Anti-Colorectal Cancer Activity Of Carnosic Acid And It’s Related Mechanism

Colorectal cancer（CRC）is a malignant tumor originating from the gastrointestinal tract and is characterized by a high incidence and high mortality rate,with the third highest incidence and the second highest mortality rate in the world.The occurrence and development of CRC are associated with various factors,such as genetics,poor lifestyle habits,and inflammatory bowel diseases,among which the imbalance of the intestinal microbiota caused by multiple external factors is one of the main causes of CRC.The current treatment for CRC is based on surgery,radiotherapy,chemotherapy,and targeted therapy.However,as a malignant tumor,CRC is inherently prone to metastasis and a high recurrence rate,while the side effects of radiotherapy and chemotherapy are significant and easily resistant.Therefore,according to the etiology and clinical manifestations of CRC,it is imperative to find a highly effective natural therapeutic agent with few side effects to prevent and control the occurrence of CRC.Carnosic acid（CA）,a naturally occurring phenolic diterpene derived from plants in the Labiatae family,such as sage and rosemary,is a naturally occurring active substance with a high safety profile.Available studies have shown that it has various pharmacological effects,including antibacterial,antioxidant,antitumor,and anti-inflammatory.However,no studies have been conducted to investigate the activity of CA in inhibiting CRC using the Apc ^Min/+mouse model of colorectal cancer.This experimental study aimed to evaluate the anti-CRC efficacy of CA through in vivo experiments on the Apc ^Min/+mouse model and to explore the possible mechanisms of the anti-colorectal cancer activity of CA in Apc ^Min/+mice in combination with gut macrobiotics,serum metabolomics,and tumor proteomics.First,in vivo validation of the CRC inhibitory activity of CA was performed using Apc ^Min/+mice.CA gavage treatment of mice for 56 consecutive days revealed that CA significantly reduced the number of tumors in the colorectal tissues of Apc ^Min/+mice without affecting body weight or organ index,suggesting that CA can inhibit the development and progression of colorectal cancer.Subsequently,we sequenced the contents of the cecum of Apc ^Min/+mice using 16S amplicons and performed bioinformatics analysis,which showed that CA altered the composition of intestinal microorganisms in Apc ^Min/+mice and increased the number of anti-inflammatory bacteria of the genera Faecalibacterium,Blautia,Subdoligranulum,and Bifidobacterium;and decreased the abundance of harmful bacteria such as Lachnospiraceae＿NK4A136＿group,Alistipes and Desulfovibrio.Using UHPLC-Q-TOF MS to detect metabolites in the serum of Apc ^Min/+mice,23 serum metabolite levels were found to be altered in the untreated and CA-treated groups,with CA up-regulating the levels of 7 metabolites and down-regulating the levels of 16 metabolites and a high correlation between these 23 significantly different metabolites.Protein levels in untreated and CA-treated colorectal tumor tissues were analyzed using Label-free quantitative proteomic assays and 27 differentially expressed proteins were screened for correlation.Protein interaction analysis using STRING revealed 65 protein interactions between these 27 differentially expressed proteins.GO enrichment and KEGG enrichment analysis revealed that the differentially expressed proteins were mainly involved in biological processes such as inflammatory response,and immune regulation,as well as KEGG pathways such as the Interleukin（IL）-17 signaling pathway,complement and coagulation cascade.Validation analysis of IL-17-related inflammatory cytokines using the ELISA showed that CA significantly reduced IL-17A,chemokine（CX-C motif）ligand 1（CXCL1）,and IL-1βlevels and significantly increased interferon-α（IFN-α）levels in the colorectal tissues of Apc ^Min/+mice.Finally,the protein expression in colorectal tumor tissues of Apc ^Min/+mice was further examined by Western blot,which revealed that CA inhibited the expression of Wnt/β-catenin signaling pathway-related proteins and S100 calcium binding protein A9（S100A9）/Toll-like Receptor 4（TLR4）/Nuclear Factor kappa-B（NF-κB）signaling pathway.In addition,CA can inhibit the inflammatory response by decreasing IL-6,CXCL1,and IL-17 levels and increasing IFN-αlevels in the colorectum of Apc ^Min/+mice,thus exerting an anti-CRC effect.In summary,CA inhibited the development of CRC in Apc ^Min/+mice,probably by modulating the gut microbiota and serum metabolites,suppressing IL-17 expression in the colorectum tumor tissue of Apc ^Min/+mice,and modulating the S100A9/TLR4/NF-κB signaling pathway thereby showing anti-CRC properties.These results provide an experimental basis for future CA treatment of CRC.