Medicated Serum of Bupleuri Radix Regulates HFL1 Apoptosis and Fibroblast-myofibroblast Transition via Smad3/Rheb Axis
10.13422/j.cnki.syfjx.20222438
- VernacularTitle:柴胡含药血清通过Smad3/Rheb轴调节HFL1细胞凋亡和肌成纤维细胞转化
- Author:
Da LI
1
;
Xiao SHEN
2
;
Quhui WU
2
;
Lei ZHENG
2
;
Silin ZHAO
1
;
Zhaohui JIN
1
;
Xuefei XIAO
1
;
Fuyuan FAN
1
Author Information
1. The First Hospital of Hunan University of Chinese Medicine, Changsha 410007, China
2. Hunan University of Chinese Medicine, Changsha 410208, China
- Publication Type:Journal Article
- Keywords:
idiopathic pulmonary fibrosis;
Bupleuri Radix;
fibroblast;
Smad3;
RAS homolog enriched in brain;
fibroblast-myofibroblast transition
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2023;29(11):89-96
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTransforming growth factor-β1 (TGF-β1) was used to stimulate human fetal lung fibroblast 1 (HFL1) for simulating the pathological process of idiopathic pulmonary fibrosis (IPF) and thereby the effects and mechanism of medicated serum of Bupleuri Radix against IPF were investigated. MethodTGF-β1 (10 μg·L-1) was employed to stimulate HFL1, and cells were treated with medicated serum of Bupleuri Radix (5%, 10%, 15%, 20%) for 24 h. Then cell proliferation rate was determined with cell counting kit-8 (CCK-8). Subsequently, cells were classified into the control group (20% blank serum), TGF-β1 group (20% blank serum and 10 μg·L-1 TGF-β1), TGF-β1 + medicated serum of Bupleuri Radix group (5% blank serum, 15% medicated serum, and 10 μg·L-1 TGF-β1), and TGF-β1 + SIS3 group (3 μmol·L-1 SIS3, 20% blank serum, 10 μg·L-1 TGF-β1). Based on in situ end labeling (TUNEL) staining, the apoptosis rate was examined, and mRNA expression of apoptosis-related proteins B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax) and myofibroblast marker α-smooth muscle actin (α-SMA) was detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). The protein expression of α-SMA, Ras homolog enriched in brain (Rheb), and phosphorylated (p)-Smad3 was determined by immunofluorescence. Expression of Rheb, p-Smad3, and Smad3 was examined by Western blot. ResultThe cell proliferation rate of TGF-β1 group increased compared with that of the control group (P<0.05). The cell proliferation rate of TGF+15% medicated serum of Bupleuri Radix group and TGF+20% medicated serum of Bupleuri Radix group decreased compared with that of the TGF-β1 group (P<0.01). Compared with the control group, TGF-β1 group showed decrease in apoptosis rate, increase in mRNA expression of Bcl-2 and α-SMA, reduction in Bax mRNA expression, and rise of α-SMA and Rheb protein expression and p-Smad3 level (P<0.05). Compared with TGF-β1 group, TGF-β1 + medicated serum of Bupleuri Radix group and TGF-β1 + SIS3 group demonstrated high apoptosis rate, low Bcl-2 and α-SMA mRNA expression, high Bax mRNA expression, and low α-SMA and Rheb protein expression and p-Smad3 level (P<0.05). ConclusionMedicated serum of Bupleuri Radix can inhibit TGF-β1-induced HFL1 proliferation and fibroblast-myofibroblast transition and promote fibroblast apoptosis by regulating the Smad3/Rheb axis.