| Objective: To research the effects of the intestinal flora disturbance on ALI(acute lung injury)in mice,and analyzing TLR4 / NF-κB signaling pathway,preliminarily discuss the potential mechanism of its influence on lung disease.Methods: The 6-8weeks-old of C57 BL / 6J mice were divided into three groups by random assignment:(1)NS group,(2)LPS group and(3)FMT group,adaptive feeding for 1 week with normal diet and freely drank water.Mice in the NS group were gavaged with sterile PBS solution for 8 consecutive days,followed by endotracheal intubation and intratracheal injection of 25mg/kg LPS for 4 days.At the same time,persistently gavaged with sterile PBS solution until the end of the model establishment.Mice in the LPS group were injected with four broad-spectrum antibiotics(0.10mg/g neomycin;0.10mg/g ampicillin,0.05mg/gvancomycin,0.10mg/g metronidazole)by gavage for the last eight days and 2 times a day,the rest of the treatment was same with NS group,sterile PBS solution was gavaged continuously for 4 days.The operation of mice in FMT group was similar to the LPS group,successive lavage with broad-spectrum antibiotics and intratracheal injection of LPS(lipopolysaccharide),then given the fresh fecal bacteria liquid for four days.The mice were sacrificed in 13 th day,Lung tissue and Intestinal tissue were collected in each group.W/D value of each group were measured by weighing,and the degree of pathological inflammatory response in tissues was evaluated after HE staining.IL-6(Interleukin-1),IL-1(Interleukin-1β)and TNF-α(Tumor necrosis factor-α)were measured with RT-q PCR.Meanwhile,the protein expression level of TLR4/NF-κB signaling pathway was detected by Western blot.The structure and distribution of microflora in the intestinal tract of mice were calculated by high-throughput sequencing.Results: Compared with NS group,the level of the W/D in LPS group was significantly increased(P<0.05),pulmonary edema was more sereve.He staining showed that a mass quantity of diffuse infiltration of inflammatory cells in the alveolar ducts and alveoli,increasedly haemocytes lost,obvious thickening of alveolar septum,focal atelectasis and lung consolidation,the pathological injury was serious.However,compared with the LPS group,the severity of lung injury,inflammatory cells and bleeding were reduced,the pulmonary edema was also significantly alleviated in FMT group(P<0.05).RT-q PCR result found that the gene expression of IL-1β,IL-6 and TNF-α in LPS group were significantly higher than those in NS group(P<0.05),while these inflammatory cytokines in FMT group were down-regulated after faecal bacteria transplantation.WB result indicated that the expression of TLR4 was significantly up-regulated in LPS group compared with that in NS group,the phosphorylation of NF-κB increased and the pathway was activated.Compared with the LPS group,the protein of the pathway in FMT group were decreased(P < 0.05).Intestinal HE staining revealed that the integrity of intestinal mucosa in the LPS group was destroyed,some villi were shed,and intestinal lining was edema.16 Sr DNA sequencing result showed that the diversity of bacteria in LPS group were decreased,and the dominant flora changed.Compared with the sequencing results of the FMT group,after treating with fresh fecal bacteria transplantation,it discovered that the diversity and dominant flora of intestinal microorganism had a remarkable regression.Conclusion:Intestinal flora dysregulation can aggravate the inflammatory response of lung tissue by regulating the TLR4/NF-κB signaling pathway,but healthy intestinal flora has a protective effect on LPS-induced ALI. |
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