Intestinal Microbiota Aggravate The Pathogenesis Of Mechanical Trauma And Its Mechanism

Objective:The establishment of mechanical injury model of Drosophila melanogaster and Mice,further explore the potential mechanism of intestinal microbiota in the pathogenesis of mechanical trauma.Methods:1.Drosophila melanogaster was used as the experimental subjects to establish the model of mechanical trauma with the method of free-falling.C57BL6 male mice were used as the experimental subjects,and the mechanicaltrauma model was established by free-falling method.20 mice in the control group and20 mice in the experimental group were tested for the survival rate of 24 h and 48 h after mechanical trauma respectively.2.The CR flies was used as the experimental subjects,and the motor ability was measured with the device of measuring the mechanical trauma.C57BL6 male mice were used as experimental subjects,5 mice in the controlgroup and 20 mice in the experimental group.The running speed and climbing frequency of mice in the control group,20 min after mechanical trauma and 40 min after mechanical trauma were measured,respectively.3.C57BL6 male mice were used as experimental subjects,5 mice in the control group and 20 mice in the experimental group.Colonic tissue samples of the control group,24 h and 48 h after mechanical trauma were taken respectively.Colonic tissue length and width were measured by Image J software respectively.4.CR,GF,OAC and E.coli flies were selected as subjects and divided into control group and experimental group.After mechanical trauma,the intestinal bacteria loads of flies were detected by concentration gradient dilution plate method.C57BL6 male mice were selected as experimental subjects,5 mice in the control group and 20 mice in the experimental group.Colonic tissue was sampled 24 h after mechanical trauma and 48 h after mechanical trauma.Five tissue samples in each group were frozen in liquid nitrogen,cryopreserved in dry ice,and 16S rDNA sequenced.5.CR,GF,OAC and E.coli flies were used as subjects to determine the mortality index at 24 hours after mechanical trauma.6.CR,GF,OAC and E.coli flies were selected as subjects and divided into control group and experimental group respectively.Dye permeability assay was used to detect intestinal barrier function after mechanical trauma.7.CR,GF,OAC and E.coli flies were selected as subjects and divided into control group and experimental group.The expression of Attacin-C and Diptericin B genes was detected by real-time PCR after mechanical trauma.C57BL6 male mice were selected as experimental subjects and divided into control group and experimental group.The expression of inflammatory cytokines TNF-?,lL-6,lL-10 and tight junction protein Zo-1and Occludin in colon tissues of mice detected by real-time PCR after mechanical trauma.8.The CR flies were divided into control group and experimental group after mechanical trauma.The control group was treated with 5%sucrose and the experimental group was treated with mixed antibiotics.After 24 hours of treatment,indicators such as mortality index,locomotion ability,SI₂₄,intestinal bacteria load and the expression of Attacin-C and Diptericin B were determined respectively.Results:1.The model of free-falling trauma is established,which has the advantages of strong representativeness,simple operation and high repeatability.2.The mortality of flies was increased by free-falling trauma,and the mortality index increased with the increase of the height of free-falling and the number of mechanical trauma,and the difference was statistically significant?P<0.001?.The height of mechanical trauma model with 15 m free fall was optimized.The survival rates at 24 h and 48 h after mechanical trauma were 85%and 75%respectively.3.The model of mechanical trauma reduces locomotion ability.The results showed that the running velocity and climbing velocity of CR flies decreased after mechanical injury?P<0.01,P<0.001?.C57BL6 male mice showed a decrease in running velocity and climbing frequency at the instant after mechanical trauma and at 20 and 40 min after mechanical trauma?P<0.01,P<0.01,P<0.01?.At the moment after mechanical trauma,the climbing frequency of mice was 0,and at 20 min and 40 min after mechanical trauma,and the difference was statistically significant?P<0.001,P<0.001?.4.Colonic tissue width measurement at 24 h and 48 h after mechanical trauma in C57BL6 male mice.Image J software results showed that colonic tissue width increased with the extension of time after mechanical trauma?P<0.05,P<0.001?,and there was no statistical difference in colonic tissue length?P>0.05?.5.16S rDNA sequencing showed that the abundance of Firmicutes increased within24 h after mechanical trauma?P<0.05?and decreased within 48 h after mechanical trauma,but still higher than the control group.The abundance of Bacteroides decreased within 24 h after mechanical trauma?P<0.05?,and increased within 48 h compared with24 h after trauma,but still lower than the control group.The abundance of Verruca microflora reached the highest level 24 h after mechanical trauma?P<0.05?.There was no significant change in the abundance of Actinomycetes.The abundance of Proteobacteria increased at 24 h and 48 h after mechanical trauma.PCA analysis showed that the bacterial community distribution in the control group,24 h and 48 h after mechanical trauma was significantly different,but the community distribution of samples in each group was similar.The results showed that in the top 50 species of total abundance at genus level,Firmicutes and Proteobacteria were closely related to other species.Bacteroides and Actinomycetes are less closely related to other species.Intestinal bacterial load results showed that intestinal bacterial load increased in CR,OAC and E.coli after mechanical trauma.The intestinal bacterial load of each fly in the CR group was?20.73±2.41?×10⁶ CFU?P<0.001?.The intestinal bacterial load of each fly in the OAC experimental group was?2.46±0.36?×10⁶ CFU?P<0.01?.E.coli experimental group was?24.67±0.94?×10⁶ CFU?P<0.01?.6.The mortality index of GF flies after mechanical injury was lower than that CR group,while OAC or E.coli increased the mortality index of GF flies?P<0.001,P<0.001?,indicating that intestinal microbiota aggravated the severity of mechanical injury.7.The results of dye permeability assay showed that the percentage of Smurf in CR flies was?38.00±2.86?%at 24 h after mechanical trauma?P<0.001?;Compared with the control group,the percentage of Smurf in GF,OAC and E.coli flies have a significant statistical difference?P<0.01,P<0.01,P<0.001?,indicating that mechanical trauma caused intestinal barrier dysfunction and resulting in increased intestinal barrier permeability.The SI₂₄4 percentage of CR,OAC and E.coli flies was statistically different from that of GF flies?P<0.001,P<0.01,P<0.01?,indicating that intestinal microbiota aggravated mechanical trauma-induced intestinal barrier dysfunction and increased intestinal barrier permeability.8.The results of real-time PCR showed that the expression Attacin-C and Diptericin B in GF flies were significantly higher than those in the control group after mechanical trauma?P<0.05,P<0.01?;The expression of both genes in CR,OAC and E.coli flies were increased.The expression of inflammatory cytokines TNF-?,lL-6 and lL-10 in mice colon tissues were significantly higher than those in the control group?P<0.01,P<0.05,P<0.05?,indicating that mechanical trauma caused inflammatory response of intestinal tissue.The expression of tight junction proteins ZO-1 and Occludin were significantly higher than those of the control group?P<0.001,P<0.01?,indicating that mechanical trauma caused increased intestinal barrier permeability in mice.9.The results of antibiotic treatment showed that the mortality index of CR flies decreased?P<0.001?.The velocity of running and climbing increased compared with the control group?P<0.001,P<0.05?;the amount of bacteria in the intestine of flies decreased?P<0.01?;SI₂₄4 decreased?P<0.001?,indicating that the treatment of antibiotics reduced the damage of intestinal barrier by mechanical trauma.The expression of Attacin-C and Diptericin B decreased?P<0.05,P<0.01?.These indicators show that t antibiotic intervention reduces the severity of mechanical trauma.Conclusion:In summary,we established a model of mechanical trauma in Drosophila and Mice by free-falling method.And further explore the pathogenesis and potential mechanism of intestinal microbiota aggravating mechanical injury.The results showed that mechanical trauma caused pathological changes in the organism and further caused changes in the composition and function of intestinal microbiota.Intestinal microbiota aggravate mechanical injury-induced intestinal barrier dysfunction and stimulate the innate immune response and intestinal inflammation induced by mechanical trauma.Antibiotic can alleviate the pathology of mechanical trauma.