| Objective:To investigate the feasibility,efficacy,as well as potential mechanism for the treatment of modified Buyang Huanwu decoction,a representative formula of Buqi Wenyang Tongluo method,in the pathogenesis of pulmonary fibrosis.1.A rat model of pulmonary fibrosis was established by intratracheal instillation of bleomycin to explore the effect of modified Buyang Huanwu decoction on histopathology in rats with pulmonary fibrosis;2.Different concentrations of modified Buyang Huanwu decoction were used to intervene rats with pulmonary fibrosis to further investigate their effects on pulmonary fibrosis-related factors,hepatocyte growth factor(HGF),epithelial-mesenchymal transition(EMT),and the TGF-β1/Smads signaling pathway;3.For cells in vitro,different concentrations of modified Buyang Huanwu decoction were used to intervene A549 cells for exploring the mechanism of their effects on pulmonary fibrosis-related factors,HGF,EMT,and the TGF-β1/Smads signaling pathway,extending and supplementing the results of animal experiments;4.For cells in vitro,silencing HGF gene expression with si RNA to further explore the exact mechanism of HGF in modified Buyang Huanwu decoction.Methods:1.In vivo experiments: 36 clean Wistar rats(male,weight 200±20g)were randomly divided into 6 groups according to the random number table after one week of routine feeding: the control group(Control,n=6),model group(BLM,n=6),pirfenidone group(BLM+Pre,n=6),modified Buyang Huanwu decoction low dose group(BLM+JBT-L group,n=6),modified Buyang Huanwu decoction medium dose group(BLM+JBT-M group,n=6),and modified Buyang Huanwu decoction high dose group(BLM+JBT-H group,n=6),established rat models of pulmonary fibrosis by intratracheal instillation of bleomycin.24 hours after modeling,the Control and BLM groups were given normal saline by gavage,the BLM+Pre group was given pirfenidone suspension(162mg/kg·d)by gavage,the BLM+JBT-L group was given modified Buyang Huanwu decoction(10.18g/kg·d)by gavage,BLM+JBT-M group was given modified Buyang Huanwu decoction(20.36g/kg·d)by gavage,and BLM+JBT-H group was given modified Buyang Huanwu decoction(40.72g/kg·d)by gavage.After 28 days,the rats were sacrificed and lung tissues were obtained.HE staining and Masson staining were used to observe the pathological changes and fibrosis degree of lung tissue in rats with pulmonary fibrosis.The protein and m RNA expressions of HGF,pulmonary fibrosis markers α-smooth muscle actin(α-SMA),collagen Ⅰ,and fibronectin,EMT molecular markers E-Cadherin,N-cadherin,and Vimentin,and the TGF-β1/Smads signaling pathway related TGF-β1,Smad2,and Smad3,were detected by immunohistochemistry,westernblot(WB),and realtime PCR(RT-PCR).2.In vitro cell experiment 1: Combined with the previous study,5ng/ml TGF-β1 was used to induce EMT of alveolar epithelial cell A549,and the serum containing modified Buyang Huanwu decoction(40.72g/kg·d)with volume fraction of 5%,10%,and 20% were respectively as the low,medium and high intervention concentrations for subsequent experiments.The experiment was randomly divided into 6 groups: the control group(Control),TGF-β1 model group(TGF-β1),low-dose modified Buyang Huanwu decoction group(TGF-β1+5%JBT),medium-dose modified Buyang Huanwu decoction group(TGF-β1+10%JBT),high-dose modified Buyang Huanwu decoction group(TGF-β1+20%JBT),pirfenidone group(TGF-β1+Pre).EDU kit was used to detect cell proliferation ability in each group.Flow cytometry was used to detect cell apoptosis ability in each group.The levels of protein and m RNA expression of HGF,α-SMA,collagen Ⅰ,fibronectin,EMT marker E-Cadherin,N-cadherin,vimentin,and TGF-β1,Smad2,Smad3 were detected by WB and RT-PCR.3.In vitro cell experiment 2: TGF-β1 was used to induce EMT of A549 cells,and si-NC and si-HGF were transfected into A549 cells to construct low HGF expresseed A549 cell lines.The experiment was randomly divided into 5 groups: the control group(Control),TGF-β1 model group(TGF-β1),modified Buyang Huanwu decoction group(TGF-β1+20%JBT),si RNA-NC control group(TGF-β1+20%JBT+si-NC),and modified Buyang Huanwu decoction si RNA-HGF group(TGF-β1+20%JBT+ si-HGF).The cell proliferation ability of each group was detected by EDU kit,the cell apoptosis ability of each group was detected by flow cytometry.Using WB,RT-PCR to detect the protein and m RNA levels of α-SMA,collagen Ⅰ,fibronectin,and EMT molecular markers E-Cadherin,N-cadherin,vimentin,and TGF-β1,Smad2,Smad3.Results:1.From vivo experiments: HE staining and Masson staining showed that the lung tissue of rats with pulmonary fibrosis was damaged and fibrotic tissue was formed,suggesting that the rat models of pulmonary fibrosis were successfully established.According to the Mikawa score and Ashcroft score,modified Buyang Huanwu decoction can significantly reduce the inflammation and fibrosis of lung tissue in rats with pulmonary fibrosis.The efficacy of the BLM+JBT-H group was slightly lower than that of the BLM+Pre group,but the difference was not significant.In addition,compared with the model group,modified Buyang Huanwu decoction can significantly up-regulate the protein and gene expressions of HGF.Modified Buyang Huanwu decoction can also down-regulate the protein and gene expressions of BLM induced pulmonary fibrosis markers such asα-SMA,collagen Ⅰ,and fibronectin,significantly elevate the protein and gene expressions of E-Cadherin,reduce the expressions of N-cadherin and Vimentin,as well as suppress the protein and gene expressions of TGF-β1,Smad2 and Smad3,all in a dose-dependent manner.Compared with western drug pirfenidone in control group,difference is unobvious in the BLM+JBT-H group.2.From vitro cell experiment 1: The post-intervention results showed that,from EDU and Flow cytometry assays,modified Buyang Huanwu decoction was found to promote the proliferation and inhibit the apoptosis of A549 cells in a dose-dependent manner.WB and RT-PCR experiments showed that the expressions of HGF in each concentration group of serum containing modified Buyang Huanwu decoction increased with drug concentration.Compared with the model group,each concentration group and the TGF-β1+Pre group can down-regulate the expressions of α-SMA,collagen Ⅰ,and fibronectin,inhibit the expressions of N-cadherin and Vimentin,elevate the expressions of E-Cadherin,and suppress the protein and m RNA expressions of TGF-β1,Smad2,and Smad3.The differences were more obvious in the TGF-β1+10%JBT group,the TGF-β1+20%JBT group,and the TGF-β1+Pre group,and no significant difference in the TGF-β1+20%JBT group compared with the TGF-β1+Pre group.3.From vitro cell experiment 2: Compared with the TGF-β1+20%JBT group,the TGF-β1+20%JBT+si-HGF group had a lower effect on promoting the proliferation and inhibiting the apoptosis of A549 cells.At the same time,after HGF gene silencing,the inhibitory effect of modified Buyang Huanwu decoction on pulmonary fibrosis-related factors α-SMA,collagen I,and fibronectin in A549 cells was reduced,as well as the weakened effect on elevating E-Cadherin and the reduced inhibitory effect on N-cadherin and vimentin.It also reversed the inhibitory effect on protein and m RNA expressions of TGF-β1,Smad2,and Smad3 related to the TGF-β1/Smads signaling pathway.Conclusion:1.Modified Buyang Huanwu decoction,a prescription based on Buqi Wenyang Tongluo method,has a significant protective effect on BLM-induced rat model and TGF-β1-induced A549 cells.It may improve the EMT process and delay pulmonary fibrosis by up-regulating HGF and inhibiting the TGF-β1/Smads signaling pathway.2.There may be interactions among HGF,EMT,and the TGF-β1/Smads signaling pathway,suggesting that HGF may play a molecular structural basis for inhibiting EMT through the TGF-β1/Smads signaling pathway.3.HGF is an important target of modified Buyang Huanwu decoction in the treatment of pulmonary fibrosis.Silencing HGF can reverse the anti-pulmonary fibrosis effect of modified Buyang Huanwu decoction.As a pleiotropic factor,HGF plays an important role in the recovery of lung function,implying that the intensive study of HGF may have a great significant for the treatment of pulmonary fibrosis. |
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