[Investigation of inflammatory responses of pulmonary microvascular endothelial cells induced by lipopolysaccharide and mechanism].

OBJECTIVE

Lipopolysaccharide (LPS) can activate pulmonary vascular endothelial cells (PMVECs) and induce inflammatory injury. Toll-like receptor-4 (TLR-4) is integrally involved in LPS signaling and has a requisite role in the activation of NF-κB. NF-κB is a key intercellular signaling event that mediates cell inflammatory responses. The aim of the study was to investigate in an in vitro model the inflammatory responses of PMVECs induced by LPS and the probable mechanism underlying the observed inflammatory responses.

METHODS

The present study was performed on isolated PMVECs from Sprague-Dawley rats. After being identified, PMVECs were divided into 2 groups: a control group, and a LPS (0.01, 0.1, 1, 10 mg/L) intervention group. ICAM-1, TNF-α and IL-8 were detected by ELISA or radioimmunological methods. The expression of TLR-4 mRNA was detected by real time PCR. The activation of NF-κB was detected by Western blot (proteins of I-κBα and NF-κB p65) and immunocytochemical staining (NF-κB p65).

RESULTS

Compared with the control group, cytokines secreted from PMVECs-stimulated by LPS were increased in a dose-dependent manner. When stimulated with LPS 10 mg/L for 2, 6 and 12 h, cytokines measured were all increased. ICAM-1 and TNF-α were significantly increased and peaked after 2 h before gradually declining at 6 and 12 h. IL-8 was higher after 2 h, which continued up to 12 h. The expression of TLR-4 mRNA was significantly higher and peaked after 2 h and continued to 12 h (4.34 ± 1.42, 3.62 ± 1.45, 3.32 ± 1.36), which were all higher than that of the control group (1.00 ± 0.00, P < 0.05). Meanwhile, NF-κB was activated at 0.5, 2, 6 and 12 h indicated by the significant degradation of IκB-α and the significant increased release of NF-κB P65 and its subsequent translocation into the nucleus with approximately synchronized.

CONCLUSION

Taken together, the results demonstrated that LPS was able to induce PMVECs inflammatory injury via activating TLR-4 and subsequently activating NF-κB.