The Protective Effects Of Bactericidal/neutralizing-endotoxin Peptide(BNEP) On Murine Acute Lung Injury Induced By Lipopolysaccharides

Objective: Lipopolysaccharides (LPS), usually called endotoxin, is a major component of the outer leaflet of gram-negative bacterial outer membrane and has been demonstrated to be a key molecule in the pathophysiology of systemic inflammatory response syndrome (SIRS), sepsis and multiple organ dysfunction syndrome (MODS). Despite of intensive efforts focused on the treatment of sepsis mediated by LPS and various inflammatory cytokines, none of the safe and potent LPS-neutralizing agents has been applied in clinical therapy so far. Bactericidal/permeability increasing protein (BPI),a kind of acid cationic protein found in the primary granules of human and mammalian neutrophils, exerts the bactericidal activities and the effects of neutralization of LPS. An 14-aminoacid peptide, named bactericidal/neutralizing-endotoxin peptide (BNEP), was designed and synthesized by the researchers in our institute based on the crystal structure of BPI. Many our previous experiments have demonstrated that BNEP exerted strong antibacterial and LPS-neutralizing activities in vivo and vitro. As we know, lung is one of early target organ in MODS induced by LPS, so to define the treatment role of BNEP on acute lung injury (ALI) induced by LPS is the aim of the study.Materials and methods: Healthy BALB/C mice were used to replication ALI models by intranasal instillation of LPS. At selected time points, the lungs were excised for lung weight and histopathology measurement. Pulmonary edema caused by LPS was determined by calculating the ratio increase of lung weight by use of normal lung weights (negative control) as the reference weight. All above scores were assigned to confirm the reliable of ALI induced by LPS. Then the mice were divided randomly into three groups: control, LPS and BNEP group. The control mice were administered intranasally with 60 μ l LPS-free saline, LPS group with 500ul/kg LPS. In BNEP group, each mouse received 10mg/kg BNEP via tail vein after instillation of LPS. After 48 hours, the lungs were excised for thefollowing measurement^) the ratio of wet lung weight Vs dry lung weight;(2) the permeability of pulmonary capillary vessel(Ewans Blue); (3) histopathology. Immunohistochemistry were used for detecting the expression of TLR2 and TLR4 in the lung tissue.Results: The wet lung weight, which indicate the lung edema, increased initially at 4h(0.8%) after LPS instillation and increase continuously at 8h, 12h and 24h, and decrease to 14.3% at 72h after reached peak at 48h(51.9%). The histopathology results showed obviously lung hyperemia, edema, and hemorrhage in a time manner dependent. Instillation of LPS in mice increased in inflammatory cell count, mainly neutrophils. Aggregation of inflammatory cells was found in alveolar and alveolar walls. In control group, the lung structures are integral and there were no inflammatory reaction appeared at 48h after saline administration. In LPS group, the classic histopathology of ALI was observed, include the increased ratio of wet lung weight Vs dry lung weight, and the permeability of pulmonary capillary vessel. In BNEP group, the lung injuries were attenuated significantly compared with that of LPS group. The immunohistochemistry experiments showed that there were a few of Toll-like receptor-2(TLR2) and TLR4 in the lung of control group mice, and usually major expressed in alveolar macrophage, alveolar type II epithelial cell, endothelial cell and alveolar type I epithelial cell. In LPS group, the TLR2 and TLR4 up-regulated and can also been seen on neutrophil. In BNEP group, down-regulated expression was determined significantly compared with that of LPS group.Conclusions: (1) the murine model of ALI by intranasal instillation of LPS is feasible and reliable. The time-dependently change of lung edema, Hyaline membrane forming and inflammatory cells influx into the lung tissue were obvious and familiar with pathology of classic ALI. So this ALI model is well suited for study of pathogenesis of ALI and preliminary pharmacological trial for therapeutic agents. (2) BNEP can relieve lung edema, decrease the permeability of pulmonary capillary vessel and reduce the amount of neutrophil influx into the lung tissue, which induced by LPS instillation. (3) BNEP can down-regulate the the expression of TLR2 and TLR4 in the lung tissue, which were stimulated by LPS. So, it suggested that BNEP could block the inflammatory response and protect the lung from inflammatory reaction-induced damage. Based on the previous testimony and these research results, BNEP, as a artificial peptide derived from BPI, wouldhave the possibility to develop as a new drug used for block biological activities of endotoxin.