鲎抗内毒素因子模拟肽CLP-19诱导RAW264.7细胞内毒素耐受的实验研究
发布时间：2018-08-22 14:32
【摘要】：研究背景及目的： 脓毒症(Sepsis)是临床上一种常见的危重病症，是指由感染引起的全身炎症反应综合征(Systemic inflammatory response syndrome，SIRS)，具有发病率高、死亡率高的特点。作为革兰氏阴性菌细菌细胞壁外层的主要成分之一的内毒素(Lipopolysaccharide,LPS)除了能引发脓毒症外，还可通过诱导“内毒素耐受”，降低各种原因引起的炎症反应，从而预防潜在脓毒症的发生。内毒素耐受是指动物或免疫细胞等在给予小剂量LPS预处理后，对LPS再次刺激时无反应或反应性明显降低。因此，寻找一种可诱导内毒素耐受同时又不诱导机体产生炎性反应的药物是抗感染及抗炎药物研究的新策略。 环状鲎肽(Cyclic limulus peptide，CLP-19)是经美洲鲎抗内毒素因子(Limulusanti-lipopolysaccharide factor, LALF)结构改造后得到的一条无溶血性、对巨噬细胞等无致炎作用的环肽，具有较理想中和LPS及抗菌活性。课题组前期研究表明，提前给予CLP-19可显著降低血清肿瘤坏死因子-α (Tumour Necrosis Factor, TNF-α)水平、提高大肠杆菌致急性腹膜炎模型鼠的生存率；预防性给予CLP-19能显著减少大肠杆菌致急性腹膜炎模型鼠体内的细菌量。在细胞水平，CLP-19预刺激30min后，以磷酸盐缓冲盐水(Phosphate buffered saline, PBS)清洗细胞，再用LPS进行刺激，结果发现，CLP-19预处理组同样可显著降低LPS刺激所引起的TNF-α的升高。上述现象表明，CLP-19可诱导“内毒素耐受”，然而其机制有待深入研究。 因此，为明确CLP-19诱导内毒素耐受效应及分子机制，本实验通过建立RAW264.7细胞内毒素耐受模型，利用逆转录聚合酶链式反应(Reverse TranscriptionPolymerase Chain Reaction, RT-PCR)、免疫印迹法(Western Blotting, WB)等比较研究巨噬细胞炎症信号通路中关键因子的mRNA及蛋白的变化，初步探讨CLP-19诱导内毒素耐受的分子机制。通过以上研究，拟明确CLP-19诱导小鼠“内毒素耐受”的作用及其途径，并为其用于脓毒症预防提供理论依据。 实验方法： 第一部分：CLP-19诱导内毒素耐受的时间效应和剂量效应研究 1. ELISA分析LPS诱导内毒素耐受的时间效应和剂量效应 不同浓度(0ng/ml、0.05ng/ml、0.1ng/ml、0.2ng/ml、0.5ng/ml、1ng/ml、5ng/ml、7ng/ml、10ng/ml)的LPS作用于RAW264.7细胞，培养不同时间(10h、20h、24h)后，新鲜培养基培养2h，再给予10ng/ml的LPS作用于细胞，4h后取上清液，酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)观察不同时间、不同剂量下TNF-α的表达量，从而确定LPS诱导内毒素耐受的最佳时间和剂量。 2. ELISA分析CLP-19诱导内毒素耐受的时间效应和剂量效应 用不同浓度(0μg/ml、0.1μg/ml、1μg/ml、5μg/ml、10μg/ml、20μg/ml、50μg/ml、100μg/ml)的CLP-19作用于RAW264.7细胞，培养不同时间(10h、20h、24h)后，新鲜培养基培养2h，再加入LPS使其终浓度为10ng/ml，，4h后取上清液，测定不同时间、不同剂量下TNF-α的表达量和IL-10的表达量，从而确定CLP-19诱导内毒素耐受的最佳时间和剂量。以LPS诱导内毒素耐受的最佳时间和剂量为对照组。 3. CLP-19诱导内毒素耐受后IL-6和IL-10表达变化 分别以5ng/ml LPS、50μg/ml CLP-19孵育细胞20h，用PBS反复清洗后以新鲜培养基培养2h，再加入LPS使其终浓度为10ng/ml，4h后取上清液测定IL-6和IL-10表达变化。 第二部分：CLP-19诱导RAW264.7内毒素耐受的分子机制研究 1. CLP-19诱导内毒素耐受后胞内TLR4、myD88、TRAF6的mRNA表达变化 用50μg/ml CLP-19预处理细胞20h，再用培养基培养2h，向各组中均加入10ng/mlLPS处理细胞不同时间(0h、1h、3h、6h、12h)后，提取细胞RNA，通过RT-PCR检测细胞因子Toll样受体4(Toll like receptor, TLR4)、髓样细胞分化因子88(mydoidMyD88，MyD88)、肿瘤坏死因子受体相关因子6(TNF receptor-associated factor6，TRAF6)的mRNA变化。以培养基和5ng/ml LPS预处理作为对照。 2. CLP-19诱导内毒素耐受后胞内和胞膜TLR4的蛋白表达变化 用50μg/ml CLP-19预处理细胞20h，用培养基培养2h后，再向各组中加入10ng/mlLPS作用4h后，分别提取胞膜和胞内蛋白，Western Blot检测胞膜和胞内TLR4蛋白的表达变化。以培养基预处理作为对照。 3. CLP-19诱导内毒素耐受后P38的磷酸化水平变化 用50μg/ml CLP-19预处理细胞20h，用培养基培养2h后，向各组中均加入10ng/mlLPS分别作用不同时间(0min、10min、30min、60min)后，提取细胞总蛋白，Western Blot检测促分裂原活化蛋白激酶(Mitogen-activated protein kinase, MAPK)通路中P38的磷酸化水平。以培养基和5ng/ml LPS预处理作为对照。 4. CLP-19诱导内毒素耐受后Iκ-B的蛋白表达变化 用50μg/ml CLP-19预处理细胞20h，用培养基培养2h后，再向各组中加入10ng/mlLPS作用不同时间(0min、5min、15min、30min、60min)后，提取细胞总蛋白，WesternBlot检测核因子κB抑制蛋白(I-kappa-B，IκB)的蛋白表达变化。以培养基预处理作为对照。 结果： 第一部分：CLP-19诱导内毒素耐受的时间效应和剂量效应研究 5ng/ml LPS预处理RAW264.7细胞20h为LPS诱导内毒素耐受的最佳剂量和时间。50μg/ml CLP-19预处理RAW264.7细胞20h为CLP-19诱导内毒素耐受的最佳剂量和时间。50μg/ml CLP-19预处理巨噬细胞20h后，炎症因子IL-6表达下调，抗炎因子IL-10表达增加，与培养基预处理组相比，均有显著差异(P0.01)。 第二部分：CLP-19诱导RAW264.7细胞内毒素耐受的分子机制研究 1. CLP-19诱导内毒素耐受后胞内TLR4、myD88和TRAF6的mRNA表达变化 RT-PCR结果显示，与培养基预处理细胞组比较，CLP-19预处理细胞组中，随着刺激时间的延长，TLR4mRNA表达下调，差异有统计学意义(P0.01)。而MyD88和TRAF6的mRNA表达均无明显变化(P㧐0.05)。 2. CLP-19诱导内毒素耐受后胞内和胞膜TLR4蛋白表达降低 Western Blot结果显示，与培养基预处理组相比，CLP-19诱导的内毒素耐受组中，胞膜和胞内TLR4的蛋白表达均下调，有显著差异(P0.05)。 3. CLP-19诱导内毒素耐受后MAPK通路中P38的磷酸化被抑制 Western Blot结果显示，培养基预处理细胞组中，P38的磷酸化水平在刺激15min后明显增加，30min后达到高峰。与培养基预处理细胞组相比，CLP-19诱导的耐受组中，P38的磷酸化水平均被显著抑制(P0.01)。 4. CLP-19诱导内毒素耐受后IκB未被降解 Western Blot结果显示，培养基预处理细胞组中，IκBα在大剂量LPS刺激30min内迅速降解，IκBβ在大剂量LPS刺激60min后迅速降解。与培养基预处理细胞组相比，CLP-19诱导的耐受组中，IκBα和IκBβ表达水平有显著差异(P0.01)。 结论： 1.50μg/ml CLP-19预处理RAW264.7细胞20h为CLP-19诱导内毒素耐受的最佳剂量和时间。 2.鲎抗内毒素因子模拟肽CLP-19可通过降低TLR4在胞膜和胞内的表达诱导内毒素耐受。 3.鲎抗内毒素因子模拟肽CLP-19可通过抑制P38磷酸化诱导内毒素耐受。 4.鲎抗内毒素因子模拟肽CLP-19可抑制IκBα和IκBβ的降解，从而抑制NF-κB转位进入胞核，进而诱导内毒素耐受。
[Abstract]:Background and purpose:
Sepsis is a common and critical disease in clinic. It refers to the systemic inflammatory response syndrome (SIRS) caused by infection. It has the characteristics of high morbidity and high mortality. In addition to causing sepsis, endotoxin tolerance can also be induced to reduce inflammation caused by various causes, thereby preventing the occurrence of potential sepsis. Endotoxin tolerance refers to the fact that animals or immune cells do not respond to LPS after pretreatment with low doses of LPS, or their reactivity to LPS is significantly reduced. Drugs that induce endotoxin tolerance without inflammation are new strategies for the study of anti-infective and anti-inflammatory drugs.
Cyclic Limulus peptide (CLP-19) is a non-hemolytic, non-inflammatory cyclic peptide derived from the structural modification of Limulus anti-lipopolysaccharide factor (LALF), which has ideal neutralizing LPS and antimicrobial activity. Previous studies of the research group showed that early administration of CLP-19 can significantly improve the hemolytic activity of the peptide. To reduce the level of serum tumor necrosis factor-alpha (TNF-alpha) and increase the survival rate of E.coli-induced acute peritonitis model mice, CLP-19 could significantly reduce the bacterial count in E.coli-induced acute peritonitis model mice. At the cellular level, CLP-19 was pretreated with phosphate buffered saline (Phos) after 30 minutes. Phase buffered saline (PBS) was used to clean the cells and stimulate them with LPS. The results showed that CLP-19 pretreatment could also significantly reduce the elevation of TNF-alpha induced by LPS stimulation.
Therefore, in order to clarify the effect and molecular mechanism of endotoxin tolerance induced by CLP-19, the cytotoxic tolerance model of RAW264.7 cells was established. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB) were used to study the inflammatory signaling pathway of macrophages. To explore the molecular mechanism of CLP-19 inducing endotoxin tolerance, the changes of mRNA and protein of the key factors in CLP-19 inducing endotoxin tolerance were studied.
Experimental methods:
Part I: time and dose effects of endotoxin tolerance induced by CLP-19
1. ELISA analysis of the time effect and dose effect of LPS induced endotoxin tolerance
LPS of different concentrations (0 ng/ml, 0.05 ng/ml, 0.1 ng/ml, 0.2 ng/ml, 0.5 ng/ml, 1 ng/ml, 5 ng/ml, 7 ng/ml, 10 ng/ml) acted on RAW264.7 cells. After cultured in different time (10 h, 20 h, 24 h), fresh medium was cultured for 2 h, then LPS of 10 ng/ml acted on the cells, supernatant was taken out after 4 h, and enzyme-linked immunosorbent assay (ELISA) was used. To determine the optimal time and dose of LPS-induced endotoxin tolerance, the expression of TNF-alpha was measured at different time and dosage.
2. ELISA analysis of the time effect and dose effect of CLP-19 induced endotoxin tolerance
RAW264.7 cells were treated with CLP-19 at different concentrations (0,0.1,1,5,10,20,50,100,100,etc.) for 2 hours in fresh medium (10,20,24 hours). Then the final concentration of LPS was 10 ng/ml. After 4 hours, the expression of TNF-alpha and IL-10 in the supernatant were measured at different time and dosage. The optimal time and dosage of CLP-19 inducing endotoxin tolerance were determined by the expression level. The optimal time and dosage of LPS inducing endotoxin tolerance were used as control group.
Expression of IL-6 and IL-10 after endotoxin tolerance induced by 3. CLP-19
The cells were incubated with 5 ng/ml LPS and 50 ug/ml CLP-19 for 20 hours. After repeated washing with PBS, the cells were cultured in fresh medium for 2 hours. The final concentration of LPS was 10 ng/ml. The expression of IL-6 and IL-10 in the supernatant was determined after 4 hours.
The second part: molecular mechanism of CLP-19 induced endotoxin tolerance in RAW264.7.
1. the expression of TLR4, myD88 and TRAF6 mRNA after endotoxin tolerance induced by CLP-19
Cells were pretreated with 50 ug/ml CLP-19 for 20 hours, then cultured in medium for 2 hours. The cells were treated with 10 ng/ml LPS for different time (0 h, 1 h, 3 h, 6 h, 12 h). RNA was extracted. Toll-like receptor 4 (TLR4), myeloid cell differentiation factor 88 (mydoid MyD88, MyD88), tumor necrosis factor receptor phase were detected by RT-PCR. The mRNA changes of TNF receptor-associated factor 6 (TRAF6) were compared with that of medium and 5 ng/ml LPS pretreatment.
2. the expression of TLR4 protein in intracellular and membrane after induction of endotoxin tolerance by CLP-19
Cells were pretreated with 50 ug/ml CLP-19 for 20 hours, cultured in medium for 2 hours, then treated with 10 ng/ml LPS for 4 hours. The membrane and intracellular proteins were extracted respectively. The expression of TLR4 protein was detected by Western Blot.
Changes of P38 phosphorylation level after endotoxin tolerance induced by 3. CLP-19
Cells were pretreated with 50 ug/ml CLP-19 for 20 hours, cultured in medium for 2 hours, and then treated with 10 ng/ml LPS for different time (0 min, 10 min, 30 min, 60 min) respectively. Total protein was extracted. The phosphorylation level of P38 in mitogen-activated protein kinase (MAPK) pathway was detected by Western Blot. Pretreatment with 5ng/ml LPS was used as a control.
Protein expression of I kappa -B after endotoxin tolerance induced by 4. CLP-19
Cells were pretreated with 50 ug/ml CLP-19 for 20 hours, cultured in medium for 2 hours, and then treated with 10 ng/ml LPS for different time (0 min, 5 min, 15 min, 30 min, 60 min). Total protein was extracted and the expression of nuclear factor kappa-B inhibitor protein (I-kappa-B, I-kappa-B) was detected by Western Blot.
Result:
Part I: time and dose effects of endotoxin tolerance induced by CLP-19
5 ng/ml LPS preconditioning RAW264.7 cells for 20 hours was the best dose and time for LPS-induced endotoxin tolerance. 50 ug/ml CLP-19 preconditioning RAW264.7 cells for 20 hours was the best dose and time for CLP-19-induced endotoxin tolerance. After 20 hours preconditioning macrophages with 50 ug/ml CLP-19, the expression of inflammatory factor IL-6 was down-regulated and the expression of anti-inflammatory factor IL-10 was increased. Compared with the pretreatment group, there was a significant difference (P0.01).
The second part: the molecular mechanism of endotoxin tolerance induced by CLP-19 in RAW264.7 cells.
1. the expression of TLR4, myD88 and TRAF6 mRNA after endotoxin tolerance induced by CLP-19
The results of RT-PCR showed that the expression of TLR4 mRNA in CLP-19 pretreated cells was down-regulated with the prolongation of stimulation time (P 0.01), while the expression of MyD88 and TRAF6 mRNA was not significantly changed (P 0.05).
2. the expression of TLR4 protein in intracellular and membrane after induction of endotoxin tolerance by CLP-19
Western Blot results showed that the expression of TLR4 protein in the endotoxin tolerance group induced by CLP-19 was down-regulated compared with that in the medium pretreatment group (P 0.05).
3. the phosphorylation of P38 in MAPK pathway was inhibited after CLP-19 induced endotoxin tolerance.
Western Blot results showed that the phosphorylation level of P38 increased significantly after 15 min stimulation and reached its peak after 30 min stimulation. Compared with the tolerance group induced by CLP-19, the phosphorylation level of P38 was significantly inhibited (P 0.01).
After 4. CLP-19 induced endotoxin tolerance, I kappa B was not degraded.
Western Blot results showed that I-kappa B-alpha rapidly degraded within 30 minutes after high-dose LPS stimulation, and I-kappa B-beta rapidly degraded after 60 minutes of high-dose LPS stimulation. Compared with the tolerance group induced by CLP-19, the expression levels of I-kappa B-alpha and I-kappa B-beta were significantly different (P 0.01).
Conclusion:
The optimal dose and time of 20h pretreatment for RAW264.7 cells induced by 1.50 RAW264.7 g/ml CLP-19 were endotoxin tolerance.
2. Limulus antiendotoxin factor mimetic peptide CLP-19 can induce endotoxin tolerance by decreasing the expression of TLR4 on the cell membrane and in the cell.
3. the Limulus anti endotoxin factor mimetic peptide CLP-19 can induce endotoxin tolerance by inhibiting P38 phosphorylation.
4. Limulus anti-endotoxin factor mimic peptide CLP-19 can inhibit the degradation of I-kappa B-alpha and I-kappa B-beta, thereby inhibiting the translocation of NF-kappa B into the nucleus and inducing endotoxin tolerance.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R96;R459.7
本文编号：2197382
[Abstract]:Background and purpose:
Sepsis is a common and critical disease in clinic. It refers to the systemic inflammatory response syndrome (SIRS) caused by infection. It has the characteristics of high morbidity and high mortality. In addition to causing sepsis, endotoxin tolerance can also be induced to reduce inflammation caused by various causes, thereby preventing the occurrence of potential sepsis. Endotoxin tolerance refers to the fact that animals or immune cells do not respond to LPS after pretreatment with low doses of LPS, or their reactivity to LPS is significantly reduced. Drugs that induce endotoxin tolerance without inflammation are new strategies for the study of anti-infective and anti-inflammatory drugs.
Cyclic Limulus peptide (CLP-19) is a non-hemolytic, non-inflammatory cyclic peptide derived from the structural modification of Limulus anti-lipopolysaccharide factor (LALF), which has ideal neutralizing LPS and antimicrobial activity. Previous studies of the research group showed that early administration of CLP-19 can significantly improve the hemolytic activity of the peptide. To reduce the level of serum tumor necrosis factor-alpha (TNF-alpha) and increase the survival rate of E.coli-induced acute peritonitis model mice, CLP-19 could significantly reduce the bacterial count in E.coli-induced acute peritonitis model mice. At the cellular level, CLP-19 was pretreated with phosphate buffered saline (Phos) after 30 minutes. Phase buffered saline (PBS) was used to clean the cells and stimulate them with LPS. The results showed that CLP-19 pretreatment could also significantly reduce the elevation of TNF-alpha induced by LPS stimulation.
Therefore, in order to clarify the effect and molecular mechanism of endotoxin tolerance induced by CLP-19, the cytotoxic tolerance model of RAW264.7 cells was established. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB) were used to study the inflammatory signaling pathway of macrophages. To explore the molecular mechanism of CLP-19 inducing endotoxin tolerance, the changes of mRNA and protein of the key factors in CLP-19 inducing endotoxin tolerance were studied.
Experimental methods:
Part I: time and dose effects of endotoxin tolerance induced by CLP-19
1. ELISA analysis of the time effect and dose effect of LPS induced endotoxin tolerance
LPS of different concentrations (0 ng/ml, 0.05 ng/ml, 0.1 ng/ml, 0.2 ng/ml, 0.5 ng/ml, 1 ng/ml, 5 ng/ml, 7 ng/ml, 10 ng/ml) acted on RAW264.7 cells. After cultured in different time (10 h, 20 h, 24 h), fresh medium was cultured for 2 h, then LPS of 10 ng/ml acted on the cells, supernatant was taken out after 4 h, and enzyme-linked immunosorbent assay (ELISA) was used. To determine the optimal time and dose of LPS-induced endotoxin tolerance, the expression of TNF-alpha was measured at different time and dosage.
2. ELISA analysis of the time effect and dose effect of CLP-19 induced endotoxin tolerance
RAW264.7 cells were treated with CLP-19 at different concentrations (0,0.1,1,5,10,20,50,100,100,etc.) for 2 hours in fresh medium (10,20,24 hours). Then the final concentration of LPS was 10 ng/ml. After 4 hours, the expression of TNF-alpha and IL-10 in the supernatant were measured at different time and dosage. The optimal time and dosage of CLP-19 inducing endotoxin tolerance were determined by the expression level. The optimal time and dosage of LPS inducing endotoxin tolerance were used as control group.
Expression of IL-6 and IL-10 after endotoxin tolerance induced by 3. CLP-19
The cells were incubated with 5 ng/ml LPS and 50 ug/ml CLP-19 for 20 hours. After repeated washing with PBS, the cells were cultured in fresh medium for 2 hours. The final concentration of LPS was 10 ng/ml. The expression of IL-6 and IL-10 in the supernatant was determined after 4 hours.
The second part: molecular mechanism of CLP-19 induced endotoxin tolerance in RAW264.7.
1. the expression of TLR4, myD88 and TRAF6 mRNA after endotoxin tolerance induced by CLP-19
Cells were pretreated with 50 ug/ml CLP-19 for 20 hours, then cultured in medium for 2 hours. The cells were treated with 10 ng/ml LPS for different time (0 h, 1 h, 3 h, 6 h, 12 h). RNA was extracted. Toll-like receptor 4 (TLR4), myeloid cell differentiation factor 88 (mydoid MyD88, MyD88), tumor necrosis factor receptor phase were detected by RT-PCR. The mRNA changes of TNF receptor-associated factor 6 (TRAF6) were compared with that of medium and 5 ng/ml LPS pretreatment.
2. the expression of TLR4 protein in intracellular and membrane after induction of endotoxin tolerance by CLP-19
Cells were pretreated with 50 ug/ml CLP-19 for 20 hours, cultured in medium for 2 hours, then treated with 10 ng/ml LPS for 4 hours. The membrane and intracellular proteins were extracted respectively. The expression of TLR4 protein was detected by Western Blot.
Changes of P38 phosphorylation level after endotoxin tolerance induced by 3. CLP-19
Cells were pretreated with 50 ug/ml CLP-19 for 20 hours, cultured in medium for 2 hours, and then treated with 10 ng/ml LPS for different time (0 min, 10 min, 30 min, 60 min) respectively. Total protein was extracted. The phosphorylation level of P38 in mitogen-activated protein kinase (MAPK) pathway was detected by Western Blot. Pretreatment with 5ng/ml LPS was used as a control.
Protein expression of I kappa -B after endotoxin tolerance induced by 4. CLP-19
Cells were pretreated with 50 ug/ml CLP-19 for 20 hours, cultured in medium for 2 hours, and then treated with 10 ng/ml LPS for different time (0 min, 5 min, 15 min, 30 min, 60 min). Total protein was extracted and the expression of nuclear factor kappa-B inhibitor protein (I-kappa-B, I-kappa-B) was detected by Western Blot.
Result:
Part I: time and dose effects of endotoxin tolerance induced by CLP-19
5 ng/ml LPS preconditioning RAW264.7 cells for 20 hours was the best dose and time for LPS-induced endotoxin tolerance. 50 ug/ml CLP-19 preconditioning RAW264.7 cells for 20 hours was the best dose and time for CLP-19-induced endotoxin tolerance. After 20 hours preconditioning macrophages with 50 ug/ml CLP-19, the expression of inflammatory factor IL-6 was down-regulated and the expression of anti-inflammatory factor IL-10 was increased. Compared with the pretreatment group, there was a significant difference (P0.01).
The second part: the molecular mechanism of endotoxin tolerance induced by CLP-19 in RAW264.7 cells.
1. the expression of TLR4, myD88 and TRAF6 mRNA after endotoxin tolerance induced by CLP-19
The results of RT-PCR showed that the expression of TLR4 mRNA in CLP-19 pretreated cells was down-regulated with the prolongation of stimulation time (P 0.01), while the expression of MyD88 and TRAF6 mRNA was not significantly changed (P 0.05).
2. the expression of TLR4 protein in intracellular and membrane after induction of endotoxin tolerance by CLP-19
Western Blot results showed that the expression of TLR4 protein in the endotoxin tolerance group induced by CLP-19 was down-regulated compared with that in the medium pretreatment group (P 0.05).
3. the phosphorylation of P38 in MAPK pathway was inhibited after CLP-19 induced endotoxin tolerance.
Western Blot results showed that the phosphorylation level of P38 increased significantly after 15 min stimulation and reached its peak after 30 min stimulation. Compared with the tolerance group induced by CLP-19, the phosphorylation level of P38 was significantly inhibited (P 0.01).
After 4. CLP-19 induced endotoxin tolerance, I kappa B was not degraded.
Western Blot results showed that I-kappa B-alpha rapidly degraded within 30 minutes after high-dose LPS stimulation, and I-kappa B-beta rapidly degraded after 60 minutes of high-dose LPS stimulation. Compared with the tolerance group induced by CLP-19, the expression levels of I-kappa B-alpha and I-kappa B-beta were significantly different (P 0.01).
Conclusion:
The optimal dose and time of 20h pretreatment for RAW264.7 cells induced by 1.50 RAW264.7 g/ml CLP-19 were endotoxin tolerance.
2. Limulus antiendotoxin factor mimetic peptide CLP-19 can induce endotoxin tolerance by decreasing the expression of TLR4 on the cell membrane and in the cell.
3. the Limulus anti endotoxin factor mimetic peptide CLP-19 can induce endotoxin tolerance by inhibiting P38 phosphorylation.
4. Limulus anti-endotoxin factor mimic peptide CLP-19 can inhibit the degradation of I-kappa B-alpha and I-kappa B-beta, thereby inhibiting the translocation of NF-kappa B into the nucleus and inducing endotoxin tolerance.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R96;R459.7
【共引文献】
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