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

Inonotus hispidus (Bull.: Fr.) P. Karst. spore powder (IHS) contains polyphenols and triterpenoids with pharmacological effects. Here, we analyzed its composition, and we investigated the effects of IHS on colorectal cancer (CRC) in B6/JGpt-Apcem1Cin(min)/Gpt (ApcMin/+) mice and its potential mechanisms by analyzing gut microbiota and serum metabolomics. The enzyme-linked immunosorbent assays and Western blotting were used to confirm the changes in the cytokine and protein levels associated with IHS administration. The IHS affected the abundance of gut microbiota and the level of L-arginine (L-Arg). Furthermore, the IHS influenced T cells in ApcMin/+ mice by increasing the interleukin (IL)-2 and decreasing the IL-5, -6, and -10 levels, thus suppressing tumor development. Overall, IHS showed anti-CRC properties in ApcMin/+ mice by affecting the gut microbiota and serum metabolites, which in turn affected the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, and regulated the abundance of CD8+ T cells. These results provide experimental support for the potential future treatment of CRC with IHS.


Introduction
As the second leading cause of cancer deaths, 1.9 million new cases of colorectal cancer (CRC) (including anal cancer) and 935,000 CRC-related deaths occurred in 2020, worldwide [1]. The patients with CRC may be susceptible to coronavirus disease (COVID-19) [2]. Genetics, unhealthy living and eating habits, such as a high-fat and low-fiber diet, are the main predisposing factors for CRC [3,4]. Fiber can be fermented by the gut microbiota to produce short-chain fatty acids, which positively affect the immune system and lower the risk of CRC [4]. The changes in the composition of the gut microbiota may be important etiological factors in CRC development and progression [5]. 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 [6]. The T lymphocyte-mediated immune functions play important roles in CRC [7]. In particular, CD8 + T cell infiltration is independently favorable for CRC prognosis [8]. The CRC patients with high levels of CD8 + T cells at the center of the tumor or at the invasive margins typically show a longer survival time [9].
Inonotus hispidus (Bull.: Fr.) P. Karst. is a medicinal fungus of the family Hymenochaetaceae and is the source of the traditional Chinese herb "Sanghuang" [10]. I. hispidus was listed in the ancient Chinese book The Herbal Classic of Shen Nong as a treatment for gynecological diseases. In modern research, I. hispidus has been artificially cultivated by Chinese scientists [10] and was shown to contain physiologically active triterpenes and polyphenols [11].
I. hispidus substrates and their extracts have immunomodulatory [12], anti-tumor [13], antibacterial [14], and antioxidant [15] properties. In H22 tumor-bearing mice, I. hispidus solid fermentation powder can inhibit tumor development by regulating immune functions [13]. In humans, I. hispidus extract enhances T cell activation while apoptosis occurs [12]. However, the current studies have focused on I. hispidus substrates and their extracts, whereas Inontus hispidus (Bull.: Fr.) P. Karst. spore powder (IHS) has received less attention, thus its composition and effects with respect to CRC are unclear.
The B6/JGpt-Apc em1Cin(min) /Gpt (Apc Min/+ ) mice were used as the CRC models, due to an adenomatous polyposis coli (Apc) mutation. The Apc gene is a colorectal cancer oncogene, and in over 70% of the CRC cases it shows mutations which lead to the constitutive activation of the Wnt/β-catenin pathway in the intestinal epithelial cells [16,17]. The signal transducer and activator of transcription 3 (STAT3) promotes the initial stages of CRC formation in Apc Min/+ mice [18]. The activated Janus kinase (JAK)/STAT pathway observed in most solid tumors can affect CRC development by modulating the cell growth, survival, invasion, and migration [19], and some of its antagonists impede the progression of malignancies [20]. The interleukins (IL)-2 and -6 are the essential mediators of T cell differentiation and function during immune responses related to the activation of the JAK/STAT pathway [21,22]. In our previous study, calf thymus polypeptides effectively increased the IL-2 and CD8 + T cell levels to inhibit CRC growth in Apc Min/+ mice [23]. Similarly, the knockout of the monocyte chemo-attractant protein 1 in Apc Min/+ mice increased the abundance of CD8 + T cells and decreased the IL-6 expression, thus alleviating the development of CRC [24].
In this study, the suppressive effects of IHS on CRC development in Apc Min/+ mice were confirmed. According to the gut microbiota and serum metabolomics, the IHS treatment restored the imbalance in the gut microbiota and the levels of serum metabolites, which then regulated the abundance of CD8 + T cells related to JAK/STAT signaling.

IHS Component Analysis
The IHS was collected in Linqing Sanghuang Town (Linqing, China) and was identified by Prof. Y. Li from Jilin Agriculture University.

Nutrients
The Kjeldahl method was used to measure the total nitrogen content, and a conversion factor of 6.25 was used to calculate the total protein content [25]. The phenol sulfate method was used to determine the total sugar content. Fat was extracted from the dried IHS samples using petroleum ether and a Soxhlet apparatus, and the fat content was assessed, using the residual method [25]. The gravimetric method was used to assess the total dietary fiber content [25], and the total polyphenols [25], alkaloids [26], flavonoids [26], sterols [27], triterpenoids [28], and saponins [29] were assessed using UV spectrophotometry.

Animal Experimental
All of the procedures were approved by the Institutional Animal Ethics Committee of Jilin University (SY202103008), and all of the experimental procedures involving animals were performed in strict accordance with the institutional guidelines. Eight-weeks-old Apc Min/+ mice purchased from GemPharmatech Co., Ltd., Jiangsu, China, (SCXK [SU] 2018-0008) were housed in a controlled environment at 23 ± 1 • C and 50-60% humidity under a 12/12 h light/dark cycle. A rodent diet with 60 kcal% fat (#D12492; Research Diet, New Brunswick, NJ, USA) and water were provided ad libitum.
After one week of adaptive feeding, twelve Apc Min/+ mice were randomly assigned to two groups (n = 6, each), and 100 mg/kg IHS or vehicle only was orally administered for six weeks. The standard procedures of intragastric gavage were on individuals of both groups to account for the effects of gavage stress. The body weights were monitored weekly. After the last administration, Apc Min/+ mice were fasted for 12 h, and blood samples were collected from the tail vein. The mice were killed through intraperitoneal injection with 150 mg/kg sodium pentobarbital, after which the feces were collected from the cecum in a sterile environment. The organs including the colorectum, heart, liver, spleen, lungs, and kidneys were collected for organ index calculations and biochemical and pathological assessment ( Figure 1A).

Animal Experimental
All of the procedures were approved by the Institutional Animal Ethics Committee of Jilin University (SY202103008), and all of the experimental procedures involving animals were performed in strict accordance with the institutional guidelines. Eight-weeksold Apc Min/+ mice purchased from GemPharmatech Co. Ltd., Jiangsu, China, (SCXK [SU] 2018-0008) were housed in a controlled environment at 23 ± 1 °C and 50-60% humidity under a 12/12 h light/dark cycle. A rodent diet with 60 kcal% fat (#D12492; Research Diet, New Brunswick, NJ, USA) and water were provided ad libitum.
After one week of adaptive feeding, twelve Apc Min/+ mice were randomly assigned to two groups (n = 6, each), and 100 mg/kg IHS or vehicle only was orally administered for six weeks. The standard procedures of intragastric gavage were on individuals of both groups to account for the effects of gavage stress. The body weights were monitored weekly. After the last administration, Apc Min/+ mice were fasted for 12 h, and blood samples were collected from the tail vein. The mice were killed through intraperitoneal injection with 150 mg/kg sodium pentobarbital, after which the feces were collected from the cecum in a sterile environment. The organs including the colorectum, heart, liver, spleen, lungs, and kidneys were collected for organ index calculations and biochemical and pathological assessment ( Figure 1A).

Hematoxylin and Eosin (H&E) Staining
According to a previous study [31], after fixation with 4% paraformaldehyde and dehydration, the tissues were embedded in paraffin and were exposed to gradient ethanol. The specimens were then cut into standard 5-µm sections, and were stained using hematoxylin and eosin (H&E), and examined using a microscope (Eclipse E100; Nikon, Tokyo, Japan).

Gut Microbiota Analysis
As in our previous study [33], the total microbial genomic DNA samples were isolated from cecal content (n = 4) using an OMEGA Soil DNA Kit (#M5635-02; Omega Bio-Tek, Norcross, GA, USA), and extraction was performed using a DNeasy PowerSoil Kit (QI-AGEN, Inc., Venlo, The Netherlands). The quantity and quality of the extracted DNAs were measured, using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and agarose gel electrophoresis, respectively. The V3-V4 regions of the bacterial 16S rRNA gene were amplified through polymerase chain reaction (PCR) with primers 338F (5 -ACTCCTACGGGAGGCAGCA-3 ) and 806R (5 -GGACTACHVGGGTWTCTAAT-3 ). The PCR products were purified, using Agencourt AMPure beads (Beckman Coulter, Indianapolis, IN, USA) and were quantified using a PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The amplicons were pooled at equal amounts, and the sequencing was performed on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). The data were analyzed as previously described [34].

Metabolomics Analysis
Metabolomics analysis was performed on the serum samples (n = 4) which were added to a pre-cooled methanol/acetonitrile/water solution (2:2:1, v/v), followed by sonication at a low temperature for 30 min and centrifugation at −20 • C for 10 min; the supernatant was dried under vacuum. An aqueous acetonitrile solution (100 µL, acetonitrile: water = 1:1, v/v) was added, and after centrifugation, the supernatant was used for mass spectrometry analysis through ultra-high-performance liquid chromatography (1290 Infinity LC; Agilent Technologies, Santa Clara, CA, USA) coupled to a quadrupole time-of-flight (Q-TOF, TripleTOF 6600; Sciex, Framingham, MA, USA) mass spectrometer at Shanghai Applied Protein Technology (Shanghai, China). The detailed detection parameters were the same as those used in our previous study [35]. Univariate statistical analysis, multidimensional statistical analysis, differential metabolite screening, differential metabolite correlation analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed, as described previously [36].

Western Blot
The tumor and spleen tissues were lysed, as described in Section 2.8. The proteins (40 µg) were electrophoresed using an Omni-Easy™ One-Step PAGE Gel Fast Preparation Kit (#PG212, EpiZyme), and were transferred to polyvinylidene difluoride membranes (0.45 µm; Merck, Darmstadt, Germany). After blocking in NcmBlot blocking buffer (#P30500; NCM Biotech) at 4 • C for 30 min, the membranes were incubated with primary antibodies (Table S1, Supplementary Materials) at 4 • C overnight. After washing with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated separately at 4 • C for 4 h with the appropriate horseradish peroxidase-conjugated secondary antibodies (Table S1, Supplementary Materials). The protein bands were visualized using electrochemiluminescence detection kits (Merck Millipore, Billerica, MA, USA) and a Tanon 5200 gel imaging system (Tanon Science & Technology, Shanghai, China). The band intensity was assessed using ImageJ software v1.8.0 (National Institutes of Health, Bethesda, MD, USA).

Statistical Analyses
One-way analysis of variance was performed, followed by post-hoc multiple comparisons (Tukey's test) using DSS 25.0 software (version 25.0; IBM Corporation, Armonk, NY, USA). The statistical significance is reported at p < 0.05. The graphs were generated using GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA).

IHS Restricts Tumor Growth in Apc Min/+ Mice
The IHS reduced the volume and abundance of colorectal tumors ( Figure 1B) and colorectal indices (p < 0.05; Figure 1C) without influencing the organ indices ( Figure S1A-E, Supplementary Materials) in the Apc Min/+ mice. According to the H&E staining, the tumor cells in the mucosal layer of the colorectum of the Apc Min/+ mice were heteromorphic, with a high nucleoplasm ratio and glandular tubular arrangement, all of which was reversed by IHS treatment ( Figure 1D). Furthermore, the inflammatory cell infiltration in the heart and a large amount of hepatocyte hydropic degeneration with loose and lightly stained cytoplasm in the liver of Apc Min/+ mice were ameliorated by IHS treatment (Figure S1F, Supplementary Materials).

IHS Alters the Gut Microbiota of Apc Min/+ Mice
An imbalance in the gut microbiota is closely associated with the development of colorectal cancer [5]. According to the produced Venn diagram, 7630 OTUs were detected in the two experimental groups; however, only 971 (12.73%) OTUs occurred in both of the groups (Figure 2A), suggesting significant differences in the composition of the microbial communities. A nonmetric multidimensional scaling plot (beta diversity) obtained by the weighted UniFrac distance matrix (stress value = 0.0000772) showed a significantly different clustering of gut microbiota between the vehicle-and IHS-treated mice ( Figure 2B). The IHS administration increased the abundance of Oscillospira, Odoribacter, Rikenella, Dehalobacterium, and Coprococcus and reduced the abundance of Allobaculum and Mucispirillum ( Figure 2C,D; Table S2, Supplementary Materials). According to an analysis based on the KEGG database of metabolic pathways, the amino acid metabolism pathway was involved in IHS-mediated anti-CRC activity ( Figure 2E).

IHS Influences Serum Metabolites in Apc Min/+ Mice
The metabolites in the blood are associated with the gut microbiota under various physiological or pathological conditions [37]. The orthogonal partial least squares discriminant analysis (OPLS-DA) showed that the metabolite profiles were completely clustered into two groups, suggesting significantly different serum metabolic profiles in each group ( Figure 3A). OPLS-DA VIP > 1 and p < 0.05 were used to identify differential metabolites. Compared with the vehicle-treated Apc Min/+ mice, the IHS treatment enhanced the level of L-arginine (L-Arg) and reduced the levels of docosahexaenoic acid, cholesteryl sulfate, phosphorylcholine, galactonic acid, lumichrome, and uracil in the serum ( Figure  3B; Table S3, Supplementary Materials). According to the KEGG analysis, 10 potential targeted metabolism pathways were screened, of which L-Arg participated in six metabolic pathways ( Figure 3C; Table S4, Supplementary Materials). L-Arg was a key metabolite identified among the differential metabolites. L-Arg is the precursor for the biosynthesis of NO [38], and Arg-1 limits the production of NO by the catabolism of Arg [39]. A high expression of IL-2 induced a high production of L-Arg and NO [40]. The IHS increased the levels of Arg (p < 0.05; Figure 3D) and NO (p < 0.01; Figure 3F) and reduced the levels of Arg-1(p < 0.05; Figure 3E) in tumors. The IHS increased the levels of IL-2 (p < 0.05; Figure  3G) in tumors and in the spleen of Apc Min/+ mice. The increment in the levels of IL-2 by IHS in tumors and in the spleen of Apc Min/+ mice was further confirmed by IHC (p < 0.01; Figure  3H; Figure S2A, Supplementary Materials), and Western blotting (p < 0.01; Figure 3I

IHS Influences Serum Metabolites in Apc Min/+ Mice
The metabolites in the blood are associated with the gut microbiota under various physiological or pathological conditions [37]. The orthogonal partial least squares discriminant analysis (OPLS-DA) showed that the metabolite profiles were completely clustered into two groups, suggesting significantly different serum metabolic profiles in each group ( Figure 3A). OPLS-DA VIP > 1 and p < 0.05 were used to identify differential metabolites. Compared with the vehicle-treated Apc Min/+ mice, the IHS treatment enhanced the level of L-arginine (L-Arg) and reduced the levels of docosahexaenoic acid, cholesteryl sulfate, phosphorylcholine, galactonic acid, lumichrome, and uracil in the serum ( Figure 3B; Table S3, Supplementary Materials). According to the KEGG analysis, 10 potential targeted metabolism pathways were screened, of which L-Arg participated in six metabolic pathways ( Figure 3C; Table S4, Supplementary Materials). L-Arg was a key metabolite identified among the differential metabolites. L-Arg is the precursor for the biosynthesis of NO [38], and Arg-1 limits the production of NO by the catabolism of Arg [39]. A high expression of IL-2 induced a high production of L-Arg and NO [40]. The IHS increased the levels of Arg (p < 0.05; Figure 3D) and NO (p < 0.01; Figure 3F) and reduced the levels of Arg-1(p < 0.05; Figure 3E) in tumors. The IHS increased the levels of IL-2 (p < 0.05; Figure 3G) in tumors and in the spleen of Apc Min/+ mice. The increment in the levels of IL-2 by IHS in tumors and in the spleen of Apc Min/+ mice was further confirmed by IHC (p < 0.01; Figure 3H; Figure S2A, Supplementary Materials), and Western blotting (p < 0.01; Figure 3I; Figure S2B, Supplementary Materials).

IHS Increases the Abundance of CD8 + T Cells
IL-2 stimulates the cytotoxic activity of CD8 + T cells by binding to the IL-2 receptor and activating the JAK/STAT signaling pathway [41]. In the IHS-treated mice, an increased abundance of the CD3e + CD8a + cells (p < 0.05) was observed in the blood; however, no significant changes were found regarding the abundances of CD3e + CD19 − , CD3e − CD19 + , and CD3e + CD4 + cells in the blood ( Figure 4A). The IHC showed that the IHS treatment increased the positive area of CD8 (p < 0.05) of tumor tissues, indicating an increase in the abundance of CD8 + cells in the tumor ( Figure 4B); thus, IHS may mediate the tumor cell-killing effect by CD8 + T cells through increasing their abundance.

IHS Increases the Abundance of CD8 + T Cells
IL-2 stimulates the cytotoxic activity of CD8 + T cells by binding to the IL-2 receptor and activating the JAK/STAT signaling pathway [41]. In the IHS-treated mice, an increased abundance of the CD3e + CD8a + cells (p < 0.05) was observed in the blood; however, no significant changes were found regarding the abundances of CD3e + CD19 − , CD3e − CD19 + , and CD3e + CD4 + cells in the blood ( Figure 4A). The IHC showed that the IHS treatment increased the positive area of CD8 (p < 0.05) of tumor tissues, indicating an increase in the abundance of CD8 + cells in the tumor ( Figure 4B); thus, IHS may mediate the tumor cell-killing effect by CD8 + T cells through increasing their abundance.

Discussion
Here, we report for the first time the inhibitory effects of IHS on tumor growth in Apc Min/+ mice, which was related to its regulation of gut microbiota-mediated immune function, especially through the regulation of the abundance of CD8 + T cells. Encouragingly, the tumor cells in the mucosal layer of the colorectum of Apc Min/+ mice were heteromorphic, with a high nucleoplasm ratio and glandular tubular arrangement, all of which were reversed by IHS.
The nutritional composition of IHS was systematically analyzed, and the content of dietary fiber, polyphenols, and triterpenoids was assessed, which provided a basis for identifying its anti-CRC activity. A low intake of dietary fiber is one of the main factors of CRC susceptibility [4]. Natural polyphenols are a rich source of natural antioxidants, prebiotics, and dietary polyphenols, which serve as immunomodulators and can inhibit the etiology and pathogenesis of CRC [42]. Additionally, triterpenoids can inhibit the proliferation of tumor cells by inhibiting glycolysis, thus acting as anti-CRC agents [43].
The dysbiosis of the human gut microbiota is closely associated with the development of CRC [5], as evidenced by the alteration of Ruminococcus, Clostridium, Coprococcus, Oscillospira, Odoribacter, Bifidobacterium, and Lactobacillus in patients with CRC [44][45][46]. Corresponding changes were observed in Apc Min/+ mice and were improved after IHS administration. Ruminococcus can degrade natural polysaccharides that are not digestible by humans [47,48]. Bifidobacterium and Lactobacillus help produce lactate from carbohydrate substrates [48]. However, an accumulation of D-lactate is life-threatening in cases with short bowel syndrome [49]. The acidification caused by lactate increases the expression of Arg-1, which hydrolyzes L-Arg in the macrophages [50,51]. A depletion of L-Arg leads to impaired CD3 ζ-chain expression, especially in CD8 + T cells, resulting in cell growth inhibition [52]. The IHS increased the levels of L-Arg in the tumor and spleen of the Apc Min/+ mice, suggesting an important role of the gut microbiota regarding the anti-CRC effects of IHS.

Discussion
Here, we report for the first time the inhibitory effects of IHS on tumor growth in Apc Min/+ mice, which was related to its regulation of gut microbiota-mediated immune function, especially through the regulation of the abundance of CD8 + T cells. Encouragingly, the tumor cells in the mucosal layer of the colorectum of Apc Min/+ mice were heteromorphic, with a high nucleoplasm ratio and glandular tubular arrangement, all of which were reversed by IHS.
The nutritional composition of IHS was systematically analyzed, and the content of dietary fiber, polyphenols, and triterpenoids was assessed, which provided a basis for identifying its anti-CRC activity. A low intake of dietary fiber is one of the main factors of CRC susceptibility [4]. Natural polyphenols are a rich source of natural antioxidants, prebiotics, and dietary polyphenols, which serve as immunomodulators and can inhibit the etiology and pathogenesis of CRC [42]. Additionally, triterpenoids can inhibit the proliferation of tumor cells by inhibiting glycolysis, thus acting as anti-CRC agents [43].
The dysbiosis of the human gut microbiota is closely associated with the development of CRC [5], as evidenced by the alteration of Ruminococcus, Clostridium, Coprococcus, Oscillospira, Odoribacter, Bifidobacterium, and Lactobacillus in patients with CRC [44][45][46]. Corresponding changes were observed in Apc Min/+ mice and were improved after IHS administration. Ruminococcus can degrade natural polysaccharides that are not digestible by humans [47,48]. Bifidobacterium and Lactobacillus help produce lactate from carbohydrate substrates [48]. However, an accumulation of D-lactate is life-threatening in cases with short bowel syndrome [49]. The acidification caused by lactate increases the expression of Arg-1, which hydrolyzes L-Arg in the macrophages [50,51]. A depletion of L-Arg leads to impaired CD3 ζ-chain expression, especially in CD8 + T cells, resulting in cell growth inhibition [52]. The IHS increased the levels of L-Arg in the tumor and spleen of the Apc Min/+ mice, suggesting an important role of the gut microbiota regarding the anti-CRC effects of IHS.
Furthermore, low levels of Arg have been observed in patients with CRC [53], and L-Arg metabolism can regulate the immune responses [54]. Clostridium converts Arg to ornithine, which facilitates Arg transport [55]. L-Arg is a precursor of NO synthesis [56], which is associated with a strong induction of IL-2 in humans and other species [57]. In a feedback loop, the high expression of IL-2 induces increased L-Arg and NO production [40], and Lactobacillus significantly induces IL-2 [58]. IL-2 promotes CD8 + T cells differentiation and expansion during immune regulation [59], which contributes to the effective restoration of T cell functioning during CRC [60]. The cytokines are key components of the immune barrier, some of which act on epithelial tissues, especially in the gut [61]. The CD8 + T cells inhibit IL-5 synthesis to reduce the eosinophil infiltration in models of experimental lung disease [62]. IL-6 induces strong immunosuppression in the CRC microenvironment by recruiting immunosuppressive cells, impairing T cell infiltration, and reducing the abundance of CD8 + T cells [63]. Additionally, IL-10 suppresses the immune response by inhibiting the T cell-proliferation and inducing T cell depletion [64]. A high abundance of CD8 + T cells was confirmed in the blood and at the tumor sites of the IHS-treated Apc Min/+ mice. Taken together, the CD8 + T cells are involved in IHS-mediated anti-CRC processes related to the regulation of immune responses [7].
The JAK/STAT3 pathway is closely associated with the CD8 + T cell differentiation and maturation [65,66], and is a potential target for the CRC treatment [19]. IL-2 can activate several downstream signaling molecules, including the JAKs, by binding to their receptors [20]. The JAKs mediate the recruitment of STAT1, 3, and 5 [20]. The sustained activation of STAT3 increases the tumor cell proliferation, survival, and invasion, thereby accelerating the tumor formation process [67]. IL-6, a pleiotropic cytokine, can promote Arg-1 expression through STAT3 [68,69]. In contrast, IL-6 activates the JAK/STAT signaling through glycoprotein 130 [22]. The IHS inhibits the activation of JAK/STAT and affects the abundance of the CD8 + T cells, regulates the immune response, and inhibits CRC development.
This study has some limitations. Owing to production restrictions, IHS is currently difficult to commercialize, and little research has been conducted on its pharmaceutical effects. IHS comprises a complex mixture of nutrients, thus further research is required to identify its active substances. Moreover, different from our study, some of the research performed the metataxonomics in dirty colon tissue and the metabolites in the clean colon tissue of CRC mice [70,71], which should be followed in our future studies.

Conclusions
In this study, we confirmed for the first time the inhibitory effects of IHS on CRC development in Apc Min/+ mice, and through combining gut microbiota analyses and serum metabolomics, this effect was confirmed to be associated with the regulation of CD8 + T cell abundance through JAK/STAT signaling.