The Effects And Mechanisms Of CXCR4Antagonist-N15P Peptide On LPS-induced Inflammation

N15P peptide (sequence: LGASWHRPDKCCLGY) is a new a CXCR4chemokinereceptor antagonist which independently developed by our group, and it has obtainedthe national invention patent authorization (authorization number: CN1872879B). Asa pathological phenomena closely related to the human variety diseases,inflammation is one of today's hot medical research field, but most of theinflammatory drugs have side effects. Because of chemokines and their receptorsclosely related to the inflammatory process, so this project is proposed to researchthe effects and mechanisms of CXCR4antagonist-N15P peptide on LPS-inducedinflammation.Object: To build the endotoxin (LPS) model of inflammation in vitro and in vivo,and use N15P peptide in the endotoxin (LPS) model of inflammation in vitro and invivo. To research the effects and mechanisms of CXCR4antagonist-N15P peptide onLPS-induced inflammation and set up the base for the N15P peptidethe as an newanti-inflammatory drug.Methods: The main contents of this subject consisted of two parts:(1) In vitrostudies: Using endotoxin (LPS) to stimulate peripheral blood mononuclear cells(PBMC), we builded the model of endotoxin inflammatory cells. The differentconcentrations of N15P peptide were acted on the model(control drugs:dexamethasone). By ELISA and real-time PCR method, we detected inflammatorycytokines TNF-α, IL-6, IL-8protein and mRNA content; Using classic Griessreagent method,we detected the change of NO content. At the same time, Usingwestern blot method, we detected the change of inflammatory mediators NF-kBprotein content. Using real-time PCR method, we detected the inflammatorymediators(NF-kB, COX-2, TLR4, MyD88, PI3K, AKT) mRNA content changes.From protein and mRNA levels, we explored the effect and mechanism of N15Ppeptide in vitro model of endotoxin inflammation. (2) In vivo studies: Endotoxin (LPS) was injected intraperitoneally into SPF mice tobuild inflammation in animal models of endotoxin. N15P peptide was acted on themodel (control drugs: dexamethasone).16h after LPS injection, we got the bloodfrom the mice's eyes and the mice were killed. We got the mice's liver, lung organsby routine conservation method. Blood of mice was determined by automatedhematology analyzer (WBC count, the total number of red blood cells, hemoglobin,neutrophil cell%, lymphocytes%, hematocrit, platelets); separation of serum wasdetermined with aspartate aminotransferase (GOT), alkaline phosphatase (ALP),alanine aminotransferase (GPT) by using the automatic biochemical analyzer. Wemeasured the serum inflammatory cytokines TNF-a, IL-6, IL-8content by ELISA;Using classic Griess reagent method, we detected serum NO content. Usingconventional production biopsy in mice liver and lung, we observed the extent oforgan loss; By immunohistochemical staining of NF-KB P65in expression in theliver cells and Western blot, we detected changes of NF-kB content in the liver cell;By RT-PCR reaction, we detected liver tissue of inflammatory cytokines and media(NF-kB,COX-2and TLR4) mRNA expression changes. In terms of the degree oforgan damage, blood biochemical parameters, inflammatory factors and mediumprotein and mRNA levels, we explored the effect and mechanism of N15P peptide inendotoxin inflammation in vivo mouse model.Results:(A) The contents of In vitro studies part:(1) A final concentration of10μg/mL LPS in PBMC24h, could effectively stimulatethe secretion of inflammatory cytokines TNF-α, IL-6, IL-8.(2) ELISA assay results showed that compared with the LPS group, the middle groupconcentration (5ng/ml) and high concentration group (15ng/ml) N15P peptide anddexamethasone could effectively inhibit TNF-α, IL-6IL-8protein expression (P<0.05).(3) PCR results showed that, compared with LPS group, N15P peptide anddexamethasone could be effective in reducing the cells of inflammatory cytokinesTNF-α, IL-6, IL-8mRNA expression (P <0.05). (4) NO detection kit results showed that the N15peptide drug group anddexamethasone in the control group, the NO content compared with LPS stimulation,significantly decreased (P <0.05).(5) Western blot and real-time PCR results showed that the N15peptide drug groupand dexamethasone in the control group could be effective in reducing the proteincontent and mRNA expression of NF-kB in the cells, compared with LPSstimulation, there was significant difference (P <0.05).(6) Real-time PCR results showed that dexamethasone medication in the controlgroup did not significantly reduce the cells of COX-2mRNA expression comparedwith LPS stimulation, it was no significant difference (P>0.05). N15peptide druggroup was able to inhibit COX-2mRNA expression in cells, compared with LPSstimulation, there was significant difference (P <0.05). The N15peptide wasdifferent of the mechanism with dexamethasone.(7) PCR results showed that the N15peptide treatment group and dexamethasonemedication in the control group, could effectively inhibit the mRNA expression ofTLR4-MyD88and PI3K-AKT. These two information channel media, comparedwith LPS stimulation, they were significant difference (P <0.05). It was showed thatthe N15peptide could effectively influence the two major inflammatory signalingpathway (TLR4-MyD88and PI3K-AKT), which inhibit the role of inflammation inthe development process.(B) The contents of In vivo studies part:(1) Using LPS (20μg/kg) by intraperitoneal injection in mice, endotoxin mousemodel of inflammation could be successfully established. LPS infection of the liverand lungs of the mice was subject to more serious injury.(2) From blood and biochemical parameters, N15P peptide and dexamethasone couldeffectively inhibit the inflammatory symptoms of the mice, and could effectivelyprotect the liver of mice, reduce endotoxin to its damage.(3) ELISA results showed that the N15peptide and the dexamethasone groupsignificantly inhibited levels of TNF-α, IL-6, IL-8protein secretion in serum.(4) N15peptide and the dexamethasone group were significantly inhibited the NO secretion in the mice serum.(5) By HE staining to semi-quantitatively assessed the degree of tissue necrosis, theN15P peptide medication group and the dexamethasone group, the liver, lung tissue,the degree of necrosis were significantly improved than LPS infection group,indicating that the N15P peptide and dexamethasone group plays a protective role inthe mice liver and lung.(6) The immunohistochemical results showed that in LPS-infection group, NF-KBP65was in strong positive expression,N15P peptide drug group and dexamethasonegroup were weakly positive.(7) Western blot results showed that the liver cells of the N15P peptide drug groupand dexamethasone group of NF-KB P65were significantly decreased in expressionthan LPS infection group (P <0.05). N15P peptide and dexamethasone couldeffectively inhibit the NF-KB P65in protein expression in liver tissue.(8) RT-PCR results showed that N15P peptide drug group and dexamethasone groupin the liver cells of NF-KB P65, TLR4mRNA expression was significantly reducedthan LPS infection group (P <0.05); N15P peptide drugs COX-mRNA decreasedsignificantly than LPS infection group (P <0.05), while dexamethasone COX-2mRNA in the infected group no significant difference with LPS group(P>0.05).Conclusion: N15P peptide either endotoxin model of inflammatory cells, or theinternal toxins mouse model of inflammation, have good suppression ofinflammatory factors (such as: TNF-α, IL-6, IL-8, NO) and inflammatory mediators(NF-kB and COX-2) protein and mRNA expression capabilities, and effectiveinhibition of TLR4-MyD88and PI3K-AKT the two major inflammatory signalingpathway expression. It can also protect mouse liver and lung. With the role ofprotecting the body to reduce inflammation damage, N15P peptide is a promisingnew anti-inflammatory drug.