In vitro Culture Method for Intestinal Epithelial Cells of Takifugu fasciatus and the Cortisol Effect on its Physiological Characteristic

Abstract

Stress is a nonspecific response that occurs when the body is stimulated by internal and external stimuli. When the stress levels exceed the body´s tolerance threshold, physiological and biochemical changes occur that affect homeostasis within the internal environment. Takifugu fasciatus is an important cultured fish species in China, and its demand in the aquatic market has increased in recent years. Stress is extremely harmful to T. fasciatus farming, and cortisol is considered an essential marker for determining the stress response of fish, as it changes significantly within the organism under stress. The intestine is a contact medium between fish and the external environment and can respond to physiological stress levels. Although the study of fish intestinal responses to stress is a global research focus, the role of the T. fasciatus intestinal tract in response to stress remains unclear. In this study, intestinal epithelial cells of T. fasciatus were cultured using four different methods: Trypsin (0.25%) with DMEM, trypsin (0.25%) with RPMI 1640, typeⅠcollagenase (1 mg/mL) in DMEM, and typeⅠcollagenase (1 mg/mL) in RPMI 1640 to establish an in vitro culture method for primary intestinal epithelial cells of T. fasciatus. The effects of different concentrations of cortisol on oxidative stress, apoptosis, and lipid metabolism were investigated. The study methodology involved disinfecting of T. fasciatus with 75% alcohol and subsequently anesthetizing with MS-222. Furthermore, the intestines were removed and cleansed using PBS containing 100 IU/mL penicillin and 100 μg/mL streptomycin. The intestines were then sectioned into 1.5 mL centrifuge tubes with digestive solutions and digested at 28 ℃ for 30 min. Cells were subsequently centrifuged at 1 000 r/min for 5 min at 4 ℃, and resuspended in a new complete medium (DMEM containing 20% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin). The proliferation rate of intestinal epithelial cells under the four culture methods was determined within 5 d using the CCK-8 method, and cell growth was observed under a microscope after 24 h of culture. Cortisol solutions at varying concentrations were diluted with complete medium, and the cell viability of T. fasciatus was measured using the CCK-8 method after cortisol treatment (0, 100, 1 000, 2 000, 3 000, and 5 000 nmol/L) for 24 h. The morphological structure of intracellular mitochondria was observed through transmission electron microscopy after cortisol treatment (0, 10, 100, and 1 000 nmol/L) for 24 h. After cortisol treatment (0, 10, 100, and 1 000 nmol/L) for 3, 6, 12, and 24 h, the expression patterns of oxidative stress-related genes, apoptosis-related genes, and lipid metabolism-related genes in intestinal epithelial cells were measured by real-time fluorescence-based quantitative PCR (qRT-PCR). Cell apoptosis was measured using the Annexin V-FITC/PI method, and the contents of triglycerides, total cholesterol, and free fatty acids were measured using kits from Nanjing Jiancheng Co. The results showed that the intestinal epithelial cells cultured with trypsin (0.25%) digestion and DMEM had the highest cell proliferation rate among the four culture methods, with predominantly fibroblast-like cell morphology and the best apposition. At a cortisol concentration of 1 000 nmol/L, the intestinal epithelial cell viability was 0.8 times that of the control group, with no significant difference between the 1 000 nmol/L cortisol-treated group and the 2 000 nmol/L cortisol-treated group. However, the cell viability significantly decreased when the cortisol concentration exceeded 2 000 nmol/L (P < 0.05). Thus, 1 000 nmol/L or lower was selected for cortisol treatment. Transmission electron microscopy revealed that the mitochondrial structure of intestinal epithelial cells was altered in all the cortisol-treated groups, with an increase in the number of mitochondria. The expression of oxidative stress-related genes (sod, cat, and gsh-px) and apoptotic index significantly increased with increasing cortisol treatment concentration and time. Pro-apoptotic genes (caspase-3, caspase-7, caspase-9, bax and p53) increased significantly, while anti-apoptotic gene bcl-2 expression decreased significantly with the increase of cortisol treatment concentration (P < 0.05). The expression of lipid synthesis-related genes (g6pd, 6gpd, pparγ, fas and acc) decreased significantly and lipolysis-related genes (hsl, pparα, lpl and cpt-1) increased significantly with increasing cortisol treatment concentration (P < 0.05). Triglyceride and total cholesterol contents decreased significantly, whereas the free fatty acids content increased significantly with increasing cortisol treatment concentration and time (P < 0.05). These results indicate that trypsin (0.25%) digestion with DMEM is optimal for culture of primary intestinal epithelial cells of T. fasciatus. Cortisol promotes oxidative stress, apoptosis, and lipid decomposition but suppresses lipid synthesis in intestinal epithelial cells of T. fasciatus. This study established the optimal isolation and culture method for primary intestinal epithelial cells of T. fasciatus and investigated the mechanism of the intestinal response to cortisol stress in T. fasciatus, thus providing a theoretical basis for the subsequent anti-stress culture of fish.

Keywords

• takifugu fasciatus
• intestinal epithelial cell
• cortisol
• oxidative stress
• cell apoptosis
• lipid metabolism