Effect of Banxia Xiexintang-containing Serum on Epithelial-mesenchymal Transition of Peritoneal Mesothelial Cells Induced by Gastric Cancer-derived Exosomes
10.13422/j.cnki.syfjx.20212126
- VernacularTitle:半夏泻心汤含药血清对胃癌来源外泌体诱发腹膜间皮细胞上皮间质转化的影响
- Author:
Jun-gang DONG
1
;
Xi-ping LIU
1
;
Pei-qing LI
1
;
Qing-miao WANG
1
;
Zhong-bo ZHU
1
;
Ling HE
1
;
Yong-yu LI
1
;
Yu WANG
1
Author Information
1. Gansu University of Chinese Medicine， Lanzhou 730000， China
- Publication Type:Research Article
- Keywords:
gastric cancer;
exosomes （Exo）;
human peritoneal mesothelial cells;
epithelial-mesenchymal transition （EMT）;
Banxia Xiexintang
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2021;27(21):19-26
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of Banxia Xiexintang on the epithelial-mesenchymal transition （EMT） of human peritoneal mesothelial cell line （HMrSV5） induced by gastric cancer-derived exosomes （Exo）. Method:Banxia Xiexintang-containing serum was prepared and the human gastric cancer NCI-N87-derived exosomes （NCI-N87-Exo） were extracted， followed by their identification by transmission electron microscopy and Western blotting and labeling with 1，1-dioctadecyl-3，3，3，3- tetramethylindocarbocyanine perchlorate （Dil）. The cells were divided into the blank group， model group， and low-， medium-， and high-dose （13.5，27，54 g·kg-1） Banxia Xiexintang groups. HMrSV5 cells in the blank group were cultured alone， the ones in the model group with 100 mg·L-1 NCI-N87-Exo， and those in the low-， medium-， and high-dose Banxia Xiexintang groups with 100 mg·L-1 NCI-N87-Exo plus low-， medium-， and high-dose 10% Banxia Xiexintang-containing serum， respectively. Confocal laser microscope was used to observe the uptake of NCI-N87-Exo by HMrSV5 cells at 24 h， 48 h and 72 h. Seventy-two hours later， the morphological changes in HMrSV5 cells were observed. The protein expression levels of E-cadherin， cytokeratin 19 （CK19）， α-smooth muscle actin （α-SMA）， elastin， and transforming growth factor-β1 （TGF-β1）， Smad2/3， and p-Smad2/3 were assayed by Western blot. Result:It was observed under the transmission electron microscope that NCI-N87-Exo showed an oval or dish-shaped vesicle structure with a particle size ranging from 40 to 80 nm. Exo marker proteins CD9 and CD63 were highly expressed while calreticulin was not expressed， implying that the NCI-N87-Exo was confirmed. After 24 h， 48 h， 72 h of co-culture， it was observed under the fluorescence microscope that NCI-N87-Exo were taken up by HMrSV5 cells， which was positively correlated with time. Compared with the blank group， Banxia Xiexintang significantly inhibited the uptake of NCI-N87-Exo by HMrSV5 cells， with better effect noticed in the middle- and high-dose Banxia Xiexintang groups（P<0.05，P<0.01）. After intervention with Banxia Xiexintang-containing serum， the HMrSV5 cells were arranged densely， and the intercellular space was significantly reduced， with the most obvious changes present in the high-dose Banxia Xiexintang group. Western blot revealed that the protein expression levels of E-cadherin and CK19 in HMrSV5 cells after being intervened with the medium- and high-dose Banxia Xiexintang-containing serum were increased significantly as compared with those in the blank group， whereas the levels of α-SMA and Elastin were decreased significantly （P<0.01）. Banxia Xiexintang-containing serum at the low， medium， and high doses remarkably down-regulated TGF-β1 and p-Smad2/3 protein expression（P<0.05，P<0.01）. However， there was no significant change in Smad2/3. Conclusion:NCI-N87-Exo can be taken up by HMrSV5 cells to induce EMT. Banxia Xiexintang can inhibit the uptake of NCI-N87-Exo by HMrSV5 cells and the resulting EMT induced by NCI-N87-Exo， which is related to the regulation of TGF-β1/Smads signaling pathway.
