| Objective:to investigate the mechanism of the disease In order to improve the pulmonary fibrosis of Mycoplasma pneumoniae,qinbaiqingfei concentrated pill was used as the research background.The active monomers in qinbaiqingfei concentrated pill were determined by fishing technology.The monomers were used to act on pulmonary fibrosis factors,and the mechanism of qinbaiqingfei concentrated pill inhibiting pulmonary fibrosis of Mycoplasma pneumoniae was elucidated,to fill the shortage of clinical drugs in the treatment of mycoplasma pneumonia.Method:qinbaiqingfei concentrated pill solution was fished by Biacore,enriched,regenerated,and recovered with TGF-β1.The active monomer was identified as baicalein by ultra performance liquid chromatography-mass spectrometry(uplc-q-tof-ms).The Affinity Kinetics of baicalein was analyzed by using Biacore,and its pharmacological mechanism was elucidated by pharmacodynamics in vivo.Forty SPF BALB/c mice weighing 20-22g were randomly divided into 4 groups:blank group,model group(mycoplasma infection group),baicalein group and azithromycin group(positive control group),with 10 mice in each group;It staining was used to observe the pathological changes of lung tissue after Mycoplasma pneumoniae infection;Masson collagen staining was used to observe the pathological changes of pulmonary fibrosis after Mycoplasma pneumoniae infection;Flow cytometry was used to observe the effect of baicalein on apoptosis of lung tissue after Mycoplasma pneumoniae infecti on;RT-PCR,Western blot and immunohi stochemi stry were used to detect pulmonary fibrosis factors.The mRNA and protein expression levels of TGF-β1,MMP2,TIMP2 were changed.Result:the purity of TGF-β1 was verified by gel electrophoresis.The purity of the protein is qualified and can be used for subsequent experiments;In Biacore fishing experiment,qinbaiqingfei concentrated pill solution was mixed with TGF-β1.By using uplc-q-tof-ms and comparing with the previously established database of qinbaiqingfei concentrated pills and baicalein standard,the content of TGF-β1 in the recovered samples of qinbaiqingfei concentrated pills could be determined.Baicalein was the product of protein binding;The affinity of baicalein with TGF-βi was tested,the binding peak of baicalein with TGF-β1 was 161.ORU.The combination of baicalein and TGF-β1 was confirmed.The results of affinity analysis showed that different concentrations of baicalein could interact with TGF-β1.The results of two experiments with baicalein concentration of 116.5nm also showed that the experiment had good repeatability.Under the above concentration gradient,the signal response intensity of baicalein solution and TGF-β1 was fitted.The relationship between baicalein and TGF-β1 was confirmed.The affinity constant(KD)was 1.218 × 10-5.These results indicate that baicalein interacts with TGF-β1 in vitro.Baicalein is a kind of transforming growth factor.The effective binding component of protein;It staining showed that in the model group,the lung tissue was the most serious,most of the alveoli collapsed,a large area of alveolar wall congestion and inflammatory cell infiltration,bronchial epithelial shedding;In the blank group,the pulmonary alveoli were normal,and there was no congestion and inflammatory cell infiltration in the alveolar wall.In the baicalein group,there was a small amount of congestion and inflammatory cell infiltration in the alveolar wall.Masson collagen staining results showed that the lung tissue of the blank group was arranged regularly with few inflammatory cells infiltration,and the deposition of filamentous fibers was almost invi sible.The lung tissue of the model group had larger blue area(collagen fiber cells)with filamentous and dark color,disordered morphology and structural arrangement,wide alveolar septum and a large number of inflammatory cells infiltration,compared with the model group,the color of baicalein treatment group was lighter,the area of blue collagen fiber cells was significantly reduced,the lung tissue morphology was more regular,and a small amount of inflammatory cells infiltrated;The results of flow cytometry showed that the apoptosis of blank group was the least;Compared with the model group,the apoptosis rate of baicalein group was significantly reduced,and the difference was statistically significant(#P<0.05);Through the above pharmacodynamic experiments in vivo,MPP model is more successful,and its lung tissue meets the requirements of subsequent experiments;RT-PCR analysis showed that compared with the model group,baicalein group could reduce the mRNA expressions of TGF-β1,Mmp2,Timp2.There were significantly different among groups(#P<0.05);The results of We stern blot showed that baicalein could reduce in protein expression of TGF-β1,Mmp2,Timp2.There were significant differences among groups(#P<0.05);Immunohi stochemical results showed that the tissue Timp2 staining in each group was ob served under 200 times microscope.Compared with the model group,the pulmonary fibrosis in baicalein group was significantly reduced,and the difference between each group was statistically significant(#P<0.05).Conclusion:Baicalein is an effective active monomer of qinbaiqingfei concentrated pills;Baicalein can interact with TGF-β1,reduce the expression of Mmp2,Timp2,inhibit the process of pulmonary fibrosis,play the advantage of treatment of MPP. |
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