Study Of Cytoprotection Of Gastric Mucosal And Intervention By Platelet Activating Factor Receptor Antagonist During Endotoxemia In Baby Rats

IntroductionAcute gastric mucosal injury is a common complication of serious infection, trauma,shock,etc.the incidence rate of the above diseases may be as high as 60～100%and the massive bleeding rate is 15%among patients without being treated,so it often endangers life.Pathogenesis is that protective mechanism of gastric mucosa is impaired,which makes injury factor strengthen relatively.In the recent years,the study finds that the peptides have important effect on the defence and repair of gastric mucosal injury.Growing,regenerative and repara tive processes of gastric mucosal epithelium are controlled strictly and the mech anisms of regulation and control mainly come from mucosa itself,have many kinds of peptides joining and the restorability may relate with expression level. Serious symptom infection is common reason of acute gastric mucosal injury in the dangerous and serious symptoms for the department of pediatrics.It is un clear now how the peptides protect and repair the gastric mucosal injury in infec ting endotoxemia.There is few study and report whether or not the peptides have an effect through traditional prostaglandin way.pS2(trefoil factor family 1,TFF1)is a member of trefoil peptide family. Its structure is 6 cysteine residues forming 3 intrastrand disulphide bridges,pS2 makes gastric mucosa have the functions of defence and repair through regulating cell proliferation and has the effect of promoting mitosis for different cell lines. Expression change and protective and reparative mechanisms of pS2 for gastric; mucosal injury in baby rats with endotoxemia aren't clear.Transforming growth factor alpha(TGF-α)is a main regulative peptide of repairing gastric mucosal injury in the epidermal growth factor family that can promote the metabolism and growth of gastric mucosal epithelium.Gastric muco sa itself can synthesize TGF-α,so TGF-αis the most important peptide maintaining the integrality of gastric mucosa.The expressions' sequence of TGF -α,pS2 gene and TGF-αgene still needs studying.Cyclooxygenaxe-2(COX-2)is induction of COX isozyme and an impor tant rate-limiting enzyme during the process of synthesizing PGs.Prostaglandin is a kind of lipid which mainly be synthesized in seminal vesicle,renal medulla, lung and gastrointestinal tract.Continuous production and release of PG play the protective role and improve the healing of gastric mucosal ceils.Expression change and regulation of COX-2,and the concentration change of PGE arent very clear in the baby rats with endotoxemia.Inducible nitric oxide synthase (iNOS)is induction of NOS isozyme and output of endogenous NO depends on iNOS expression content,iNOS expression change and regulation,NO change and effect on gastric mucosa aren' t very clear in the baby rats with endotox emia.Proliferation cell nuclear antigen(PCNA)is a kind of nucleoprote with mo lecular weight of 35712u(36000 Dalton),existing in different positions of cell nucleus,which is the necessary factor synthesizing cell DNA.The occurrence of PCNA has the intimate relation with cell proliferation cycle and PCNA is an im portant biological indicator reflecting cell proliferation activity.The PCNA change isnt reported in the baby rats with endotoxemia.Platelet activating factor(PAF)is the strongest endogenous helcosis meson and plays potentiation role to other inflammatory cell factors.PAF receptor an tagonist can obviously change hemodynamics and tissue injury induced by endo toxin or PAF.Materials and Methods1.MaterialsPreparing endotoxemia model of eighteen-day -old Wistar rats with injec ting intraperitoneally endotoxin(5 mg/kg 055;B5 lipopolysaccharide)and same content normal saline as control group,with injecting intraperitoneally PAF antagonist(5 mg/kg BN52021 Ginkgolide B)at 0.5h before injecting endotoxin as prevention group and with injecting intraperitoneally PAF antagonist(5 mg/kg BN52021 Ginkgolide B)in 0.5h after injecting endotoxin as treatment group. The animals were killed in 1.5,3,6,24,48 and 72h respectively after endo toxin injection.Observe the injury conditions of gastric mucosa and record ulcer index.0.5cm 0.5cm gastric tissue in glandular stomach region of greater curva ture was put in 4%formaldehyde solution to store;0.1 cm 0.5cm gastric mucosa was cut into lmm³ and put in 2.5%glutaraldehyde solution to store;0.5cm 0. 5cm gastric mucosa was stayed to weigh and others were washed 3 times with normal saline and put into the tubes deprived of RNA enzyme,frozen with liquid nitrogen and stored in -70℃low temperature refrigerator.8 rats from every group were used every time point.2.METHODSDecide ulcer index of gastric mucosa according to Guth standard integral. For gastric mucosa,pathological change was checked by H-E(hematoxylin eosin)staining,ultrastructure change was observed by transmission electric mi croscopy,PGE₂ concentration was measured by radioimmunoassay method,NO content was measured by chemical colorimetry using nitric acid reductase,the expressions of pS2,TGF-α,COX-2,iNOS and PCNA were measured by im munohistochemistry SP method and the expressions of pS2.TGF-α,COX-2 and iNOS mRNA were measured by reverse transcription polymerase chain reac tion(RT-PCR)method.Experimental results were analyzed statistically by SPSS 10.0 software system and analysis of variance was used in the comparison among groups.RESULTS1.Changes of gastric mucosal epithelium cell,parietal cell ultrastruc ture and effect of PAF receptor antagonist in baby rats with endotoxemiaGastric mucosal injury was most serious in 6h after injecting endotoxin for LPS group.On the surface,massive anabrosis,bleeding and cord necrosis par alleling with gastric longitudinal axis can be seen mainly in glandular stomach region.Mucosal superficial epithelium dropped out abroad in light microscopy; it had the.bleeding,inflammatory cell infiltration,fragmentation and pycnosis of nuclear,apoptosis corpus appearing and gland injury in the mucosa.The UI score of gastric mucosa was highest and the differences were very significant(P＜0.01)in the comparison with the control group or between the groups.Extents of gastric mucosal injury were obviously alleviated in PAF antagonist prevention group and treatment group and UI store obviously decreased.Observation with transmission electric microscopy,intercellular space of superficial epithelium was broadened,surface of cavity and mucosa was damaged,inter cells connect ed tightly without continuity,smallish microvillus decreased,mitochondria seri ously swelled,ridge crack even disappeared with vacuolar degeneration,rough endoplasmic reticulum distended,ribosome dropped,chromatin concentration existed in the nuclear circumference and pyknosis and clearance of nuclear membrane broadening could be seen.Parietal cells swelled,hydrochloric acid tubule distending could be seen and partial cells was destroyed showing vacuole change,mitochondria increased and partial cells showed vacuole change,cyto plasm ribosome dropped and smooth endoplasmic reticulum expanded.2.Changes of gastric mueosal COX-2,iNOS,PGE₂ and NO and effect of PAF receptor antagonist in baby rats with endotoxemiaThe PGE₂ concentration of gastric mucosa was lowest in 6h after injecting endotoxin for LPS group and the difference of PGE₂ concentration was significant (P＜0.01)between LPS group and control group,the difference was significant (P＜0.01)among PAF antagonist prevention group,treatment group and LPS group,and the difference was significant(P＜0.05)among PAF antagonist pre vention group,treatment group and control group.The NO content of gastric mucosa was highest in 6h after injecting endotoxin for LPS group and the differ ence was significant(P＜0.01)between LPS group and control group,the difference was significant(P＜0.01)among PAF antagonist prevention group, treatment group and LPS group,and the difference was significant(P＜0.05). among PAF antagonist prevention group,treatment group and control group.No the expressions of COX-2,iNOS protein or mRNA was seen in gastric mucosal tissues of control group.COX-2 protein expressed in the cytoplasm of gastric mucosal tissue in 6h after injecting endotoxin for LPS group,obviously increased in 48h and the mRNA level also ascended,COX-2 protein expres sion obviously increased and mRNA level also obviously ascended in 6h in PAF antagonist prevention group and treatment group.Cytoplasm iNOS protein of gas tric mucosal tissue expressed in 1.5h after injecting endotoxin for LPS group, obviously increased in 6h,it was highest in 24h,began to decrease in 48h and didnt recover normal in 72h;the mRNA level also increased and decreased,iN OS protein began to express in 3h in PAF antagonist prevention group and treat ment group,it obviously increased in 6h,decreased in 48h and was the same as control group in 72h;the mRNA level also increased and decreased.3.Expression changes of gastric mucosal pS2,TGF-αand PCNA and effect of PAF receptor antagonist in baby rats with endotoxemiaTissue nucleus of gastric mucosa had many PCNA positive staining in con trol group.PCNA expression obviously decreased in 3h after injecting endotoxin forLPS group,it was lowest in 6h,had some recovery in 24h and didnt com pletely recover normal in 72h.PCNA positive staining of gastric mucosal tissue nucleus in PAF antagonist prevention group and treatment group didnt decrease obviously.The cytoplasm of gastric mucosal tissue had the expressions of pS2 mRNA and TGF-αmRNA in control group,pS2 gene expression of gastric mu cosa weakened in 1.5h after injecting endotoxin for LPS group,it began to re cover in 3h and was the same as control group in 6h,was obviously higher than that of control group and highest in 24h,decreased in 48h～72h,but it was still higher than that control group,pS2mRNA expression obviously strengthened in 3h in PAF antagonist prevention group and treatment group and recovered nor mal level in 72h.TGF-αmRNA expression of gastric mucosa obviously strengthened in 6h after injecting endotoxin for LPS group,it was strongest in 6h～24h,decreased in 48h～72h,but it was still higher than that of control group.TGF-αmRNA expression recovered normal level in 48h in PAF antago nist prevention group and treatment group.Immunohistochemistry method proved that pS2 and TGF-αexpressions of gastric mucosa changed. CONCLUSIONS1.Injecting intraperitoneally endotoxin can result in acute gastric mucosal injury in baby rats and the changes took place on gastric mucosal epithelium and parietal cell ultrastructure.PAF antagonist can obviously alleviate acute gastric mucosal injury in baby rats with endotoxemia and PAF may be main factor that results in acute gastric mucosal injury in baby rats with endotoxemia.2.The mRNA expressions of COX-2 and iNOS of gastric mucosa increase, and protein expressions of COX-2 and iNOS strengthen for the endotoxemia. PAF antagonist can improve the expression of COX-2,decrease the expression level of iNOS and alleviate acute gastric mucosal injury and help the healing of gastric mucosal injury.Acute gastric mucosal injury can be induced when PGE₂ of gastric mucosa decreases and NO content increases for endotoxemia.PAF receptor antagonist can influence PGE₂ concentration and NO content of gastric mucosa and can as sist in protecting gastric mucosa.3 PCNA expression of gastric mucosa reduces and that of gastric mucosal injury repair also increases for endotoxemia.At the same time,the expressions of pS2,mRNA and TGF-αmRNA increase,protein expressions of pS2 and TGF-αstrength,perhaps to join the defence and repair of acute gastric muco sal cells through mediating cell proliferation.