| The intestine is not only the organ of digestion and absorption,but also has the functions of immune defense,nerve and endocrine.It is closely related to other tissues and organs.When intestine get damaged,it will not only lead to intestinal dysfunction,but also involve extraintestinal organs.Intestinal injury will affects the occurrence,development,outcome and prognosis of diseases.Ischemia/reperfusion is one of the main causes of intestinal injury.The mechanism of this injury is generally believed to be related to the strong oxidative stress.Recent studies have found a link between changes in gut microbes and gut injury under ischemia/reperfusion conditions.Cardiovascular surgery performed under the support of cardiopulmonary bypass(CPB)often leads to ischemia/reperfusion injury in multiple organs in the body,of which the intestine is the most sensitive organ to ischemia/reperfusion.In the early stage of the laboratory,it was found that 35.05% of patients will show gastrointestinal complications after CPB assisted cardiovascular surgery(hereinafter referred to as CPB surgery).Gastrointestinal bleeding is one of the most serious gastrointestinal complications with low incidence but high mortality.It is a high-risk factor for poor prognosis in patients after CPB.Besides,Gut microbiota of patients after CPB changed significantly compared with that before CPB.Especially,there were also significant differences in the composition of gut microbiota between patients with postoperative gastrointestinal bleeding and patients without gastrointestinal bleeding.However,the causal relationship between the difference of gut microbiota and lower gastrointestinal bleeding after CPB is not clear.This study amis to reveal the casual relationship and related mechanism between gut microbiota disorder and lower gastrointestinal bleeding in patients after CPB.The main research contents and results are as follows:The first study: Effect of fecal bacteria in patients with gastrointestinal bleeding after cardiopulmonary bypass on the intestine of miceTo study the effect of fecal bacteria on the intestine of mice in patients with lower gastrointestinal bleeding after cardiopulmonary bypass surgery,48 male C57BL/6 mice aged 6-8 weeks were randomly divided into 4 groups with 12 mice in each group.Bacterial solution were prepared from preoperative feces of patients with low gastrointestinal bleeding(Pre-GIB),postoperative feces of patients with low gastrointestinal bleeding(Post-GIB),preoperative feces of patients without gastrointestinal bleeding(Pre-NGIB),postoperative feces of patients without gastrointestinal bleeding(Post-NGIB).Gavage of the above four groups of bacterial solution was performed on the first 3 days and the 7th day during the experiment.On the day 35 of the experiment,the mice were slaughtered and sampled to detect the gut microbiome and other indexes.All indicators were analyzed by Two-way ANOVO,the main are as follows:1.Gut microbiome: the microbial Chao1 index and Observed＿species index of mice in Post-NGIB group and Post-GIB group(hereinafter referred to as Post group)were significantly lower than those in Pre-NGIB group and Pre-GIB group(hereinafter referred to as Pre group)(P < 0.05).The microbial Shannon index and Pielou＿e index of mice in Pre-GIB group and Post-GIB group(hereinafter referred to as GIB group)were significantly lower than those in Pre-NGIB group and Post-NGIB group(hereinafter referred to as NGIB group)(P < 0.05).Besides,the diversity of gut microbiota in each group was significantly different(P < 0.05).The difference analysis found that,at the phylum level,Pre-GIB group was rich in Verrucomicrobia,While Post-GIB group was rich in Firmicutes and Proteobacteria.At the genus level,the specific bacteria in Pre-GIB group were Akkermansia,Allobacuium,Exiguobacterium,Brevundimonas,the specific bacteria in Post-GIB group were Bacteroides,Shigella,Helicobacter,Owenweeksia,Robinsoniella,Anaerotruncus,the specific bacteria in Pre-NGIB group were Blautia,Sphingobium,Roseivirga,the specific bacteria in Post-NGIB group were Parabacteroides,Anaerostipes.These results indicated that after the mice were gavaged with postoperative fecal bacteria in patients,especially in patients with lower gastrointestinal bleeding,the gut microbiota diversity was reduced,the microbiota composition was disordered,harmful microbiota were increased and beneficial microbiota were decreased.2.Fecal short chain fatty acids: the fecal propionate levels of mice in Post group were significantly lower than those in Pre group(P < 0.05).The levels of acetate,propionate,butyrate and total short-chain fatty acids in feces of mice in GIB group were significantly lower than those in NGIB group(P < 0.05).These results indicated that the ability of gut microbiota to produce short chain fatty acids was significantly reduced after CPB surgery,especially in patients with lower gastrointestinal bleeding after CPB.3.Intestinal cytokine expression: the gene expression of Il-17 in cecum of mice in Post group was significantly higher than that in Pre group(P < 0.05).The gene expressions of Il-1b and Il-17 in cecum of mice in GIB group were significantly higher than those in NGIB group(P < 0.05).These results indicated that fecal bacteria in patients after CPB surgery and fecal bacteria in patients with gastrointestinal bleeding triggered a more severe inflammatory response in the cecum of mice.4.Intestinal tight junction protein expression: compared with Pre group,the gene expressions of Occludin,ZO-2,Claudin-4 in jejunum and ZO-2 in colon of mice in Post group were significantly reduced(P < 0.05).Compared with NGIB group,the gene expression of ZO-1 in cecum of mice in GIB group was significantly reduced(P < 0.05).In addition,compared with Post-NGIB group,the gene expression of Occludin in cecum of mice in Post-GIB group was significantly reduced(P < 0.05).These results indicated that the fecal bacteria in patients after CPB surgery significantly inhibited the gene expression related to the structure of jejunum and colon barrier in mice,while the fecal bacteria in patients with lower gastrointestinal bleeding,especially the fecal bacteria after surgery,significantly inhibited the gene expression related to the structure of cecum barrier in mice.5.Red blood cell and hemoglobin parameters: compared with Pre group,MCH and MCHC in the blood of mice in Post group were significantly reduced(P < 0.05).Compared with Pre group,RBC,HGB and MCH of mice in GIB group were significantly increased(P < 0.05).These results indicated that fecal bacteria in patients after CPB surgery and fecal bacteria in patients with lower gastrointestinal bleeding caused a certain degree of anemia in mice.6.Systemic inflammatory response: compared with Pre group,WBC,Neu of mice in Post group were significantly increased(P < 0.05).Compared with NGIB group,Neu,Neu%,Eos,Eos% of mice in GIB group were significantly increased,and Lym% was significantly decreased(P < 0.05).In addition,compared with Post-NGIB group,Neu and Eos of mice in Post-GIB group were significantly increased,and Lym% was significantly decreased(P < 0.05).These results indicated that the fecal bacteria in patients after CPB surgery and fecal bacteria in patients with lower gastrointestinal bleeding caused systemic inflammatory response in mice,and the inflammatory response caused by postoperative fecal bacteria in patients with lower gastrointestinal bleeding was more serious.Since the mice did not show symptoms of lower gastrointestinal bleeding under this experimental condition,it is necessary to further confirm the causal relationship between lower gastrointestinal bleeding and gut microbiota under ischemia/reperfusion conditions.The second study: Causal relationship between gut microbiota and lower gastrointestinal bleeding under intestinal ischemia/reperfusionTo confirm the causal relationship between gut microbiota and lower gastrointestinal bleeding after cardiovascular cardiopulmonary bypass surgery,72 male C57BL/6 mice aged 6-8 weeks were randomly divided into 3 groups with 24 mice in each group.Bacterial solution were prepared from preoperative feces of patients with low gastrointestinal bleeding(Pre-GIB-I,treatment 1 group)and postoperative feces of patients with low gastrointestinal bleeding(Post-GIB-I,treatment 2 group).Gavage of the above two groups of bacterial solution and PBS solution(PBS-I,control group)was performed on the first 3 days and the 7th day during the experiment.On the day 14 of the experiment,12 mice in each of the three groups were subjected to intestinal ischemia/reperfusion surgery,and then all mice were slaughtered and sampled to detect the expression of intestinal inflammatory cytokines and other indexes.The main are as follows:1.Weight change and intestinal bleeding: the body weight of mice in treatment 2group was significantly higher than that of treatment 1 group on day 1 of faecal bacteria transplantation,while the body weight of mice in treatment 2 group was always lower than that of treatment 1 group and control group after day 2(P < 0.05),indicating that postoperative fecal bacteria in patients with lower gastrointestinal bleeding caused more weight loss in mice.After intestinal ischemia/reperfusion surgery,the intestines of mice in control,treatment 1,and treatment 2 group were filled with blood,and intestinal osmotic bleeding occurred.2.Intestinal cytokine expression:compared with control group,the m RNA level of cecal Il-12 b,IFN-γ in treatment 1 group was significantly increased(P < 0.05).Compared with control group,the gene expression of jejunal Il-1b,Il-17,TNF-α,IFN-γ,ileal IFN-γ,cecal Il-1b,Il-12 b,IFN-γ,in treatment 2 group was significantly increased(P < 0.05).In addition,compared with treatment 1 group,the gene expression of duodenal Il-22,jejunal Il-1b,Il-17,IFN-γ,ileal Il-1b,Il-17,Il-22,IFN-γ and cecal Il-1b in treatment 2 group was significantly increased(P < 0.05).These results indicated that under the condition of intestinal ischemia/reperfusion,fecal bacteria in patients with lower gastrointestinal bleeding lead to more serious intestin Fal inflammatory reaction,in which the inflammatory reaction of jejunum and ileum was more obvious,and the influence of postoperative fecal bacteria was more severe.3.Intestinal tight junction protein expression: compared with control group,the gene expression of duodenal ZO-1,ZO-2,jejunal ZO-1,ZO-2,Occludin,Claudin-4,cecal Occludin in treatment 1 group was significantly reduced(P < 0.05),the gene expression of duodenal ZO-1,ZO-2,Occludin,jejunal ZO-2,Occludin,Claudin-4,ileal ZO-2,Occludin,Claudin-4 in treatment 2 group was significantly reduced(P < 0.05).In addition,compared with treatment 1 group,the gene expression of ileal ZO-2,Occludin in treatment 2 group was significantly reduced(P < 0.05).These results indicated that under the condition of intestinal ischemia/reperfusion,fecal bacteria in patients with lower gastrointestinal bleeding significantly inhibited the expression of genes related to intestinal barrier structure in mice,and the influence of postoperative fecal bacteria was stronger.4.Intestinal antioxidant enzyme activity: compared with treatment 1 group,the cecal superoxide dismutase activity and jejunal catalase activity of treatment 2 group were significantly decreased(P < 0.05),indicating that under the condition of intestinal ischemia/reperfusion,postoperative fecal bacteria in patients with lower gastrointestinal bleeding weakened the intestinal antioxidant capacity.5.Red blood cell and hemoglobin parameters: compared with control group,MCH of mice in treatment 2 group was significantly reduced(P < 0.05).Compared with control group,MCH and MCHC in treatment 2 group were significantly reduced(P < 0.05).These results indicated that postoperative fecal bacteria in patients with lower gastrointestinal bleeding exacerbated the anemia caused by intestinal ischemia/reperfusion.6.Systemic inflammatory response: compared with control group and treatment 1group,Neu% of mice in treatment 2 group was significantly increased and Lym% was significantly decreased(P < 0.05),indicating that postoperative fecal bacteria in patients with lower gastrointestinal bleeding exacerbated the bacterial infection caused by intestinal ischemia/reperfusion.In summary,the main conclusion of this study is:1.Cardiovascular cardiopulmonary bypass assisted surgery causes gut microbiota disorder,significantly reduces short chain fatty acids,increases the inflammatory response of each intestinal segment,and leads to structural damage to intestinal epithelial barrier,which is an important cause of systemic bacterial infection and anemia.2.Disorder of fecal microbiota caused by cardiovascular cardiopulmonary bypass assisted surgery will aggravate intestinal ischemia/reperfusion injury,increase the inflammatory response of the jejunum and ileum,seriously damage the intestinal epithelial barrier function,and has a causal relationship with postoperative lower gastrointestinal bleeding. |
