鲎抗内毒素因子模拟肽XS10/REMP1/REMP2抗菌抗内毒素活性研究
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摘要
细菌内毒素(endotoxin)或称脂多糖(lipopolysaccharide,LPS),是所有革兰阴性细菌(G-)细胞壁外膜上的主要结构成分,是G-细菌主要的致病因子。临床上严重烧伤、创伤患者往往面临感染的威胁,甚至发展成为脓毒症或多器官功能衰竭。目前尚无有效拮抗LPS的药物应用于临床。已发现存在于生物体内的一些中和LPS活性成分具有潜在的药用开发价值,如鲎抗内毒素因子(limulus antilipopolysaccharide factor, LALF),LALF是从鲎蓝色血液中分离鉴定的一种具有抗菌及中和LPS作用的蛋白,我们在前期工作中以LALF为模板通过计算机模拟设计,应用固相化学合成得到了多条模拟肽,并进行了活性初筛,本研究将继续对其中3条环化肽XS10/REMP1/REMP2进行抗LPS及抗菌活性实验检测。
第一部分、XS10/REMP1/REMP2体外中和内毒素及抗菌活性研究。
方法:
1. XS10 /REMP1 /REMP2直接中和LPS实验:以多黏菌素B(PMB)为对照,不同浓度模拟肽溶液分别与LPS工作标准品溶液直接混合反应,应用EDS-99细菌LPS测定系统,测定反应后溶液中LPS含量,计算中和率。
2. XS10 /REMP1 /REMP2对LPS介导的小鼠RAW264.7细胞产生TNF-α的抑制作用:以PMB为对照,将PMB及各模拟肽梯度浓度溶液和LPS溶液同时加入细胞培养液中,孵育一定时间后用ELISA试剂盒检测细胞上清液内的TNF-α含量。
3. XS10 /REMP1 /REMP2最低抑菌浓度(minimal inhibitory concentration,MIC)测定:采用琼脂稀释法,对大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌等标准菌株及临床分离菌株进行MIC测定。
4. REMP1 /REMP2对E.coli ATCC 25922形态的影响:分别将一定浓度的REMP1和REMP2加入大肠杆菌标准菌株E.coli ATCC 25922培养液中,作用一定时间后,透射电镜下观察E.coli ATCC 25922的形态学变化。
5. XS10 /REMP1 /REMP2细胞毒性实验:采用MTT实验测定。
结果:
1. XS10 /REMP1 /REMP2均有不同程度的直接中和LPS的作用.并且随浓度升高而增强,中和能力从强到弱依次为REMP2 >REMP1> XS10,以REMP2最强,在0.01～100μM浓度下对1.0EU/ml的LPS中和率分别为:(5.66±2.08)%～(88.66±5.03)%,接近PMB中和能力。
2. XS10 /REMP1 /REMP2在10μM、20μM、40μM、80μM浓度下,均能不同程度抑制LPS介导的TNF-α产生,与阳性对照组比较P<0.05,且随着浓度增高,TNF-α的分泌量逐渐减少。呈现出显著的浓度梯度效应。
3.三种模拟肽均表现出一定的抗菌活性,对各种细菌的MIC为16～256μg/ml,抑菌活性从强到弱分别为REMP1 > REMP2 > XS10, REMP1和REMP2还对属于革兰阳性细菌的金黄色葡萄球菌表现出一定的抑菌活性。
4.电镜下REMP1或REMP2作用后大肠杆菌内外膜变模糊、毛糙,菌体肿胀,胞质空泡变性,有的菌体裂解破碎。
5. XS10 /REMP1 /REMP2三种模拟肽对RAW264.7细胞无毒性作用。第二部分XS10 / REMP1 / REMP2体内抗内毒素活性研究
方法:
1. D-氨基半乳糖(D-Gal)致敏昆明小鼠LPS休克模型的建立:不同剂量的D-Gal和LPS先后1h腹腔注射于昆明小鼠,找出LD100的最佳剂量组合。
2. XS10 /REMP1 /REMP2对LPS休克小鼠保护的剂量效应:复制昆明小鼠LPS休克模型,观察三种模拟肽在0.5mg/kg、1.0mg/kg、2.0mg/kg剂量下与LPS同时注射时对小鼠的保护率。
3.不同时间注射REMP2对LPS休克小鼠的保护效应:观察LPS攻击前1h和LPS攻击后1h注射REMP2(2.0mg/kg)对LPS休克小鼠的保护率。
4. XS10 / REMP1 / REMP2对LPS休克小鼠血清TNF-α的影响:用ELISA法检测模拟肽作用后LPS休克小鼠血清TNF-α的变化。
5. REMP1、REMP2对LPS休克小鼠重要脏器的病理学影响:REMP1、REMP2与LPS同时注射于D-Gal致敏小鼠后5h,取小鼠心脏、肺脏、肝脏、小肠、肾脏常规病理学检查。
结果:
1.预先注射D-Gal可使昆明小鼠对LPS敏感性极大增强,D-Gal 500mg/kg + LPS 250μg /kg组及D-Gal 600mg/kg + LPS 50μg/kg组小鼠72h内死亡率达100%。模型复制采用D-Gal 600mg/kg + LPS 50μg/kg剂量组合。
2. 0.5、1.0、2.0mg/kg三种剂量下,XS10、REMP1、REMP2对LPS休克小鼠均有一定保护作用,REMP2保护作用最强,保护率依次为30%、60%和90%,与PMB比较P>0.05。
3. LPS攻击前1h和攻击后1h注射REMP2对LPS休克小鼠的保护率分别为30%和50%,保护效果均低于同时注射组。
4. LPS攻击后75分钟TNF-α升高至6365±2087 pg/ml,PMB及模拟肽作用组TNF-α水平降低,REMP1和REMP2组分别为3811±1236 pg/ml和905±264 pg/ml,与LPS组比较P<0.05,REMP2组与PMB组比较P>0.05。
5. D-Gal组除肝脏有轻微病理改变外,其余脏器无病理改变,LPS组小鼠各脏器均出现炎症性病理改变,尤以肺脏和肝脏明显,REMP1和REMP2组各脏器病变程度减轻。
结论
1.环状鲎抗内毒素因子模拟肽XS10、REMP1、REMP2在溶液内可以直接中和LPS,使溶液内所能检测到的LPS减少。
2.XS10、REMP1、REMP2对LPS刺激下小鼠RAW264.7细胞所产生的炎症介质TNF-α有抑制作用。
3.XS10、REMP1、REMP2对临床常见的革兰阴性杆菌具有一定杀菌作用,而且还对金黄色葡萄球菌(革兰阳性菌)有一定抑制作用。
4.MTT实验表明XS10、REMP1、REMP2没有细胞毒性。
5.XS10、REMP1、REMP2可以降低LPS休克小鼠的死亡率。
6.XS10、REMP1、REMP2能够降低LPS休克小鼠血清TNF-α水平。
7.REMP1、REMP2可以减轻LPS休克小鼠心脏、肺脏、肝脏、肾脏、小肠的炎性病理变化。
8.三种模拟肽各自在体外和体内的抗LPS及抗菌活性表现一致,抗LPS活性从强到弱依次为REMP2 >REMP1 > XS10,抗菌活性从强到弱依次为REMP1 > REMP2 > XS10。
9.模拟肽REMP2体内外的抗LPS活性与PMB相当,并且无细胞毒性,具有潜在的药用开发价值。
Lipopolysaccharide (LPS), also called endotoxin, is a major component of the outer membrane of gram-negative bacteria. It is considered to be an important mediator of many of the responses manifested during the development of gram-negative bacterial sepsis and septic shock, which often leads to resultant multiple organ dysfunction and death in intensive care patients. The promising strategies targeting sepsis are preventing LPS from binding to its receptors, blockage of LPS intracellular signal transduction, antagonism of cytokine and application of glucocorticoids,but unfortunately all these therapies got little clinical benefits. There is no effective and safe drugs available till now.
The Limulus anti-lipopolysaccharide factor and Tachypleus anti-lipopolysaccharide factor(LALF,TALF)from both limulus polyphemus and Tachypleus tridentatus Leach have been proved striking anti-gram negative bacillus as well as anti-LPS effects in vitro and in vivo. Based on the analysis of LALF and TALF structural features as well as their functional domains by way of bioinformatics methods, we designed and synthetized a series of peptides by computational molecular modeling. In this study we will investigate the bactericidal and antiendotoxic properties of synthetic cyclic peptides XS10, REMP1 and REMP2.
Section 1. Bactericidal and antiendotoxic activities of XS10/REMP1/REMP2 in vitro.
Methods:
1. The LAL(Limulus Amebocyte Lysate)test was to determine the ability of XS10/ REMP1/ REMP2 to neutralize LPS in vitro. Various concentrations of the peptides and Polymyxin B (PMB) were incubated with LPS respectively , The kinetic turbidity was measured using a endoxin detecte system. The LPS neutralizing rates were calculated.
2. Mouse RAW264.7 macrophages were cultured and then 10μl LPS (100 ng/ml) and equal volume peptides and PMB solutions (10, 20, 40 and 80μM) were added simultaneously. After cells were incubated for 4 h, the levels of TNF-αin the supernatants were analyzed using the ELISA kits.
3. The minimal inhibitory concentration (MIC) of XS10 \REMP1 \REMP2 for the Escherichia coli, Pyocyaneum bacterium, Staphylococcus aureus, et al were determined by agar dilution.
4. The morphological changes of E.coli ATCC 25922 affected by REMP1 and REMP2 were observed by transmission electron microscope.
5. Cytotoxicity of XS10, REMP1 and REMP2 to the Mouse RAW264.7 macrophages was established using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.
Results:
1. XS10,REMP1,REMP2 and PMB exhibited neutralizing activities on LPS in a dose-dependent manner. The activities decrease progressively from REMP2, REMP1 to XS10. REMP2 was the best with the neutralizing rates from 5.66% to 88.66%, which were similar to PMB.
2. Treatments of cells with XS10, REMP1, REMP2 and PMB both inhibited the TNF-αrelease in response to the presence of LPS in a dose-dependent manner. The TNF-αdecreased significantly compared with the control group (p<0.05).
3. All the peptides have Bactericidal activities. the MIC were from 16μg/ml to 256μg/ml for the bacteria in test. The activities decrease progressively from REMP1, REMP2 to XS10. moreover REMP1 and REMP2 have bactericidal activities to Staphylococcus aureus which is belong to gram positive coccus.
4. Under the electron microscope the outer and interior membranes of Escherichia coli. affected by REMP1 and REMP2 was obscure, the bacterium swelled,vacuoles formation and even ruptured.
5. XS10, REMP1 and REMP was not observed to be toxic to mouse macrophages at the concentrations tested in the MTT test. Section 2. Antiendotoxic activities of XS10/REMP1/REMP2 in vivo.
Methods:
1. To generate the murine endotoxin shock model induced by D-galactosamine- sensitized KunMing mice, Kunming mice were stimulated with intraperitoneal(i.p.) injection of different dosage of D-galactosamine(D-Gal) and LPS one hour later. Mice lethality was recorded at 72 h. the dose of LD100 were chosed.
2. Generate the murine endotoxin shock models. groups of 10 mice were injected with the peptides and PMB(0.5, 1.0 and 2.0 mg/kg) intraperitoneally, together with LPS. Survivals was recorded every 6 h during the period of 72 h.
3. Generate the murine endotoxin shock models. groups of 10 mice received an i.p. injection of peptides and PMB (2.0 mg/kg) at indicated time points: (1) 1h before LPS; (2) 1h after LPS. Survivals was recorded every 6 h during the period of 72 h.
4. Mice endotoxin shock models was made. Seventy-five minutes after intra-peritoneal injection of peptides, PMB(2.0 mg/kg) and LPS, blood samples were collected from the sacrificed mice. The levels of TNF-αin serum samples were analyzed using the ELISA kits.
5. Mice endotoxin shock models was made. Mice were injected intraperitoneally with REMP1 and REMP2 (2.0 mg/kg) together with the LPS. Five hours later the mice were sacrificed and the lung, heart, liver, intestine and kidney removed for histopathology.
Results:
1. Treatment of mice with D-galactosamine increased remarkably their sensitivity to the lethal effects of LPS. The dosage of D-Gal 500mg/kg + LPS 250μg /kg or D-Gal 600mg/kg + LPS 50μg/kg could produc 100% mortality in 72 h.
2. The survival rates of LPS- attacked mice increased significantly with peptides intervention at the three dosages (p < 0.05). REMP2 have show the highest rescue rates with 30%, 60% and 90% at the dosages of 0.5, 1.0 and 2.0mg/kg.
3. The survival rates of LPS-challenged mice after 72 h were 30%, and 50% when receiving REMP2 injection 1h before or 1h after LPS respectivel. The protection effects were less than that of injection together with LPS.
4. the TNF-αconcentrations of mice challenged by LPS alone increased fiercely to 6365±2087 pg/ml, but decreased significantly after treatment with the peptides. The inhibitory efficacy of REMP2(905±264 pg /m l) was nearly at the equal grade with PMB(p > 0.05).
5. LPS-attacked mice produced acute inflammation in the lung, heart, liver, intestine and kidney in mice of control group. In contrast, with REMP1 and REMP2 treatment, there was significantly abatement of inflammation.
Conclusions:
1. The synthetic cyclic peptides XS10/REMP1/REMP2 based on LALF can directly neutralize the LPS in solutions.
2. XS10, REMP1 and REMP2 can inhibit the TNF-αrelease in response to the presence of LPS in Mouse RAW264.7 macrophages.
3. XS10, REMP1 and REMP2 have bactericidal activities to some of the bacteria obtaine from clinical patients including the Staphylococcus aureus which is belong to gram positive coccus.
4. XS10, REMP1 and REMP2 have not Cytotoxicity.
5. XS10, REMP1 and REMP2 can increase survival rates of mice challenged by lethal LPS.
6. XS10, REMP1 and REMP2 can decrease the TNF-αlevels in endotoxin shock mice.
7. REMP1 and REMP2 can decrease inflammation in the lung, heart, liver, intestine and kidney in mice of endotoxin shock.
8. The bactericidal and antiendotoxic activities of XS10, REMP1 and REMP2 in vivo are in line with that in vitro. The antiendotoxic activities degree are REMP2 > REMP1 > XS10 and the bactericidal activities degree are REMP1 > REMP2 > XS10。
9. REMP2 have the antiendotoxic activities nearly at the equal grade with PMB, but have less bactericidal activities than that of PMB.
第一部分、XS10/REMP1/REMP2体外中和内毒素及抗菌活性研究。
方法:
1. XS10 /REMP1 /REMP2直接中和LPS实验:以多黏菌素B(PMB)为对照,不同浓度模拟肽溶液分别与LPS工作标准品溶液直接混合反应,应用EDS-99细菌LPS测定系统,测定反应后溶液中LPS含量,计算中和率。
2. XS10 /REMP1 /REMP2对LPS介导的小鼠RAW264.7细胞产生TNF-α的抑制作用:以PMB为对照,将PMB及各模拟肽梯度浓度溶液和LPS溶液同时加入细胞培养液中,孵育一定时间后用ELISA试剂盒检测细胞上清液内的TNF-α含量。
3. XS10 /REMP1 /REMP2最低抑菌浓度(minimal inhibitory concentration,MIC)测定:采用琼脂稀释法,对大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌等标准菌株及临床分离菌株进行MIC测定。
4. REMP1 /REMP2对E.coli ATCC 25922形态的影响:分别将一定浓度的REMP1和REMP2加入大肠杆菌标准菌株E.coli ATCC 25922培养液中,作用一定时间后,透射电镜下观察E.coli ATCC 25922的形态学变化。
5. XS10 /REMP1 /REMP2细胞毒性实验:采用MTT实验测定。
结果:
1. XS10 /REMP1 /REMP2均有不同程度的直接中和LPS的作用.并且随浓度升高而增强,中和能力从强到弱依次为REMP2 >REMP1> XS10,以REMP2最强,在0.01～100μM浓度下对1.0EU/ml的LPS中和率分别为:(5.66±2.08)%～(88.66±5.03)%,接近PMB中和能力。
2. XS10 /REMP1 /REMP2在10μM、20μM、40μM、80μM浓度下,均能不同程度抑制LPS介导的TNF-α产生,与阳性对照组比较P<0.05,且随着浓度增高,TNF-α的分泌量逐渐减少。呈现出显著的浓度梯度效应。
3.三种模拟肽均表现出一定的抗菌活性,对各种细菌的MIC为16～256μg/ml,抑菌活性从强到弱分别为REMP1 > REMP2 > XS10, REMP1和REMP2还对属于革兰阳性细菌的金黄色葡萄球菌表现出一定的抑菌活性。
4.电镜下REMP1或REMP2作用后大肠杆菌内外膜变模糊、毛糙,菌体肿胀,胞质空泡变性,有的菌体裂解破碎。
5. XS10 /REMP1 /REMP2三种模拟肽对RAW264.7细胞无毒性作用。第二部分XS10 / REMP1 / REMP2体内抗内毒素活性研究
方法:
1. D-氨基半乳糖(D-Gal)致敏昆明小鼠LPS休克模型的建立:不同剂量的D-Gal和LPS先后1h腹腔注射于昆明小鼠,找出LD100的最佳剂量组合。
2. XS10 /REMP1 /REMP2对LPS休克小鼠保护的剂量效应:复制昆明小鼠LPS休克模型,观察三种模拟肽在0.5mg/kg、1.0mg/kg、2.0mg/kg剂量下与LPS同时注射时对小鼠的保护率。
3.不同时间注射REMP2对LPS休克小鼠的保护效应:观察LPS攻击前1h和LPS攻击后1h注射REMP2(2.0mg/kg)对LPS休克小鼠的保护率。
4. XS10 / REMP1 / REMP2对LPS休克小鼠血清TNF-α的影响:用ELISA法检测模拟肽作用后LPS休克小鼠血清TNF-α的变化。
5. REMP1、REMP2对LPS休克小鼠重要脏器的病理学影响:REMP1、REMP2与LPS同时注射于D-Gal致敏小鼠后5h,取小鼠心脏、肺脏、肝脏、小肠、肾脏常规病理学检查。
结果:
1.预先注射D-Gal可使昆明小鼠对LPS敏感性极大增强,D-Gal 500mg/kg + LPS 250μg /kg组及D-Gal 600mg/kg + LPS 50μg/kg组小鼠72h内死亡率达100%。模型复制采用D-Gal 600mg/kg + LPS 50μg/kg剂量组合。
2. 0.5、1.0、2.0mg/kg三种剂量下,XS10、REMP1、REMP2对LPS休克小鼠均有一定保护作用,REMP2保护作用最强,保护率依次为30%、60%和90%,与PMB比较P>0.05。
3. LPS攻击前1h和攻击后1h注射REMP2对LPS休克小鼠的保护率分别为30%和50%,保护效果均低于同时注射组。
4. LPS攻击后75分钟TNF-α升高至6365±2087 pg/ml,PMB及模拟肽作用组TNF-α水平降低,REMP1和REMP2组分别为3811±1236 pg/ml和905±264 pg/ml,与LPS组比较P<0.05,REMP2组与PMB组比较P>0.05。
5. D-Gal组除肝脏有轻微病理改变外,其余脏器无病理改变,LPS组小鼠各脏器均出现炎症性病理改变,尤以肺脏和肝脏明显,REMP1和REMP2组各脏器病变程度减轻。
结论
1.环状鲎抗内毒素因子模拟肽XS10、REMP1、REMP2在溶液内可以直接中和LPS,使溶液内所能检测到的LPS减少。
2.XS10、REMP1、REMP2对LPS刺激下小鼠RAW264.7细胞所产生的炎症介质TNF-α有抑制作用。
3.XS10、REMP1、REMP2对临床常见的革兰阴性杆菌具有一定杀菌作用,而且还对金黄色葡萄球菌(革兰阳性菌)有一定抑制作用。
4.MTT实验表明XS10、REMP1、REMP2没有细胞毒性。
5.XS10、REMP1、REMP2可以降低LPS休克小鼠的死亡率。
6.XS10、REMP1、REMP2能够降低LPS休克小鼠血清TNF-α水平。
7.REMP1、REMP2可以减轻LPS休克小鼠心脏、肺脏、肝脏、肾脏、小肠的炎性病理变化。
8.三种模拟肽各自在体外和体内的抗LPS及抗菌活性表现一致,抗LPS活性从强到弱依次为REMP2 >REMP1 > XS10,抗菌活性从强到弱依次为REMP1 > REMP2 > XS10。
9.模拟肽REMP2体内外的抗LPS活性与PMB相当,并且无细胞毒性,具有潜在的药用开发价值。
Lipopolysaccharide (LPS), also called endotoxin, is a major component of the outer membrane of gram-negative bacteria. It is considered to be an important mediator of many of the responses manifested during the development of gram-negative bacterial sepsis and septic shock, which often leads to resultant multiple organ dysfunction and death in intensive care patients. The promising strategies targeting sepsis are preventing LPS from binding to its receptors, blockage of LPS intracellular signal transduction, antagonism of cytokine and application of glucocorticoids,but unfortunately all these therapies got little clinical benefits. There is no effective and safe drugs available till now.
The Limulus anti-lipopolysaccharide factor and Tachypleus anti-lipopolysaccharide factor(LALF,TALF)from both limulus polyphemus and Tachypleus tridentatus Leach have been proved striking anti-gram negative bacillus as well as anti-LPS effects in vitro and in vivo. Based on the analysis of LALF and TALF structural features as well as their functional domains by way of bioinformatics methods, we designed and synthetized a series of peptides by computational molecular modeling. In this study we will investigate the bactericidal and antiendotoxic properties of synthetic cyclic peptides XS10, REMP1 and REMP2.
Section 1. Bactericidal and antiendotoxic activities of XS10/REMP1/REMP2 in vitro.
Methods:
1. The LAL(Limulus Amebocyte Lysate)test was to determine the ability of XS10/ REMP1/ REMP2 to neutralize LPS in vitro. Various concentrations of the peptides and Polymyxin B (PMB) were incubated with LPS respectively , The kinetic turbidity was measured using a endoxin detecte system. The LPS neutralizing rates were calculated.
2. Mouse RAW264.7 macrophages were cultured and then 10μl LPS (100 ng/ml) and equal volume peptides and PMB solutions (10, 20, 40 and 80μM) were added simultaneously. After cells were incubated for 4 h, the levels of TNF-αin the supernatants were analyzed using the ELISA kits.
3. The minimal inhibitory concentration (MIC) of XS10 \REMP1 \REMP2 for the Escherichia coli, Pyocyaneum bacterium, Staphylococcus aureus, et al were determined by agar dilution.
4. The morphological changes of E.coli ATCC 25922 affected by REMP1 and REMP2 were observed by transmission electron microscope.
5. Cytotoxicity of XS10, REMP1 and REMP2 to the Mouse RAW264.7 macrophages was established using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.
Results:
1. XS10,REMP1,REMP2 and PMB exhibited neutralizing activities on LPS in a dose-dependent manner. The activities decrease progressively from REMP2, REMP1 to XS10. REMP2 was the best with the neutralizing rates from 5.66% to 88.66%, which were similar to PMB.
2. Treatments of cells with XS10, REMP1, REMP2 and PMB both inhibited the TNF-αrelease in response to the presence of LPS in a dose-dependent manner. The TNF-αdecreased significantly compared with the control group (p<0.05).
3. All the peptides have Bactericidal activities. the MIC were from 16μg/ml to 256μg/ml for the bacteria in test. The activities decrease progressively from REMP1, REMP2 to XS10. moreover REMP1 and REMP2 have bactericidal activities to Staphylococcus aureus which is belong to gram positive coccus.
4. Under the electron microscope the outer and interior membranes of Escherichia coli. affected by REMP1 and REMP2 was obscure, the bacterium swelled,vacuoles formation and even ruptured.
5. XS10, REMP1 and REMP was not observed to be toxic to mouse macrophages at the concentrations tested in the MTT test. Section 2. Antiendotoxic activities of XS10/REMP1/REMP2 in vivo.
Methods:
1. To generate the murine endotoxin shock model induced by D-galactosamine- sensitized KunMing mice, Kunming mice were stimulated with intraperitoneal(i.p.) injection of different dosage of D-galactosamine(D-Gal) and LPS one hour later. Mice lethality was recorded at 72 h. the dose of LD100 were chosed.
2. Generate the murine endotoxin shock models. groups of 10 mice were injected with the peptides and PMB(0.5, 1.0 and 2.0 mg/kg) intraperitoneally, together with LPS. Survivals was recorded every 6 h during the period of 72 h.
3. Generate the murine endotoxin shock models. groups of 10 mice received an i.p. injection of peptides and PMB (2.0 mg/kg) at indicated time points: (1) 1h before LPS; (2) 1h after LPS. Survivals was recorded every 6 h during the period of 72 h.
4. Mice endotoxin shock models was made. Seventy-five minutes after intra-peritoneal injection of peptides, PMB(2.0 mg/kg) and LPS, blood samples were collected from the sacrificed mice. The levels of TNF-αin serum samples were analyzed using the ELISA kits.
5. Mice endotoxin shock models was made. Mice were injected intraperitoneally with REMP1 and REMP2 (2.0 mg/kg) together with the LPS. Five hours later the mice were sacrificed and the lung, heart, liver, intestine and kidney removed for histopathology.
Results:
1. Treatment of mice with D-galactosamine increased remarkably their sensitivity to the lethal effects of LPS. The dosage of D-Gal 500mg/kg + LPS 250μg /kg or D-Gal 600mg/kg + LPS 50μg/kg could produc 100% mortality in 72 h.
2. The survival rates of LPS- attacked mice increased significantly with peptides intervention at the three dosages (p < 0.05). REMP2 have show the highest rescue rates with 30%, 60% and 90% at the dosages of 0.5, 1.0 and 2.0mg/kg.
3. The survival rates of LPS-challenged mice after 72 h were 30%, and 50% when receiving REMP2 injection 1h before or 1h after LPS respectivel. The protection effects were less than that of injection together with LPS.
4. the TNF-αconcentrations of mice challenged by LPS alone increased fiercely to 6365±2087 pg/ml, but decreased significantly after treatment with the peptides. The inhibitory efficacy of REMP2(905±264 pg /m l) was nearly at the equal grade with PMB(p > 0.05).
5. LPS-attacked mice produced acute inflammation in the lung, heart, liver, intestine and kidney in mice of control group. In contrast, with REMP1 and REMP2 treatment, there was significantly abatement of inflammation.
Conclusions:
1. The synthetic cyclic peptides XS10/REMP1/REMP2 based on LALF can directly neutralize the LPS in solutions.
2. XS10, REMP1 and REMP2 can inhibit the TNF-αrelease in response to the presence of LPS in Mouse RAW264.7 macrophages.
3. XS10, REMP1 and REMP2 have bactericidal activities to some of the bacteria obtaine from clinical patients including the Staphylococcus aureus which is belong to gram positive coccus.
4. XS10, REMP1 and REMP2 have not Cytotoxicity.
5. XS10, REMP1 and REMP2 can increase survival rates of mice challenged by lethal LPS.
6. XS10, REMP1 and REMP2 can decrease the TNF-αlevels in endotoxin shock mice.
7. REMP1 and REMP2 can decrease inflammation in the lung, heart, liver, intestine and kidney in mice of endotoxin shock.
8. The bactericidal and antiendotoxic activities of XS10, REMP1 and REMP2 in vivo are in line with that in vitro. The antiendotoxic activities degree are REMP2 > REMP1 > XS10 and the bactericidal activities degree are REMP1 > REMP2 > XS10。
9. REMP2 have the antiendotoxic activities nearly at the equal grade with PMB, but have less bactericidal activities than that of PMB.
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