Research On The Mechanism Of Antagonism Of Buyanghuanwu Decoction In The Pulmonary Fibrosis Base On TGF-β/Smad/ERK Signal Transmission Path

Pulmonary fibrosis (PF) is a kind of the disease of fiber hyperplasia as a common pathological basis process of interstitial lung disease (ILD) caused by various reasons, its diseased tissues involve pulmonary interstitium, alveolar epithelial cells and ulmo-nary vascular, characterized with over reproduction of lung fibroblast (LFB) and excess deposition of extracellular matrix (ECM) contained collagen protein. As result of its pathogenesis is not thoroughly clear so far, there is no perfect therapeutic method in clinic. Since most of lung diseases could transform into pulmonary fibrosis as their final outcome, And as the air pollution, environment deterioration and other factors, the incidence rate of PF is increasing constantly in the world, it is becoming a severe threat to human health. Therefore, it is a very important value in scientific to study the pathogenesis of PF and effective medicine to treat this disease. Research shows that transforming growth factor-β1(TGF-β1) is the key cytokine of leading to PF, the effect of causing fibrosis of TGF-β1/Smad signal path-mediated and the effect of promoting proliferation of ERK signal path-mediated are indispensable to the development of PF. PF is under the category of "lung atrophy","lung impediment" in traditional Chinese medicine, its mechanism due mostly to qi deficiency with blood stasis, so we believes that tonifying qi and activating blood is the basically therapeutic method. Our preliminary research on the effect of tonifying qi and activating blood’ method on PF found that Buyanghuanwu decoction can efficiently regulate expression levels of TGF-β1in the lung tissues, reduce the hydroxylproline (HYP) in bronchoalveolar lavage fluid (BALF) and tumor necrosis factor-α (TNF-α) content in alveolar macrophage (AM) of pulmonary fibrosis rats, which suggests we inhibitory mechanism of Buyanghuanwu decoction on pulmonary fibrosis could be associated with regulating the TGF-β/Smad/ERK signal path. Based on the above study, we use the key point of TGF-β/Smad/ERK signal path, and make our target of research accurately position in the key pathological links of PF’ formation, to discuss the associated mechanism of action of Buyanghuanwu decoction in the pulmonary fibrosis, in order to wide the application range, and lay the foundation for the clinical study.In this study, we apply BLM to induce the PF model on rats as an appraisal carrier, to explore the mechanism of Buyanghuanwu decoction regulating the TGF-β/Smad/ERK signal path from the perspective of pathology and molecular biology. The experimental research consists of four parts as following:1. The effects of Buyanghuanwu decoction on lung index and pathomorphology in the lung tissue of PF rats by BLM.Established PF model rats, on the second day after the models were successfully made, treated with Buyanghuanwu decoction. At different points in time, though observing general status of PF rats (such as the condition of mental, diet, hair, behavioral activity, and weight, etc), the changes of lung index, to measure alveolar catarrh and pulmonary fibrosis degrees in lung tissue dyed with HE and Masson, discussed the effect of Buyanghuanwu decoction on PF.Results:①General status:The rats of sham operation group’s behavioral activity, glossiness of hair and diet were unchanged; model group had the phenomena of less eating, messy or sparse hair, even hair removal, movement decreasing, slow increase of weight, other medicated groups’s general status have different improvements with time, increased gradually weight;②Lung index:at all time points, a significant increase in lung index of model group were higher than that of sham operation group (p<0.01); other medicated groups had different decreases in lung index by comparison with model group (p<0.01); compared with the prednisone group, the lung index of Buyanghuanwu decoction middle-dose group showed an obviously decreased tendency on the14th and28th day (p<0.05), this condition of that in Buyanghuanwu decoction low-dose group on the28th day (p<0.05), Buyanghuanwu decoction high-dose group than in prednisone group the lung index of some high at there time points, but were significantly lower than the same period in the model group.③Pathomorphology:compared with the sham operation group, the model group and each medicated groups were mainly featured with destroyed alveoli, thickened alveoli septum and the deposition of collagen fibers on different level; the medicated groups could improve the alveolar catarrh and pulmonary fibrosis on pathomorphology in varying degree by comparison with the model group (p<0.01); the effect of reducing alveolar catarrh and pulmonary fibrosis of Buyanghuanwu decoction middle-dose group had an advantage over that of the prednisone group on the14th and28th day (p<0.05).Conclusion:The PF rat model induced by BLM was successfully duplicated; Buyanghuanwu decoction could obviously control the growth of lung index, and alleviate alveolar catarrh and pulmonary fibrosis. 2. The effects of Buyanghuanwu decoction on fibrosis index (PCⅢ、Ⅳ-C、HA、 LN) in blood serum of PF rats by BLM.Established PF model rats, on the second day after the models were successfully made, treated with Buyanghuanwu decoction.At different points in time, tested the contents of PCⅢ、Ⅳ-C、HA、LN in blood serum of experimental rats by radioimmanoassay (RIA) method, to prove the therapeutic effect of Buyanghuanwu decoction on PF.Results:①Compared with the sham operation group, the contents of PCⅢ、 IV-C、HA、LN in blood serum of other groups were gradually increased, and that of the model group was the most obvious (p<0.01);②Each medicated group had different decreases in the contents of PCⅢ、Ⅳ-C、HA、LN in blood serum by comparison with model group (p<0.01);③Compared with the prednisone group, the contents of PCⅢ、Ⅳ-C、HA、LN of Buyanghuanwu decoction middle-dose group obviously decreased on the14th and28th day (p<0.05), as well as that of Buyanghuanwu decoction low-dose group on the28th day (p<0.05).Conclusion:Buyanghuanwu decoction could effectively reduce the contents of PCⅢ、Ⅳ-C、HA、LN in blood serum of experimental rats by BLM, which proved that buyanghuanwu decoction could effectively control the reproduction of collagen fibers and excess deposition of ECM in lung tissue of PF, and played an anti-fibrosis effect.3. Research on the mechanism of regulation of Buyanghuanwu decoction in PF based on TGF-β1/Smad signal pathEstablished PF model rats, on the second day after the models were successfully made, treated with Buyanghuanwu decoction. At different points in time, tested the expressions of Col-1、TβRⅠ、TβRⅡ、Smad3、Smad7in lung tissue by immunohistochemical staining, measured the contents of TGF-β1、Smad3、Smad7mRNA in lung tissue by reverse transcription polymerase chain reaction (RT-PCR), to discuss the Buyanghuanwu decoction’s mechanism of anti-lung fibrosis though regulating TGF-β1/Smad signal path.Results:①Compared with the sham operation group, the expressions of Col-Ⅰ、 TβRⅠ、TβRⅡ、TGF-β1mRNA、Smad3mRNA in lung tissue of model group were obviously increased (p<0.01); the expressions of Smad7、Smad7mRNA were gradually decreased (p<0.01);②Each medicated group had different decreases in the expressions of Col-Ⅰ、TβRⅠ、TβRⅡ、TGF-β1mRNA、Smad3mRNA in lung tissue by comparison with model group (p<0.01or p<0.05), the expressions of Smad7、 Smad7mRNA of medicated groups had different increases (p<0.01or p<0.05);③The expressions of Col-Ⅰ、TβRⅠ、TβRⅡ、TGF-β1mRNA Smad3mRNA of buyanghuanwu decoction were lower than that of the prednisone group (p<0.01or p<0.05); on the contrary, the expressions of Smad7、Smad7mRNA showed a increased tendency,, with statistical significance (p<0.05).Conclusion:Buyanghuanwu decoction could effectively regulate the cell factors’ expression levels of exerting effect in TGF-β1/Smad signal path, those were Col-Ⅰ、TβRⅠ、TβRⅡ、TGF-β1、Smad3, meanwhile promote the expressions of Smad7,which played an opposite role to regulate the signal path, which proved that the therapeutic effect of Buyanghuanwu decoction in PF were associated with regulating the TGF-β/Smad signal path.4. Research on the mechanism of regulation of Buyanghuanwu decoction in PF based on TGF-β1/ERK signal pathEstablished PF model rats, on the second day after the models were successfully made, treated with Buyanghuanwu decoction. At different points in time, tested the expressions of TGF-β1、ERK1/2、p-ERK in lung tissue by Western blot method, to discuss the Buyanghuanwu decoction’s mechanism of anti-lung fibrosis though regulating TGF-β1/ERK signal path.Results:①Compared with the sham operation group, the expressions of TGF-β1、ERK1/2、p-ERK in lung tissue of model group were significantly increased (p<0.01);②Each medicated group had different decreases in the expressions of TGF-β1、ERK1/2、p-ERK in lung tissue by comparison with model group (p<0.01or p<0.05);③Compared with the prednisone group, the expressions of TGF-β1、 ERK1/2、p-ERK of Buyanghuanwu decoction middle-dose group obviously decreased on the14th and28th day (p<0.05),as well as that of Buyanghuanwu decoction low-dose group on the28th day (p<0.05).Conclusion:Buyanghuanwu decoction could effectively suppress the excess expressions of TGF-β1、ERK1/2、p-ERK, which proved that Buyanghuanwu decoction could regulate the expressions of the key cell factors in TGF-β1/ERK signal path, and had a good effect of controlling proliferation.In conclusion, we established animal model of PF by a classic method though tracheofistulization with BLM, based on TGF-β/Smad/ERK signal path, to describe the effect and mechanism of Buyanghuanwu decoction on PF from integral level using the methods and techniques of molecular biology. The results prove that Buyanghuanwu decoction could obviously alleviate alveolar catarrh and pulmonary fibrosis in lung tissue of experimental rats by BLM, reduce the contents of PCⅢ Ⅳ-C、HA、LN in blood serum,suppress the excess expressions of Col-Ⅰ、TβRⅠ、 TβRⅡ、TGF-β1、Smad3、ERK1/2、p-ERK,meanwhile promote the viabilities of Smad7, which proved that Buyanghuanwu decoction could efficiently regulate the effect of causing fibrosis of TGF-β1/Smad signal path-mediated and the effect of promoting proliferation of ERK signal path-mediated, and had a good effect of anti-lung fibrosis.