摘要Objective:To explore the preventive and therapeutic effects of Dahuang Zhechong Pill(DZP)on pulmonary fibrosis and the underlying mechanisms.Methods:The first key rate-limiting enzyme hexokinase 2(HK2)of glycolysis was silenced and over-expressed through small interfering RNA and lentivirus using lung fibroblast MRC-5 cell line,respectively.The cell viability,migration,invasion and proliferation were detected by cell counting kit-8,wound healing assay,transwell assay,and flow cytometry.The mRNA and protein expression levels of HK2 were detected by RT-PCR and Western blotting,respectively.The contents of glucose,adenosine triphosphate(ATP)and lactate in MRC-5 cells were determined by enzyme-linked immunosorbnent assay(ELISA).Then,the relationship between miR-29b-2-5p and HK2 was explored by luciferase reporter gene assay.Pulmonary fibrosis cell model was induced by transforming growth factor-β1(TGF-β1)in MRC-5 cells,and the medicated serum of DZP(DMS)was prepared in rats.MRC-5 cells were divided into control,TGF-β1,TGF-β1+10％DMS,TGF-β1+10％DMS+miR-29b-2-5p inhibitor,TGF-β1+10％DMS+inhibitor negative control,TGF-β1+10％DMS+miR-29b-2-5p mimic and TGF-β1+10％DMS+mimic negative control groups.After miR-29b-2-5p mimics and inhibitors were transfected into MRC-5 cells,all groups except control and model group were treated with DMS.The effect of DMS on MRC-5 cells were detected using aforementioned methods and immunofluorescence.Similarly,the contents of glucose,ATP and lactate in each group were measured by ELISA.Results:The mRNA and protein expressions of HK2 in MRC-5 cells were successfully silenced and overexpressed through si-HK2-3 and lentiviral transfection,respectively.After silencing HK2,the mRNA and protein expressions of HK2 were significantly decreased(P＜0.01),and the concentrations of glucose,ATP and lactate were also significantly decreased(P＜0.05).The proliferation,migration and invasion of MRC-5 cells were significantly declined(P＜0.05 or P＜0.01),while the apoptosis of MRC-5 cells was significantly increased(P＜0.01).After overexpressing HK2,the mRNA and protein expressions of HK2 were significantly increased(P＜0.05),and the concentrations of glucose,ATP and lactate were also significantly increased(P＜0.05 or P＜0.01).The proliferation,migration and invasion of MRC-5 cells were significantly increased(P＜0.05 or P＜0.01),while the apoptosis of MRC-5 cells was significantly decreased(P＜0.05).The relative luciferase activity of 3'UTR-WT+hsa-miR-29b-2-5p transfected with HK2 was significantly decreased(P＜0.01).After miR-29b-2-5p mimic and inhibitor were transfected into the MRC-5 cells,DMS intervention could significantly reduce the concentration of glucose,ATP and lactate,and the mRNA and protein expressions of HK2,phosphofructokinase and pyruvate kinase isoform M2(P＜0.05 or P＜0.01).The proliferation,migration and invasion of MRC-5 cells were alleviated(P＜0.05 or P＜0.01),and the deposition of fibronectin,α-smooth muscle actin,and collagen Ⅰ were significantly decreased(P＜0.05 or P＜0.01).Conclusions:Glycolysis is closely related to pulmonary fibrosis.DZP reduced glycolysis and inhibited fibroblasts' excessive differentiation and abnormal collagen deposition through the miR-29b-2-5p/HK2 pathway,which played a role in delaying the process of pulmonary fibrosis.