Huang Qin decoction (HQD) has a similar composition as previously mentioned. Pan et al. (2022) treated AOM/DSS induced mice with different doses of HQD aqueous extract (0.5, 1.0, and 1.5 g/kg) for 7 weeks. Aspirin (40 mg/kg) was used as the positive control drug. HQD administration at a minimum dose of 1.0 g/kg was found to inhibit intestinal tumor growth in a dose-dependent manner. Aspirin also had a tumor suppressor effect similar to that of HQD at 1.5 g/kg. HQD repaired the intestinal mucosal barrier by upregulating Occludin and zona occludens-1 (ZO-1). After HQD treatment, neutrophils and CD11b⁺Ly6C⁺ monocytes increased in the mice, whereas the lymphocytes increased. In addition, HQD downregulated the expression of programmed cell death protein 1 (PD-1) in CD8⁺T-cells. PD-1 can induce, establish and maintain immune tolerance in the TME and help cancer achieve immune escape (Han et al., 2020), an important immune checkpoint (Xu-Monette et al., 2018). Therefore, HQD enhanced immune surveillance of CD8⁺ T-cell. Network pharmacology analysis revealed that peptidyl arginine deaminase 4 (PAD4) might be the key target of HQD. PAD4 is activated in neutrophils and can mediate the formation of neutrophil extracellular traps (NETs) (Hawez et al., 2019). Recent studies indicate that the formation of NETs in IBD increases significantly (Mortensen et al., 2022) and is associated to the occurrence of CRC (Chen et al., 2022). NETs markers decreased after HQD treatment. This indicates that HQD can slow down intestinal inflammation, inhibit tumor proliferation and reduce NETs aggregation, which has a potential regulatory effect on tumor immune microenvironment.

Xian-Lian-Jie-Du decoction (XLJDD) is an empirical prescription for the treatment of CRC developed by Qihuang scholar Professor Cheng Haibo. It comprises 4 herbal medicines, including Agrimonia pilosa Ledeb. [Rosaceae; Agrimoniae herba] and Coptis chinensis Franch. [Ranunculaceae; Coptidis rhizoma]. Yu et al. (Yu et al., 2022) intervened AOM/DSS and high-fat diet-induced mice for 17 weeks by gavage with XLJDD aqueous extract (12.9 g/kg, the best intervention dose determined in previous studies) and its five main components, respectively. Folic Acid was used as the positive control drug (the dose is unknown). They found that XLJDD exerted better inhibition on tumors than every single group. RNA-seq analysis of tumor tissues in each group revealed that XLJDD treatment was associated with inflammatory factor reactions and cell death-related pathways, and IFN-related pathways were significantly enriched. IFN can induce tumor cell death (Chawla-Sarkar et al., 2003). Subsequent experiments confirmed the occurrence of apoptosis and revealed that XLJDD significantly improved the proportion of B-cell clusters in the tumor immune microenvironment, which was associated with the ferroptosis pathway. Key genes, including Mfsd2a and Ccdc85c in the XLJD group, were identified through multi-group analysis. The former was up-regulated in CAC tissue and significantly down-regulated after XLJDD intervention, whereas the latter showed the opposite trend. Based on previous studies, Mfsd2a has a protective effect on colitis (Ungaro et al., 2017), whereas Ccdc85c is related to malignant proliferation (Tanaka et al., 2017). This study shows that XLJDD can promote cell death, regulate immune microenvironment and prevent CAC. Although the key gene of XLJDD in treating CAC has been obtained through multi-omics screening, its molecular mechanism is still unclear due to the lack of target verification experiments.

Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS) is derived from Jin-gui-yao-lue and comprises 3 herbs, including Coix lacryma-jobi var. ma-yuen (Rom.Caill.) Stapf [Poaceae; Coicis semen] and Aconitum carmichaelii Debeaux [Ranunculaceae; Aconiti lateralis radix praeparata], which are recorded in medical books for treatment of intestinal carbuncles. Zhang et al. (Zhang et al., 2022) treated AOM/DSS induced mice with different doses of YYFZBJS aqueous extract (3.825, 15.3 g/kg) for 12 weeks. Aspirin (30 mg/kg) was used as the positive control. They discovered that YYFZBJS with two doses significantly inhibits the inflammation-to-cancer transformation of IBD and regulates the mucosal immune response. No difference in hepatorenal toxicity was noted between treatment groups, indicating that the herbal doses were safe. RNA-seq analysis revealed that genes related to the hypoxia-inducible factor (HIF) -1α signaling pathway were significantly downregulated in the YYFZBJS high-dose group, and the phenotypic prediction was closely associated with Treg differentiation. Subsequent experiments confirmed that YYFZBJS significantly down-regulated the level of HIF-1α and increased the infiltration of CD4⁺ CD25⁺ cells in the tumor. In vitro experiments confirmed that the level of HIF-1α expressed by Tregs was substantially increased in a hypoxic environment. Additionally, HIF-1α overexpression reversed the inhibitory effect of Tregs on the proliferation of colon cancer cells, with no change in the expression of forkhead box protein P3 (Foxp3⁺) Tregs. Of note, Foxp3⁺ is a marker of naturally occurring Tregs (nTregs) (Zhang and Zhou, 2019). Therefore, HIF-1α may regulate IBD-CAC transformation by targeting peripheral Tregs (pTregs) rather than the polarization of nTregs. Subsequently, CRC cell proliferation was improved through the addition of HIF-1α-transfected Tregs in a co-culture system of CRC cells and normal epithelial cells. In contrast, the YYFZBJS extract delayed CRC cell proliferation, down-regulated phosphatidylinositide 3-kinases (PI3K), p-STAT3, and p-NF-κB expressions, closely related to malignant proliferation. Further in vivo verification in a Treg-depleted mouse model revealed that the antitumor effect of YYFZBJS was suppressed by the absence of Tregs. The study is relatively rigorous in design. It indicates that YYFZBJS improves the tumor immune microenvironment via HIF-1α-induced Treg polarization, thereby preventing the occurrence and development of CAC.

Shenling Baizhu San (SBS) is derived from Tai-ping-hui-min-he-ji-ju-fang and consists of 10 herbs, including Panax ginseng C.A.Mey. [Araliaceae; Ginseng radix et rhizoma] and Poria cocos (Schw.) Wolf [Polyporaceae; Poria]. SBS is a commonly used prescription for the treatment of chronic diarrhea. Lin et al. (2015) intervened AOM/DSS induced mice by gavage with aqueous extract of SBS (7.28 g/kg). It could not find the intervention time of SBS clearly in the paper. Positive control was not set. They found that it exerted anti-inflammatory and anticancer effects and could reduce the infiltration of CD11b⁺Ly6C⁺ cells and serological level of MDSCs effector molecule TGF-β1. CD11b⁺Ly6C⁺ is a phenotype of MDSCs with immunosuppressive effects (Zhao et al., 2016), producing chemokines such as TGF-β1 to induce EMT (Taki et al., 2021). SBS increased E-cadherin, an EMT epithelial phenotype; reduced N-cadherin, fibronectin, vimentin and the transcription factor Snail, as well as Wnt5a; upregulated the expression of axis inhibition (Axin), dishevelled 2 (Dvl2) and p-GSK-3β. SBS and its component Atractylenolide-1 (AT1) inhibit the proliferation of colon cancer cells in a dose-dependent manner in vitro. In addition, SBS reduced TGF-β1-induced activation of CRC cell matrix signal Wnt5a and reversed the down-regulation of E-cadherin. In this study, drug concentration gradient and positive control drug were not set, and the intervention time of SBS was not clear, which reduced the reliability of conclusions.

Shaoyao decoction (SYD) has the same composition as described before. Lin et al. (2014) intervened AOM/DSS induced mice by gavage with SYD aqueous extract (7.12 g/kg). We were unable to identify the intervention time of SYD in the thesis. No positive controls were set for this study. SYD effectively improved CAC symptoms and reduced tumor incidence, tumor burden, and expression of oncogenic proteins. In vitro experiments have also confirmed that both SYD and its component paeonol can suppress the proliferation of CRC cells and the tumor proliferation-related proteins PCNA, β-catenin, and p53 in a dose-dependent manner. This indicates that SBS may inhibit the malignant progression of IBD-CAC. In animal experiments, SYD repressed the EMT transcription factor Snail and regulated the expression levels of downstream EMT-related proteins including E-cadherin, N-cadherin, and Vimentin. SYD downregulated the levels of the serum inflammatory factors IL-1β, IL-6, TNF-α, expression of NF-κB p65, and the TAMs marker F4/80+. This suggests that SYD can prevent and treat CAC, inhibit the infiltration of TAMs and the occurrence of EMT induced by Snail. There was no concentration gradient of SYD in this study, and the research design was not rigorous enough.

Huoxiang Zhengqi (HXZQ), derived from Tai-ping-hui-min-he-ji-ju-fang, comprise 13 herbs, including Atractylodes macrocephala Koidz. [Asteraceae; Atractylodis macrocephalae rhizoma] and Citrus × aurantium f. deliciosa (Ten.) M. Hiroe [Rutaceae; Citri reticulatae pericarpium]. HXZQ has been made into Chinese patent medicine and is broadly applied for the treatment of diarrhea. Dong et al. (2022) treated AOM/DSS induced mice with different doses of HXZQ (0.45, 1.35 g/kg) for 6 weeks. Positive control was not set. They found that HXZQ could suppress genes and proteins related to the Nrf2/NF-κB/NLRP3 signaling pathway, release of inflammatory factors and the expression of oxidative stress markers and inhibit the occurrence and development of CAC. The dosage of 1.35 g/kg was better than 0.45 g/kg. At the phylum level, HXZQ increased Bacteroidetes and decreased the proportion of Firmicutes unlike in the model group. At the genus level, HXZQ reduced the abundance of Candidatus Arthromitus, Dorea, and other genera; it also increased the abundance of butyric acid-producing bacteria including Anaerotruncus, Prevotella, Butyricimonas, and other genera. Butyrate is a four-carbon SCFAs produced by the fermentation of dietary fiber by specific flora with anti-inflammatory properties that can improve intestinal mucosal immunity and barrier function (Liu et al., 2018). This indicates that HXZQ can inhibit Nrf2/NF-κB/NLRP3 signaling pathway, resist oxidative stress and positively regulate intestinal microbiota. This may be the potential mechanism of HXZQ in preventing and treating CAC.

Chang-wei-qing (CWQ) is a prescription developed by Professor Fan Zhongze, a famous TCM expert, for treating malignant tumors of the digestive tract. It comprises 8 herbs, including Astragalus mongholicus Bunge [Fabaceae; Astragali radix] and Atractylodes lancea (Thunb.) DC. [Asteraceae; Atractylodis rhizoma]. Wan et al. (2019) intervened AOM/DSS induced mice with different intervention doses of CWQ (5, 10 mg/kg) for 89 days. Bifico (4.2 g/kg) was used as the positive control. Bifico or CWQ at different doses can restore colon length, and minimize inflammation and the number and volume of tumors. CWQ demonstrated a dose-dependent effect. After treatment with Bif or CWQ, no statistical difference was noted in the number of total bacteria in the mouse feces, unlike the model group, whereas the levels of Faecalibacterium prausnitzii (F. prausnitzii) and bacterial metabolite β-glucuronidase (BGUSs) were down-regulated compared to the model group. BGUSs are involved in the catabolism of carbohydrates. They are known to prevent elimination of certain carcinogens from the digestive tract of the host, which increases the risk of CRC (Zhang et al., 2019a). It has been shown that Bif or CWQ reduces the levels of d-lactose and LPS in peripheral blood, both of which are metabolites of intestinal microflora. Moreover, Bif or CWQ can down-regulate TLR4, NF-κB p65, P-STAT3, and Bcl-2 expression levels. Therefore, CWQ might inhibit CAC progression by regulating the gut microflora and their metabolites such as BGUSs and LPS. Due to the lack of target verification, it is impossible to draw exact conclusions about the molecular mechanism of CWQ. Unlike many studies, F. prausnitzii is a well-known butyric acid-producing bacteria of the Firmicutes phylum and is a protective factor for IBD and CRC (Lopez-Siles et al., 2017). Here, F. prausnitzii abundance was however downregulated with the improvement of the disease after treatment. We believe that this requires further study and explanation by the researchers.

San-Wu-Huang-Qin decoction (SWHQ) is derived from Jin-gui-yao-lue comprising 3 drugs, i.e., Scutellaria baicalensis Georgi [Lamiaceae; Scutellariae radix] and Sophora flavescens Aiton [Fabaceae; Sophorae flavescentis radix], often used for the treatment of CRC. Zhou et al. (2022) intervened AOM/DSS induced mice with SWHQ aqueous extracts (1.82, 3.64 g/kg) for 11 weeks. Aspirin (1.4 mg/kg) was used as the positive control. The results showed that SWHQ or Aspirin could effectively reduce the tumor burden. SWHQ was dose-dependent. 7.28 g/kg SWHQ was superior to Aspirin. The result indicated that SWHQ exhibits a significant antitumor effect. TEM and detection of tight junction proteins revealed that SWHQ restored the integrity of the intestinal mucosal barrier. 16S rRNA analysis of feces revealed that SWHQ could reverse the imbalance of Firmicutes and Bacteroides caused by the AOM/DSS model, upregulate the abundance of the beneficial bacteria Akkermansia and Faecalibaclum, and reduce the abundance of the pathogenic bacteria Bacteroides, Entero Coccus and Escherichia-Shigella. PICRUSt analysis showed a significant reduction in bacterial LPS synthesis in the SWHQ group. The intestinal microbiota of the model and SWHQ groups were colonized in the sham-sterile model mice via fecal microbiota transplantation (FMT), and the findings revealed that the antitumor and mucosal protection effects of the FMT-SWHQ group were similar to that of the donor group. 16S rRNA also showed similar results to that of the donor group, indicating that changes in the intestinal flora of the donor were partially replicated in the receptor. Based on the analysis of intestinal microflora, targeting the intestinal microflora, particularly the LPS-producing flora, is an important mechanism of SWHQ against tumors. Of interest, both species identified by 16S rRNA are related to butyric acid in colitis. Faecalibaclum is a butyric acid-producing bacterium and has a negative correlation with the activity of UC (Machiels et al., 2014). Akkermansia has anti-inflammatory properties in DSS-induced colitis and is positively correlated with the concentration of butyric acid (Wan et al., 2021). Further experiments showed that both SWHQ and FMT-SWHQ significantly down-regulate TLR4/NF-κB activation and the expression of inflammatory factors in the mouse colon tissue. Taken together, this study was well designed, and verified that intestinal flora was the target of SWHQ in the intervention of CAC.

Wu Mei Wan (WMW) has a similar composition to WMP. Jiang et al. (2020) intervened AOM/DSS induced mice with WMW aqueous extract (5.8 g/kg) for 10 weeks. Positive control was not set. They discovered that WMW could significantly reduce AOM/DSS-induced weight loss, tumor number, and tumorigenic grade. Mechanistic studies have shown that WMW can reduce the expression of NF-kB, p65, P-STAT3, and IL-6 in colon tissue and downregulate the serum IL-6 level. Previous studies have shown that the activation of STAT3 and NF-κB signals is important to the development of CAC (Waldner and Neurath, 2015). Fecal 16S rDNA sequencing analysis showed that after WMW intervention, Bacteroides decreased and Firmicutes increased at the phylum level; Bacteroides_S24-7_group decreased, whereas Lachnospiraceae increased at the family level, unlike the model group. The role of Bacteroidales_S24-7_group has always been controversial. Lachnospiraceae is a typical obligate anaerobe-producing butyrate (Leth et al., 2022), promoting colonization resistance of the host against intestinal pathogens and is beneficial to the body (Sorbara et al., 2020). This study shows that WMW can regulate intestinal flora and inhibit the transformation of IBD from inflammation to cancer. Due to the lack of targeted verification, it cannot be explained that intestinal flora is the target of WMW in the treatment of CAC.

Canmei formula (CMF) was derived from Ben-cao-gang-mu and comprise 2 drugs, i.e., Prunus mume (Siebold) Siebold and Zucc. [Rosaceae; Mume fructus] and Bombyx mori Linnaeus [Bombycidae; Bombyx batryticatus]. CMF was used to treat hematochezia. Zhang et al. (Zhang et al., 2019b) used CMF ethanol extract (CMF-A 0.657 g/kg) and CMF aqueous extract (CMF-L 3.65 g/kg, CMF-H 7.3 g/kg) to treat AOM/DSS and high-fat-diet-induced C57BL/6 mice for 68 days, respectively. Aspirin (1.4 mg/kg) was used as the positive control. The results indicated that CMF observably suppressed the occurrence and growth of tumors in mice, whereas aspirin only inhibited the growth of large (d ≥ 3 mm) colon adenomas. In addition, the CMF-A, CMF-L/CMF-H, and aspirin groups significantly reduced the expression of IL-17C and NF-κB. The 16S rDNA results revealed a decrease in bacterial population diversity in the model group and a significant increase after treatment with CMF-A, whereas aspirin did not display this capacity. Muribaculaceae, Bacteroides, and Preceptellaceae were the most abundant flora at the family level. After treatment with CMF, the abundance of Desulfovibrionaceae was significantly reduced compared to the model. One study has shown that Desulfovibrionaceae is significantly enriched in mice fed with a high-fat diet (Zhang et al., 2021a) and that the high-fat diet can drive AOM/DSS-induced CAC (Choi and Snider, 2019), hence Desulfovibrionaceae may be the key driving CAC. After treatment with CMF-A, the levels of Bacteroidaceae and Staphylococcaceae significantly changed from family to genus, and Turicibacter, Bacteroides, Bacteroidaceae, and Faecalibaculum were significantly enriched at the genus level. Of interest, Zhong et al. (2015) used dietary fiber to regulate the intestinal flora of high-fat diet-fed rats, and the results showed that the abundance of Turicibacter positively correlated with butyric acid concentration. This is consistent with the modeling method and findings in this study. There is a likelihood that CMF positively regulates intestinal inflammation via flora and its metabolites butyric acid, which requires further experimental verification.

Qingchang Wenzhong decoction (QCWZD) was developed by Li and consists of 8 Chinese medicinal materials, including Coptis chinensis Franch. [Ranunculaceae; Coptidis rhizoma] and Zingiber officinale Roscoe [Zingiberaceae; Zingiberis rhizoma praeparatum]. QCWZD is used to treat UC (Xia et al., 2022). Ren et al. (2022) intervened AOM/DSS induced C57BL/6/6 mice with different doses of QCWZD (0.7, 1.4 g/kg) for 15 weeks. QCWZD at 1.4 g/kg was used to intervene in a high-fat diet combined with an Apc^min/+ induced mouse model for 14 weeks. Positive control group was not set. The results indicated that QCWZD at 1.4 g/kg could significantly reduce the tumor burden of the two models, as well as improve the histological inflammatory injury and repair the intestinal mucosal barrier. QCWZD also suppressed the expression of two models pyroptosis-related proteins NLR pyrin domain containing 3 (NLRP3), gasdermin E (GDSME), high-mobility group box 1 protein (HMGB1), IL-1β and IL-18. The intestinal bacterial metabolites LPS and nigericin et al. can activate NLRP3, cut the scorch death-inducing protein GDSME, and promote the conversion of pro-IL-1β and pro- IL-18 into their active forms, resulting in pyroptosis of inflammatory cells (Wang et al., 2021a). Fecal 16S rRNA sequencing showed that QCWZD could significantly alter the overall composition of intestinal flora in the two model mice. In the AOM/DSS induction study group, Desulfobacterota was present only in the model group at the phylum level. At the genus level, the abundance of Bacteroides, Colidextribacter, and Muribaculum was significantly downregulated, whereas that of Bosea, Caulobacter, and Ralstonia was significantly upregulated compared to the model group. The high expression of Muribaculum is regarded as a potential marker of metabolic disorder of bile acids (BAs) (Marion et al., 2020). Secondary BAs, such as deoxycholic acid and lithocholic acid, are risk factors for IBD and CRC (Zeng et al., 2019). In the high-fat diet + Apc^min/+ induction study group, the QCWZD high dose group up-regulated the abundance of Desulfobacterota, Anaerotruncus, and Butyricoccus at phylum level and down-regulated the abundance of Erysipelotrichaceae_unclassified, unlike the model group. The Anaerotruncus and Butyricimonas upregulated after treatment were all butyric acid-producing bacteria. Interestingly, previous studies indicate that a decrease in the abundance of Desulfobacterota is a common feature of acute and chronic disease stages in patients with UC (Zhu et al., 2022). Desulfobacterota was increased after AOM/DSS modeling, as well as significantly after high-dose QCWZD intervention in Apc^min/+ mice, yielding a controversial finding on whether Desulfobacterota is beneficial or pathogenic. This difference may also be caused by tumors with different phenotypes caused by different modeling methods. Based on the above results, it can be concluded that QCWZD can treat CAC and small intestinal tumors. This effect may be achieved by mediating intestinal flora and cell death, but it needs more rigorous and perfect experimental verification.

Pai-Nong-San (PNS) is derived from Jin-gui-yao-lue and consists of 3 herbal medicines, including Citrus × aurantium L. [Rutaceae; Aurantii fructus immaturus] and Paeonia lactiflora Pall. [Paeoniaceae; Paeoniae radix alba]. PNS is used for the treatment of carbuncles and pus. Zhang et al. (2021b) intervened AOM/DSS induced mice with different doses of PNS aqueous extract (0.8, 1.6, 3.2 g/kg) for 9 weeks. Aspirin (13 mg/kg) was used as the positive control drug. PNS at 0.8 g/kg was found to be the lowest dose effective in preventing tumorigenesis. PNS exhibited a dose-dependent behavior. PNS could effectively prevent tumor occurrence. Additionally, PNS and Aspirin up-regulated anti-inflammatory factors, down-regulated pro-inflammatory factors, and HIF-α levels. At the same time, the infiltration of CD4⁺ and CD8⁺T lymphocytes in colonic tissues was significantly reduced. The effect of PNS was dose-dependent. 16S rRNA sequencing results showed that at the phylum level, PNS treatment adjusted the modeling-induced abundance abnormalities of Bacteroides, Proteobacteria, and Firmicutes to normal levels, however, aspirin only corrected the abundance abnormalities of Bacteroides. At the genus level, PNS significantly increased the abundance of Lactobacillus. Lactobacillus is the most common probiotic genus in the human intestinal tract. It can effectively decompose sugars and produce lactic acid, enhance the intestinal antioxidant stress capacity, and inactivate carcinogenic factors (Eslami et al., 2019). Lactobacillus positively correlated with a butyric acid concentration in UC rats (Wang et al., 2022b). Clustering analysis showed that the PNS group separated from the CAC group and was close to the normal group, indicating that PNS had a positive regulatory effect on the intestinal flora. In contrast with the model, PNS suppressed the expression of p-GSK-3β, β-catenin, and c-Myc, indicating that PNS could suppress the activation of the Wnt/β-catenin signaling pathway. This study shows that PNS might prevent the progression of IBD-CAC by inhibiting the Wnt/β-catenin pathway, CD4⁺, and CD8⁺ T-lymphocyte infiltration, as well as positively regulating the intestinal flora. Certainly, this also needs further targeted verification.

Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS) is the same composition as described before. Chai et al. (2021) intervened AOM/DSS induced mice with YYFZBJS ethanol extract (3.825, 7.65, and 15.3 g/kg) for 9 weeks at different administration doses. Aspirin (30 mg/kg) was used as the positive control drug. They discovered that YYFZBJS significantly reduced the occurrence of CAC in a dose-dependent manner. 16S rRNA sequencing showed that YYFZBJS could enhance the α diversity of the intestinal flora. Unlike the CAC model, YYFZBJS increased the abundance of Lactobacillus, Ruminococcaceae, and Clostridium, but decreased the abundance of enterotoxigenic Bacteroides fragilis (ETBF) in a dose-dependent manner. Interestingly, all three added bacteria could produce butyric acid (Guo et al., 2022; Ávila et al., 2023). Ruminococcaceae can regulate secondary BAs, promote the malignant progress of inflammation (Sinha et al., 2020), and may play a role in anti-tumor immunity (Peng et al., 2020). The research of Zhong et al. (2015) shows that Clostridium is significantly related to the metabolite succinic acid, which is an index to inhibit EMT of CRC stem cells (Terasaki et al., 2018). AOM/DSS-induced sterile mice were infected with ETBF, and ETBF infection promoted tumor proliferation. Subsequently, Ray Biotech revealed that ETBF activated the expression of M2-type macrophage cytokines in sterile mice. In contrast with the ETBF group, the number of adenomas was significantly reduced after intervention with YYFZBJS, and the expression of M2 markers was downregulated, whereas the secretion of M1-related protein was upregulated, suggesting that YYFZBJS might prevent and treat CAC by regulating macrophage polarization with ETBF. In vitro assay, ETBF was co-cultured with colon cancer cells and significantly up-regulated JNK, STAT3, NF-κB, and Arg-1. M2-type BMDMs were isolated from mice treated with ETBF and ETBF + YYFZBJS, respectively, to interfere with colon cancer cells in vitro. Consequently, BMDMs in the ETBF + YYFZBJS group suppressed the proliferation of colon cancer cells and reduced the expression of p-STAT3, MMP-2, MMP-9, cyclinD1, and c-Met; conversely, STAT3 was activated in the ETBF group. This study was designed in detail, and the results showed that the mechanism of YYFZBJS treating CAC might be achieved by regulating intestinal flora and reducing ETBF-mediated polarization of M2 macrophages.

Sini decoction (SND), originating from Shang-han-lun, comprises 3 herbal medicines, including Aconitum carmichaelii Debeaux [Ranunculaceae; Aconiti lateralis radix praeparata] and Glycyrrhiza uralensis Fisch. ex DC. [Fabaceae; Glycyrrhizae radix et rhizoma], and is suitable for the treatment of yang deficiency syndrome in TCM. Wang et al. (2021b) intervened AOM/DSS induced mice with SND aqueous extract (3.5 g/kg) for 90 days. Positive control drug was not set. They discovered that SND could effectively prevent and treat CAC. Immunohistochemical results showed that SND increases CD8⁺ T-cells in mouse colon tissue and decreases the infiltration of CD4⁺ T-cells. Meanwhile, SND increased Occludin and downregulated the expression of IL-6, IL-17, TNF-α, IFN-γ, and CRC markers. 16S rRNA sequencing showed that, unlike the model group, SND reduced the abundance of Bacteroidetes and increased the abundance of Firmicutes at the phylum level. At the genus level, Lactobacillus gasseri and Bacillus coagulans were significantly enriched in the SND group, whereas Bacteroides fragilis was significantly enriched in the CAC group. One study found that the abundance of Bacteroides fragilis increases in patients with colonic precancerous lesions and early cancer, suggesting that it may be a risk factor and biomarker for CRC (Zamani et al., 2019). After SND treatment, the abundance of Bacteroides fragilis decreased, whereas the abundance of beneficial bacteria including Bacillus coagulans, Akkermansia muciniphila, and Bifidobacterium increased. Bacillus coagulans is the producer of hyperbutyrate (Shi et al., 2022), and Lactobacillus gasseri and Akkermansia muciniphila also belong to the butyric acid-producing bacteria. Bifidobacterium is a lactic acid-producing probiotic that can also work with other microorganisms to increase the rate of butyrate production (Rivière et al., 2015). Sivan A et al. (Sivan et al., 2015) have found that oral administration of Bifidobacterium can promote the anti-tumor immunity of tumor patients. One study has shown that the abundance of Lactobacillus, Bifidobacterium, and Akkermansia could be increased following Lactobacillus gasseri intervention in AOM/DSS induced mice (Oh et al., 2020). The Result was similar to those in this study, indicating the protective effect of Lactobacillus gasseri on CAC. This study shows that SND may inhibit the occurrence and development of CAC by mediating the crosstalk between some specific flora such as Bacteroides fragilis and immune cells CD4⁺ CD8⁺. Since there is no drug concentration gradient, the dose-effect relationship of SND cannot be obtained.