A Comparative Study Of Two Mouse Pulmonary Fibrosis Models Based On The Idea Of ??"correspondence Between Prescriptions And Syndromes"

Objective:To observe the dynamic changes of a new mouse model of pulmonary fibrosis induced by trans-resveratrol(TR)within 8 weeks.Its characteristics are summarized with the help of comparison with a reference group,another mouse pulmonary fibrosis model induced by bleomycin(BLM).To analyse the relationship between the pulmonary fibrosis-related indexes and the main elements of traditional Chinese medicine(TCM)syndromes,and to clarify the syndrome characteristics of the two models,by comparing the treatment results of Zhigancao decoction and Maimendong decoction on the fibrosis-related indexes of the two modelsMethods:1.Duplication of animal models.Two models of pulmonary fibrosis were replicated with normal ICR mice and observed the changes for 8 weeks.For the TR model,intragastric administration was continued for 4 weeks;for the BLM model,a one-time intratracheal injection was used.The observation time points were 1,2,4,5,6 and 8 weeks after the experiment started.Animal weight,lung organ coefficient and mortality were counted to evaluate the animal's growth and development.lung lesions and living conditions.The pathological sections of hematoxylin-eosin(HE)and Masson staining were made to judge whether the models were successfully replicated.The hydroxyproline(HYP)content in the lung tissue was measured by the alkaline hydrolysis method to determine the collagen content in the lung tissue.2.Dynamic changes and characteristics of the two models.By collecting mouse alveolar lavage fluid(BALF)and using a fully automatic blood analyzer to classify and count cells,the numbers and changes of different types of white blood cells in BALF were counted to evaluate the models' inflammation.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX)and malondialdehyde(MDA)in lung tissues were measured to evaluate the oxidative stress level of the models.The protein expression levels of fibrotic cytokines TNF-?,IFN-?,IL-4,IL-12p70,and IL-13 in lung tissues were quantitatively measured by ELISA method.3.Comparison of the effects of Zhigancao Decoction and Maimendong Decoction on two models.Mice after 4 weeks of modeling were administrated with Zhigancao Decoction and Maimendong Decoction decoction for 4 weeks separately.The measurement and analysis of indicators were the same as Methods 1 and 2.Results:1.The TR model was successfully replicated.(1)In the TR model,mice lost weight,the mortality rate was 0%,and the lung coefficient was not significantly different from that of the control group.(2)The results of HE staining showed that the lung tissues of the TR model mice had mild hemorrhage,thickening of the alveolar septum,infiltration of the alveolar wall inflammatory cells and fibrosis,and the trend exaggerated with time.Masson staining results showed that in the TR model,collagen deposited in the interstitial lung.Alveolar septum,and the area around the bronchi and collagens gradually increased;the fibrosis score of the TR model was significantly higher than that of the control group(P<0.0001).(3)The results of HYP content showed that the collagen content in the lung tissue of the TR model increased significantly,compared with the control group(P<0.0001).2.Compared with the BLM model,the TR model has lighter inflammation,and the development of fibrosis was mild and stable.(1)The number of white blood cells in the TR model's BALF,which was almost equal to that of the control group,was lower than the BLM model.And the proportion of monocytes,lymphocytes and neutrophils remained stable overall.(2)The SOD and GSH-PX activity levels of the TR model were significantly lower than those of the control group(P<0.0001);its MDA level increased in the 5-8 weeks,which was significantly higher than that of the BLM model during the same period(P<0.01).(3)The protein's expression levels of IFN-y,IL-13,and IL-12p70 in the two models were different from those in the control group(P<0.01),but the overall trend was complex and irregular.As for IL-4 and TNF-?,there was no significant differences among the control group and the two model groups.The IL-12p70/IL-13 ratios of the two models were always below 1,and both were lower than the control group;the IFN-?/IL-4 ratio was not obviously different among the three groups.3.For the TR model,the treatment effect of Zhigancao Decoction was better than Maimendong Decoction;for the BLM model,the treatment effects of the two prescriptions were not as good as the TR model:(1)Body weight of the TR model mice in Maimendong Decoction group was significantly higher than that of the BLM model group under the same treatment(P<0.01).After the treatment of decoctions,the lung coefficient of the TR model increased,while that of the BLM model showed the opposite trend.(2)Pathological section staining results showed that pathological changes in both models were alleviated by treatments,manifesting as alveolitis and fibrosis decreased,alveolar septum thinned,and pleural lesions reduced from diffuse to focal.In the BLM model,the degree of these improvements was more obvious.However,the fibrosis scores showed no significant difference among the four treatment groups.(3)HYP content results in the lung tissue changed little in the four treatment groups.Instead,the HYP content of the treatment groups under Maimendong Decoction increased comparing with the control group(P<0.0001).(4)The cell classification and counting results in BALF showed that after the treatment of Zhigancao Decoction,the amount of white blood cells in BALF of the TR model mice did not change significantly,and was lower than that of the BLM model under the same treatment(P<0.0001).After the treatment of Maimendong Decoction,however,the result of the TR model appeared counter,though the difference was not statistically significant.And monocyte,lymphocyte,and neutrophil count results were consistent with the white blood cell tendency.(5)Zhigancao Decoction did not significantly increase SOD activity in TR model group,but Maimendong Decoction was opposite(P<0.01),while both the two decoctions decreased the SOD activity in the BLM model.As for the GSH-PX activity level,the TR model-Zhigancao decoction group were lower than that of the TR model-Maimendong decoction group,while that of the two treated BLM model groups decreased significantly comparing with the control group(P<0.0001).Both the two decoctions could reduce the levels of MDA in the lung tissues of the two models,and the TR model had more significant changes than the BLM model(P<0.0001).(6)The changes of fibrosis-related cytokines showed that there was little change in IFN-y expression levels of all treated groups,while the levels of IL-13 decreased,especially in the treated TR model group(P<0.05).The levels of IL-4 slightly increased in the TR model treated with Zhigancao Decoction,and decreased in that with Maimendong Decoction,but significantly decreased in the treated BLM model groups(P<0.05).The levels of IL-12p70 decreased significantly in all the treated groups,in which the BLM model-Zhigancao decoction group had the greatest distinction compared with the untreated group(P<0.0001).The levels of TNF-? of the TR model slightly decreased with Zhigancao Decoction and increased with Maimendong Decoction,while the treated BLM model group showed a significant decrease compared with the control group(P<0.05).Zhigancao Decoction reduced the ratio of IFN-?/IL-4 but Maimendong Decoction increased it in both models,yet without statistically significant differences.On the ratio of IL-12p70/IL-13,Zhigancao Decoction and Maimendong Decoction effected differently to different models,that is,in the TR model,the result of Zhigancao decoction group was lower than that of Maimendong decoction,and the results was opposite in the BLM model.(7)In general,Zhigancao Decoction had a better therapeutic effect on the two models than Maimendong Decoction,especially on the TR model,and the TR model could recover better than the BLM model.Conclusions:1.The TR model manifests a stabler pulmonary fibrosis with milder symptoms than the BLM model.This new model does not recover on its own within 8 weeks and has no obvious inflammation,which are similar with the early subclinical stages of idiopathic pulmonary fibrosis in human.In the TCM syndrome elements,the TR model's performance in the recovery period is more inclined to the yang deficiency and the yin deficiency supplemented;the BLM model mice's performance is inclined to the yin deficiency.2.Zhigancao Decoction and Maimendong Decoction have different therapeutic effects on the same model;the TR model and BLM model respond differently to the same decoction.Zhigancao Decoction is superior to Maimendong Decoction in improving lung tissue fibrosis,but inferior to that in relieving inflammation and oxidative stress levels.3.The performance of the TR model is more in line with the indications of Zhigancao Decoction,and is also closer to the early hidden symptoms of idiopathic pulmonary fibrosis;Maimendong Decoction has a slightly better therapeutic effect on the BLM model.Again,this suggests that the TR model and BLM model have corresponding differences in TCM syndrome elements.If the TR model can be analyzed more thoroughly,it will become another animal model to choose for human IPF in TCM research.