Identifying Oral And Gut Microbiota In Gastric Cancer Before And After Chemotherapy:Clinical Study And Animal Experiment

BACKGROUND:Many links between human diseases and microbiota has been found,yet the concept of 'common flora' remained unclear.Stomach cancer and microbiota related researches have been focusing on the gastric region due to H.pylori.However,recent discoveries indicate other bacteria may also participate in the process of carcinogenesis.In order to get a comprehensive understanding of co-existing bacteria in gastric cancer patients,and to explore the influence of chemotherapy,probiotic and nutrition therapy on microbiota,we performed several clinical studies and animal experiments.In this article,we used 16S rDNA sequencing to identify saliva and fecal microbiota in healthy individuals and gastric patients,analyzed the changes in microflora after chemotherapy,and found preliminary evidence that probiotics therapy might attenuate the side effects of chemotherapy on mice.Part 1.Identifying the difference in oral and gut microbiota behveen healthy individuals and gastric cancer patientsOBJECTIVE:To explore the difference in oral and gut microbiota between gastric cancer patients and healthy individualsMETHODS:Saliva and feces of 26 healthy people(group:Hsal,Hfec)and 19 gastric cancer patients(group:GCsal,GCfec)were collected,then 16S rDNA,V3-V4 region of bacteria was amplified and sequenced.Clustered OTUs were annotated using Silva 132 online database,and relative abundance(RA)of each taxa was calculated.RESULTS:The four major phylums in all samples are Firmicutes(RA:50-70%)Bacteroidetes(15-20%),Proteobacteria(9-18%)and Actinobacteria(2-9%).In GCsal,relative abundance of Actinomycetaceae,Peptostrptococcaceae was higher compared to Hsal.Whereas in GCfec,Streptococcaceae,Prevotellaceae an Bjfiobacteriaceae had higher abundance than in Hfec.Non-parametric multivariate analysis(PCA.NMDS and Anosim)on OTUs revealed significant difference between fecus and saliva,Hfec and GCfec.Linear discriminal analysis identified Streptococcaceae as a biomarker enriched in GCfec.CONCLUSION:The four major phylums(RA sum>90%)in human saliva and fecus were Firmicutes,Bacteroidetes,Proteobacteria and Actinobacteria.Microbiota structure was significantly differerent between saliva and fecus samples,and between Hfec and GCfec.Higher abundance of Streptococcaceae,Prevotellaceae and Bifidobacteriaceae was observed in GCfec,and the increase in Streptococcaceae may indicate colonization of oral flora in colon.Part ?:Changes in microbiota before and after neoadjuvant chemotherapy in patients with gastric cancerOBJECTIVE:To investigate the changes of saliva and fecal flora in patients ·ith gastric cancer before and after neoadjuvant chemotherapy.METHODS:Saliva and fecal samples of seven gastric cancer patients were collected before and after 1-2 courses of neoadjuvant chemotherapy.16S rDNA,V3-V4 region of bacteria was amplified and sequenced.Clustered OTUs were annotated using Silva 132 online database,and relative abundance(RA)of each specimen was calculated.RESULTS:After chemotherapy,the abundance of Veillonella in saliva increased.In feces,the abundance of Lactobacillus,Shigella,and Streptococcus increased,and the abundance of Faecalibacterium,Bifidobacterium and Bacteroides decreased.Changes in Veillonella and Bacteroides were statistically significant by LefSe analysis.CONCLUSION:After neoadjuvant chemotherapy,the abundance of Veillonella in saliva increases,the abundance of Bacteroides in feces decreases in gastric cancer patients.Chemotherapy may lead to the reduction of normal gut flora,the increase of pathogens,disrupting the mucosal barrier.Part ? The protective effect of probiotics and prebiotics on intestinal barrier of 5-Fu chemotherapy miceObjective:To investigate the protective effects of probiotics and enteral nutrition containing prebiotics on intestinal barrier of 5-Fu chemotherapy mice.METHODS:25 Balb/c male mice were randomly divided into 1-control group,2-chemotherapy group.3-prebiotic nutrient group,4-probiotic group,5-probiotic+prebiotic nutrient group(n=5).Give blank control,5-Fu chemotherapy,5-Fu+enteral nutrition with dietary fiber,5-Fu+mixed probiotics(B.logwn,L.plantarum and E.faecalis),5-Fu+mixed probiotics+enteral nutrition with dietary fiber.Lactic acid bacteria in the feces of the mice before the experiment was cultured.The body weight,daily food consumption,diarrhea score,fecal flora were assessed during the experiment,and the thickness of colon mucosa and the microbiota in jejunal content were measured at the end of the experiment.RESULTS:The fecal flora of healthy mice was mainly composed of Bacteroidetes and Firmicutes(the sum of abundance>90%).and the live bacteria concentration of Lactobacillus was 1.39×109 CFU/g,which mainly contained L.animalis,L.johnsonii and L.murinus.Body?eight loss of group 5 of mice:Group 5(-0.23±0.84g)<Group 1(0.85±0.32g)<Group 4(1.04±0.99g)<Group 3(3.58±1.22g)<Group 2(3.86±1.14g),P=0.024<0.05.The average food intake of mice:Group 3(6.55±1.82g)<Group 2(8.52±1.27g)<Group 4(11.88±0.58g)<Group 5(10.82±1.97g)<Group 1(15.85±0.73g),P<0.001.The probiotic nutrient group developed diarrhea on the first day of chemotherapy.The mean values of the thickness of colon villus mucosa in each group were:Group 2(119.35±3.83)<Group 3(120.72±6.8 8)<Group 4(125.68±5.78)<Group 1(133.00±7.86)<Group 5(134.23±6.24)),P=0.344.During chemotherapy,the abundance of Firmicutes reduced in group 2 mice,but then automatically returned to previous level on post chemotherapy day 4.CONCLUSION:(1)The fecal flora of healthy mice is mainly composed of Bacteroides and thick-walled bacteria.The live bacteria concentration of Lactobacillus in feces is 1.39×109 CFU/g,mainly containing Lactobacillus animalarum and Lactobacillus johnsonii.3 kinds of Lactobacillus pneumoniae;(2)5-Fu chemotherapy can reduce the appetite,weight loss,thinning of the colonic villus mucosa,and reduce the abundance of thick-walled bacteria,but can recover after 4 days of chemotherapy.Bifidobacterium triplet bacteria can improve the appetite of mice and restore the thickness of colonic mucosa.The combination of probiotics and enteral nutrition with prebiotics can maintain the body weight of chemotherapy mice and protect the intestinal mucosal barrier.Part ?:Anxiety,depression and disease cognition of patients with gastric cancer OBJECTIVE:To investigate the anxiety,depressive symptoms and illness perception of newly diagnosed patients with gastric cancerMETHODS:The newly diagnosed gastric cancer patients(n=27)were investigated with the Self-inspection Scale for Depression(SDS)and Self-inspection Scale for Anxiety(SAS).The standard scores were calculated and the incidence of depression and anxiety symptoms were calculated.The Chinese version of the disease cognition questionnaire was used.(IPQ-70)investigated the newly diagnosed patients with gastric cancer(n=23).statistically identified the factors with the highest incidence of symptom recognition and etiological cognition,and calculated the average scores of each dimension item of disease cognition,and compared it with the middle score of 3 points.RESULTS:The detection rate of depression in the newly diagnosed patients with gastric cancer was 11.1%,the detection rate of anxiety was 0%,and the three patients detected depressive had mild symptoms.The factors that the patient thinks are most likely to cause illness are food or eating habits(13/23),followed by poor medical care in the past or not being treated promptly(9/23),and the third place is bacterial or viral infection,smoking,immunity(all 5/23).Patients' answers were highly counsistent with the current recognized risk factors for gastric cancer.IPQ-70 score:7 cases of disease course,consequence cognition,consistency,periodicity,emotional characterization lower than the middle score,treatment control,personal control score exceeded the middle scoreCONCLUSION:The detection rate of depression(11.1%)and anxiety(0%)in patients with newly diagnosed gastric cancer is low.The newly diagnosed patients have a certain degree of correct understanding of the disease and holds a more optimistic attitude.