Effects Of Changes In Intestinal Microbiota Under Antibiotic Pressure On CRISPLD2 Expression And Inflammatory Levels In Septic Mice

Background and objective:Sepsis is characterized by infection,inflammatory response and impaired organ function,and is one of the most intractable problem facing acute and critical diseases around the world,with a global mortality rate as high as 25%-30%.Antibiotic treatment is widely recognized as an important component of sepsis treatment,and epidemiological surveys have shown that 64%-71%of patients in intensive care units(ICUs)receive antibiotic treatment,with 10%-51%suffering from hospital-acquired infections(HAIs).However,clinical studies have shown that antibiotics can not completely control the development of inflammation and the further deterioration of organ damage,and can even induce the growth of drug-resistant bacteria in the intestine,with long-term harmful effects on the host.As a natural LPS-binding protein in serum,CRISPLD2 is highly expressed in intestinal and blood immune cells,and can competitively inhibit the binding of LPS to circulating immune cells TLR4,thus antagonizing LPS-induced waterfall inflammation.This study aims to explore the regulatory effect of intestinal flora on CRISPLD2 protein and the effect of CRISPLD2 on the inflammatory response and survival rate of LPS-induced sepsis mice under long-term use of antibiotics.Method:1.Mice were randomly divided into different cage positions and divided into control group and mixed antibiotic treatment group(penicillin,vancomycin,imipenem,metronidazole,100mg/kg/d)for 2 weeks.The fecal samples of small intestine of mice were sampled and measured by 16s rDNA sequencing.Cluster analysis,difference analysis and other bioinformatics analysis methods were used to classify and quantify the microorganisms of different samples to reveal the influence of broad-spectrum antibiotics on intestinal flora.2.Mice were exposed to the combination of antibiotics for 14 consecutive days.Realtime-PCR and Western blot were used to detect CRISPLD2 transcription and expression levels in small intestine of mice,and ELISA was used to detect CRISPLD2 content in serum of mice.The concentrations of CRISPLD2 in serum of germ-free mice were determined and compared with those in the conventional feeding group.RT-PCR was used to detect CRISPLD2 transcription in different segments of the small intestine of mice under conventional feeding conditions.3.The mice were divided into four groups:Control group,LPS-induced sepsis group,antibiotic exposure LPS-induced sepsis group,CRISPLD2-treated antibiotic exposure LPS-induced sepsis group.Serum inflammatory cytokines were detected 24h later,and survival curves of sepsis mice except control group were recorded.Result:1.Effects of long-term application of broad-spectrum antibiotics on small intestinal flora of miceA total of 670,551 optimized sequences and 407 OTU were detected in all small intestine contents samples,among which 7 OTU were in the core.Alpha diversity in the small intestine decreased,and species diversity in the intestinal tract decreased significantly,which was reflected in Shannon index and Simpson index.Changes in the structure and relative abundance of small intestinal contents in the two groups at different levels.At the phylum level,the control group was mainly composed of Bacteroidetes(65.74%),Firmicutes(23.75%),Proteobacteria(9.44%).Proteobacteria was the main pathogen in the antibiotic mixed group,accounting for 93.91%.At the level of genus classification,the unclassified species accounted for the most in the control group,followed by Turicibacter(13.6%),Parasutterella(9.18%),Faecalibaculum(4.52%)and Lactobacillus(1.94%).Enterobacter(88.25%),Rhodococcus(2.91%)and Dietzia(1.11%)were the dominant species in the mixed antibiotic group.2.Regulation effect of intestinal flora on CRISPLD2 in miceAfter long-term exposure to the above broad-spectrum antibiotics,we found that the expression of CRISPLD2 was significantly upregulated in the small intestinal,but the concentration of CRISPLD2 in the serum was decreased.Meanwhile,serum CRISPLD2 concentrations in germ-free mice were significantly lower than those in conventionally fed mice.We also compared the expression of CRISPLD2 in the small intestine of mice under conventional feeding conditions,and found that the expression of CRISPLD2 in the distal region of the small intestine(ileum)was significantly higher than that in the proximal region(duodenum).3.The effect of CRISPLD2 on LPS-induced inflammatory response and the therapeutic effect of sepsis mice under long-term antibiotic useIntravenous supplementation of CRISPLD2 protein down-regulated inflammatory cytokines(TNF-A,IL-6)in serum of septic mice exposed to antibiotics for a long time.However,there was no significant difference in the prognosis among the groups.The 9d survival rate of LPS-induced sepsis mice was 40%,that of LPS-induced sepsis mice exposed to antibiotics was 40%,and that of CRISPLD2 protein treated mice was 50%.Conclusion:1.Long-term exposure to broad-spectrum antibiotics significantly altered the structure of intestinal microbiota and significantly reduced the diversity of the microbiota.2.The expression of CRISPLD2 in mouse intestinal tissue and serum is regulated by intestinal microbiota.3.Treatment with the recombinant CRISPLD2 protein reduced inflammatory responses in mice with LPS-induced sepsis that had been exposed to broad-spectrum antibiotics for a long time,but unfortunately,treatment with this protein did not translate into survival benefits in mice with prolonged exposure to antibiotic sepsis.