Effects of cyclophosphamide on the metabolism of primary hepatocytes in mice
Description
Cyclophosphamide (CTX) is widely used as a chemotherapeutic agent in various cancers and in the mobilization and conditioning regimens for blood and marrow transplantation. However, hepatotoxicity resulting from CTX could contribute to prominent morbidity, with the underlying mechanism obscure. Since the liver is the central organ for metabolism, we thereby aimed to investigate the exact effect of CTX on the primary mouse hepatocytes and whether the metabolic pathways and metabolites are involved. The primary mouse hepatocytes were divided in the CTX group and the control group. CCK8, Annexin V and ELISA assays were employed. Our results indicated that CTX inhibited the vitality, impaired the morphology, reduced the numbers and accelerated the early apoptotic rate of the primary mouse hepatocytes. Moreover, CTX could lead to the change of key metabolic enzymes and metabolites involved in the three metabolisms pathways the in primary hepatocytes, which eventually caused the inhibition of anabolism in primary hepatocytes and promoted their catabolism. In conclusion, CTX exerted toxic effects on the primary mouse hepatocytes and affected the key metabolic enzymes and metabolites involved in the three metabolisms pathways in the mouse liver.
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Experimental animal C57BL / 6 male mice were obtained from Slake, Shanghai; SPF mice (8 to 10 weeks old, weighing 25-30g,) were bred with specialized diet (purchased from Slake) and sterile water in the constant temperature and humidity barrier (IVC) system of Xuzhou Medical University. The present study was approved by the Animal Ethics Committee of Xuzhou Medical University. Isolation of primary mouse hepatocytes Isolation of primary mouse hepatocytes was as described previously, with minor modifications (Charni-Natan and Goldstein 2020). Briefly, following anesthesia of C57BL/6 male mice with 1% sodium pentobarbital, the abdominal cavity was incised, with the inferior vena cava cannulated and the hepatic portal vein severed. Subsequently, the liver was perfused with the chelate calcium at a flow rate of 15 mL/min till the liver became yellow-white. Then, collagenase was perfused to the liver in order to dissociate extracellular matrix. After dissection of liver, the isolated liver tissues were transferred to a petri dish containing high glucose medium (HDMEM), where the liver cells were released into hepatocyte suspension, the hepatocyte suspension was then centrifuged at 500 rpm/min for 1 min at 4℃. After aspiration of the supernatant, the hepatocyte suspension was re-suspended with addition of HDMEM as appropriate. 100 mesh screen filtering was employed to filter the connective tissue and cell clumps. The filtered hepatocyte suspension was re-suspended and added to the SIP (SIP: 0.96 mL 10 PBS, in the Percoll), which were mixed by stirring and agitation for 5 times, and re-suspended and recentrifuged. Cell activity was also estimated with trypan blue staining. The hepatocytes were cultured in a humidified 5% CO2 incubator at 37℃. After 6h plating, the medium was replaced with warm maintenance media and double antibodies were added, with the medium replaced every 24 hours thereafter. Cell culture Cells were plated at a density of 5 ×103 cells / mL in 96-well plates and 2-2.5 ×105 cells / mL in 6-well plates, respectively. Type I collagen was mounted on the 6-well plates and 96-well plates. According to the instructions for Type I mouse tail collagen, the original concentration was 5 mg/mL. The collagen was diluted to 0.012 mg/mL with 0.006 mol/L (0.36 g/L) of sterile acetic acid, and was diluted again with the final coating concentration of 2 μg/cm2, followed by incubation at 37℃ and aspiration of excess liquid. Afterwards, cells were rinsed 3 or 4 times with PBS, and were left to dry in a sterile bench for 10 min before usage. After 6 hours of plating, the cells were observed under an inverted microscope, with the morphology and nuclear structures of hepatocytes photographed and recorded.
Institutions
- Xuzhou Medical University