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Inhibition of glucose 6-phosphate dehydrogenase alleviates pulmonary Inhibition of glucose 6-phosphate dehydrogenase alleviates pulmonary
fibrosis
f i b r o s i s
a
Hak Su Kim
S
u
H
k
K
i
m

Veterans Medical Research Institute, Veterans Health Service Medical Center, Seoul, Republic of Korea
V e t e r a n s M e d i c a l R e s e a r c h I n s t i t u t e , V e t e r a n s H e a l t h S e r v i c e M e d i c a l C e n t e r , S e o u l , R e pu bl i c o f K o r e a
BACKGROUND AIM
Pentose phosphate (PP) pathway is one of the major metabolic pathways associated with glucose This study aims to investigate the
metabolism. Glucose 6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of PP pathway and its role of G6PD in idiopathic pulmonary
function is inhibited by 6-aminonicotinamide (6-AN). However, the role of PP pathway in idiopathic fibrosis (IPF).
pulmonary fibrosis (IPF) remain unexplored.
METHODS
The metabolites of PP pathway were analyzed with Agilent 7890/5975 GC/MSD system and HP-5 MS column in the human lung tissues. The roles
of G6PD were evaluated using fibrotic markers in fibroblasts or epithelial cells treated with transforming growth factor-β1 (TGF-β1). The antifibrotic
role of 6-AN was assessed in a bleomycin-induced lung fibrosis mice model. The levels of proteins in cell lysates or tissues were measured by
western blot assays or RT-PCR.
RESULTS
Fig. 1. PP pathway metabolites are increased in the lung tissues Fig. 2. The 6-AN effect on the TGF-β1– induced epithelial
of patients with IPF mesenchymal transition in epithelial cells (Beas-2b cells)

(A) Metabolites of pentose phosphate pathway, 6-phosphogluconate (6PG), ribulose-5-phosphate (R5P), ribulose-
1,5-bisphosphate (R1,5P), sedoheptulose-7-phosphate (S7P) (B) Quantification of in IPF (n=31) and control lung
tissues (n=20) was performed using gas chromato graphy-mass spectrometry. ** indicates p<0.01, *** indicates
p<0.01.
Fig. 3. The 6-AN effect on the TGF-β1– induced activation of MRC-5 Beas-2b cells were exposed to 5 ng/ml TGF-β1 for 24 hours with/without 6-AN (100-400 μM). (A) Total cell extracts
were prepared and subjected to western blot assays. Densitometry was used to analyze fold changes. (B) Analyzed
cells mRNA expression levels of E-cadherin, snail and slug which are key regulators of EMT induced by TGF-β1.
Fig. 4. G6PD-knockdown attenuated the activation of fibroblasts.

MRC-5 Cells were exposed to 5 ng/ml TGF-β1 for 24 hours with/without 6-AN (100-400 μM). (A) The protein levels of
collagen type 1 and α-SMA were measured by Western blot analyses. Densitometry was used to analyze fold changes in the
levels of collagen 1/actin (B) and α-SMA/Actin (C). The mean values from three independent experiments were used in the (A, B) siG6PD-treansfectied fibroblasts were exposed to 5 ng/ml TGF-β1 for 24 hours with/without 6-AN (100-400 μM).
graphs. * indicates p<0.05. TGF-β1 stimulation significantly increased collagen type 1 and α-SMA protein expression, which Total cell extracts were prepared and subjected to western blot assays. (c) Densitometry was used to analyze fold
was inhibited by 6-AN treatment. changes in the levels of collagen 1/actin (and α-SMA/Actin . The mean-values from three independent experiments were
used in the graphs. * indicates p<0.05
Fig. 5. 6-AN induced antifibrotic effects via AMPK signaling. Fig. 6. The 6-AN effect on bleomycin-induced pulmonary
fibrosis in a mouse model

MRC-5 cells were exposed to 6-AN (100-400uM) or vehicle for 24 hours and 5ng/ml TGF-β1 for 1 hours. (A) 6-AN increased 6-AN (10mg/kg) were administered by intraperitoneal (i.p) at once in 2 days for 2 weeks from day 7 after
phosphorylation of AMPK and ACC protein. (B) AMPK signaling stimulation by AICAR, AMPK activator, has been reported to intatracheal infection of bleomycin (Bleo, 3U/kg). (A) The body weight of the mice (B) Histopahologiclal analysis.
reduce fibrotic activity. (C) AMPK-DN effectively down-regulated the increased phosphorylation levels of ACC and ( C) There was a significant increase of hydroxyproline contents in the bleomycin group compared with the control
attenuated the anti-fibrotic effects by 6-AN. group. However, 6-AN treated group showed a tendency of reducing hydroxyproline levels.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
Our findings indicate that inhibition of G6PD may have anti- 1. Tzouvelekis, A. Chest 156(2), 383-391 (2019). This study was supported by a grant from the Basic Science
fibrotic effects on pulmonary fibrosis caused by abnormal 2. Yu, G., Matrix biol 68-69, 422-434 (2018). Research Programme by the National Research Foundation of
activation of PP pathway in IPF is related to pathogenesis, 3. Mendoza-Milla, C. Euro resp j 42, 1309-1321 (2013). Korea (2020R1F1A1049629) and by a VHS Medical Center
4. Saunier, C., Respiration 40, 69-75 (1980). Research Grant, Republic of Korea (grant number:
suggesting that G6PD is implicated as a potential therapeutic 5. Adegunsoye, A. Chest 150, 1371-1386 (2016). VHSMC21001)
target in IPF.