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Effect of Banxia Xiexintang-containing Serum on Epithelial-mesenchymal Transition of Peritoneal Mesothelial Cells Induced by Gastric Cancer-derived Exosomes / 中国实验方剂学杂志
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 19-26, 2021.
Article in Chinese | WPRIM | ID: wpr-905953
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<sup>-1</sup>) Banxia Xiexintang groups. HMrSV5 cells in the blank group were cultured alone, the ones in the model group with 100 mg·L<sup>-1</sup> NCI-N87-Exo, and those in the low-, medium-, and high-dose Banxia Xiexintang groups with 100 mg·L<sup>-1</sup> 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-cadherincytokeratin 19 (CK19), <italic>α</italic>-smooth muscle actin (<italic>α</italic>-SMA), elastin, and transforming growth factor-<italic>β</italic><sub>1</sub> (TGF-<italic>β</italic><sub>1</sub>), 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(<italic>P</italic><0.05,<italic>P</italic><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 <italic>α</italic>-SMA and Elastin were decreased significantly (<italic>P</italic><0.01). Banxia Xiexintang-containing serum at the low, medium, and high doses remarkably down-regulated TGF-<italic>β</italic><sub>1</sub> and p-Smad2/3 protein expression(<italic>P</italic><0.05,<italic>P</italic><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-<italic>β</italic><sub>1</sub>/Smads signaling pathway.

Full text: Available Index: WPRIM (Western Pacific) Type of study: Prognostic study Language: Chinese Journal: Chinese Journal of Experimental Traditional Medical Formulae Year: 2021 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Type of study: Prognostic study Language: Chinese Journal: Chinese Journal of Experimental Traditional Medical Formulae Year: 2021 Type: Article