The Effect Of Epigallocatechin-3-gallate In Organs Infiltration Of Polymorphonuclear Neutrophilsw At The Early Phase Of SIRS

Background and ObjectiveEpigallocatechin-3-gallate(EGCG),which is known as catechin monomer,is a kind of tea polyphenols compounds and has the biologic activities of anti-lipid peroxidation, cleaning of free radical and antimutation.A great quantity of former experiments have validated the effectiveness of a series of catechin monomers,such as EGCC,on the regards of chemical prophylaxis of tumors,as they participate to regulate the activity of oxidoreduction-sensitive transcription factor in tumorigenic cell signal pathway.And nuclear factorκB(NF-κB)and activator protein 1(AP-1)are the key transcription factors, which are commonly related in the process of development and invasion of various inflammations and tumors.Thus,generous researches about catechin on chemical prophylaxis of tumors have simultaneously revealed its great potential in anti-inflammatory treatment.Catechin family has been proved to have the positive efficacy in chronic inflammation diseases such as arteriosclerosis and arteriosclerosis. And EGCG has also been proved to be the catechin monomer which has the most conspicuous influence upon composition of inflammatory cytokines,but there are not much studies on intervention in acute inflammatory process,opposite to chronic inflammation.Systemic Inflammatory Response Syndrome(SIRS)is the important pathologic and physiologic developing pathway of Multiple Organ Dysfunction Syndrome(MODS). Polymorphonuclear neutrophilsw(PMNs)is the key cell which causes tissue damages, even MODS.PMNs' activation,aggregation at target tissues and liberation of large amounts of destructive enzymes is the main path which induces tissue damages and MODS.In this process,immune chemotatic factors and intercellular adhesion molecules have played important roles.Chemotatic factors composited by regional tissues and stable adhesion between PMNs and endothelial cells(ECs)have their great irreplaceable contributions to PMNs' adherence,emigration,directional migration and considerable sequestration into target organs.Combining the lipopolysaccharides(LPS)-induced mouse acute lung injury model and TNF-α-induced endothelial cell inflammation model,this study carries out a series of researches in vivo and vitro,in order to investigate the effects of EGCG,as a natural anti-oxidant with high potency,upon synthesis of chemotatic factors,and upon stable adhesion of PMNs/ECs,at the early phase of systemic inflammation.The thesis also evaluates the infiltration degree of PMNs in lung,which is represented by the activity of myeloperoxidase(MPO),thereby explores the EGCG's effect upon PMNs infiltration to remote organs at the early phase of SIRS in several aspects.Methods1.Animals preparation and cell culture6-week old ICR mice of clean grade,were pretreated by EGCG hypodermic injection at abdominal walls with doses of 10mg/kg,20mg/kg or 40mg/kg.Endotoxemia was caused by means of vena caudalis LPS injection with dose of 10mg/kg.The animal model of systemic inflammatory response was duplicated.The mice were executed respectively 6h and 18 h after LPS challenge according to the study need and lung samples were preserved. Human umbilical vein endothelial cells(HUVEC)were cultured in high glucose DMEM supplemented with 10%foetal bovine serum(FBS)and placed in an incubator under condition of 37℃and 5%CO₂.The medium were removed and replaced respectively by fresh one with EGCG final concentration of 10μmol/1,20μmol/1 and 50μmol/1,when HUVEC were adheringly cultured enough to cover 80%bottom area.After 1 h pretreatment,TNF-αwere put in with final concentration of 10 ng/ml and the model of endothelial cell inflammation was duplicated.According to the study need,after 4 h and 8 h TNF-αadministration,cells were respectively digested and collected for detections.2.To analyze the gene expression levels of murine macrophage inflammatory protein 2(MIP-2)and ICAM-1 through RT-PCRAfter 5 h LPS attack,different groups of mice were executed and lungs were reserved. HUVEC were collected after EGCG pretreatment and 4 h TNF-αinduction.Total RNA was extracted by TRIZOL.After reverse transcription,PCR and semiquantitative analysis were performed,taking miceβ-Actin and human GAPDH as reference.3.Immune histochemistry dyeing and morphology observationAfter 18 h LPS attack,different groups of mice were executed and lungs were reserved and fixed.Then the tissus were paraffin imbedded,sliced and incubated in anti-mouse MIP-2 polyclonal antibody and HRP labeled IgG successively,with DAB coloration,hematoxylin afterstain test under microscope and taking photos.4.To analyze the protein expression levels of MIP-2 and ICAM-1 through Western BlotAfter 18 h LPS attack,different groups of mice were executed and lungs were reserved,homogenated and split.HUVEC were collected after EGCG pretreatment and 8 h TNF-αinduction.Total protein was extracted,performed SDS-PAGE,and transferred onto PVDF membrane,which was incubated with the fast antibody,the second antibody, and ECL mixture successively.Then the membrane was processed by exposal and develop.The integral absorbance of the bands were measured withβ-Actin as reference. 5.The metry on MPO vigor of lung tissusAfter 18 h LPS attack,different groups of mice were executed and lung tissus were reserved and weighed accurately.The yellow compound which is the remains of O-dianisidine after the donation caused by MPO has the maximum absorption peak at the point of 460nm.The products were detected by spectrophotometric method,according to the manufacturer's instructions of the MPO detecting kit.6.The viability of HUVEC determined by MTT assayWhen adheringly cultured enough to cover 75%bottom area of 96-well plant, HUVEC were processed by different concentrations of EGCG pretreatment and 8 h TNF-αinduction.Then the medium were removed and replaced by fresh one with MTT. After 4 h incubation,the supernatant was abandoned and the DMSO was added in.After 30 m incubation,the OD value was determined at the wave length of 490nm.7.The adhesion tests of HUVEC and PMNs10ml peripheral blood of a healthy adult was drawn and treated with demixing effusion of Ficoll-Hypaque by density gradient centrifugation,to produce purer PMNs. The viability of the PMNs were determined.The partes aequales PMNs were added in the HUVEC which were adheringly cultured at the bottom of 24-well plant,after EGCG pretreatment and 8 h TNF-αinduction.After 1 h incubation,PMNs which were not adherent were rinsed and the eluant was collected.PMNs before the incubation and in the eluant were counted respectively and the adhesion ratios were calculated.8.Statistical evaluationThe results were expressed as mean±S.D.;Statistical comparisons were made among groups using a one-way analysis of variance.P-value less than 0.05 was considered to be statistically significant difference.SPSS10.0 was used for statistical processing of data.Results1.EGCG made the mRNA expressions and protein synthesis of MIP-2 descend dose-dependently in mice lungs with endotoxin-induced acute lung injury.The differences had statistical significance(P＜0.05).2.Immune histochemistry and histopathology demonstrated that EGCG made the protein expressions of MIP-2 decrease in mice lungs with endotoxin-induced acute lung injury, and lessened the situation of engorgement in lung interstitial tissues and Leukocytic infiltrate.3.EGCG made the MPO vigor descend in mice lungs with endotoxin-induced acute lung injury dose-dependently.The differences had statistical significance(P＜0.05).4.EGCG with the concentration of 50μmol/1 led to the increasing of Apoptosis of TNF-α-induced HUVEC and normal growing HUVEC(P＜0.05),while EGCG with the concentration of 10μmol/1 and 20μmol/1 did not result in the changing of HUVEC's viability(P＞0.05).5.EGCG made the mRNA expressions and protein synthesis of ICAM-1 descend in TNF-α-induced HUVEC dose-dependently.The differences had statistical significance (P＜0.05).6.EGCG made the ratios of adhesion between TNF-α-induced HUVEC and PMNs descend dose-dependently.The differences had statistical significance(P＜0.05).ConclusionThe study in vivo finds that EGCG dose-dependently inhibits the synthesis of chemotatic factors in mice's lungs with endotoxin-induced systemic inflammatory reaction and reduces the infiltration degree of PMNs in lung.And the experiments in vitro finds that EGCG dose-dependently inhabits the TNF-α-induced synthesis of the important adhesion molecules on the surface of HUVEC membrane as well as the adhesion between HUVEC and PMNs.Consequently,the experiment has certified that anti-oxidant EGCG possesses the inhibitive effect on the immune adherence and chemotaxis at the early phase of SIRS and accordingly may have the positive precaution significance on the function damage of target organs at the late phase.