Inhibitory Effect of Verapamil on the Growth of Human Airway Granulation
Fibroblasts
Authors: Li Jing , Zhang Yunhui , Cai Bo , Yuan Bing , Cui Jinghua 
Published: May 2022

Objectives:
To explore the inhibitory effect of verapamil, a calcium channel blocker, on
the growth of human airway granulation fibroblasts to provide an experimental basis for the clinical
use of calcium channel blockers in preventing and treating benign airway stenosis.

Methods:
Primary human airway normal fibroblasts and human airway granulation fibroblasts
were cultured by tissue block attachment culture method, and the experimental studies were carried
out using 3-8 generation cells. Cell Counting Kit-8 (CCK-8) was used to test the proliferation of
human normal airway fibroblasts and human airway granulation fibroblasts and the semi-inhibitory
concentration of verapamil on normal airway fibroblasts and airway granulation fibroblasts. A
scratch test detected the migration effect of verapamil on human airway granulation fibroblasts.
The mRNA relative expression levels of related factors were detected by PCR to compare the differences
between normal airway fibroblasts and airway granulation fibroblasts. Western blot was
used to detect the relative amount of related proteins and compare the differences between normal
airway fibroblasts and granulation airway fibroblasts. After 48 hours of treatment with half of the
inhibitory concentration of Vera Pammy for granulation airway fibroblasts, the relative expression
levels of related factors on mRNA and protein were observed.

Results:
Human normal airway fibroblasts and human airway granulation fibroblasts with a purity
of more than 95% could be obtained from primary culture by tissue block adherence method. CCK8
results showed that the proliferation rate of human airway granulation fibroblasts was faster than that
of the normal human airway fibroblasts. The semi-inhibitory concentration of verapamil on human
normal airway fibroblasts was 92.81 ug/ml, while the semi-inhibitory concentration on human airway
granulation fibroblasts was 69.57 ug/ml. The scratch test indicated that the cell migration rate of human
airway granulation fibroblasts treated with verapamil decreased significantly (P < 0.05). PCR
results showed that the mRNA relative expression levels of TGFβ1, COL1A1, Smad2/3, VEGFA,
IL6, and IL8 in human airway granulation fibroblasts were significantly higher than those in normal
human airway fibroblasts (P < 0.05). The mRNA relative expressions of TGFβ1, smad2/3, and
COL1A1 in human airway granulation fibroblasts treated with semi-inhibited verapamil for 48h were
down-regulated (P < 0.05), while the mRNA relative expressions of VEGFA, IL6 and IL8 had no
significant changes (P > 0.05). WB test showed that the relative protein expressions of TGFβ1,
Smad2, and VEGFC in human airway granulation fibroblasts were upregulated (P < 0.05) but downregulated
after verapamil treatment compared with before treatment (P < 0.05).

Conclusion:
Calcium channel blockers can inhibit the proliferation of human airway granulation
fibroblasts through TGFβ1/ Smad pathway, which may be a method to prevent and treat benign
airway stenosis.