Bifidobacterium Infantis Ameliorates Chemotherapy-Induced Intestinal Mucositis Via Regulating T Cell Immunity In Colorectal Cancer Rats

Colorectal cancer(CRC)is the most common malignant tumor in the digestive system.The occurrence and the development of CRC are closely related to genetic heterogeneity,environmental factors,dietary habits and other factors.There are some reasons contribute to the malignant transformation of intestine mucosa and tumor progression,such as internal environment,heredity and so on.Mutations of multiple genes and abnormal DNA repair are all related to the occurrence and development of colorectal cancer.In addition,the composition of intestinal microorganisms is also an important factor affecting the occurrence of tumor.The change of intestinal flora had a complicated effect on the treatment and prognosis of colorectal cancer.For the treatment of CRC,chemotherapy is still an important treatment in CRC precision clinical treatment currently.However,the intestinal mucositis(IM)is a commonly encountered side effect in cancer patients receiving chemotherapy.It is also associated with intestinal flora imbalance.Therefore,the studies about the intestinal flora structure,chemotherapy-induced intestinal mucositis and the intestinal immunity system have been the hot spots recently.The intestinal microorganism is an important part of intestinal mucosal barrier.Bifidobacterium as the important member of intestinal flora,could be benefit for body healthy.Among them,Bifidobacterium infantis(B.infantis)is a commensal microbe isolated from the human gastrointestinal mucosa and has shown beneficial effects on gastrointestinal disease by modulating the immune function and has shown efficient in treating inflammatory bowel disease(IBD).B.infantis also can ameliorate trinitro-benzene-sulfonic acid(TNBS)-induced colitis and 5-Fluorouracil(5-FU)-induced intestinal mucositis in a normal mouse model.In our study,we used the CRC rat model to explore whether B.infantis could ameliorate the development of severe 5-FU combined with Oxaliplatin induced intestinal mucositis and to explore the mechanism.AimsIntestinal mucositis is a commonly encountered side effect in cancer patients receiving chemotherapy.To compare the severity of intestinal mucosal inflammation of CRC rats among three groups(Control group,Chemotherapy group,B.infantis group).Furthermore,to explore whether B.infantis could ameliorate the development of severe 5-FU combined with Oxaliplatin induced intestinal mucositis in rats with colorectal cancer and to explore the potential mechanism.Methods(1)To make the animal model(CRC rats model):The rats were injected with dimethylhydrazine(DMH)subcutaneously weekly for 10 weeks,and then injected with SW480 cells in rectal mucosa to create a CRC model.Rats were randomly divided into 3 groups(n=10): Control group(saline ~+ saline),Chemotherapy group(saline ~+ 5-FU~+Oxaliplatin),and B.infantis group(B.infantis ~+ 5-FU~+Oxaliplatin).After 2 weeks,the rats were orally administrated B.infantis(B.infantis group)or suspension saline(Control group and Chemotherapy group)daily for 11 days.On the 8th day,5-fluorouracil and Oxaliplatin(Chemotherapy group and B.infantis group)or saline(Control group)was injected intraperitoneally(i.p.)for 3 days.(2)To record and analyze the body weight of the CRC rats.(3)To record and analyze the body weight of the CRC rats.(4)Histological Analysis: to measure the intestinal villus height and the crypt depth,and to evaluate the severity of the IM.(5)ELISA analysis: to measure the pro-inflammatory cytokines(IL-6,TNF-α,IL-1β)of intestinal tissue and evaluate the severity of the chemotherapy-induced IM.(6)Flow cytometry analysis: to measure T cell subsets(CD4~+ IL17A~+ cells and CD4~+ CD25~+ Foxp3~+ Tregs)in mesenteric lymph nodes(MLNs)and evaluate the severity of the chemotherapy-induced IM.(7)Reverse transcription-polymerase chain reaction(RT-PCR): to measure T cell subsets related cytokine profiles(IL-2 m RNA,IL-12 m RNA,IFN-γ m RNA,T-bet m RNA,IL-17 m RNA,IL-21 m RNA,IL-23 m RNA,RORγt m RNA,IL-10 m RNA,Foxp3 m RNA and TGF-β m RNA)and evaluate the severity of the chemotherapyinduced IM.(8)Statistical analysis: All experiment results were analyzed with SPSS software(version 21.0).Data of rats were analyzed by Wilcoxon rank sum test,KruskalWallis H test and Nemenyi test.P values less than 0.05 were considered statistically significant.Results(1)The study showed that after chemotherapy treatment,those rats in Chemotherapy group had higher body weight(BW)loss than those in Control group.However,the B.infantis group demonstrated a higher BW compared to the Chemotherapy group.Therefore,the BW loss was partially prevented in rats receiving B.infantis.(2)The study showed that rats in Chemotherapy group had more serious diarrhea than those in Control group.However,the B.infantis group demonstrated a lower Diarrhea score compared to the Chemotherapy group.Therefore,the severity of diarrhea was clearly attenuated in those rats treated with B.infantis.(3)The results showed that villus height was significantly reduced in all rats receiving chemotherapy compared to the Control group.However,B.infantis-treated rats showed a higher villus height compared to Chemotherapy group.Besides,chemotherapy significantly lengthened crypt depth of the intestine compared with the Control group.On the contrary,the crypt depth was significantly restored by B.infantis treatment.(4)ELISA analyses revealed that increased plasma IL-6,IL-1β and TNF-α levels were detected in all rats in the Chemotherapy group compared to Control group.However,the level of IL-6,IL-1β and TNF-α were decreased in the B.infantis group compared to the Chemotherapy group.The level of pro-inflammatory factors were decreased in those rats treated with B.infantis.(5)Flow cytometric analyses revealed that chemotherapy increased the percentage of CD4~+CD17A~+T cells and decreased the percentage of CD4~+CD25~+Foxp3~+ cells in MLNs compared with saline control.B.infantis feeding decreased the percentage of CD4~+CD17A~+T cells and increased the percentage of CD4~+CD25~+Foxp3~+ cells in MLNs compared with Chemotherapy group.(6)We measured the relative m RNA expression of different T cell cytokines by RTPCR.Compared with the Control group the relative m RNA expression of Th1 cytokines(IL-2 m RNA,IL-12 m RNA,IFN-γ m RNA and T-bet m RNA)and Th17 cytokines(RORγt m RNA,IL-17 m RNA,IL-21 m RNA,IL-23 m RNA)increased in Chemotherapy group.However,the relative m RNA expression of Foxp3~+ Tregs cytokines(IL-10 m RNA,Foxp3 m RNA,TGF-β m RNA)decreased in Chemotherapy group compared with the control group.Administration of B.infantis reversed upregulation of Th1-and Th17-responses and downregulation of Foxp3~+Treg responses in chemotherapy-induced IM in rats.ConclusionB.infantis effectively attenuates chemotherapy-induced intestinal mucositis by decreasing Th1 and Th17 response and increasing CD4~+ CD25~+ Foxp3~+ Tregs response.