鱟抗內(nèi)毒素因子模擬肽CLP-19誘導RAW264.7細胞內(nèi)毒素耐受的實驗研究
發(fā)布時間：2018-08-22 14:32
【摘要】：研究背景及目的： 膿毒癥(Sepsis)是臨床上一種常見的危重病癥，是指由感染引起的全身炎癥反應(yīng)綜合征(Systemic inflammatory response syndrome，SIRS)，具有發(fā)病率高、死亡率高的特點。作為革蘭氏陰性菌細菌細胞壁外層的主要成分之一的內(nèi)毒素(Lipopolysaccharide,LPS)除了能引發(fā)膿毒癥外，還可通過誘導“內(nèi)毒素耐受”，降低各種原因引起的炎癥反應(yīng)，從而預防潛在膿毒癥的發(fā)生。內(nèi)毒素耐受是指動物或免疫細胞等在給予小劑量LPS預處理后，對LPS再次刺激時無反應(yīng)或反應(yīng)性明顯降低。因此，尋找一種可誘導內(nèi)毒素耐受同時又不誘導機體產(chǎn)生炎性反應(yīng)的藥物是抗感染及抗炎藥物研究的新策略。 環(huán)狀鱟肽(Cyclic limulus peptide，CLP-19)是經(jīng)美洲鱟抗內(nèi)毒素因子(Limulusanti-lipopolysaccharide factor, LALF)結(jié)構(gòu)改造后得到的一條無溶血性、對巨噬細胞等無致炎作用的環(huán)肽，具有較理想中和LPS及抗菌活性。課題組前期研究表明，提前給予CLP-19可顯著降低血清腫瘤壞死因子-α (Tumour Necrosis Factor, TNF-α)水平、提高大腸桿菌致急性腹膜炎模型鼠的生存率；預防性給予CLP-19能顯著減少大腸桿菌致急性腹膜炎模型鼠體內(nèi)的細菌量。在細胞水平，CLP-19預刺激30min后，以磷酸鹽緩沖鹽水(Phosphate buffered saline, PBS)清洗細胞，再用LPS進行刺激，結(jié)果發(fā)現(xiàn)，CLP-19預處理組同樣可顯著降低LPS刺激所引起的TNF-α的升高。上述現(xiàn)象表明，CLP-19可誘導“內(nèi)毒素耐受”，然而其機制有待深入研究。 因此，為明確CLP-19誘導內(nèi)毒素耐受效應(yīng)及分子機制，本實驗通過建立RAW264.7細胞內(nèi)毒素耐受模型，利用逆轉(zhuǎn)錄聚合酶鏈式反應(yīng)(Reverse TranscriptionPolymerase Chain Reaction, RT-PCR)、免疫印跡法(Western Blotting, WB)等比較研究巨噬細胞炎癥信號通路中關(guān)鍵因子的mRNA及蛋白的變化，初步探討CLP-19誘導內(nèi)毒素耐受的分子機制。通過以上研究，擬明確CLP-19誘導小鼠“內(nèi)毒素耐受”的作用及其途徑，并為其用于膿毒癥預防提供理論依據(jù)。 實驗方法： 第一部分：CLP-19誘導內(nèi)毒素耐受的時間效應(yīng)和劑量效應(yīng)研究 1. ELISA分析LPS誘導內(nèi)毒素耐受的時間效應(yīng)和劑量效應(yīng) 不同濃度(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細胞，培養(yǎng)不同時間(10h、20h、24h)后，新鮮培養(yǎng)基培養(yǎng)2h，再給予10ng/ml的LPS作用于細胞，4h后取上清液，酶聯(lián)免疫吸附法(enzyme linked immunosorbent assay, ELISA)觀察不同時間、不同劑量下TNF-α的表達量，從而確定LPS誘導內(nèi)毒素耐受的最佳時間和劑量。 2. ELISA分析CLP-19誘導內(nèi)毒素耐受的時間效應(yīng)和劑量效應(yīng) 用不同濃度(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細胞，培養(yǎng)不同時間(10h、20h、24h)后，新鮮培養(yǎng)基培養(yǎng)2h，再加入LPS使其終濃度為10ng/ml，，4h后取上清液，測定不同時間、不同劑量下TNF-α的表達量和IL-10的表達量，從而確定CLP-19誘導內(nèi)毒素耐受的最佳時間和劑量。以LPS誘導內(nèi)毒素耐受的最佳時間和劑量為對照組。 3. CLP-19誘導內(nèi)毒素耐受后IL-6和IL-10表達變化 分別以5ng/ml LPS、50μg/ml CLP-19孵育細胞20h，用PBS反復清洗后以新鮮培養(yǎng)基培養(yǎng)2h，再加入LPS使其終濃度為10ng/ml，4h后取上清液測定IL-6和IL-10表達變化。 第二部分：CLP-19誘導RAW264.7內(nèi)毒素耐受的分子機制研究 1. CLP-19誘導內(nèi)毒素耐受后胞內(nèi)TLR4、myD88、TRAF6的mRNA表達變化 用50μg/ml CLP-19預處理細胞20h，再用培養(yǎng)基培養(yǎng)2h，向各組中均加入10ng/mlLPS處理細胞不同時間(0h、1h、3h、6h、12h)后，提取細胞RNA，通過RT-PCR檢測細胞因子Toll樣受體4(Toll like receptor, TLR4)、髓樣細胞分化因子88(mydoidMyD88，MyD88)、腫瘤壞死因子受體相關(guān)因子6(TNF receptor-associated factor6，TRAF6)的mRNA變化。以培養(yǎng)基和5ng/ml LPS預處理作為對照。 2. CLP-19誘導內(nèi)毒素耐受后胞內(nèi)和胞膜TLR4的蛋白表達變化 用50μg/ml CLP-19預處理細胞20h，用培養(yǎng)基培養(yǎng)2h后，再向各組中加入10ng/mlLPS作用4h后，分別提取胞膜和胞內(nèi)蛋白，Western Blot檢測胞膜和胞內(nèi)TLR4蛋白的表達變化。以培養(yǎng)基預處理作為對照。 3. CLP-19誘導內(nèi)毒素耐受后P38的磷酸化水平變化 用50μg/ml CLP-19預處理細胞20h，用培養(yǎng)基培養(yǎng)2h后，向各組中均加入10ng/mlLPS分別作用不同時間(0min、10min、30min、60min)后，提取細胞總蛋白，Western Blot檢測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。以培養(yǎng)基和5ng/ml LPS預處理作為對照。 4. CLP-19誘導內(nèi)毒素耐受后Iκ-B的蛋白表達變化 用50μg/ml CLP-19預處理細胞20h，用培養(yǎng)基培養(yǎng)2h后，再向各組中加入10ng/mlLPS作用不同時間(0min、5min、15min、30min、60min)后，提取細胞總蛋白，WesternBlot檢測核因子κB抑制蛋白(I-kappa-B，IκB)的蛋白表達變化。以培養(yǎng)基預處理作為對照。 結(jié)果： 第一部分：CLP-19誘導內(nèi)毒素耐受的時間效應(yīng)和劑量效應(yīng)研究 5ng/ml LPS預處理RAW264.7細胞20h為LPS誘導內(nèi)毒素耐受的最佳劑量和時間。50μg/ml CLP-19預處理RAW264.7細胞20h為CLP-19誘導內(nèi)毒素耐受的最佳劑量和時間。50μg/ml CLP-19預處理巨噬細胞20h后，炎癥因子IL-6表達下調(diào)，抗炎因子IL-10表達增加，與培養(yǎng)基預處理組相比，均有顯著差異(P0.01)。 第二部分：CLP-19誘導RAW264.7細胞內(nèi)毒素耐受的分子機制研究 1. CLP-19誘導內(nèi)毒素耐受后胞內(nèi)TLR4、myD88和TRAF6的mRNA表達變化 RT-PCR結(jié)果顯示，與培養(yǎng)基預處理細胞組比較，CLP-19預處理細胞組中，隨著刺激時間的延長，TLR4mRNA表達下調(diào)，差異有統(tǒng)計學意義(P0.01)。而MyD88和TRAF6的mRNA表達均無明顯變化(P㧐0.05)。 2. CLP-19誘導內(nèi)毒素耐受后胞內(nèi)和胞膜TLR4蛋白表達降低 Western Blot結(jié)果顯示，與培養(yǎng)基預處理組相比，CLP-19誘導的內(nèi)毒素耐受組中，胞膜和胞內(nèi)TLR4的蛋白表達均下調(diào)，有顯著差異(P0.05)。 3. CLP-19誘導內(nèi)毒素耐受后MAPK通路中P38的磷酸化被抑制 Western Blot結(jié)果顯示，培養(yǎng)基預處理細胞組中，P38的磷酸化水平在刺激15min后明顯增加，30min后達到高峰。與培養(yǎng)基預處理細胞組相比，CLP-19誘導的耐受組中，P38的磷酸化水平均被顯著抑制(P0.01)。 4. CLP-19誘導內(nèi)毒素耐受后IκB未被降解 Western Blot結(jié)果顯示，培養(yǎng)基預處理細胞組中，IκBα在大劑量LPS刺激30min內(nèi)迅速降解，IκBβ在大劑量LPS刺激60min后迅速降解。與培養(yǎng)基預處理細胞組相比，CLP-19誘導的耐受組中，IκBα和IκBβ表達水平有顯著差異(P0.01)。 結(jié)論： 1.50μg/ml CLP-19預處理RAW264.7細胞20h為CLP-19誘導內(nèi)毒素耐受的最佳劑量和時間。 2.鱟抗內(nèi)毒素因子模擬肽CLP-19可通過降低TLR4在胞膜和胞內(nèi)的表達誘導內(nèi)毒素耐受。 3.鱟抗內(nèi)毒素因子模擬肽CLP-19可通過抑制P38磷酸化誘導內(nèi)毒素耐受。 4.鱟抗內(nèi)毒素因子模擬肽CLP-19可抑制IκBα和IκBβ的降解，從而抑制NF-κB轉(zhuǎn)位進入胞核，進而誘導內(nèi)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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R96;R459.7
本文編號：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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R96;R459.7
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