Jiang Zheng, Hong Zhou, Guo Wei, Xiaoyun Gong, Gengfa Lu, Yongning Lu, Guangxia Xiao
Medical Research Center, Southwestern Hospital, Third Military Medical University, Chongqing 400038, China.
Int Immunopharmacol. 2004 Apr;4(4):527-37. doi: 10.1016/j.intimp.2004.02.004.
Lipopolysaccharide (LPS [endotoxin]), a structural component of gram-negative bacteria, is implicated in the pathogenesis of septic shock. Lipid A is an evolutionarily conserved region of LPS that has been identified as the toxic component of LPS. Therapeutic strategies for the treatment of septic shock in humans are currently focused on neutralization of LPS. Here, the anti-endotoxin activity of BNEP, a synthetic peptide derived from the human bactericidal/permeability-increasing protein (BPI; aa 148-161) was investigated in vitro and in experimental animal endotoxemia models in vivo. The ability of BNEP to bind LPS from Escherichia coli O55:B5 and lipid A from Salmonella Re 595 was tested using an affinity sensor assay, and its ability to neutralize LPS was tested using a sensitive Limulus amebocyte lysate (LAL) assay. Polymyxin B (PMB) was used as the positive control in the in vitro experiments and in mouse experiments. We found that BNEP and PMB bound LPS with a similar affinity (Kd values of 25.4 and 25.8 nM, respectively). In contrast, BNEP bound lipid A with a slightly lower affinity than that of PMB (Kd values of 8 and 5.6 nM, respectively). The exact capacity of BNEP binding to LPS was approximately 0.53 microg peptide per 1 ng of LPS, as shown by affinity sensor assay. The LAL test showed that 256 microg of BNEP almost completely neutralized 2 ng LPS. In vivo, mice were randomized, intravenously injected with BNEP (0.5-10 mg/kg) or 1 mg/kg PMB, and then lethally challenged with 20 mg/kg LPS. We found that 5 mg/kg BNEP significantly protected mice from LPS challenge. In an endotoxemia rat model, animals were co-treated with 5 or 10 mg/kg BNEP and 10 mg/kg LPS via cardiac catheter. BNEP treatment resulted in significant reduction of tumor necrosis factor alpha (TNF-alpha) and IL-6, compared with LPS-only control animals. In addition, 10 mg/kg BNEP-treated animals showed a significant decrease in plasma endotoxin levels in comparison to animals treated with LPS alone. These results provide evidence that BNEP effectively neutralizes LPS in vitro and in vivo, and could protect animals from the lethal effects of LPS via decreasing plasma endotoxin and proinflammatory cytokines. Our work suggests that this peptide is worthy of further investigation as a possible novel treatment for septic shock.
脂多糖（LPS[内毒素]）是革兰氏阴性菌的一种结构成分，与脓毒性休克的发病机制有关。脂质A是LPS中一个进化上保守的区域，已被确定为LPS的毒性成分。目前，治疗人类脓毒性休克的策略主要集中在中和LPS上。在此，我们对一种源自人杀菌/通透性增加蛋白（BPI；氨基酸148 - 161）的合成肽BNEP的抗内毒素活性进行了体外和体内实验动物内毒素血症模型研究。使用亲和传感器测定法测试BNEP与大肠杆菌O55:B5的LPS以及鼠伤寒沙门氏菌Re 595的脂质A结合的能力，并使用灵敏的鲎试剂（LAL）测定法测试其中和LPS的能力。在体外实验和小鼠实验中，多黏菌素B（PMB）用作阳性对照。我们发现BNEP和PMB与LPS的结合亲和力相似（解离常数Kd值分别为25.4和25.8 nM）。相比之下，BNEP与脂质A的结合亲和力略低于PMB（Kd值分别为8和5.6 nM）。亲和传感器测定法显示，BNEP与LPS的确切结合能力约为每1 ng LPS结合0.53 μg肽。LAL试验表明，256 μg BNEP几乎能完全中和2 ng LPS。在体内实验中，将小鼠随机分组，静脉注射BNEP（0.5 - 10 mg/kg）或1 mg/kg PMB，然后用20 mg/kg LPS进行致死性攻击。我们发现5 mg/kg BNEP能显著保护小鼠免受LPS攻击。在一个内毒素血症大鼠模型中，通过心导管给动物联合注射5或10 mg/kg BNEP和10 mg/kg LPS。与仅用LPS处理的对照动物相比，BNEP处理导致肿瘤坏死因子α（TNF-α）和白细胞介素-6显著降低。此外，与仅用LPS处理的动物相比，10 mg/kg BNEP处理的动物血浆内毒素水平显著降低。这些结果证明BNEP在体外和体内均能有效中和LPS，并可通过降低血浆内毒素和促炎细胞因子来保护动物免受LPS的致死作用。我们的研究表明，作为脓毒性休克一种可能的新型治疗方法，该肽值得进一步研究。
