| Objective:To study the effects of Mycoplasma neumonia on the levels of inflammatory cytokines TNF-?,TGF-?,IL-1?,IL-8 and its down-regulation of inflammatory factors.To explore the mechanism of apoptosis of mouse alveolar epithelial cells and human pulmonary microvascular endothelial cells induced by Caspase-3/8 and Fas/FasL pathway and the protective effect of Qingfeitongluo Recipe on apoptosis of mouse alveolar epithelial cells and human pulmonary microvascular endothelial cells induced by Mycoplasma pneumoniae.Methods:Seventy-two 4-week-old,16±2g,SPF-grade,female Balb/c mice were randomly divided into three groups.24 rats in each group:1)Group A was the normal control group;2)Group B was the model group;3)Group C was the Qingfei Tongluo group.After anesthetizing each group of animals with 0.5%sodium pentobarbital 13 uL/g.The normal group was intranasally instilled with 50 ul of 0.9%saline.The other groups were slowly instilled into the nasal cavity with 50 ul of MP solution(containing 1×107 CFU/ml)for 2 days.Treatment begins on the first day of infection.Groups A and B were intragastrically administered with normal saline 0.25 ml/20 g;group C was administered with Qingfei Tongluo Recipe 0.25 ml/20 g once daily for 7 days.On the third and seventh days of the experiment,the mice were sacrificed in batches and taken.The lung tissue morphology,tracheal and perivascular inflammatory cell infiltration,alveolar wall thickening and destruction,airway stenosis and thrombosis were observed under light microscope.The levels of TNF-?,TGF-?,IL-1? and IL-8 in the serum of each group were detected by ELISA.The counts and classification of PMVEC and AT-? cells in BALF of each group were detected by flow cytometry.Real Time-PCR and Western Blot were used to detect the expression differences of Fas,FasL,Caspase-3,Caspase-8,upstream factor Apaf-1,cytochrome C,downstream factor PARP and protein kinase C in each group.Result:(1)The expression of MP DNA in lung tissue of each group was detected by PCR.The results showed that MP DNA expression was found in the lung tissue of mice infected by model group and Qingfei Tongluo Recipe on the 3rd day of the experiment,which indicated that the MP infection was small.The mouse model was successful;(2)The pathological observation showed that the lung tissue structure of the control mice was basically intact,no obvious lesions were found in the bronchi and alveoli,and no inflammatory cell infiltration was observed.In the model group,a large number of inflammatory cells infiltrated in the peri-bronchial and interstitial components of the lung tissue,and the alveolar wall was capillary.The vasodilatation is filled,the alveolar wall is thickened and destroyed,the alveolar space is thickened,the bronchiole wall is thickened,and the bronchial lumen is narrowed.In the lung tissue of the Qingfei Tongluo Recipe group,a small amount of inflammatory cell infiltration is observed in the peribronchial and interstitial cells.The alveolar wall capillaries are slightly dilated and filled,a small part of the alveolar wall is thickened,the bronchial epithelial edema is reduced,and the bronchial lumen is narrowed;(3)The levels of TNF-?,TGF-?,IL-1? and IL-8 in the serum of each group were detected by ELISA.Compared with the control group,the serum levels of the factors in the model group were higher than those in the control group,P<0.01.The difference was statistically significant.Compared with the model group,the serum levels of the factors in the treatment group were lower than those in the model group,P<0.01,the difference was statistically significant.The QTF intervention was compared with the 7-day test results.The levels of each factor in the serum of the mice in each group were lower than those in the intervention for 3 days,P<0.05,and the difference was statistically significant;(4)The counts and classification of PMVEC and AT-II cells in BALF of each group were detected by flow cytometry.Compared with the control group,the cell count of BALF in the model group was higher than that of the control group,P<0.01,the difference was statistically significant.Compared with the model group,the cell count of BALF in the treatment group was lower than that in the model group,P<0.01,the difference was statistically significant.Compared with the 7-day test results of QTF intervention,the BALF of each group of mice was intervened for 7 days.The cell counts were lower than the intervention for 3 days,P<0.05,and the difference was statistically significant;(5)The expression levels of apoptosis-related proteins Fas,FasL,Caspase-3,Caspase-8,upstream factor Apaf-1,cytochrome C and downstream factor PARP and protein kinase C were detected by Western-Blot method.Compared with the control group,the model group was small.The expression of each protein in the lung tissue of rats was significantly increased,P<0.01,the difference was statistically significant.Compared with the model group,the expression of each protein in the lung tissue of the treatment group was significantly decreased,P<0.01,the difference was statistically significant.Compared with the 7-day test results of QTF intervention,the expression of each protein in the lung tissue of each group was lower than that of the intervention for 3 days,P<0.05,the difference was statistically significant;(6)The expression levels of apoptosis-related proteins Fas,FasL,Caspase-3,Caspase-8,upstream factor Apaf-1,cytochrome C,downstream factor PARP and protein kinase C mRNA were detected by RT-PCR.Compared with the control group,the model group was small.The expression of each protein gene in the tissue increased significantly,P<0.01,the difference was statistically significant.Compared with the model group,the expression of each protein gene in the lung tissue of the treatment group was significantly decreased,P<0.01,the difference was statistically significant.Compared with the 7-day test results of QTF intervention,the expression of each protein gene in the lung tissue of each group was lower than that of the intervention for 3 days.P<0.05,the difference was statistically significant.Conclusion:(1)MP can up-regulate the expression of inflammatory factors(TNF-?,TGF-?,IL1?,IL-8)in mouse serum,and QTF can alleviate the level of inflammation caused by MP infection;(2)MP can induce apoptosis of mouse alveolar epithelial cells and pulmonary microvascular endothelial cells through Caspase-3/8 and Fas/FasL pathway,and QTF can inhibit apoptosis induced by MP infection;(3)After 7 days of QTF treatment,the effects of down-regulation of inflammation and inhibition of apoptosis were higher than those of the 3-day treatment group. |
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