[Unfractionated heparin inhibits lipopolysaccharide-induced expression of granulocyte colony-stimulating factor in human endothelial cells through Toll-like receptor 4 signaling pathway].

OBJECTIVE: To determine the effect of unfractionated heparin (UFH) on lipopolysaccharide (LPS)-induced expression of granulocyte colony-stimulating factor (G-CSF), and the role of Toll-like receptor 4 (TLR4) signaling pathway in this process.

METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were cultured in vitro, and the cells between passages 3 and 5 were used in the experiments. Experiment I: the cells were divided into four groups as follows: control group, LPS stimulation group (LPS 10 μg/mL), LPS + 0.1 U/mL UFH group, and LPS + 1 U/mL UFH group. HPMECs in UFH groups were treated with 0.1 U/mL or 1 U/mL UFH 15 minutes before LPS stimulation, and HPMECs in control group were treated with an equal volume of phosphate-buffered saline (PBS) instead. The concentrations of interleukin-6 (IL-6) and G-CSF in cell culture supernatants were determined by enzyme linked immunosorbent assay (ELISA) 24 hours after LPS challenge to detect the effect of UFH on HPMECs. Experiment II: HPMECs were treated with 5 μg/mL of rhodobacter sphaeroides LPS (LPS-RS, antagonist for TLR4) 4 hours before the addition of PBS or LPS. The concentrations of IL-6 and G-CSF in cell culture supernatants were determined 24 hours after LPS stimulation to detect the effect of TLR4 on LPS-induced HPMEC injury. Experiment III: HPMECs were divided into four groups as before: control group, LPS stimulation group, LPS + 0.1 U/mL UFH group, LPS + 1 U/mL UFH group. Treatments to cells were the same as experiment I. The protein expression of TLR4 in HPMECs was determined by Western Blot 1 hour after LPS stimulation to detect the effect of UFH on TLR4.

RESULTS: (1) Compared with control group, the levels of IL-6 and G-CSF in LPS stimulation group were increased [IL-6 (ng/L): 655.9±58.3 vs. 75.5±18.2, G-CSF (ng/L): 388.7±36.2 vs. 35.3±12.6, both P < 0.05]. Compared with those of LPS stimulation group, in LPS + 0.1 U/mL UFH group and LPS + 1 U/mL UFH group, the levels of IL-6 and G-CSF were significantly decreased [IL-6 (ng/L): 518.2±64.6, 489.1±75.6 vs. 655.9±58.3, G-CSF (ng/L): 298.8±41.0, 273.4±33.2 vs. 388.7±36.2, all P < 0.05]. The results indicated that 1 U/mL UFH had better results, though there was no statistical significance between the results of two UFH groups. (2) LPS-induced up-regulation of IL-6 and G-CSF levels was prevented by LPS-RS [IL-6 (ng/L): 139.1±37.6 vs. 655.9±58.3, G-CSF (ng/L): 73.7±19.7 vs. 388.7±36.2, both P < 0.05]. LPS-RS alone had no effect on cytokines [IL-6 (ng/L): 118.2±42.1 vs. 75.5±18.2, G-CSF (ng/L): 48.4±26.8 vs. 35.3±12.6, both P > 0.05]. (3) Compared with control group, the protein expression of TLR4 (grey value) in LPS stimulation group was significantly upregulated after 1 hour (0.87±0.23 vs. 0.36±0.12, P < 0.05). UFH with 0.1 U/mL and 1 U/mL lowered TLR-4 protein expression induced by LPS (0.68±0.18, 0.62±0.26 vs. 0.87±0.23, both P < 0.05).

CONCLUSIONS: The expressions of IL-6 and G-CSF were increased obviously in LPS treated HPMECs. UFH might take its therapeutic effect through TLR4-dependent pathway.