Dahuang Zhechong Pill Improves Pulmonary Fibrosis Through miR-29b-2-5p/HK2 Pathway

Ethnopharmacological relevance: Dahuang Zhechong Pills (DZP) is a classic Chinese medicinal prescription in 'The Synopsis of Prescriptions of the Golden Chamber (Jin Gui Yao Lüe)'. In China, it was officially approved by the Chinese Association of integrative medicine for the prevention and treatment of liver fibrosis. At present, it was officially recorded in Chinese Pharmacopoeia and is clinically used to treat gynecopathy, hepatic diseases, atherosclerosis and multiple organ fibrosis.Aim of the study: Based on the early clinical observation that DCP can effectively improve the lung function of patients with pulmonary fibrosis, this study aims to further explore the preventive and therapeutic effects of DZP against pulmonary fibrosis and the underlying mechanisms.Materials and methods: This study first verified the relationship between glycolysis and pulmonary fibrosis using lung fibroblast MRC-5 cell line, silenced the first key rate-limiting enzyme hexokinase 2 (HK2) of glycolysis through small interfering RNA, and over-expressed HK2 through lentivirus. CCK8, Wound Healing, Transwell, Flow cytometry, RT-PCR and Western blot were used to detect the effect on MH-S cells. In addition, the contents of glucose, ATP and lactic acid in MRC-5 cells were also measured. Then, the relationship between miR-29b-2-5p and HK2 was explored by Luciferase reporter gene experiment. Finally, DZP was then used to inhibit TGF-β1-induced MRC-5 cells via the miR-29b-2-5p/HK2 pathway. After MRC-5 cells were transfected with miR-29b-2-5p mimic and inhibitor, DCP intervened in each group, similarly, CCK8, Wound Healing, Transwell, Flow cytometry, RT-PCR, Western blot and Immunofluorescence were used to detect the effect on MH-S cells. The contents of glucose, ATP and lactic acid in MRC-5 cells were also measured, simultaneously.Results: In this study, we demonstrated that after silencing and overexpression of HK2, the important metabolic enzymes and metabolites in the glycolysis pathway and the proliferation, migration, invasion and apoptosis of lung fibroblasts changed significantly. And the 3'UTR of HK2 interacts with miR-29b-2-5p. DZP could decrease glucose intake, adenosine triphosphate (ATP), and lactic acid concentration, reduce the key enzymes of glycolysis pathway HK2, phosphofructokinase (PFK), and pyruvate kinase (PKM2), and also lighten the proliferation, migration, and invasion of MRC-5 cell line, but increase cell apoptosis through miR-29b-2-5p/HK2 axis. It also significantly declines fibronectin (FN), alpha-smooth muscle actin (α-SMA), and collagen I (COL I) deposition.Conclusion: To sum up, this study displayed that DZP reduced glycolysis and inhibited fibroblasts' excessive differentiation and abnormal collagen deposition through the miR-29b-2-5p/HK2 axis, which played a role in delaying the process of pulmonary fibrosis.