| Objective:Legionella pneumonia is one of the three common severe pneumonias in community environment.The incidence rate is 2-9%,and the mortality rate is 10%.Serum type 1 Legionella pneumophila(Lp)is the most virulent to humans,accounting for 84%of Legionella infection.Immunosuppressive status is an important risk factor for Legionella infection,and immunosuppressed hosts are more susceptible to Lp than normal immune hosts and easily develop severe pneumonia with a mortality rate of20-70%.The characteristic pathophysiological changes of severe pneumonia are increased pulmonary capillary permeability and pulmonary edema.At present,immunosuppressed host infection Lp is mostly reported in clinical cases,and there is a lack of comparison with the severity of Lp disease to normal immune host infection.At the same time,patients with Legionella pneumonia are prone to multiple organ system damage,such as abnormal liver function,diarrhea,etc,and there is no relevant basic research.This study aims at establishing a model of immunosuppressed guinea pig combined with Lp infection to study the changes of pulmonary capillary permeability,intestinal flora imbalance,translocation and liver injury.Methods:This study is divided into two parts.The first part studies the mechanism of pulmonary capillary permeability changes and the role of mean pulmonary artery in the formation of pulmonary edema in guinea pigs with immunosuppression combined with Lp infection.The experimental animals were divided into four groups:control group,Lp-infected group,immunosuppressed group and immunosuppressed Lp-infected group.There were 3 time points in the experiment:24h,48h and 72h after Lp infection.Immunosuppressed guinea pigs were injected triamcinolone acetonide and cyclophosphamide,then the main airway was inoculated with Lp to obtain immunosuppressed Lp-infected guinea pigs.The immunosuppressed model was successfully established by detecting peripheral blood leukocytes,lymphocytes,neutrophils,CD4~+T cells and CD8~+T cells.By measuring the lung weight of each group at 24h,48h and 72h after Lp infection,the degree of pulmonary edema in vivo was confirmed.The changes of isolated lung weight in each group at 24h,48h and 72h after Lp infection were observed by the method of isolated lung perfusion.The difference of pulmonary capillary permeability changes was confirmed.Finally,the mechanism of the difference in pulmonary capillary permeability changes was clarified by HE,immunohistochemistry,immunofluorescence and transmission electron microscopy.The second part studies the imbalance of intestinal flora,translocation and liver injury in immunosuppressed Lp-infected guinea pigs.The grouping of experimental animals and immunosuppressed model were the same as the first part.The bacteria 16S rDNA of the intestine contents of each group were sequenced and analyzed to confirm the intestinal flora imbalance.The bacteria 16S rDNA of lung,liver and blood was sequenced and analyzed to identify the existence and timing of intestinal bacterial translocation.The presence of intestinal bacterial translocation was further confirmed by lung,liver Gram staining and bacterial fluorescence in situ hybridization.Through the detection of programmed cell death-related proteins in liver cells,the mechanisms of liver damage by translocation bacteria were investigated.Results:The number of peripheral blood leukocytes,neutrophils,lymphocytes,and CD8~+T cells were significantly decreased in immunosuppressed group.Pulmonary capillary permeability changes of immunosuppressed Lp-infected guinea pig.After Lp infection,the lung weight of guinea pigs in Lp-infected group and immunosuppressed Lp-infected group increased continuously.The lung weights of the two groups were similar at 24h and 48h,and the lung weight of former increased more significantly at 72h.In Lp-infected group,the weight of the isolated lung gradually increased with the perfusion time,and the increase was most obvious at 48h.In immunosuppressed Lp-infected group,the weight of the isolated lung gradually decreased with the perfusion time at 24h,partially decreased and increased with the perfusion time at 48h,gradually increased the most significant at 72h.In Lp-infected and immunosuppressed Lp-infected group,the VEGFA,VEGFR2 and VE-cadherin were lower than that in control group within the lung tissue.Transmission electron microscopy(TEM)showed that in Lp-infected group the endothelial cells were swollen,and the cell junction was partially open at 24h,the cell junction was completely open at 48h,the capillary endothelial cells shrunk,the cell connections were blurred and open at 72h.In immunosuppressed Lp-infected group alveolar epithelial cells and capillary endothelialcells were swollen,the basement membrane was blurred,the cell junction density was reduced,partially open at 24h,capillary endothelial cells were swollen,the number of cell connections was reduced,the density of cell connections was intermittently reduced,and part of the cells connections were open,Lp was observed at48h,the ultrastructural destruction of lung tissue was obvious,the alveolar epithelial cells were swollen,the membrane was incomplete,the capillary endothelial cells were swollen,the membrane was incomplete,the nuclei was swollen,the nucleus was gathered,and no clear cell connections were observed at 72h.HE staining of lung tissue in Lp-infected group showed that the injury gradually worsened,the structure of lung tissue was disordered,the structure of alveolar and alveolar wall was unclear,and a large number of inflammatory cells mainly neutrophils exuded at 72h.The pathological changes of lung tissue in immunosuppressed Lp-infected group were constantly aggravated.A large-scale lung tissue disorder was observed,a large number of alveolar spaces were filled with inflammatory exudates,and the alveolar wall was extensively thickened and reduced in number at 72h.Study on imbalance and translocation of intestinal flora in guinea pigs with immunosuppressed Lp-infected.In control and immunosuppressed group,no bacterial16S rDNA was extracted from the lung,liver and blood.In control group 14 cases,immunosuppressed group 15 cases were successfully extracted 16S rDNA from the small intestine contents.In Lp-infected group 9 cases of lung,3 cases of liver,6 cases of blood,and 14 cases of small intestine contents were successfully extracted bacteria 16S rDNA.In immunosuppresed Lp-infected group 9 cases of lung,2 cases of liver,1 case of blood,16 cases of small intestine contents were successfully extracted bacteria 16S rDNA.Order-level the dominant bacteria in the small intestine of the control group and the immunosuppressed group was Bacteroidales.Order-level the dominant bacteria in the small intestine of the Lp-infected group was Bacteroidales at 24h,Bacillales at 48h,and Enterobacteriales at 72h.Order-level the dominant bacteria of the immunosuppressed Lp-infected group was Bacteroidales in the small intestine at 24h and 48h,and Enterobacteriales at 72h.The dominant bacteria in blood,liver and lung(2 cases in 24h and 48h in Lp-infected group and 2 cases in 24h in immunosuppressed Lp-infected group)was Anoxybacillus kestanbolensis 43.57±2.95%,Geobacillus vulcani 25.72±2.07%and Klebsiella oxytoca 16.00±1.99%,while in the remaining lung Lp was the absolute dominant bacteria.The gram staining revealed positive bacteria and bacterial probe fluorescence in situ hybridization(BP-FISH)revealed positive expression of the above bacteria in lung and liver of Lp-infected group and immunosuppressed Lp-infected group.Liver injury caused by intestinal translocation bacteria in immunosuppressed Lp-infected group.In control group and immunosuppressed group,there were sporadic TUNEL positive cells in liver.A large number of TUNEL-positive liver cells were observed in Lp-infected group and immunosuppressed Lp-infected group,and most of the TUNEL-positive cells were mainly distributed under the liver capsule.The expression of C Caspase-3 and C Caspase-8 in Lp-infected group was significantly higher than that in the control group,and the expression was highest at 48h,and decreased significantly at72h.The expression of C Caspase-3 and C Caspase-8 in immunosuppressed Lp-infected group increased gradually.The expression of p-RIP-3 was transiently increased in Lp-infected group,while the expression of p-MLKL was continuously increased.The expression of p-RIP-3 was increased in immunosuppressed Lp-infected group,while the expression of p-MLKLwas the highest at 24h,which decreased at 48h and increased significantly at 72h.Conclusion:1.The injury of pulmonary capillary endothelial cells and the permeability of pulmonary capillaries in immunosuppressed Lp-infected guinea pigs were more serious than those in Lp-infected guinea pigs and the mean pulmonary artery pressure plays an important role in the process of pulmonary edema in immunosuppressed Lp-infected guinea pig.2.The degree of increased pulmonary capillary permeability,rather than the degree of pulmonary edema,reflects the severity of immunosuppressed Lp-infected guinea pigs.3.Intestinal flora imbalance and intestinal bacteria translocation occurred in Lp-infected and immunosuppressed Lp-infected group,and intestinal bacteria translocation induced apoptosis and necroptosis of lver cells. |
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