| Objective(1)To observe the effects of Yifeijianpi Recipe on lung histopathology,collagen deposition and EMT in IPF rats,we established a BLM-induced IPF rat model;(2)Based on the Wnt3a/β-catenin signaling pathway,the EMT molecular mechanism of Yifeijianpi Recipe on BLM-induced IPF model rats was explored.MethodsThere were 84 male rats in the SPF level Wistar,12 rats were selected as the blank(Con-trol)group,according to the random number table method,and saline(5 mg/kg)was admini-stered,and the rest rats were induced with BLM solution(5 mg/kg)at a concentration of 5mg/m L to establish the IPF rat model.7 rats died after 3 days,and after 5 rats were randomly excluded,the remaining 60 rats were divided into 5 groups according to the random number table method,namely Model group,YFJP-G group,YFJP-M group,YFJP-D group and PIRF group,12 rats in each group.The rats were given the corresponding drugs for 28 consecutive days,and were observed for mental activity,hair color,bleeding,food intake and other general conditions.After gavage on the 28 th day,chest CT and lung function tests were performed on each group of rats.The serum and lung tissues were collected immediately;HE staining and Masson staining were used to observe the pathological morphology and collagen deposition of lung tissue respectively,and to score the degree of pulmonary fibrosis;HYP content in rat serum was detected by alkaline hydrolysis;E-cadherin and Vimentin expression levels in lung tissue were detected by immunohistochemistry;the expression of Wnt3 a andβ-catenin in lung tissue was detected by WB;the expression of Wnt3 a and β-catenin m RNA in lung tissue was detected by PCR.Results(1)General status of the rat: The rats in the Control group were treated with saline drip through the oral trachea,and their respiratory rate increased on the first 1-2 days,with some rales appearing.After drug administration,the status of rats in each group tended to be stable,and the status of the drug administration group was better than that of the Model group,and the status of the YFJP-D group gradually improved;the status of the PIRF group,YFJP-M group and YFJP-G group improved more quickly.During the administration period,there was no mortality in the rats of all groups.(2)Feeding: Compared with the Control group,the feeding intake of rats in the Model group was significantly reduced at all time periods(P<0.01).Compared with the Model group,all dosing groups could significantly improve the food intake of rats,and the differences were statistically significant.Compared with the PIRF group,the food intake of YFJP-M group on day 7,YFJP-D group on day 14,YFJP-D group on day 28 and YFJP-G group was significant-ly reduced(P<0.05),the food intake of rats in YFJP-D group on day 7 and 21 was significant-ly reduced(P<0.01),the food intake of YFJP-M group on days 14,21 and 28 was lower than that of PIRF group,and the food intake of YFJP-G group on days 7,14,21 and 28 was higher than that of PIRF group.None of the differences were statistically significant.(3)Lung function: PIF,PEF,TV,EV,and EF50 were significantly lower in the Model group compared with the Control group(P < 0.01).Compared with the Model group,PIF,TV,EV,EF50 in each administration group and PEF in the YFJP-G group,were significantly increased(P < 0.01);compared with the PIRF group,the YFJP-M group was similar to its effect,and PEF,TV,and EV in the YFJP-G group were increased.(4)Chest CT: Control group rats had clear lung texture,no disorder or thickening,no thickening of lobular septa,no solid changes;Model group had thickened texture in both lungs,visible diffuse irregular thickening of lobular septa,and extensive subpleural foveal and grid-like changes,accompanied by dilated drawn bronchi and abnormal vascular bundles,suggesting fibrosis formation in the lungs;YFJP-D group had scattered ground.In the YFJP-D group,scattered ground glass density images were seen in both lungs,with subpleural more obvious;in the PIRF group,lobular septal thickening was seen,and intralobular vascular thickening was more obvious due to wall thickening,and scattered ground glass images existed,but the overall improvement was compared with the Model group;in the YFJP-M group,scattered stripe-like changes were seen in both lungs,and no abnormalities were seen in the bronchovascular bundles,which was better than the Model and PIRF groups overall;in the YFJP-G group,lung In the YFJP-G group,there were a few striated densities in the lungs,no abnormalities in the bronchovascular bundles,and no pleural thickening.(5)HE staining and Masson staining results showed that: the morphological structure of lung tissue in the Control group was intact and clear,with a small amount of collagen deposition;in the Model group,the alveolar structure was disordered,collapsed,or even fused,the alveolar cavity became smaller,the alveolar wall was significantly thickened,and a large amount of collagen fiber deposition was visible in the lung parenchyma and alveolar wall,with obvious fibrotic lesions.In comparison with the Model group,the degree of alveolar structural damage,alveolar wall septal thickening and collagen deposition in the PIRF and YFJP groups decreased to different degrees as the drug concentration increased in each administration group.(6)Serum HYP levels were measured by alkali hydrolysis: serum HYP levels were significantly higher in the Model group compared to the Control group(P<0.01).Compared with the Model group,all the administered groups showed different degrees of decrease(P<0.01).Compared with the PIRF group,there were no differences in each herbal group.(7)The expression of E-cadherin and Vimentin in lung tissue was detected by immunohistochemistry: the positive expression of E-cadherin was stronger in the Control group;the overall positive signal was weaker in the Model group than in the Control group,the PIRF and YFJP-M groups were comparable and both stronger than the Model group,and the overall positivity was slightly stronger in the YFJP-D and YFJP-G groups than in the Model group.The positive expression of Vimentin was weaker in the Control group;a large number of positive signals were seen in the Model group,and the overall positivity was stronger in the PIRF and YFJP-M groups,which were both weaker than the Model group,and the overall positivity was slightly weaker in the YFJP-D and YFJP-G groups than the Model group.(8)WB method was used to detect the protein expression of Wnt3 a and β-catenin in lung tissue: the relative expression levels of Wnt3 a and β-catenin proteins were significantly enhanced in the Model group compared with the Control group(P < 0.01).Compared with the Model group,the relative expression of Wnt3 a and β-catenin was significantly down-regulated in all dosing groups(P < 0.01).Compared with the PIRF group,the relative expression levels of Wnt3 a protein were lower in the YFJP-M and YFJP-G groups(P < 0.01),and the relative expression levels of β-catenin protein were higher in the YFJP-D group(P <0.01)and lower in the YFJP-G group(P < 0.01)than in the PIRF group.(9)The expression of Wnt3 a and β-catenin mRNA in lung tissue was detected by PCR:the relative expression levels of Wnt3a and β-catenin m RNA were significantly enhanced in the Model group compared with the Control group(P < 0.01).Compared with the Model group,each administration group significantly downregulated the relative expression of Wnt3 a and β-catenin m RNA(P < 0.01).Compared with the PIRF group,the relative expression levels of Wnt3 a and β-catenin m RNA in each herbal medicine administration group were significantly different(P < 0.01),among which the expression levels in the YFJP-G group were lower than those in the PIRF group and the rest of the groups were higher than those in the PIRF group.Conclusion(1)The Yifei Jianpi Recipe the lung and strengthening the spleen can reduce BLM-induced lung histopathological damage,improve collagen deposition and EMT,and delay the progression of fibrosis in IPF model rats,which has a protective effect on IPF model rats.(2)The mechanism of improvement of IPF by Yifei Jianpi Recipe may be related to the inhibition of Wnt3a/β-catenin pathway activation. |
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