| In this study,we established a rat model of pulmonary fibrosis and used Baofeikang Granule as a therapeutic drug to evaluate the effects of Baofeikang Granule on Wnt/?-catenin signaling pathway and epithelial mesenchymal transition in the model rats.Methods:After adaptively feeding 50 healthy rats for one week,8 rats were randomly selected as blank group,and the other 42 rats were used to establish the rat model of pulmonary fibrosis.The preparation method was that bleomycin sulfate solution(5mg/kg)was injected into the trachea after routine anesthesia,and the blank group was injected with the same amount of normal saline by the same operation.On the 7th day after the establishment of the model,one of the remaining 33 surviving bleomycin model rats was randomly removed,and the other 32 rats were randomly divided into model group(n=8),prednisone acetate group(n=8),Baofeikang low-dose group(n=8)and Baofeikang high-dose group(n=8).Two rats in each group were randomly selected without intervention and were killed on the 21st day after the establishment of the model.The lung tissue was taken for paraffin-embedded section and HE staining to observe the pathological changes and evaluate the model.Other rats were given corresponding intervention:Baofeikang high and low dose groups were given Baofeikang granule 27.18 g/kg/d and 13.59g/kg/d,prednisone acetate group was given prednisone acetate 1.17mg/kg/d,control group and model group were given the same amount of normal saline,once a day for 21 days,the general characteristics of rats were observed,and the samples were taken 24 hours after the last administration.(1)The pulmonary ventilatory function,compliance,histomorphology and pathological changes of rats in each group were compared:pulmonary ventilation function and compliance were measured,and forced vital capacity(FVC),forced maximum expiratory flow rate(PEF),total pulmonary resistance(RL)and dynamic compliance(Cydn)were recorded.After the rats were killed,the morphological changes of both lungs were observed,and the left lung was fixed and embedded in paraffin.The pathological changes of lung tissue in each group were observed by HE and Masson staining,and the severity of pulmonary fibrosis was evaluated.(2)To compare the related indexes of Wnt/?-catenin signal pathway and EMT in each group:the blood was collected from abdominal aorta and the right lung was lavaged to obtain BALF solution,and the contents of IL-1 ?,IL-6,TGF-? 1,TNF-? and HYP in serum and BALF were detected by Elisa.The fixed and paraffin embedded sections of left lung were taken out,and the AOD values of Wnt3a.?-catenin,MMP-7,?-SMA and E-cadherin were determined by immunohistochemical method.The upper,middle and lower lobes of the right lung were taken to detect the content of Col ? by Elisa method,the middle lobe was used to detect the protein expression levels of Wnt3a,?-catenin,MMP-7,?-SMA and E-cadherin by WB.and the lower lobe was used to detect the mRNA expression levels of ?-catenin,Gsk-3 ?,Myc,MMP-7,?-SMA and Col ?Results:(1)Model evaluation:HE staining of rat lung tissue showed that most of the lung tissue of the model group was seriously damaged,the alveolar structure was destroyed,and the alveolar septum disappeared,replaced by a large fibrotic focus,a small part of the alveolar cavity expanded,the alveolar wall became thinner,and there were inflammatory cell infiltration around the bronchi.The model was successfully prepared.Within 7 days after the establishment of the model,the bleomycin model rats were dispirited,the activity decreased,the respiratory rate increased significantly,accompanied by obvious abdominal muscle contraction,dark color of mouth,nose and claws,part of oral and nasal cavity bleeding,messy and dull fur.slow or loss of body weight,and 9 died one after another.After the start of drug administration,the rats in the model group were still in poor overall state,mental malaise,mouth and nose The color of paws was purple and dark,the body was thin and the weight was slow.The mental state of the rest three groups gradually improved,the color of mouth,nose and paw gradually turned red and moist,and the best was Baofeikang high dose group;Compared with the model group.FVC,PEF,RL and Cydn of the rats in the model group were significantly decreased(P<0.05 or P<0.01).Compared with the model group,FVC.PEF and Cydn were significantly increased in the low dose group,the high dose group(P<0.05 or P<0.01),while RL had no significant difference(P>0.05),and there was no significant difference between the three groups(P>0.05).The appearance of lung tissue was observed by naked eye.The two lungs of model group were dark red with rough surface.Nodular changes,cord like grooves,old bleeding points and overall deformation could be seen.The lung volume was reduced,the texture was tough and the elasticity was poor.The changes above in prednisone acetate group were lighter than those in model group.The change of rats in low dose group was slightly lighter than that in prednisone acetate group,and that in Baofeikang high dose group was the lightest.The results of HE and Masson staining showed that most of the lungs in the model group were severely damaged,the alveolar structure was destroyed,the alveolar septum disappeared,a large number of continuous fibrotic foci,a small part of the alveolar cavity enlarged,the alveolar wall became thinner,there was inflammatory cell infiltration around the bronchus,and the fibrosis score increased significantly(P<0.01).The fibrosis scores in prednisone acetate group,Baofeikang low dose group and Baofeikang high dose group were lower than that in model group(P<0.01),and the fibrosis scores in high and low dose Baofeikang groups were even lower than that in prednisone acetate group(P<0.01).(2)Elisa detection of BALF fluid showed that the levels of IL-1 ?,IL-6,TGF-? 1 and TNF-? in the model group were significantly higher than those in the blank group(P<0.01),while the levels of IL-1 ?,IL-6,TGF-? 1 and TNF-? in the other three groups were lower than those in the model group(P<0.05 or P<0.01).The levels of IL-1,IL-6,TGF-? and TNF-? in the high and low dose Baofeikang groups were lower than those in the prednisone acetate group(P<0.05).Serum Elisa detection showed that the levels of IL-1 ?,IL-6,TGF-? 1 and TNF-? in the model group were significantly higher than those in the blank group(P<0.01),while the levels of IL-1 ?,IL-6,TGF-? 1 and TNF-? in the other three groups were lower than those in the model group(P<0.05 or P<0.01).The levels of IL-6 ?,TGF-? 1 and TNF-? in the prednisone acetate group were lower than those in the Baofeikang low dose group(P<0.05 or P<0.01).There was no significant difference in the level of IL-1 ?.The levels of IL-1 ?,IL-6,TGF-? 1 and TNF-? in Baofeikang high dose group were lower than those in prednisone acetate group(P<0.05).The Elisa detection of the level of Col ? in lung tissue showed that the level of Col ? in the model group was significantly higher than that in the blank group(P<0.01),and the level of Col ? in the other three groups was lower than that in the model group(P<0.05 or P<0.01).The level of Col ? in prednisone acetate group was not significantly different from that in Baofeikang low dose group,but higher than that in Baofeikang high dose group(P<0.01).The Elisa detection of HYP levels in serum and BALF showed that the levels of BALF and HYP in serum in the model group were significantly higher than those in the blank group(P<0.01).while the levels of BALF and serum HYP in the other three groups were lower than those in the model group(P<0.05 or P<0.01).The levels of BALF and serum HYP in the high and low dose Baofeikang groups were lower than those in the prednisone acetate group(P<0.01).The results of immunohistochemistry showed that the expression levels of Wnt3a,?-catenin,MMP-7 and ?-SMA in the model group were significantly higher than those in the blank group(P<0.01).while the expression level of E-cadherin in the model group was significantly lower than that in the blank group(P<0.01).The expression level of ?-catenin in the prednisone acetate group was lower than that in the model group(P<0.01),and the expression level of E-cadherin in the prednisone acetate group was higher than that in the model group(P<0.05).The expression levels of ?-catenin and ?-SMA in the low-dose Baofeikang group were lower than those in the model group(P<0.05),and the expression level of E-cadherin was higher than that in the model group(P<0.01).The expression levels of Wnt3a.?-catenin.MMP-7 and ?-SMA in the high-dose Baofeikang group were lower than those in the model group(P<0.01).and the expression level of E-cadherin was higher than that in the model group(P<0.01).Except for ?-catenin,the degree of decrease or increase was higher than that in the prednisone acetate group(P<0.01).The results of WB showed that the expression levels of Wnt3a,?-catenin,MMP-7 and ?-SMA in the model group were significantly higher than those in the blank group(P<0.01).while the expression level of E-cadherin in the model group was significantly lower than that in the blank group(P<0.01).The expression levels of Wnt3a,?-catenin and MMP-7 in the other three groups were all lower than those in the model group(P<0.05 or P<0.01).The expression levels of ?-catenin and MMP-7 in Baofeikang high and low dose groups were lower than those in prednisone acetate group(P<0.05 or P<0.01),but there was no significant difference in Wnt3a expression among the three groups.The expression levels of ?-SMA and E-cadherin in prednisone acetate group were not significantly different from those in model group.The expression level of ?-SMA in Baofeikang low dose group was lower than that in model group(P<0.01).There was no significant difference in the expression level of E-cadherin between the model group and the model group.The expression level of ?-SMA in the high dose Baofeikang group was lower than that in the model group(P<0.01),while the expression level of E-cadherin in the high dose group was higher than that in the model group(P<0.05).The results of PCR showed that the mRNA expression levels of ?-catenin,Gsk-3 ?,Myc,MMP-7,?-SMA and Col I in the model group were significantly higher than those in the blank group(P<0.05),while the mRNA expression levels of ?-catenin,Gsk-3 ?,Myc,MMP-7,?-SMA and Col I in the other three groups were significantly lower than those in the model group(P<0.05),and the expression levels in the high and low dose Baofeikang groups were lower than those in the prednisone acetate group(P<0.05).Conclusion:Baofeikang Granule may regulate the level of cytokines,reduce the expression of Wnt protein in epithelial cells and the accumulation of ?-catenin in cells,inhibit the signal transduction to the nucleus,thus inhibit the EMT changes of epithelial cells,slow down the process of fibrosis and collagen deposition in rat lung,improve lung compliance and lung ventilation function. |
PDF Full Text Request