Objective:

2

To explore the preventive and therapeutic effects of Dahuang Zhechong Pill (DZP) on pulmonary fibrosis and the underlying mechanisms.

Methods:

2

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-β 1 (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:

2

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:

2

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.