Causal Analysis Of Children's Pneumonia With Airway Plugging Caused By Mycoplasma Pneumoniae And Effect And Mechanism Of Bovine Lactoferrin On Acute Lung Injury Induced By MALP-2

Mycoplasma pneumoniae pneumonia (MPP) is one of the most common pneumonia of community-acquired pneumonia (CAP)in children.It can be happened scattered or irregular epidemic.It has been reported that it's periodic epidemic is about every 3-7 years.MPP accounts for 40% or more of children's CAP during the epidemic period.Clinical characteristics are varied from mild to severe.The mild ones only have symptoms such as sore throat,low heat or cough,etc.And the severe ones can be life-threatening.In recent years,there are more and more reports about the refractory mycoplasma pneumoniae pneumonia (RMPP) in children,but the mechanism remains obscure.So far,it is generally believed that it is related to the direct invasion of Mycoplasma pneumoniae (MP) and strongly inflammatory responses of the body.The structure of Mycoplasma species are different from that of bacteria.They are devoid of a cell wall,so they do not contain immunostimulants such as lipopolysaccharide,peptidoglycan or lipoteichoic acid.It is now widely considered that the lipoproteins may play an important role in the induction of inflammatory responses and the development of pneumonia in MP infections.Macrophage activated lipopeptide-2 (MALP-2) is the main component of mycoplasma's lipoproteins.It mainly binds to toll like receptor 2/6 on macrophage cell membrane,and can activate the pathways of multiple protein kinase and induce the release of inflammatory factors such as cytokines,chemotactic factors.Therefore,the study of MALP-2 is conducive to acquaint and prevent the harm of MP.Due to strongly inflammatory responses caused by MP,systemic corticosteroid such as Methylprednisolone are often used in the treatment of refractory MPP to improve the clinical symptoms in children.However,it may affect the normal immune function and increase the risk of infection by other pathogens.Consequently,it is an challenge for clinicians to find a natural substance to replace or reduce the application of corticosteroid.Lactoferrin is an iron-binding glycoprotein with a relative molecular weight of 80 kDa,which exists in a variety of mammals and contains about 700 aminoacids,with high homology among species.Some studies on the activity of lactoferrin show that it has the function of anti-bacterial and anti-inflammatory,and can be used alone or as an adjuvant of conventional antibiotics.Bovine lactoferrin(BLF) is easier to extract from milk,so it possess a high research value.The study is divided into three parts.Part one and part two are the clinical parts,the clinical characteristics of MPP in children with airway plugging were summarized,correlation analysis was performed between MP-DNA load in bronchoalveolar lavage fluid(BALF) and the main influential factors.The levels of cytokines in BALF and blood were analyzed between airway plugging and non-airway plugging patients.The third part is animal experiment,we made a model of lung injury in mice by MALP-2,and observe the protective function of bovine lactoferrin on it and explore its possible mechanism.Part One Clinical characteristics of Mycoplasma pneumonia pneumonia in children with airway plugging and its correlation with MP-DNA loadObjective:This part was performed to acquaint the clinical characteristics of Mycoplasma pneumoniae pneumonia(MPP) with airway plugging in children and analyze the cause of it,so as to make early diagnosis and timely accurate treatment of airway obstruction,shorten the course of disease and reduce sequelae.Methods:A total of 219 children with MPP treated with fiberoptic bronchoscope were enrolled from October,2015 to January,2017 in this retrospective study.According to the imaging of bronchoscopy,the patients were divided into airway plugging(AP) group and non-airway plugging(non-AP) group.The data of general information,clinical characteristics,laboratory examination and the load of MP-DNA in BALF were analyzed.Logistic regression analysis was used to observe the effect of main laboratory indicators on airway plugging.Spearman rank correlation analysis was used to analyze the correlation between MP-DNA load in BALF and major indicators such as lactate dehydrogenase(LDH),alanine transaminase(ALT),C-reactive protein(CRP) and neutrophil cells percentage(N%).With MP-DNA load as the test variable and airway plugging as the state variable,the ROC curve was obtained and its predictive value was evaluated.Results:1.73 MPP patients with AP and 146 without AP were included in thisstudy.Compared with the non-AP group,the AP patients had a longer duration of fever(P=0.005) and hospital stay (P=0.003).They had higher incidence rate of refractory MPP (P=0.000),temperature?40? (P=0.000),and pleural effusion (P=0.003).Girls are more common (P=0.035) in AP group.Laboratory index such as MP-DNA load in BALF(P=0.000),LDH (P=0.000),CRP(P=0.042),N% in peripheral blood (P=0.007) and ALT levels (P=0.000)were significantly increased in AP group.2.Logistic regression analysis showed that MP-DNA load in BALF (95%confidence interval:1.439-3.218,OR value:2.152,P=0.000) and LDH (95%confidence interval:1.003-1.007,OR value:1.005,P=0.000) were independent risk factors for airway plugging.Spearman rank correlation test showed that MP-DNA load in BALF is related to LDH (correlation coefficient 0.206;P=0.002),ALT (correlation coefficient 0.238;P=0.000),CRP(correlation coefficient 0.195;P=0.004),total fever days (correlation coefficient 0.246;P=0.000),length of stay (correlation coefficient 0.272;P=0.000).The area under the ROC curve for MP-DNA load to predict airway plugging was 0.736 (95% Cl:0.667-0.806,P=0.000).The best cut-off value was 107 of MP-DNA loads (sensitivity=69.9%,specificity=70.5%).3.Among the children with airway plugging,26 cases with bronchial casts and 47 cases with mucus plugs were included.The location of the blockage was at the segmental bronchus below.Compared with the non-AP group,more patients received methylprednisolone during the treatment in AP group(87.8% vs 49.3%,P=0.000).44 cases not only treated with bronchoalveolar lavage,but combined with cell brush or foreign body forceps.3 cases with mucosal erosion have few hemorrhages.There were no significant difference in the indicators such as duration of fever,CRP,N% in peripheral blood,MP-DNA load and ALT levels between the cases with bronchial casts and those with mucus plugs (P>0.05).Summary:1.Compared with non-AP group,cases in AP group had a longer duration of fever and hospital stay,higher levels of inflammation index such as LDH,CRP,N%,ALT.And they were easier to form high fever,pleural effusion and refractory MPP.more patients received the treatment of corticosteroid in AP-group.2.Airway plugging caused by MPP in children can be showed as mucus plugs or bronchial casts,and the location of the blockage was at the segmental bronchus below.It can be cleaned by bronchoalveolar lavage combined with cell brush or foreign body forceps,without serious complications.3.The level of MP-DNA load in BALF is remarkably higher in AP group than non-AP group,which was related to the duration of fever,hospital stay and the levels of inflammation indicators such as LDH,CRP and ALT.MP-DNA load in BALF possess some predictive value for airway plugging.The cause of airway plugging may be related to the local MP load.Part Two Analysis of inflammatory cytokines and immunity related indexes in children with airway plugging caused by Mycoplasma pneumoniae pneumoniaObjective:We aimed to observe the expression of related inflammatory cytokines of children with airway plugging(AP) and non-airway plugging(non-AP) caused by MPP in blood and BALF,and to analyze the immunity related factors between the two groups,so as to explore the pathogenesis and seek new ideas for the treatment of airway plugging caused by MPP.Mehtods:56 cases of MPP with pulmonary consolidation and atelectasis treated by bronchoscopy were included from March,2018 to November,2018.The patients were divided into AP group and non-AP group.Blood samples were collected within 24 hours after admission for immunoglobulin and lymphocyte subsets analysis.Blood and BALF were collected during the day of bronchoscopy treatment,which was used to detect Interleukin-2(IL-2),Interleukin-4(IL-4),Interleukin-6(IL-6),Interleukin-10(IL-10),Interferon-?(IFN-y) and Tumour Necrosis Factor-alpha(TNF-?),respectively.The statistical analysis of the immunity related indexes and these cytokines in blood and BALF were carried out between the two groups.Results:1.Twenty-five MPP patients with AP and thirty-one without AP were enrolled in this study.In AP group,15 males and 10 females were included,with an average age of 6.2±1.9 years and a course of 7.8±2.3 days before admission;In non-AP group,16 males and 15 females were included,with an average age of 5.7±2.3 years and a course of 7.9±2.3 days before admission.There were no significant difference in age,gender and course before admission between the two groups (P>0.05).2.Compared with the non-AP group,the levels of IL-6,TNF-? and IFN-?in BALF of children in AP group were significantly increased.(P<0.01),and the ratio of IFN-y/IL-4 was also significantly increased.(P<0.01).There were no significant difference in the concentrations of IL-2,IL-4,IL-6,IL-10,TNF-? and IFN-y in blood between the two groups(P> 0.05).3.There were no significant differences in immunity-related indicators such as CD3+,CD4+,CD8+,CD4/CD8,CD19+,CD56+,IgG,IgM and IgA between the two groups (P>0.05).Summary:1.Airway plugging caused by MPP in children may be related to the increased cytokines such as IL-6,TNF-? and IFN-y in BALF,and may not be related to the proportion of lymphocyte and IgG,IgM,IgA in peripheral blood.2.Compared with the non-AP group,the ratio of IFN-y/IL-4 in BALF of AP group was significantly increased,which suggested that local immune imbalance of Th1/Th2 was more severer in AP group.Part Three Effect and possible mechanism of bovine lactoferrin on acute lung injury induced by MALP-2 in miceObjective:To explore the protective function of lactoferrin on acute lung injury induced by MALP-2 and it's possible mechanism,so as to provide a possible drug for clinical treatment of MPP.Methods:1.Group-division of mice and interventions:36 BALB/c mice were randomly divided into 4 groups (n=9):normal saline group (NS group),MALP-2 model group (M group),bovine lactoferrin 1 group (BLF1 group),bovine lactoferrin 2 group (BLF2 group).Each BALB/c mouse in the M group was given a dose of MALP-2 1?g that diluted with 30?l NS through the trachea,and the mice were placed vertically to prevent coughing.0.1ml NS was injected intraperitoneally 0.5 hours later,once every 24 hours,three times in total.In the NS group,30?l NS was given to each mouse instead of MALP-2 through the trachea and 0.1ml NS was injected intraperitoneally in the same way as group M.In BLF1 group,each BALB/c mouse was given MALP-2 just as group M,and each mouse was intraperitoneally injected with bovine lactoferrin (100mg/kg,dissolved with 0.9% NaCl) in the same way as group M.In BLF2 group,Each BALB/c mouse was also given MALP-2 through the trachea and then each mouse was intraperitoneally injected with bovine lactoferrin (200mg/kg dissolved with 0.9% NaCl) in the same way as group M.2.Measurement indexes:the pathological changes of lung tissue were observed;the concentrations of IL-6,IL-4 and IFN-? in BALF of mice in each group were measured by enzyme-linked immunosorbent assay(ELISA);the expression of TLR2 and NF-?Bp65 in lung tissue of each group was detected by Western blot (WB).3.Statistical analysis:Data are expressed as the mean±standard deviation(SD).Differences among groups were evaluated by one-way analysis of variance (ANOVA).A value of P<0.05 was considered statistically significant.The data analyzed using SPSS software version 17.0.Results:1.Observation of lung histopathology:In NS group,the alveolar wall structure was complete,with occasional inflammatory cells infiltrating around the bronchi,blood vessels and the alveolar septum;no inflammatory exudation was found in the alveolar cavity.In M group,the damage of lung tissue was obvious,it showed partial fusion of the alveolar structure,edema of the alveolar wall,thickening of the pulmonary interstitium,and exudation of the alveolar cavity.Numerous inflammatory cells infiltrated.compared with group M,the damage of lung tissue in BLF group alleviated significantly It showed that the alveolar structure was relatively complete,the exudate in interstitial and alveolar cavity alleviated;the inflammatory cell was significantly reduced.compared with BLF2 group,the lung damage in BLF1 group were more severer.2.Measurement of cytokines in BALF:The levels of IL-6 and IFN-y in BALF of mice with lung injury induced by MALP-2 was significantly different among the groups.Compared with NS group,the levels of IL-6 and IFN-y in M group were significantly increased (431.17 ± 110.39 vs 64.65 ±4.80,P=0.000;1336.75±338.90 vs 162.25±41.14,P=0.000).Compared with M group,the levels of IL-6 and IFN-y in BLF2 group (74.03 ± 25.04 vs 431.17 ±110.39,P=0.000;142.42±20.05 vs 1336.75±338.90,P=0.000) and BLF1 group (315.0±101.39 vs 431.17 ± 110.39,P=0.016;254.53±35.29 vs±336.75±338.90,P=0.000) were significantly decreased.There were no significant difference in the levels of IL-4 in BALF among the groups (P> 0.05).3.Expression of TLR2 and NF-?B p65:Western blotting was used to detect the levels of NF-?B p65 and TLR2 in lung tissue of mice with lung injury induced by MALP-2.Compared with group M,the gray values of TLR2/?-actin was significantly decreased in BLF2 group (0.19 ± 0.05 vs 0.88± 0.16,P=0.000) and BLF1 group (0.64 ± 0.06 vs 0.88 ± 0.16,P=0.01).Compared with group M,the gray values of NF-?B p65/?-actin was significantly decreased in BLF2 group (0.87 ± 0.21 vs 0.36 ± 0.24 ?=0.007),and BLF1 group (0.87 ± 0.21 vs 0.62 ± 0.15,P=0.114) was decreased,but the difference was not statistically significant.Compared with BLF1 group,the level of NF-?B p65/?-actin in BLF2 group (0.36±0.24 vs 0.62±0.15,P=0.110)was decreased,but the difference was not statistically significant too.Summary:Bovine lactoferrin can alleviate the lung injury of mice induced by MALP-2 and decrease the levels of IL-6 and IFN-y in BALF.The mechanism may be relate to that it can inhibit the inflammatory pathway of TLR2/NF-?B.Conclusions:1.Airway plugging caused by MPP in children can be showed as mucus plugs or bronchial casts,and the location of the blockage is at segmental bronchus below.And that can be cleaned by bronchoalveolar lavage combined with cell brush or foreign body forceps,without serious complications.Patients in AP-group were easier to form refractory MPP.More girls can be seen,more patients received the treatment of corticosteroid and the levels of inflammatory indexes increased in AP-group.The formation of airway plugging may be related to the local MP load.2.Compared with non-AP group,the levels of IL-6,TNF-?,IFN-? and the ratio of IFN-y/IL-4 in BALF increased significantly in AP group.3.Bovine lactoferrin can alleviate the lung injury of mice induced by MALP-2 and decrease the levels of IL-6 and IFN-? in BALF.The mechanism may be related to that it can inhibit the inflammatory pathway of TLR2/NF-?B.