| ObjectiveCompare the effects of tetrandrine on pulmonary fibrosis in SD rats induced by one-time tracheal instillation of bleomycin under two different methods of atomization and injection.And explore the potential mechanism in vivo and vitro,which is to provide a reference for the safe and rational use of pulmonary fibrosis in clinical treatment and a basis for drug developmentMethod1 Pharmacodynamics of tetrandrine on bleomycin-induced pulmonary fibrosis in ratsOne-time tracheal instillation of bleomycin(5 mg·kg-1)was used to model SD rats.The rats were sacrificed on the 14th and 28th day by continuous atomization with tetrandrine.The general observation of lung morphology was performed dynamically,the lung coefficient was calculated,the lung function related indicators of each group of rats were recorded with a pulmonary function tester,the left lung was used for HE and MASSON staining for pathological observation,and the right lung was observed for HYP changes in the lung tissue by the ELISA method.2 Study on the mechanism of tetrandrine on bleomycin-induced pulmonary fibrosis in ratsThe ELISA method was used to detect Col-I and FN content in the lung tissue of each group of rats,and the Western Blot method was used to determine the contents of?-SMA,Vimentin and,and TGF-?1 in the lung tissue of each group of rats3 Effect of TGF-?1 stimulation on extracellular matrix in A549,MRC-5 and PMVEC cellsThe CCK-8 method was used to screen the safe concentration of TGF-?1 on three kinds of cells.After 24 hours continuous stimulation.ELISA method was used to detect the changes of Col-I and FN contents in the cell supernatant,and Western Blot method was used to determine the changes of ?-SMA and Vimentin contents in the cells.4 Study on the mechanism of tetrandrine in treating pulmonary fibrosis In vitroAccording to the data obtained in the third part,MRC-5 cells were stimulated with TGF-?1 at an appropriate concentration and tetrandrine at different concentrations.CCK-8 method was used to screen the safe concentration and time of administration.The ELISA method was used to detect the changes of Col-I and FN contents in the cell supernatant,and Western Blot method was used to determine the changes of ?-SMA and Vimentin contents in the cells.Flow cytometry was used to observe the effect on apoptosis.Result1 Tetrandrine has certain therapeutic effect on bleomycin-induced pulmonary fibrosis in ratsIntratracheal infusion of bleomycin can cause pulmonary fibrosis in rats.Comparing the model group with the sham operation group,at 14 days:the lung coefficient increased significantly to 11.14%(P<0.01),the respiratory frequency was was 96.81,and the performance was apparently accelerated(P<0.05),the airway resistance was slightly increased,but no statistical difference,and the tidal volume and dynamic lung compliance decreased to 0.82 mL and 0.13 mL·(cm H2O)-1 respectively(P<0.01),the content of HYP increased apparently to 0.154%(P<0.05).Pathology was visible that the alveolar septum was apparently thickened,inflammatory cells were apparently infiltrated,and fibrous tissue was apparently proliferated.And at 28 days:the lung coefficient was still significantly higher than the sham operation group,which was 9.09%(P<0.01),but slightly lower than the 14-day group,and all lung function indicators showed statistical differences,and tidal volume,airway resistance,and dynamic lung compliance were significantly different(P<0.01).The contents of HYP increased further,and it was significantly higher than that in the sham operation group(P<0.01),which was only 0.062%,and the pathology was visible that severe thickening of the alveolar septum,large infiltration of inflammatory cells,and severe hyperplasia of fibrous tissueCompared with the model group,continuous atomization of tetrandrine for 14 days can apparently reduce the pulmonary coefficient of rats to 7.79%(P<0.05)and apparently increase dynamic lung compliance to 0.25 mL·(cm H2O)-1(P<0.05).and other pulmonary functions such as tidal volume,respiratory frequency and airway resistance were also slightly improved,and HYP content was slightly reduced,and there was no statistical difference.Pathologically,slight inflammatory cell infiltration and slight fibrous tissue hyperplasia were observed.Continuous injection of tetrandrine for 14 days could apparently reduce pulmonary coefficient to 7.44%(P<0.05).Various pulmonary function indexes were slightly improved,and HYP content was slightly reduced,and there was no statistical difference.Pathologically,mild inflammatory cell infiltration and mild fibrous tissue hyperplasia were seen.After continuous atomization of tetrandrine for 28 days,the pulmonary coefficient of the rats did not increase further,which was apparently different from that of the model group(P<0.05).The respiratory rate of the rats was slightly reduced,lung compliance and tidal volume was slightly increased,and there was no statistical difference,and the airway resistance decreased apparently to 0.88 cm H2O·(s·mL)-1(P<0.05),and the content of HYP was 0.089%,and its expression was apparently inhibited(P<0.05),and seen pathologically,slight infiltration of inflammatory cells and slight fibrous tissue hyperplasia was shown Continuous injection of tetrandrine for 28 days,the pulmonary coefficient of the rats increased slightly,but it was still lower than the model group,and there was no statistical difference between the two groups.The airway resistance decreased apparently to 0.89 cm H2O·(s·mL)-1(P<0.05).The respiratory rate,tidal volume,and dynamic lung compliance were slightly different,but there was no statistical difference Pathological findings showed moderate inflammatory cell infiltration and moderate fibrous tissue hyperplasia.Comparison between different administration methods:the injection group was administered at a dose of 30 mg·kg-1,and the effective dose of the atomization group was 0.22 mg·kg-1,which was only 1/136 of the injection group.Compared with the atomization group:the rats in the injection group had slightly ower body weight,the pulmonary coefficients,the HYP content in the lung tissue,and the lung function of the animals were close to atomization group,and there was no statistical difference between the above indicators.But some animals in the injection group,in the later period of the experiment,had adverse reactions such as abdominal swelling,severe intestinal effusion,severe constipation,and unstable standing.And in the 28-day experiment of HE section,infiltration of inflammatory cells and infiltration of alveolar cells in the lung tissue of rats in the injection group was observed,which was apparent different with atomization group(P<0.05)2 Tetrandrine can inhibit the expression of extracellular matrix in lung tissue of rats with pulmonary fibrosis14 days after a single tracheal instillation of bleomycin,compared with the sham operation group,the level of Col-I in the lung tissue of the model group was significantly increased(P<0.01),and the content of FN and TGF-?1 was apparently increased(P<0.05).The SMA level was apparently increased(P<0.05),and the Vimentin level was significantly increased(P<0.01).After 28 days,the secretion of the above indicators was further promoted,which were apparently different from the sham operation group(P<0.05).Tetrandrine can inhibit the increase of related extracellular matrix levels in the lung tissue of rats with pulmonary fibrosis.Compared with the model group,the content of Col-I,FN,and ?-SMA in rat lung tissue was slightly inhibited at 14 days,and Vimentin and TGF-?1 level was apparently inhibited(P<0.05),and at 28 days,the content of Col-I,FN and TGF-? was apparently reduced(P<0.05),and the level of Vimentin was significantly inhibited(P<0.01),and the expression of?-SMA was slightly inhibited,but no statistical difference.3 TGF-?1 promotes the expression of extracellular matrix in A549,MRC-5 and PMVEC cellsStimulation of TGF-?1 at the appropriate concentration for 24 hours can significantly increase the content of Col-I in the supernatant of A549,MRC-5,and PMVEC cells(P<0.01),and increase the content of a-SMA and Vimentin in cells(P<0.01),can significantly promote the expression of FN in A549,MRC-5 cells(P<0.01).4 Tetrandrine can inhibit the effect of TGF-?1 stimulated MRC-5 cellsThe IC50 of MRC-5 cells,after tetrandrine and TGF-?1 co-stimulated for 24 h,was 14.07 ?M.And tetrandrine could significantly inhibit the increase of Col-I,FN,?-SMA and Vimentin in MRC-5 cells caused by TGF-?1 stimulation(P<0.01)Stimulation with TGF-?1 did not cause apoptosis in MRC-5 cells,and the apoptosis rate was 1.93%,even lower than 2.27%of the blank group.Co-stimulation with tetrandrine was added at the same time,the apoptosis rate was 17.53%-43.8%.which had a significant effect on cell apoptosis(P<0.01).Conclusion1 Tetrandrine can relieve the degree of lung tissue lesions,improve lung function,reduce lung coefficient,and reduce HYP secretion in rats with pulmonary fibrosis,and this protection also plays a role in the early stage of pulmonary fibrosis.2 Compared with injection,atomization treatment is basically consistent with its efficacy,but the effective dose of atomization is 0.22 mg·kg-1,which is only 1/136 of the dose of injection group.State,the degree of lung tissue lesions and some lung function indicators were better than the injection group3 Reduce TGF-? content,and promote activated fibroblast apoptosis to inhibit the accumulation of extracellular matrix,like Col-I,FN,and other pathways,may be the method of tetrandrine to prevent pulmonary fibrosis. |
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