The Induction Of Limulus Anti-endotoxin Factor Mimetic Peptide CLP-19to Endotoxin Tolerance In RAW264.7Cells

Sepsis is a common clinical critical illness, which is a systemic inflammatory responsesyndrome(SIRS) caused by infection, leaving characteristics with high incidence andmortality. Lipopolysaccharide (LPS), also known as endotoxin, one of the majorcomponents that presents in the cell membrane of Gram-negative bacteria, not only lead tosepsis，but also can induce endotoxin tolerance which reduce the inflammatory response ofa variety of causes for preventing the occurrence of potential sepsis. Endotoxin tolerance(ET) is a phenomenon that cells or organisms exposed to low concentrations of endotoxin(e.g. LPS) enter into a transient unresponsive state and are unable to respond to furtherchallenges with endotoxin. In other words, they develop a kind of ‘‘tolerance’’ to endotoxin.However, it is dangerous that the way preventing the potential sepsis through endotoxintolerance induced by pretreating with minute amounts of endotoxin，So new strategiesshould be developed looking for a drug that can induce endotoxin tolerance and reduceinflammatory response.In our pervious studies, a cyclic Limulus anti-endotoxin factor mimetic peptideCLP-19was obtained through analyzing the structural characteristics of functional area oflimulus anti-lipopolysaccharide factor (LALF) deriving from an amoebocyte of blood cellsin horseshoe crab. CLP-19has antibacterial activity and no haemolyticus andimmunogenicity. A good security of CLP-19was displayed Both in vitro and in vivo.Pretreatment of CLP-19for20h can efficiently protect mice from acute peritonitis causedby E. coli. bacteria and significantly suppress elevated TNF-α level stimulated by IL-1β, acytokine with different structures with LPS. Pretreatment of CLP-19for20h alsosignificantly reduce the amount of bacteria in acute peritonitis mice induced by E. coli.Pretreatment with CLP-1930min can significantly suppress elevated TNF-α level whichinduced by LPS in cell, The results indicated that CLP-19may induce endotoxin tolerance.In order to determine the function and molecular mechanisms of endotoxin toleranceinduced by CLP-19in RAW264.7cells, the model of endotoxin tolerance was established and the change of mRNA and protein of critical factor in the inflammatory signalingpathways of RAW264.7cells was analysized using RT-PCR and Western Blotting.Methods1. The time and dose effects of endotoxin tolerance induced by CLP-19in RAW264.7cells1.1The time and dose effects of endotoxin tolerance induced by LPS in RAW264.7cells by ELISA analysisRAW264.7cells were pretreat LPS with different concentrations (0ng/ml、0.05ng/ml、0.1ng/ml、0.2ng/ml、0.5ng/ml、1ng/ml、5ng/ml、7ng/ml、10ng/ml) for different times(10h,20h,24h), then were washed with PBS and cultured with fresh medium for2h. Cellswere treated with LPS of10ng/ml for4h. The supernatant was collected and TNF-α releasewas measured by ELISA.1.2The time and dose effects of endotoxin tolerance induced by CLP-19in RAW264.7cells by ELISA analysisRAW264.7cells were pretreat CLP-19with different concentrations (0μg/ml,0.1μg/ml,1μg/ml,5μg/ml,10μg/ml,20μg/ml,50μg/ml,100μg/ml) for different times(10h,20h,24h), then were washed with PBS and cultured with fresh medium for2h. Cellswere treated with LPS of10ng/ml for4h. The supernatant was collected and TNF-α andIL-10release was measured by ELISA.2. The molecular mechanism of endotoxin tolerance induced by CLP-19in RAW264.7cells2.1RT-PCR analysis of TLR4、myD88、TRAF6gene expression in RAW264.7cellsinduced endotoxin tolerance by CLP-19Pretreated with CLP-19(50μg/ml) for20h, RAW264.7cells were washed with PBSand then cultured with fresh medium for2h. RAW264.7cells were re-treated with LPS of10ng/ml for diferent time (0h,1h,3h,6h,12h), the total RNAs were extracted. The mRNAexpressions of TLR4, myD88and TRAF6were measured with RT-PCR. Meanwhile, thepretreatment of RAW264.7cell by medium and LPS(5ng/ml) were served as controls,respectively.2.2western Blot analysis of TLR4protein expression existing in the cell intracellularand membrane in after RAW264.7cells endotoxin tolerance inducing by CLP-19 Pretreated CLP-19(50μg/ml) for20h, RAW264.7cells were washed with PBS,cultured with fresh medium for2h and then treated with LPS of10ng/ml for4h. The totalproteins were extracted and TLR4protein expression existing in the intracellular andmembrane were measured with Western Blot. the pretreatment of RAW264.7cell withmedium and LPS (5ng/ml) were served as controls, respectively.2.3Western Blot analysis of p-P38phosphorylation pathways in RAW264.7cells afterendotoxin tolerance induced by CLP-19.Pretreated with CLP-19(50μg/ml) for20h, RAW264.7cells were washed with PBS,cultured with fresh medium for2h and then treated with LPS of10ng/ml for diferent time(0min,10min,30min,60min). The total protein was extracted and p-P38of MAPKphosphorylation pathways were measured by Western Blot. The pretreatment of RAW264.7cell with medium and LPS(5ng/ml) were served as controls, respectively.2.4Western Blot analysis of IκB protein expression in RAW264.7cells inducedendotoxin tolerance by CLP-19.Pretreated with CLP-19(50μg/ml) for20h, RAW264.7cells were washed with PBS,cultured with fresh medium for2h and then treated with LPS of10ng/ml for diferent time(0min,10min,30min,60min). The total protein was extracted and IκB protein expressionwere measured by Western Blot. the pretreatment of RAW264.7cell with medium andLPS(5ng/ml) were served as controls, respectively.Results1. The time and dose effects of endotoxin tolerance induced by CLP-19in RAW264.7cellsPretreatment with LPS (5ng/ml) for20h was the best dose and time for endotoxintolerance induced by LPS in RAW264.7cells, while pretreatment with CLP-19(50μg/ml)for20h was the best dose and time for endotoxin tolerance induced by CLP-19inRAW264.7cells. Compared with the pretreatment of medium, the expression of TNF-α wasno significant difference after endotoxin tolerance induced by LPS and CLP-19,respectively (P>0.05). While compared with endotoxin tolerance inducing by LPS, theexpression of TNF-α was significant increased after endotoxin tolerance inducing byCLP-19(P<0.01). 2. The molecular mechanism of endotoxin tolerance induced by CLP-19in RAW264.7cells2.1The mRNA expression of TLR4, myD88and TRAF6after endotoxin toleranceinduced by CLP-19TLR4mRNA expression was reduced, while MyD88and TRAF6mRNA expressiondid not significantly change in RAW264.7cells after endotoxin tolerance induced byCLP-19compared with the pretreatment of medium (P<0.01).2.2TLR4protein expression of cell intracellular and membrane was decreased afterendotoxin tolerance induced by CLP-19Compared with the of pretreatment of medium, TLR4protein expression were reduced,not only in cell intracellular but also in cell membrane in RAW264.7cells after endotoxintolerance induced by CLP-19(P<0.05).2.3P38of MAPK phosphorylation pathways was suppressed after endotoxin toleranceinducing by CLP-19The phosphorylation level of P38was significantly increased after restimulation withLPS in the cell of pretreatment of medium, while the phosphorylation level of P38weresignificantly suppressed after endotoxin tolerance induced by CLP-19compared with thepretreatment of medium (P<0.05).2.4The protein expression of IκB was not degradated after endotoxin toleranceinduced by CLP-19Pretreatment of medium, IκBα and IκBβ were rapidly degraded within30min and60min after restimulation with LPS (P<0.05). While IκBα and IκBβ were not degraded afterendotoxin tolerance induced by CLP-19compared with the pretreatment of medium(P>0.05).Conclusions:1. Limulus anti-endotoxin factor mimetic peptide CLP-19can induce endotoxintolerance through reducing protein expression of TLR4of cell membrane.2. Limulus anti-endotoxin factor mimetic peptide CLP-19can induce endotoxintolerance through inhibiting P38phosphorylation.3. Limulus anti-endotoxin factor mimetic peptide CLP-19can induce endotoxin tolerance through inhibiting the degradation of IκBα and IκBβ and thereby inhibitingNF-κB translocation into the nucleus.