| Background:Bronchial asthma is a common chronic inflammatory disease in the airway.There are more than 300 million asthma patients worldwide and the incidence has increased by 50%for 10 years[1,2].The pathogenesis of asthma involves a number of cell interactions.In the Th1/Th2 theory,Th1 cells mediate cellular immune responses,whereas Th2 cells mediate hormonal immune responses.The chronic inflammatory response of the airway is due to the imbalance of Th1/Th2 response[3].Acute respiratory infections can trigger asthma,and in most cases,asthma is associated with respiratory syncytial virus?RSV?infection.RSV is the main cause of lower respiratory tract infection in infants and preschool children.According to the current survey,more than 80%respiratory tract infection in children under one year old are associated with RSV infection[4,5].Early infection not only leads to chronic airway dysfunction,but also makes patients more suscesciptible to wheezing in the years after acute infection[6-9].Therefore asthma is closely related to RSV infection.For a long time,people have been concerned about the role of bacterial flora in infectious diseases.With the advent of 16srRNA sequencing,it has been shown that changes in intestinal and pulmonary microbes have an effect on the occurrence and development of asthma.Many studies have shown that the increase in the prevalence of allergic diseases is associated with changes in intestinal microbial communities[10].Microbes are essential for the maintenance and development of immune homeostasis and the induction of immune tolerance[11,12].The study of intervention strategy of intestinal microflora has become a hotspot.Probiotics are live microbes,when the ample amount is given to the host.It will introduce healthy microbes to the host[13].Clostridium butyricum is a probiotics,which is widely used in clinic,but there is no report on the effect of RSV infection on asthma.Objective:Our experiments established RSV-infected asthma mouse model.Clostridium butyricum were administered orally to RSV-infected asthmatic mice.We used FlexiVent system to evaluate the lung function of the experimental animals.Alveolar lavage fluid was analyzed under optical microscopy and white blood cells were classified and counted.enzyme-linked immunoassay Adaption test?ELISA?was used to measure the cytokines and OVA-specific immunoglobulin in serum and alveolar lavage fluid.The pathological changes of lung tissue were observed by light and transmission electron microscopy.RSV virus load was detected by fluorescence quantitative RT-PCR.The aim of this study was to investigate the effect of Clostridium butyricum on airway inflammation in asthmatic mice with RSV infection and to explore new therapies for prevention and treatment of asthma.Methods:?1?Hep-2 cells were cultured for the proliferation of RSV.?2?Virus RNA was collected and analyzed by fluorescence quantitative RT-PCR.?3?The virus titer TCID50 was determined by the virus solution identified as RSV.?4?Establishment of mouse asthmatic model:At the 0th day,the 7th day and the 14th day,the asthmatic model of RSV infection group and the group of Clostridium butyricum were injected intraperitoneally with 0.2ml solution containing 100?g OVA and 1.5mg Al?OH?3.The control group was treated with saline solution containing only Al?OH?3powder.The injection site and dose were the same as the experimental group.On the 2nd to 28th day of the experiment,Clostridium butyricum treatment group was stimulated with atomization solution containing 1%OVA solution.Two groups of mice were placed in a 5L homemade closed container,and the mice were exposed to 1%OVA aerosol once daily for 30 min.on day 29 and 30,1%OVA aerosol inhalation was given.?5?RSV virus nasal drops:except for the control group,on the 28th,30th,32th day,100ul RSV supernatant nasal drip was given to the other tow group,a total of 3 times.The mice model of RSV infection was successfully established.The control group was treated with normal saline instead of OVA atomization and nasal drip in the same manner.?6?oral Clostridium butyricum:during 0-33 days of the experiment,Clostridium butyricum solution?Clostridium butyricum concentration of 5×108 CFU/ml,that is,5grams of bacteria dissolved in 10 ml of physiological Saline mixed after dispensing,gavage when each mouse 0.2ml,-4?preservation.?gavage was given to Clostridium butyricum treatment group.The other two groups were treated with the same amount of saline instead of Clostridium butyricum solution.?7?On the 33rd day of the experiment,anesthetized mice were intubated and aerosolized with different concentrations of methacholine?0,1.5,3...100mg/ml?to measure airway hyperresponsiveness.?8?alveolar lavage fluid were collected and the white blood cells in it was analyzed.?9?The whole blood of mice was collected and serum was collected.Serum levels of cytokines and OVA specific immunoglobulin were measured.?10?Fluorescence quantitative RT-PCR was used to detect RSV viral load in mouse lung tissue.?11?The lung tissue of mice was isolated and the pathological changes of lung tissue were observed by histological method.?12?Statistical analysis.Results:?1?Hep-2 cells were successfully cultured.RSV virus within Hep-2 cell proliferated efficiently.?2?Virus RNA detection by fluorescence quantitative RT-PCR.Standard curve:slope:-3.93,Y-axis intercept:48.93,correlation coefficient:0.997,that is,equation Y=-3.93X+48.93.Results were positive,proved to be RSV;cell culture supernatant was collected for the next step.?3?In the plaque experiment,the results were analyzed by the Reed-Muench formula.RSV virus TCID50=10-4.7.That is,50%of Hep-2 cells with cytopathic RSV virus solution dilution of 10-4.7,suggesting that at least 10-4.7 dilution of the RSV suspension for the experiment,the establishment of RSV infected mouse model.?4?After RSV nasal drops,the emergence of hair erect,dull,irritability or apathetic,trembling or nodding,cough and deep breathing,rhythmic abdomen-like breathing and other positive reactions were observed,indicating the successful establishment of RSV infected asthmatic mouse model.?5?Histological analysis:HE staining of lung tissue,compared with the control group of mice,RSV infection in asthma model mice lung tissue HE staining can be seen around the bronchi and bronchioles with a large number of inflammatory cell infiltration,bronchial stenosis,Alveolar septal fracture,pulmonary bullae formation;Clostridium butyricum treatment group mice lung tissue inflammatory cells compared with the control group,the infiltration was significantly reduced,alveolar septum integrity.Masson staining showed that the collagen fibers in the airway were less in the control group,and the collagen fibers in the airway were significantly increased in the asthmatic model group.The collagen fibers in the airway of the mice treated with Clostridium butyricum were decreased.The expression of HMGB1 and TLR4 in the lung tissue of the control group was significantly lower than that in the control group?P<0.05?.The expression of HMGB1 and TLR4 in the asthmatic model group was higher than that in the control group Group weakened.?6?Airway resistance:Compared with the control group,RSV infection in mice with different concentrations of acetylcholine?basal,1.5,3,12,25,50,100mg/ml Mch?airway resistance was significantly increased;Compared with RSV-infected asthma group,Clostridium butyricum treatment group decreased the airway resistance in mice.?7?Infiltration of pulmonary inflammatory cells:Compared with control mice,the total number of white blood cells in alveolar lavage fluid of RSV-infected asthma group was significantly increased?P<0.01?,mainly in eosinophils,?P<0.01?,and the expression of eosinophils and neutrophils was significantly decreased in the alveolar lavage fluid of mice.?8?The expression of cytokines:Th1 cytokines?INF-?,IL-2,IL-12?in sera and alveolar lavage fluid of RSV-infected asthma group were significantly lower than those in the control group,and RSV-infected asthma?IL-4,IL-5,IL-13,eosinophil chemotactic factor?were significantly increased in serum and alveolar lavage fluid,and the group of Clostridium butyricum was significantly higher than that of RSV-infected asthma group The expression of Th1 cytokines was increased and the expression of Th2 cytokines was decreased in the lung serum and the lavage fluid.?9?OVA-specific immunoglobulin IgE/G1:Compared with the control group,serum IgE/G1 was significantly increased in serum of RSV-infected mice,and IgE/G1 in serum of Clostridium butyricum group was significantly decreased.?10?Fluorescence quantitative RT-PCR was used to detect RSV viral load in mouse lung tissue:The RSV viral load of lung tissue in control group was negative.The RSV viral load in the lung tissue of mice infected with RSV and the group of Clostridium butyricum was positive,and the RSV viral load in the lung tissue of Clostridium butyricum treated group was higher than that of the RSV infection model group?P<0.05?.Conclusion:Oral Clostridium butyricum can reduce the total number of inflammatory cells in BALF and serum of RSV-infected mice cells,imporove pulmonary pathological changes.It increase the secretion of Th1,IL-12,and IFN-?in mice infected with RSV to some extent,it also reduce eosinophil chemotactic factor,reduce airway inflammation in asthmatic mice.We think that Clostridium butyricum is a common probiotics that has an immune-modulating and anti-inflamation effect during the process of RSV infection.It can reduce the inflammatory response,regulate Th1/Th2 cell balance,and eventually to help the prevention and treatment of asthma. |
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