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
[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
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