Mechanism of Icariin in Regulating TGF-β1/Smad Pathway to Induce Autophagy in Human Bone Microvascular Endothelial Cells
10.13422/j.cnki.syfjx.20241828
- VernacularTitle:淫羊藿苷调控TGF-β1/Smad通路诱导人骨微内皮细胞自噬的作用机制
- Author:
Yaqi ZHANG
1
;
Yankun JIANG
1
;
Guoyuan SUN
1
;
Bo LI
1
;
Ran DING
1
;
Cheng HUANG
1
;
Weiguo WANG
1
;
Qidong ZHANG
1
Author Information
1. China-Japan Friendship Hospital， Beijing 100029， China
- Publication Type:Journal Article
- Keywords:
icariin;
steroid-induced osteonecrosis of femoral head;
bone microvascular endothelial cells;
autophagy;
transforming growth factor-β1 （TGF-β1）/Smad pathway
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2025;31(5):123-130
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo investigate the regulatory effect of icariin （ICA） on transforming growth factor-β1 （TGF-β1）/Smad pathway in bone microvascular endothelial cells （BMECs） and the effect on autophagy in BMECs. MethodsBMECs were isolated and cultured， and the cell types were identified by immunofluorescence. Cells were divided into the control group， model group （0.1 g·L-1 methyl prednisolone）， ICA group （0.1 g·L-1 methyl prednisolone +1×10-5 mol·L-1 ICA）， and TGF-β inhibitor group （0.1 g·L-1 methyl prednisolone +1×10-5 mol·L-1 ICA +1×10-5 mol·L-1 LY2157299）. Transmission electron microscopy was used to observe the ultrastructure and autophagosome number of BMECs. Autophagy double-standard adenovirus was used to monitor the confocal autophagy flow generation of each cell. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the gene and protein expression of autophagy in the TGF-β1/ Smad pathway. ResultsAfter cell separation culture， platelet endothelial cell adhesion molecule （CD31） and von willebrand factor （vWF） immunofluorescence identified BMECs. Transmission electron microscopy showed that the cell membrane was damaged， and the nucleus was pyknotic and broken in the model group. Compared with the model group， the ICA group had complete cell membranes， clear structures， with autophagy-lysosome sparsely distributed. The confocal photo showed that BMECs had autophagosomes and autophagy-lysosomes， and the autophagy expression of the ICA group was similar to that of the blank group. Compared with the blank group， in the model group and the LY2157299 group， autophagosomes and autophagy-lysosomes were barely seen in the autophagy flow. Compared with the blank group， the mRNA and protein expressions of autophagy effector protein 1 （Beclin1） and microtubule-associated protein 1 light chain 3B （LC3B） in the model group were significantly decreased （P<0.01）， and those of ubiquitin-binding protein （p62） were significantly increased （P<0.01）. The mRNA expression of TGF-β1， Smad homolog 2 （Smad2）， and Smad homolog 3 （Smad3） decreased （P<0.05， P<0.01）. The protein expressions of TGF-β1， p-Smad2， and p-Smad3 were significantly decreased （P<0.01）. Compared with those of the model group， the mRNA and protein expression of Beclin1 and LC3B in BMECs of the ICA group increased （P<0.01）， and those of p62 significantly reduced （P<0.01）. The mRNA expression of TGF-β1， Smad2， and Smad3 increased significantly （P<0.01）. The protein expression of TGF-β1， p-Smad2， and p-Smad3 increased significantly （P<0.01）. Compared with those in the model group， the mRNA and protein expressions of Beclin1， LC3B， and p62 in the inhibitor group were not statistically significant. The expression of key genes and proteins of the TGF-β1 pathway in the inhibitor group was not statistically significant. ConclusionICA can promote glucocorticoid-induced autophagy expression of BMECs， and its mechanism may be related to activating the TGF-β1/Smad signaling pathway.
