Anti-Colorectal Cancer Effects Of Inonotus Hispidus (Bull.:Fr.)P.Karst. Spore Powder Through Regulation Of Gut Microbiota-Mediated JAK/STAT Signaling

Colorectal cancer（CRC）,is the second leading cause of cancer deaths,genetics,unhealthy living,and eating habits are the main predisposing factors for CRC.The changes in the gut microbiota composition may be important etiological factors in CRC development and progression.In cases with imbalanced gut microbiota,the increased secretion of bacterial toxins and carcinogenic secondary metabolites impairs the gut barrier functions,causing immune dysregulation,which can lead to CRC.The current treatment of CRC is mainly based on surgery,supplemented by immunotherapy and adjuvant therapy such as chemotherapy after surgery.However,all these therapies have limitations to some extent,so the development of natural complexes with regulating gut microbiota activity has become one of the main research directions for anti-CRC.Inonotus hispidus（Bull.:Fr.）P.Karst.spore powder（IHS）is the natural complex ejected from the late development of Inonotus hispidus.In modern research,Inonotus hispidus contained physiologically active triterpenes and polyphenols.Inonotus hispidus substrates and their extracts have immunomodulatory,anti-tumor,antibacterial,and antioxidant properties.However,the current research on IHS has not been reported yet.In this study,we analyzed for the first time the nutritional composition and metal element content of IHS.The potential regulatory pathways of IHS on CRC treatment through regulation of gut microbiota using Apc ^Min/+mice as a CRC model.This study provides a new idea for IHS as a dietary supplement in regulating the anti-CRC activity of gut microbiota.Firstly,We analyzed the conventional material composition of IHS using mass spectrometry and UV spectrophotometric detection.The results showed that IHS contained39%carbohydrates,15.3%proteins,2.65%polyphenols,2.07%flavonoids,1.34%saponins,0.8%fats,0.39%triterpenes,0.15%sterols,and 0.07%alkaloids.According to the current Chinese standard GB2762-2017,the contents of heavy metals Pb,Cd,Hg,and As are within the safety limits,confirming the safety of IHS for consumption.Secondly,the anti-CRC activity of IHS was validated in vivo using Apc ^Min/+mice.After6 weeks of gavage administration of 100 mg/kg IHS to Apc ^Min/+mice,no significant changes were observed in the organ index,and the colorectal index decreased.H&E staining showed that the tumor size was reduced after IHS administration and the cell morphology was maintained in its original state,confirming the inhibitory effect of IHS on CRC development in Apc ^Min/+mice.Sequencing the contents of the cecum of Apc ^Min/+mice using 16S r RNA and performing gut microbiota analysis showed that the abundance of beneficial genera of Oscillospira,Odoribacter,and Coprococcus in the 100 mg/kg IHS-treated group was increased.The differential genera were predicted by The KEGG pathway to be more abundant in the amino acid metabolic pathway.Subsequently,serum metabolites from Apc ^Min/+mice were examined by using UHPLC-Q-TOF MS.Metabolomic analysis revealed that IHS administration increased serum levels of L-Arginine（L-Arg）and KEGG annotation analysis of differential metabolites was associated with the Arginine synthesis pathway.Combined with ELISA results,IHS further revealed that IHS increased Arg and nitric oxide（NO）levels and decreased the arginase 1（Arg-1）levels in colorectal tumor tissues.L-Arg is a precursor of NO synthesis,and Arg-1limits the production of NO by the catabolism of Arg.Further combination of immunohistochemistry and Western Blot results revealed that IHS increased the level of interleukin（IL）-2 in colorectal tumor tissues and spleen.A high expression of IL-2 induced a high production of L-Arg and NO,and the level of L-Arg was closely related to the proliferation of CD8 T cells.Finally,flow cytometry and immunohistochemistry showed that IHS increased the abundance of CD8 T cells in the blood and colorectal tumor tissues of Apc ^Min/+mice,while not affecting the abundance of Th cells（CD4⁺）and B cells（CD19⁺and CD20⁺）.It was confirmed that IHS may play an anti-CRC role by increasing the abundance of CD8⁺T cells,regulating immunity.Western Blot confirmed that IHS regulates JAK/STAT signaling in Apc ^Min/+mice,and the JAK/STAT3 signaling pathway plays an important role in the differentiation and maturation of CD8⁺T cells.IHS strongly suppressed the expression of IL-5,IL-6,IL-10,P-JAK1,P-JAK2,P-STAT3,and P-STAT5 and enhanced the P-STAT1expression.The reduction in IL-5,IL-6,and IL-10 in the colorectal tumor tissues and spleen of the IHS-treated Apc ^Min/+mice was further confirmed through ELISAs.In summary,the conventional substance composition of IHS was analyzed for the first time in this study.IHS had the anti-CRC effect in Apc ^Min/+mice was demonstrated at the same time.IHS affected the abundance of gut microbiota and the level of L-Arg,regulated JAK/STAT signaling,and increased the abundance of CD8⁺T cells,thus inhibiting the development of CRC.This study provides experimental data and theoretical support to reveal the composition of IHS substances and their use as active substances in treating CRC.