Synthesis of phosphatidylcholine in rats with oleic acid-induced pulmonary edema and effect of exogenous pulmonary surfactant on its De Novo synthesis

Abstract

In mammals, oleic acid (OA) induces pulmonary edema (PE), which can initiate acute lung injury (ALI) and lead to acute respiratory distress syndrome (ARDS). Pulmonary surfactant (PS) plays a key role in a broad range of treatments for ARDS. The aim of the present investigation was to assess changes in the synthesis of phosphatidylcholine (PC) from choline and determine the effect of exogenous PS on its de novo synthesis in rats with OA-induced PE. Experimental rats were randomized into three groups, including a control group, OA-induced PE group, and OA-induced group treated with exogenous PS (OA-PS). Twenty-four rats were sacrificed 4 h after induction of the OA model, and tissue was examined by light and electron microscopy to assess the severity of ALI using an established scoring system at the end of the experiment. After 15 μCi 3H-choline chloride was injected intravenously, eight rats in each group were sacrificed at 4, 8, and 16 h. The radioactivity of 3H incorporated into total phospholipid (TPL) and desaturated phosphatidylcholine (DSPC) was measured in bronchoalveolar lavage fluid (BALF) and lung tissue (LT) using a liquid scintillation counter and was expressed as counts per minute (CPM). Results showed that TPL, DSPC, and the ratio of DSPC/total protein (TP) in lung tissue decreased 4 h after challenge with OA, but the levels recovered after 8 and 16 h. At 8 h after injection, 3H-TPL and 3H-DSPC radioactivity in the lungs reached its peak. Importantly, 3H-DSPC CPM were significantly lower in the PS treatment group (LT: Control: 62327 ± 9108; OA-PE: 97315 ± 10083; OA-PS: 45127 ± 10034, P < 0.05; BALF: Control: 7771 ± 1768; OA-PE: 8097 ± 1799; OA-PE: 3651 ± 1027, P < 0.05). Furthermore, DSPC secretory rate (SR) in the lungs was significantly lower in the PS treatment group at 4 h after injection (Control: 0.014 ± 0.003; OA-PE: 0.011 ± 0.004; OA-PS: 0.023 ± 0.006, P < 0.05). Therefore, we hypothesize that exogenous PS treatments may adversely affect endogenous de novo synthetic and secretory phospholipid pathways via feedback inhibition. This novel finding reveals the specific involvement of exogenous PS in endogenous synthetic and secretory phospholipid pathways during the treatment of ARDS. This information improves our understanding of how PS treatment is beneficial against ARDS and opens new opportunities for expanding its use.

Fig 1. Alveolar structure and schematic design of isotope tracing.

³H-labeled methyl chloride choline is involved in surfactant phospholipid synthesis of alveolar type II cells. The level of ³H in phospholipids was monitored to detect the amount originating from endogenous phospholipid synthesis. The radioactivity of TPL and DSPC in BALF and LT indicates the total content of ³H-labeled choline chloride incorporated into TPL and DSPC, which reflects the newly synthesized TPL and DSPC and the body’s ability to synthesize PC. The secretory rate of TPL was expressed as the ratio of radioactivity in BALF and the whole lung (BALF+LT). The secretory rates of TPL and DSPC indicate the ability of alveolar type II epithelial cells to secrete PC into the alveolar space.

Fig 2. Histopathological characterization in rats as determined by gross morphology and hematoxylin-eosin (H-E) staining (×400).

A and D: Normal control group; B and E: OA-PE group; C and F: OA-PS treatment group. A, B, C: gross morphology; D, E, F: H-E staining.

Fig 3. Electron microscopy observations of pulmonary surfactant layer (PSL) and vascular endothelial cells.

A and D: Normal control group, B and E: OA-PE group, C and F: OA-PS treatment group. A, B, C: PSL; D, E, F: vascular endothelial cells.

Fig 4. Changes in ³H-TPL and ³H-DSPC levels in LT and BALF.

A: Changes in ³H-TPL and ³H-DSPC levels in LT. B: Changes in ³H-TPL and ³H-DSPC levels in BALF. C: Changes in TPL SR and DSPC SR.