Study On The Mechanism Of Intervention Of Formononetin In Colorectal Cancer Through Lipid Metabolism Pathway

Objective:To explore the effect and mechanism of FN on CRC through in vitro and in vivo experiments,and provide experimental basis for the clinical application of FN.Method:(1)The inhibitory effect of FN on CT-26 and HCT116 cells was determined in vitro.The anticancer effect was evaluated by MTT assay.The contents of IL-6,IL-1βand TNF-α were detected.The protein expression levels of Akt,mTOR,LC3,Beclin-1,Caspas3,Bax,Bcl-2,p-Akt and p-mTOR were detected by Western blot.(2)AOM/DSS was used to induce CRC model in mice,and two different administration regimens were designed,namely prophylactic administration and therapeutic administration.During the whole process of drug administration,the body weight,diarrhea and hematochezia of the mice were observed,and DAI score was performed.At the end of the experiment,the colon length,colon weight,tumor size,volume,spleen weight of the mice were recorded,and the colorectal cancer related histological score was performed after pathological sections.These indicators were used to preliminarily evaluate the improvement of CRC by FN.The eyeball blood was collected,and the supernatant was collected.The differential metabolites of FN in CRC mice were analyzed by non-targeted metabolomics method,and the key metabolic pathways were screened out to lay the foundation for subsequent mechanism research experiments.(3)The mechanism of lipid metabolism was studied in vivo.ELISA kits were used to measure the levels of IL-6,IL-1β,TNF-α,GSK-3,IGF-1,ACLY,A-CoA,FAS,ATGL,HSL,and FFA.The expression of ZO-1、Claudin-1、Occludin and was analyzed by immunohistochemistry.The protein expression levels of ACLY,PDE3 B,p-HSL and LC3 were detected by immunofluorescence.Western blot was used to measure the protein expression of Akt,p-Akt,GβL,Rictor,mTOR,p-mTOR,ACLY,PDE3 B,PKA,p-PKA,HSL,p-HS,LC3,Beclin-1,Caspas3,Bax,and Bcl-2.(4)Fecal samples were collected in the late stage of the AOM/DSS-induced CRC mouse model,and high-throughput sequencing was used to analyze the changes of intestinal flora in the CRC mice treated with FN.Results:(1)In vitro,FN inhibited the growth of CT-26 and HCT116 cells and reduced inflammation.The growth of CRC cells may be inhibited by enhancing the autophagy apoptosis pathway.(2)In vivo: In vivo animal experiments showed that FN could significantly inhibit colon shortening induced by AOM/DSS in CRC mice,improve DAI score,maintain survival rate of mice,relieve histopathological symptoms of CRC mice,and inhibit tumor size,number and volume.At the same time,the spleen index increased,but the body weight did not change significantly.Metabolomics analysis showed that the different metabolites were mainly concentrated in lipids and amino acids,among which lipids were the main ones,including phospholipids and fatty acids.In addition,the main metabolic pathway of FN treatment for CRC is lipid metabolism.(3)In the mechanism of lipid metabolism,FN could significantly reduce the levels of inflammatory factors IL-1β,IL-6,TNF-α,and the contents of IGF-1,ACLY,A-CoA,FAS,HSL,ATGL,and FFA,and significantly increase the content of GSK-3(P < 0.01).In addition,FN could significantly increase the expression of Occludin,Claudin-1,and ZO-1 in a dose-dependent manner.Moreover,FN decreased the expression of ACLY,PDE3 B and p-HSL in a dose-dependent manner.In addition,FN inhibited the protein expression of p-mTOR,Rictor,p-Akt,ACLY,PDE3 B,p-PKA,p-HSL and Bcl-2,and increased the autophagy proteins LC3,Beclin-1 and apoptosis proteins CL-Caspas3 and Bax.And there was significant difference(P < 0.01).(4)The results of intestinal flora showed that FN improved the diversity of intestinal flora,and at the phylum level,Bacteroidete and Firmicutes accounted for74.93%.At the family level,the proportion of Lactobacillaceae and Muribaculaceae increased,and the proportion of Enterobacteriaceae and Staphylococcaceae decreased.The main beneficial bacteria in genus were Lactobacillus and pathogenic bacteria Staphylococcus.The beneficial bacteria were Muribaculaceae and Lactobacillus,while the harmful bacteria were Staphylococcus aureus and Escherichia coli.Conclusion:(1)In vitro,FN inhibited the growth of CT-26 and HCT116 cells and reduced inflammation.It may inhibit the growth of CRC cells by enhancing the autophagy and apoptosis pathway.(2)In vivo,FN can improve CRC mice to a certain extent.It can significantly inhibit the occurrence and development of colon cancer.In addition,FN intervention in CRC mice mainly caused the production of differential metabolites of phospholipids and fatty acids,and the metabolic pathway was mainly related to lipid metabolism,which laid a foundation for subsequent mechanism verification.(3)In the mechanism study,FN can reduce the growth of CRC by inhibiting the secretion of inflammatory factors and the expression of related proteins in lipid metabolism pathways,and also block the development of CRC by activating the autophagy and apoptosis pathway.(4)FN can regulate intestinal diversity and improve the changes of microbiota in CRC.The mechanism by which it inhibits CRC development may be closely related to its microbial abundance.