The proteomics result showed that there were 251 differential variables between PEELMtreated groups and model groups.Among these metabolites,39 metabolites in the model group were obviously up-regulated in the model group and 212 metabolites were significantly downregulated.Differential proteins are mainly involved in the structure of cells and organelles,and are mainly involved in processes such as RNA processing,transport,and metabolism.PAS analysis showed that the main biological processes up-regulated were humoral immunity,cellular immunity,and antigen processing and presentation processes,while the main downregulated biological processes were cell metabolism and organic synthesis.The KEGG analysis revealed that all differential proteins involved in the following pathways: Protein processing in endoplasmic reticulum,Apoptosis-multiple species,Ras-MAPK signaling pathway and so on.West-Blot analysis was used to verify the apoptosis pathway related proteins.The results were consistent with the proteomics data.The expression level of Bid was significantly increased after administration of PEELM,while the expression of BCL-2 was decreased,and the proapoptotic proteins Bax,Cyt C and cleave Caspase3 was increased.It shows that the PEELM could increase tumor cell apoptosis mainly through the mitochondrial apoptosis pathway.The untargeted metabolomics was used to discover biomarkers and effective anti-tumor mechanisms of petroleum ether extract isolated from the solid-state fermentation of L.mongolica(PEELM).There were 37 differential variables between the model groups and normal groups.Among these metabolites,23 metabolites in the model group were obviously upregulated in the model group and 14 metabolites were significantly down-regulated in the model group.Metabolic pathway enrichment was analyzed.The top ten pathways for the differentially expressed metabolites were identified,including biosynthesis of unsaturated fatty acids,biosynthesis of amino acids,protein digestion and absorption,aminoacyl-t RNA biosynthesis,mineral absorption,serotonergic synapse,linoleic acid metabolism,pyrimidine metabolism,primary bile acid biosynthesis,and pantothenate and Co A biosynthesis.The PEELM group was distantly related to the model group and approached the normal group,showing that PEELM attenuates the pathological changes in model mice.Differential variables between the PEELMtreated group and the model group were selected.There were 37 differential variables between PEELM-treated groups and model groups.Among these metabolites,14 metabolites were obviously up-regulated and 23 metabolites were significantly down-regulated.The pathways for the differentially expressed metabolites were identified,including serotonergic synapse,pyrimidine metabolism,protein digestion and absorption,primary bile acid biosynthesis,pantothenate and Co A biosynthesis,mineral absorption,linoleic acid metabolism,aminoacyl-t RNA biosynthesis and so on.In order to further explored the anti-tumor mechanism of PEELM,16 S DNA highthroughput sequencing was used to analyze the abundance and diversity of intestinal flora among the normal group(NG),model group(MG)and PEELM group.The α-diversity analysis showed that the experimental sequencing depth was reasonable,and the data was accurate and reliable.The results of ACE index,Chaol index,simpson index,and shannon index showed that PLEEM administration could increase the abundance of intestinal flora and increase the diversity of intestinal flora in tumor-bearing mice.The result of β-diversity analysis showed that the MG group and the NG group were relatively far apart on the PCA and PCo A maps,but after PLEEM administration,the distance becomed closer,which once again indicated that the PLEEM could improve the intestinal microenvironment of mice and promote the restoration of the abundance and diversity of intestinal flora.The analysis of the difference between groups showed that at the phylum level,PEELM can increase the relative abundance of the posterior phylum and saccharified bacteria;at the family level,PEELM can increase the Lactobacillaceae,Lachnospiraceae,Relative abundance of Bacteroidales S27 family(Bacteroidales＿S24-7＿group),Prevotellaceae and Bacteroidaceae;PEELM can increase Lactobacillus,Bacteroidales＿S24-7＿group＿norank,Bacteroidaceae at the genus level(Bacteroides),Prevotellaceae＿UCG-001,Lachnospiraceae＿NK4A136＿group relative abundance.PEELM can reduce the abundance of the posterior phylum,and increase the relative abundance of the Bacteroides and saccharification phyla;at the family level,PEELM can increase the Lactobacillus and Laospirillaceae.The relative abundance of Bacteroides S27,Bacteroides and Prevotaceae decreased.Relative abundance of Lactobacillaceae,Lachnospiraceae,Bacteroidales＿S24-7＿group,Prevotellaceae and Bacteroidaceae;at the genus level PEELM It can increase the relative abundance of Lactobacillus,Bacteroidales＿S24-7＿group,Bacteroides,Lachnospiracea＿e＿NK4A136＿group,and decrease the relative abundance of Prevotellaceae＿UCG-001.In summary,this study revealed that the solid fermentation products of Leucocalocybe mongolica markedly suppressed the tumor growth in vivo at least partly by enhancing the immune response,inducing apoptosis and inhibiting angiogenesis.These findings highlight L.mongolica as a potential low-cost anti-tumor drug.