Colorectal cancer is the third most common cancer worldwide, and its prevalence is expected to rise by 60% by 2030 (Ferlay et al., 2013). Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis (UC), are chronic multifactorial gastrointestinal inflammatory disorders characterized by chronic inflammatory reaction, epithelial barrier dysfunction, diarrhea, abdominal pain, and enterohemorrhage (Zhu et al., 2019). Though the pathogenesis and etiology of IBD are not clear, dysregulation of the gut barrier and mucosal immune system may be involved in the pathogenesis (Round and Mazmanian, 2009). According to a meta-analysis, the incidence of colorectal cancer increased as the onset period of UC increased (Eaden et al., 2001). The increase in mortality and morbidity of IBD is related to the incidence of colitis-associated colorectal cancer (CAC) (Ekbom et al., 1990). Therefore, chronic IBD is an important risk factor for CAC (Shih and Targan, 2008).

Rumex japonicus Houtt (RJ) is widely distributed in East Asia. RJ is a perennial herb, and the root of RJ has been traditionally used to treat various diseases, such as constipation, jaundice, uterine bleeding, and hematemesis (Sun et al., 2020). RJ has anti-oxidative, anti-bacterial, anti-sepsis, anti-inflammatory, and anti-cancer effects (Sun et al., 2020). Moreover, RJ alleviates the disruption of tight junctions (TJs), apoptosis, and inflammation in the colons of dextran sulfate sodium (DSS)-induced colitis mouse model and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease mouse model (Kim et al., 2020; Kim et al., 2022). However, it is unclear whether RJ can suppress the incidence of CAC. Therefore, we investigated the protective effect of RJ in an azoxymethane (AOM)- and DSS-induced CAC mouse model.

The AOM/DSS model is the most commonly used model of chemically induced colon carcinogenesis because the pathogenesis of the model is similar to human colorectal cancer (De Robertis et al., 2011). The combination of AOM (tumor-inducing agent) and DSS (tumor-promoting agent) shows synergistic effects to promote colorectal cancer in the initial acute inflammation phase (De Robertis et al., 2011). Treatment of AOM/DSS induces excremental change (diarrhea and bloody feces), shortens the colon length, and increases the weight and wall thickness of the colon because DSS causes inflammation (Suzuki et al., 2006). In the present study, AOM/DSS-treated mice exhibited shorter and heavier colons than normal mice, and contained many colon tumors. However, RJ treatment inhibited morphological changes in the colon (Figures 1B–D). Moreover, the mice in the CAC group defecated with severe bloody diarrhea or black feces (blood-containing stool), but the mice in the RJ group showed fewer defecatory problems (data not shown). These results indicated that RJ suppressed AOM/DSS-induced morphological and excremental changes in the colon.

Proinflammatory cytokines, including TNF-α, IL-6, and IL-1β, play an important role in triggering carcinogenesis (Xing et al., 2017). TNF-α and IL-6 are critical tumor promotors during tumorigenesis and colonic carcinogenesis in humans and mice (Yao et al., 2019). The active form of TNF-α was detected in the colon tissues in patients with UC and colorectal cancer but not in healthy individuals (Popivanova et al., 2008). In the AOM/DSS model, the activation of TNF-α has been demonstrated to be caused by DSS rather than AOM, and TNF-α accelerates progression of colon carcinomas by increasing COX-2 expression level and inhibiting the Wnt signaling pathway (Popivanova et al., 2008). In patients with IBD or colorectal cancer, the levels of circulating and colonic IL-6 were increased, which were also found in the DSS- or AOM/DSS-treated mice (Yao et al., 2019). Also, IL-6 knockout mice treated with AOM/DSS formed fewer tumors and smaller adenomas and tumors, suggesting that IL-6 is important to the formation and growth of tumors in CAC (Grivennikov et al., 2009). In addition, TNF-α and IL-6 can affect each other; thus, the cross-regulation of these proinflammatory cytokines may be involved in CAC development to enhance inflammation and tumorigenesis (Grivennikov et al., 2009; Yao et al., 2019). The mice deficient in the anti-inflammatory cytokine IL-10 showed spontaneous gut inflammation (Sato et al., 2006), and the treatment of IL-10 decreased the level of TNF-α, IL-6, and IL-1β in an IBD animal model (Li and He, 2004). Therefore, pro- and anti-inflammatory cytokines play a critical role in inflammation and tumorigenesis in the colon. In the present study, treatment with RJ effectively regulated cytokines TNF-α, IL-6, IL-1β, and IL-10, which may contribute to alleviating the pathological changes caused by AOM/DSS in the colon (Figures 1E, 2, 3).

The intestinal barrier maintains homeostasis by inhibiting the penetration of pathogens or toxic substrates while allowing the absorption of nutrients into the intestine (Landy et al., 2016). TJs, the major components of the intestinal epithelial barrier, construct the TJ barrier by connecting neighboring epithelial cells and controlling paracellular transport (Xing et al., 2017). TJs are composed of occludin, claudins, ZO proteins, junctional adhesion molecules, tricellulin, and cingulin (Landy et al., 2016). The transmembrane proteins, occludin and claudin, are the most important controllers of gut barrier function, and the framework element ZO proteins form the central network for protein interactions (Xing et al., 2017). Occludin supports the structure of the TJ barrier and regulates paracellular permeability by binding to ZO-1 (Xing et al., 2017). Among the 27 isoforms of claudin, claudin-1 and -2 are expressed in leaky gut with inflammation (Xing et al., 2017). In IBD and colorectal cancer, the expression levels of occludin and ZO-1 are reduced, but the expression level of claudin-2 is increased (Landy et al., 2016). Claudin-2 expression level can be increased by the activation of IL-6 (Suzuki et al., 2011), and increased claudin-2 level enhances IBD-associated dysplasia and sporadic adenomas (Weber et al., 2008). When the connection of TJs is weak, the immune cells move to the intestine through the paracellular pathway and produce proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β (Kim et al., 2022). Therefore, the weakness of the TJ barrier causes epithelial inflammatory damage, such as erosion, ulceration, and apoptosis, which are major characteristics of IBD and colorectal cancer (Landy et al., 2016). In the present study, treatment of RJ effectively protected the TJ barrier and suppressed the activation of cancerous TJ like claudin-2 in the colon tissue (Figures 4, 5). In addition, in our previous studies, RJ protected the TJ barrier not only in the colon but also in the brain (Kim et al., 2020; Kim et al., 2022). Therefore, RJ may enhance the connection between TJs.

The self-renewal of intestinal epithelial cells (IECs) is important for maintaining the homeostasis of the colon, but the process cannot work properly under inflammatory conditions (Yao et al., 2019). Consequentially, inflammation and apoptosis of IECs negatively affect the normal function of the gut barrier. In fact, the inflamed sigmoid colon tissue from UC patients leaks from apoptotic foci, which may worsen depending on the severity of the inflammation (Gitter et al., 2001). A cytoplasmic protein, COX-2, catalyzes the synthesis of lipid inflammatory substrates, such as prostaglandin from arachidonic acid, and contributes to tumorigenesis by enhancing apoptosis, angiogenesis, and invasiveness of cancer cells (De Robertis et al., 2011). According to Eberhart et al., COX-2 is increased in the inflammatory area in ∼80% of colorectal cancers and 40% of colorectal adenomas (Eberhart et al., 1994). The tumor suppressor p53 plays a role in cell cycle arrest and apoptosis in the development of colorectal cancer neoplasm (De Robertis et al., 2011). The protein p53 regulates p21, which is involved in permanent and transient cell cycle arrest by suppressing the activity of cyclin-dependent kinases, preventing cell cycle transition from G1 to S phase (Pitolli et al., 2019). In addition, p53 regulates Bax, an apoptosis mediator (Pitolli et al., 2019). Caspase-3 is activated at the final step of the caspase cascade in apoptosis. In the present study, treatment with RJ regulated the expression of apoptosis-related proteins in the colon tissues of AOM/DSS-treated mice (Figure 6), which is similar to the results of our previous studies on the effect of RJ in colitis and Parkinson’s disease mouse models (Kim et al., 2020; Kim et al., 2022). These results indicate that RJ has anti-inflammatory and anti-apoptotic effects that contribute to the suppression of CAC.

This study had a few limitations. First, RJ contains various substances, and it is unclear which substances are responsible for the therapeutic effect. Second, the anti-cancer and protective effects of RJ in the colon need to be verified through clinical trials. Further studies are required to confirm these results.

In the present study, we demonstrated that RJ can protect the colon by inhibiting inflammation, TJ barrier disruption, and apoptosis in CAC mice. Moreover, RJ decreased the formation of colonic tumors. Therefore, RJ has potential for use in treating colon diseases, such as colitis, colorectal cancer, and diseases related to the intestinal immune response.