The Protective Effect And Mechanism Of Probiotics Combination On Mucositis Induced By Radiotherapy And Chemotherapy Targeting Nasopharyngeal Carcinoma

Objective:This study aimed to explore the protective of probiotic combination（Bifidobacterium animal,Lactobacillus plantarum,L.rhamnosus,and L.acidophilus）on nasopharynx and its specific mechanism through establishing randomized controlled trials of patients with nasopharyngeal carcinoma（NPC）and constructing animal models of oral mucositis（OM）.Methods:1.High throughput sequencing and real-time fluorescence quantitative polymerase chain reaction（q-PCR）were used to evaluate the diversity of gut microbiota in non cancer villagers（NT）,healthy people（HP）and tumor patients（TP）.2.After isolation and identification of bacteria from feces of volunteers in cancer-free villages,the probiotic characteristics of the combined bacteria were evaluated by the acid resistance test,bile salt resistance test,antioxidant test,pathogenic bacteria inhibition test and cell adhesion ability.3.A randomized controlled experimental study was conducted in Jiangxi Cancer Hospital from September 2017 to September 2018.Seventy-six cases of advanced nasopharyngeal carcinoma patients were enrolled.Patients were randomly divided into a probiotic combination group（38 cases）and a placebo group（38 cases）according to a 1:1 ratio,and were treated with probiotic combination or a placebo for7 weeks respectively.During this period,two hospital experts evaluated the severity of oral mucositis of patients every day,and patient plasma and feces were collected after 7 weeks.The analysis of biochemical automata and 16S rRNA were used to detect blood rOUTine,CD3⁺T cells CD4⁺T cells,CD8⁺T cells levels and gut microbiota diversity.4.Oral mucositis model using SD rats was established through chemotherapy and local oral radiation,and pretreatment with probiotic combination with 7 day.The sample of tongue,plasma,feces,and proximal colon tissue were collected on the 7^th,14^thh and 21^stt days.Q-PCR,western blot and 16s rRNA were used to detect tongue cell apoptosis and inflammatory,the expression of colon tissue permeability-related proteins（ZO-1 and Claudin-1）and tongue tissue inflammation（TLR4/NF-κB）and apoptosis（Bax/Bcl-2）,and gut microbiota diversity.Results:1.The NMDS analysis showed that samples in HP group and NT group clustered together,while deviated from samples in TP group.Besides,the results of q-PCR further confirmed that the abundance of probiotics Lactobacillus,and Bifidobacterium in the NT group were higher than that in the H and T groups（P<0.001）,while the abundance of harmful bacteria such as Clostridium,Enterococcus and Enterobacter in the NT group was reduced,compared with H and T groups（p<0.001）.2.L.plantarum ntarum MH-301,B.animalis subsp.lactis LPL-RH,L.rhamnosus nosus LGG-18,and L.acidophilus were finally chosen for their low acid resistance,high resistance to bile salts,strong oxidation resistance,broad-spectrum antibacterial ability and high cell adhesion.3.Evaluation of the combination of the probiotics on reducing the severity of OM induced by chemoradiotherapy for patients with NPC.（1）We found that patients taking t probiotic combination showed significantly reduction of OM.The incidences for Grade 0,1,2,3 and 4 OM in the placebo group and the probiotic mixture group were 0%,14.7%,38.2%,32.4%,and 14.7%and13.9%,36.1%,25%,22.2%,and 2.8%,separately.（2）Patients in the probiotic mixture group showed lower reduction rate in CD3⁺T cells（75.5%vs 81%,P<0.01）,CD4⁺T cells（64.53%vs 79.53%,P<0.01）,and CD8⁺T cells（75.59%vs 62.36%,P<0.01）compared with the placebo group.（3）The abundance of Bacteroidete and Actinobacteria were increased and the abundance of Firmicutes was decreased in the ARCP group,but,probiotics combination enriched the abundance of Firmicutes and reduced the abundance of Bacteroidetes and Actinobacteria to a normal level.The NMDS analysis found that BRCPM,ARCPM,BRCP,and HP groups were clustered together,while the ARCP group diverged from other groups and showed a scattered distribution,indicating probiotics combination restored the gut dysbiosis in NC patients with CCRT.4.Probiotics combination attenuates the severity of OM in radiotherapy and chemotherapy-induced rats possibly through down-regulating the TLR4/NF-κB signaling pathway,reducing cell apoptosis,downregulation of intestinal TJ（ZO-1and Claudin-1）proteins and regulating gut dysbiosis.（1）The mucosal index was evaluated on the 7^th,14^thh and 21^stt day.Rats in M group significantly increased the incidence of grade 3 or over than those in C group on the 7^th,14^thh and 21^stt day（76.9%vs 0%,100%vs 0%,66.7%vs 0%;p<0.01;respectively）.Otherwise,rats in T group significantly decreased the incidence of grade 3 or over than those in M group on the 7^th,14^thh and 21^stt day（23.1%vs 76.9%,50%vs 100%,0%vs 66.7%;p<0.01;respectively）.（2）Rats in M group showed a higher leukocyte concentration compared with C group on the 7^th,14^thh and 21^stt day.While,rats in T group had a lower leukocyte concentration than M group.（3）To evaluate the inflammation of oral mucosa,expression of IL-6,IL-1βand TNF-αat mRNA level in tongue tissue were measured.M group demonstrated significantly higher expression level than C group in IL-6,IL-1βand TNF-αon the 7^th,14^thh and 21^stt day,while,T group showed remarkably lower expression level than M group in IL-6,IL-1βand TNF-α)on the 7^th,14^thh and 21^stt day.（4）Probiotics combination attenuated the severity of OM possibly through down-regulating the TLR4/NF-κB signalling pathway,reducing cell apoptosis and downregulation the expression of intestinal proteins（ZO-1 and Claudin-1）.（5）16S rRNA gene sequencing analysis results showed that probiotics combination improved the microbialα-diversity and prevented the reduction of Firmicutes,Lachnospiraceae,and Ruminococcus and decreased the abundance of Bacteroidetes and Bacteroides in rats with OM caused by radiotherapy and chemotherapyConclusion:The modified probiotics combination significantly reduces the severity of OM through enhancing the immune response of patients and modifying the structure of gut microbiota.