Colorectal cancer (CRC) is a cancer occurring in the colon or rectum, with over 1.9 million new cases and 935,000 deaths estimated in 2020, ranking the third in incidence and the second in mortality (1, 2). For patients’ treatment, systemic chemotherapy is the mainstay treatment besides surgery and local radiotherapy and has been wildly used in advanced CRC (3). Currently, platinum-based chemotherapy drugs which inhibits nuclear DNA transcription and replication and initiates programmed cell death are dominating (4). It is proposed that cyclooxygenase-2 (COX-2) inhibition can hasten Caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage, as well as suppress the expressions of anti-apoptotic proteins such as surviving, thus activating tumor apoptosis (5).

Despite the anticancer effects, chemotherapy drugs lead to severe side effect include gastrointestinal toxicities, myelosuppression, immunosuppression and neurotoxicity due to poor targeting (6, 7). Chemotherapy-induced nausea and vomiting (CINV) is a common adverse effects of chemotherapy that affects patient’s life and treatment effectiveness (8, 9). It has been reported that the expression of 5-hydroxytryptamine (5-HT) in intestinal mucosa was triggered by cytotoxic chemotherapy drugs, then stimulating the 5-HT receptors on adjacent vagal afferent nerves (VAN) (10). After nerves depolarization, vomiting center in brainstem was stimulated to induce a vomiting reflex (11). Consequently, there is a pressing need for alternative treatments that are both highly effective and have minimal side effects.

The most commonly used antiemetic agents belong to neurokinin-1 receptor antagonists (NK₁-RAs), 5-hydroxytryptamine-3 receptor antagonists (5HT₃-RAs) and dexamethasone but the therapeutic effect was still not satisfactory (12, 13). Thalidomide (THD), a derivative of glutamate, was originally used to treat nausea in pregnancy, later was banned because of its teratogenicity (14, 15). However, THD, as approved drugs, has been used in the treatment of a variety of solid tumors, with good efficacy and a certain degree of safety (16). At present, the effectiveness of THD in controlling CINV has become the focus of research. The randomized control trials have clinically confirmed the effectiveness of THD by calculating and analyzing the delayed and overall complete response rates to vomiting in cancer patients (17). Subsequently, several studies have demonstrated that THD’s potent immunomodulatory activity affects the expression and activity of various cytokines to influence anti-angiogenic effects and tumor defense (18, 19). However, numerous studies have shown that patients with CRC have an altered intestinal microbiota which lost a significant amount of butyrate-producing bacteria, such as Clostridium, Roseburia, and Eubacterium spp. (20). Chemotherapeutic drugs disrupt intestinal microecology, damage intestinal mucosal barrier, and trigger gut inflammation (21). Furthermore, the use of THD will lead to gastrointestinal side effects including constipation due to the certain neurotoxicity of THD (22).

At present, the gut microbiota has been widely evidenced to be related with the occurrence, development and treatment of cancers (23). Probiotics are beneficial microorganisms exerting probiotic role by regulating the gut microbiota (24, 25). Clostridium butyricum generates short-chain fatty acids (SCFAs), especially butyrate and acetate, and affects various physiological processes contributing to host health (26, 27). C. butyricum CBM 588 has been implicated in anti-inflammation, gut epithelial barrier protection and the increased abundance of Lactobacillus, Bifidobacterium in the gut microbiota (28). A study conducted by Danfeng Chen et al. revealed that C. butyricum can inhibit the development of CRC by the stimulation of apoptosis and the reconstruction of gut microbiota (29). Moreover, C. butyricum administration was found to significantly reduce cognitive dysfunction and histopathological changes, as well as neuronal apoptosis probably via the involvement in gut-brain axis modulation (30). Therefore, this suggests a possible therapeutic role for C. butyricum in the gastrointestinal toxic side effects of chemotherapy.

In clinic, we found that the combination of C. butyricum and THD almost eliminate CINV (data unshown). Therefore, to elucidate the efficacy and mechanisms of C. butyricum+THD on treatment of CINV, we investigated the antitumor function, the preventing of nausea and vomiting, the altering of microbiota composition and the enhancement of the intestinal epithelial barrier in CRC mouse model with chemotherapy. This study presents new ideas for the prevention and treatment of CINV with C. butyricum+THD.

CRC is the third most common cancer worldwide, with high morbidity and mortality rates (35). Currently, chemotherapy, the primary treatment for advanced CRC, causes severe side effects, especially nausea and vomiting (31). CINV which threatens patient’s life and health (8, 9), leads to an urgent need to create alternative therapies that are highly effective and have minimal side effects.

Clinically, we observed that the combination of C. butyricum and THD almost eliminate CINV (data not shown), but the mechanism for the prevention of CINV remains unclear. Herein, we used AOM/DSS-induced CRC mouse models that received cisplatin chemotherapy to investigate the primary role of the combination of C. butyricum and THD in addition to its possible therapeutic mechanism. Our results indicated that AOM/DSS caused the occurrence of CRC in mice, but the mice receiving cisplatin with the combination of THD and C. butyricum showed a markedly decreasing tumor number. Our findings suggested that COX-2 and survivin in tumor are highly expressed, and cleaved Caspase-3 and cleaved PARP are lowly expressed in M group. Additionally, COX-2, which is elevated expressed in CRC, contributes to tumorigenesis by excited angiogenesis, suppressed apoptosis, and enhanced cell invasiveness (36). What’s more, survivin is an inhibitor of programmed cell death that is negatively correlated with apoptosis and directly suppresses caspase-3 activity to prevent apoptosis (37, 38). After activation of caspase-3, the DNA repair enzyme PARP was cleaved leading to loss of the ability of DNA repair resulting in apoptosis (39). Herein, we found that the combination of THD and C. butyricum can inhibit the expression of COX-2 and survivin to activate Caspase-3 and cleave PARP to induce tumor apoptosis ( Figure 1 ).

Cisplatin, a chemotherapeutic agent frequently used in clinical practice, is associated with emesis (40). Hence, we used kaolin intake measurement, an index of nausea and emesis in animal studies (41), to evaluate nausea and vomiting of mice with chemotherapy. Mice ingested cisplatin severely vomited than M group, but the combination therapy of THD and C. butyricum significantly relieved emesis. Fos in AP, a marker of neuronal activity whose expression correlates with brain stimulation resulting from the stimulation of neurotransmitter receptors on adjacent vagal afferents, was increased after cisplatin chemotherapy (42, 43). Our findings suggested that the combination of THD and C. butyricum could abundantly decrease the fos expression in the AP, confirming reduced brain neuron activation. Furthermore, it was currently reported that cisplatin-induced release of the 5-HT and Tac1 interacted with receptors (i.e., 5-HT₃R and NK-1R) of the vagus nerve in brain and gastrointestinal tract to cause the above neuronal activation (7, 44). We found that a combination of THD and C. butyricum abundantly reduces the 5-HT expression and the relative Tac1 mRNA level in brain and colon compared with MC group. Additionally, we found that the combination therapy inhibited aggregation of 5-HT₃R and NK-1R in brain of CRC mice with cisplatin chemotherapy. In summary, these results suggested that the combined administration of THD and C. butyricum reversed the up-release of neurotransmitters and activation of receptors in cisplatin-treated CRC mice model, to relieve the cisplatin-induced nausea and vomiting ( Figure 2 ).

Disordered gut microbiota is associated with weakened health (45) and is involved in gastrointestinal carcinogenesis, while cisplatin chemotherapy further destroyed its homeostasis (46, 47). In addition, Chen et al. discovered that C. butyricum is beneficial in the reconstruction of gut microbiota (48), and the reversed gut microbiota disorder further plays a role in treatment of intestinal diseases (49). Therefore, we explored whether C. butyricum could improve the intestinal microbiota disorder caused by CRC and chemotherapy via high-throughput sequencing. The results demonstrated the therapeutics of the combination treatment (MCSL group) in restoration of intestinal microbiota diversity ( Figure 3 ). Moreover, it was observed that certain genera of bacteria had reduced abundance in both M group and MC group, including Clostridium, Lactobacillus, Bifidobacterium and, Ruminococcus. Furthermore, such dysbiosis could be restored by combined treatment with THD and C. butyricum, which is consistent with the published reports about the microbiota regulatory effect of C. butyricum and ( Figures 4B–E ). Among them, the increased relative abundance of Clostridium may be related to the well intestinal colonization of C. butyricum (50). After its colonization, C. butyricum furthur regulates the richness and composition of intestinal microbiota, alleviates intestinal inflammation, further improve the efficacy of chemotherapy which may contribute to the remission of CINV (26, 51). Meng et al. discovered that L. plantarum orally administration significantly increased the abundance of Lactobacillus and Bifidobacterium in cecal content of CTX-treated mice, and markedly reduced the nausea and vomiting symptoms, suggesting that the high abundance of Lactobacillus and Bifidobacterium is positively correlated with the relief of nausea, vomiting (52). What’s more, the increased abundance of Lactobacillus limits intestinal tumor growth via gut microbiota reconstruction, tumor cell proliferation suppression and apoptosis activation (53, 54). Bifidobacterium, one of the butyrate-producing bacteria, has similarly been verified effective in decreasing pro-inflammatory cytokines and inhibiting cancer cells to suppress gastrointestinal cancer (55, 56). Notably, Ruminococcus, another strain can produce butyrate, showed a slight increase in abundance after C. butyricum administration. And there are published papers supporting that Ruminococcus could partially inhibit inflammation and protect the intestinal mucosa after cisplatin therapy in various cancer models (51, 57). At the phylum level, the findings of Burkhardt Flemer et al. (58) and our research both pointed to the decreased abundance of Firmicutes in CRC mice ( Figure 4A ). As suggested by another study, the diminished Firmicutes would be the possible mechanism responsible for cisplatin-associated side effects (47). In conclusion, the improved gut microbiota dysbiosis mediated by C. butyricum administration may be associated with anti-tumor, neurotransmitter secretion decrease and brain activation reduction, as well as anti-inflammation in colon.

In recent years, evidence has been mounting to suggest that chronic inflammation produces considerable inflammatory mediators (IL-6, IL-1β, TNF-α), activating NF-κB, thus leading to intestinal barrier function loss and colon carcinogenesis (59). Hence, we utilized H&E staining to find that the combination therapy (MCSL group) greatly suppresses colonic inflammation and attenuates histopathological changes in colon ( Figures 5A–B ). Additionally, our findings suggested that the combined treatment of THD and C. butyricum degraded the levels of IL-6, IL-1β, and TNF-α and downregulated the activity of HDAC1, TLR4, MyD88, and p-p65 ( Figures 5C–J ). Among them, histone deacetylase 1 (HDAC1) can be inhibited by butyrate to eliminate inflammation in colonic epithelial cells, subsequently suppressing colonic inflammation (60). The intestinal mucosal barrier functions are associated with intestinal tight-junction associated proteins, such as occludin (61). Besides, recent reports have suggested the importance of Trek1 in preserving the integrity of the intestinal epithelial barrier (62). We discovered that occludin and Trek1 activity in colon was significantly decreased in both M and MC groups. However, combination administration (MCSL group) substantially restored those reductions ( Figures 5K–M ). Collectively, these outcomes demonstrated that the combination of C. butyricum and THD has a notable impact on protecting intestinal barrier function and anti-inflammatory effect in CRC mice undergoing cisplatin chemotherapy.

We conclude that the combination of THD and C. butyricum improved cancer suppression efficacy of cisplatin in CRC mice by activation of caspase-3 apoptotic pathway. Moreover, our study confirmed that the inhibition of neurotransmitters (e.g., 5-HT and Tac1) and its receptor (e.g., 5-HT₃R and NK-1R), the regulation of intestinal microbiota, the inhibition of inflammation in colon, and the protection of intestinal mucosa barrier via the combination therapy are extremely important to alleviate CINV in CRC mice ( Figure 6 ). It should be noted that in this work, we only investigated the ability of C. butyricum combined with THD in the treatment of CRC and CINV via gut microbiota and vagus nerve activity modulation. However, it is important to evaluate the efficacy of additional probiotics for the development of chemotherapy adjuvant drugs. Moreover, we will fully consider the necessary of verifying the efficacy of the combination of C. butyricum and THD in CRC patients receiving chemotherapy and its chemotherapy associated side effects in the clinic.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, PRJNA941024.

This study and the animal experimental protocol were reviewed and approved by the Laboratory Animal Ethics Committee of Nanchang Leyou Biotechnology Co. Ltd. (RyE2021070902). All experiments were conducted in accordance with the guidelines.

XZ contributed with methodology, investigation, formal analysis, visualization and writing - original draft. HWu and RZ contributed with methodology, investigation and visualization. GS and JW contributed with investigation. HS and PT contributed with methodology. TC and HWe contributed with conceptualization, funding acquisition, supervision, writing – review and editing. All authors contributed to the article and approved the submitted version.