Effects of hypoxia on the expression and function of P-gp in Caco-2 cells.
10.11817/j.issn.1672-7347.2023.220448
- Author:
Anpeng ZHAO
1
;
Hongfang MU
2
;
Wanteng YAO
2
;
Xiwen CHANG
2
;
Wenbin LI
3
;
Rong WANG
4
Author Information
1. Department of Pharmacy, 940th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army, Lanzhou 730050. z_anpeng@163.com.
2. Department of Pharmacy, 940th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army, Lanzhou 730050.
3. Department of Pharmacy, 940th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army, Lanzhou 730050. yfcs2002@163.com.
4. Department of Pharmacy, 940th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army, Lanzhou 730050. wangrong-69@163.com.
- Publication Type:Journal Article
- Keywords:
Caco-2 cells monolayer;
P-glycoprotein;
drug intestinal absorption;
drug transporters;
hypoxia
- MeSH:
Humans;
ATP Binding Cassette Transporter, Subfamily B, Member 1;
Caco-2 Cells;
ATP Binding Cassette Transporter, Subfamily B;
Hypoxia;
Oxygen
- From:
Journal of Central South University(Medical Sciences)
2023;48(4):491-498
- CountryChina
- Language:English
-
Abstract:
OBJECTIVES:Hypoxia can alter the oral bioavailability of drugs, including various substrates (drugs) of P-glycoprotein (P-gp), suggesting that hypoxia may affect the function of P-gp in intestinal epithelial cells. Currently, Caco-2 monolayer model is the classic model for studying the function of intestinal epithelial P-gp. This study combines the Caco-2 monolayer model with hypoxia to investigate the effects of hypoxia on the expression and function of P-gp in Caco-2 cells, which helps to elucidate the mechanism of changes in drug transport on intestinal epithelial cells in high-altitude hypoxia environment.
METHODS:Normally cultured Caco-2 cells were cultured in 1% oxygen concentration for 24, 48, and 72 h, respectively. After the extraction of the membrane proteins, the levels of P-gp were measured by Western blotting. The hypoxia time, with the most significant change of P-gp expression, was selected as the subsequent study condition. After culturing Caco-2 cells in transwell cells for 21 days and establishing a Caco-2 monolayer model, they were divided into a normoxic control group and a hypoxic group. The normoxic control group was continuously cultured in normal condition for 72 h, while the hypoxic group was incubated for 72 h in 1% oxygen concentration. The integrity and polarability of Caco-2 cells monolayer were evaluated by transepithelial electrical resistance (TEER), apparent permeability (Papp) of lucifer yellow, the activity of alkaline phosphatase (AKP), and microvilli morphology and tight junction structure under transmission electron microscope. Then, the Papp of rhodamine 123 (Rh123), a kind of P-gp specific substrate, was detected and the efflux rate was calculated. The Caco-2 cell monolayer, culturing at plastic flasks, was incubated for 72 h in 1% oxygen concentration, the expression level of P-gp was detected.
RESULTS:P-gp was decreased in Caco-2 cells with 1% oxygen concentration, especially the duration of 72 h (P<0.01). In hypoxic group, the TEER of monolayer was more than 400 Ω·cm2, the Papp of lucifer yellow was less than 5×10-7 cm/s, and the ratio of AKP activity between apical side and basal side was greater than 3. The establishment of Caco-2 monolayer model was successful, and hypoxia treatment did not affect the integrity and polarization state of the model. Compared with the normoxic control group, the efflux rate of Rh123 was significantly reduced in Caco-2 cell monolayer of the hypoxic group (P<0.01). Hypoxia reduced the expression of P-gp in Caco-2 cell monolayer (P<0.01).
CONCLUSIONS:Hypoxia inhibits P-gp function in Caco-2 cells, which may be related to the decreased P-gp level.
