Combination of thalidomide and Clostridium butyricum relieves chemotherapy-induced nausea and vomiting via gut microbiota and vagus nerve activity modulation

Abstract

Nausea and vomiting (CINV) are distressful and widespread side effects of chemotherapy, and additional efficient regimens to alleviate CINV are urgently needed. In the present study, colorectal cancer (CRC) mice model induced by Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS) was employed to evaluate the cancer suppression and CINV amelioration effect of the combination of thalidomide (THD) and Clostridium butyricum. Our results suggested that the combination of THD and C. butyricum abundantly enhanced the anticancer effect of cisplatin via activating the caspase-3 apoptosis pathway, and also ameliorated CINV via inhibiting the neurotransmitter (e.g., 5-HT and tachykinin 1) and its receptor (e.g., 5-HT₃R and NK-1R) in brain and colon. Additionally, the combination of THD and C. butyricum reversed the gut dysbacteriosis in CRC mice by increasing the abundance of Clostridium, Lactobacillus, Bifidobacterium, and Ruminococcus at the genus level, and also led to increased expression of occludin and Trek1 in the colon, while decreased expression of TLR4, MyD88, NF-κB, and HDAC1, as well as the mRNA level of IL-6, IL-1β, and TNF-α. In all, these results suggest that the combination of THD and C. butyricum had good efficacy in enhancing cancer treatments and ameliorating CINV, which thus provides a more effective strategy for the treatment of CRC.

Keywords: CINV; Clostridium butyricum; gut- brain axis; intestinal microecology; thalidomide.

Figure 1

The combination of THD and C. butyricum promoted tumor apoptosis in CRC mice. (A) The schematic diagram of CRC modeling and treatment process in C57BL/6 mice. (B) H&E staining of colon tissue. (C) The number of tumors in colon tissue (n=11). (D) Weight of mice in different treatment groups (n=11). (E) Western blotting of COX-2, Survivin, cleaved Caspase-3 and Caspase-3 (n=3). (F–H) Relative expressions of COX-2, Survivin and cleaved Caspase-3/Caspase-3 (n=3). Significance determined using one-way ANOVA with Tukey’s multiple comparison test and expressed as mean ± SD, *P < 0.05, **P < 0.01.

Figure 2

The combination of THD and C. butyricum reduced CINV in CRC mice. (A) The kaolin consumption of mice in different treatment groups (n=3). (B) Fos protein in brain by IHC staining. (C) Fos positive cells were semiquantitatively assessed (n=3). (D) 5-HT of brain in different treatment groups (n=3). (E) the mRNA levels of Tac1 in brain (n=3). (F) 5-HT of colon in different treatment groups (n=3). (G) the mRNA levels of Tac1 in colon (n=3). (H) 5-HT₃R and NK1R protein in brain by IHC staining. (I, J) 5-HT₃R (I) and NK1R (J) positive cells were semiquantitatively assessed (n=3). Significance determined using one-way ANOVA with Tukey’s multiple comparison test and expressed as mean ± SD, *P < 0.05, **P < 0.01.

Figure 3

The combination of THD and C. butyricum reversed dysbacteriosis in CRC mice. (A) The Shannon indexes. (B) Principal coordinate analysis (PCoA). (C) Venn representation. (D) The relative abundance of the bacteria at phylum level. (E) The relative abundance of the bacteria at genus level. *P < 0.05, **P < 0.01.

Figure 4

The combination of THD and C. butyricum regulated anti-cancer related probiotics in CRC mice. (A) The relative abundance of Fimicutes (n=6). (B) The relative abundance of Clotridium (n=6). (C) The relative abundance of Lactobacillus (n=6). (D) The relative abundance of Bifidobacterium (n=6). (E) The relative abundance of Ruminococcus (n=6). Significance determined using one-way ANOVA with Tukey’s multiple comparison test and expressed as mean ± SD, *P < 0.05, **P < 0.01.

Figure 5

The combination of THD and C. butyricum inhibited colon inflammation and improved intestinal barrier in AOM/DSS-induced CRC mice. (A) H&E staining of colon tissue. (B) Histological scores of inflammations (H&E staining) (n=3). (C–E) The mRNA levels of Il6, Il1b, and Tnf (n=3). (F) Western blotting of TLR4, MyD88, p-p65, p65, HDAC1 (n=3). (G–J) The relative abundance of TLR4, MyD88, p-p65/p65, HDAC1 (n=3). (K) Western blotting of occludin and Trek1 (n=3). (L, M) The relative abundance of occludin and Trek1 (n=3). Significance determined using one-way ANOVA with Tukey’s multiple comparison test and expressed as mean ± SD, *P < 0.05, **P < 0.01.

Figure 6

Schematic diagram of the underlying mechanisms of the combination of THD and C. butyricum in cancer treatments and CINV amelioration. The combination of THD and C. butyricum enhanced anti-tumor effects and ameliorated CINV via activating caspase-3 apoptosis pathway, inhibiting neurotransmitter (e.g., 5-HT and Tac1) and its receptors (e.g., 5-HT₃R and NK-1R), and reversing intestinal dysbiosis in Azoxymethane/Dextran Sodium Sulfate induced colorectal cancer (CRC) mice. THD, thalidomide; C. butyricum, Clostridium butyricum; CINV, chemotherapy-induced nausea and vomiting; 5-HT, 5-hydroxytryptamine; Tac1, tachykinin 1; 5-HT₃R, 5-hydroxytryptamine-3 receptor; NK1R, neurokinin-1 receptor.