Study On Regulation And Mechanisms Of Control Inflammatory Responses Of Glycine In Burn Rats With Sepsis

Sepsis after Burn Injury is one of the commen a complication of serious burn injury,which can make a further progress in a short time.Sepsis can induce SIRS andMODS,and is the mainly cause of death after serious burn injury.Substantial evidences showed that glycine(Gly)could protect cells on different levels. Substantial evidences showed that Gly could blunt activation of Kupffer cells, alveolar macrophages,and neutrophils by activating a glycine-gated chloride channel.Objectives Explore protection of glycine on sepsis rats after burn,and the regulation and mechanisms of out of control inflammatory responses of Glycine.Methods1.The animal model of endotoxin after burn injury:Totally 96 wistar rats of either gender:The depilatory area was immersed into warm bath for 20%TBSA superficial second degree burn injury.Recover all rats by sodium lactate ringer's injection injected into abdominal cavity(40ml/kg)6h later.Injected LPS 10mg/kg 3h later,and gave 1g/3ml Gln or Gly to different groups.The rats were sacrificed at 3,6,12,24h.All 96 rats were randomly and equally divided into 4 groups: simple burn group(A),sepsis after burn injury group(B),Gln group(C),Gly group(D),and each group was regrouped for 3,6,12,24h(n=6).2.Determination of inflammatory cytokines:The changes of TNF-αand IL-10 in serum were determined by enzyme-linked immunospecific assay at different time points,and also the changes of TNF-α/IL-10;The chromogenic substrate limulus amebocyte lysate(LAL)apply to detect level of LPS in serum.3.Preparation of PMφ(Peritoneal macrophages):PMφwas prepared and cultured: intraperitoneal injected and aspirated Hanks solution 20ml.Rotation speed 1500 r/min and time 10 min.Mixed with RPM11640(10%FBS),and cultured in sample cell for 4h(5%C0₂,37℃).Liquidate and added fresh RPM11640(10%FBS).and incubation for 12h.5.Inspection of PMφ[Ca²⁺]_i:â‘ Load fluorescent calcium indicator:PMφwas measured flurometrically using the fluorescent calcium indicator dye fura-2(2μmol/L-2/AM,m-HBSS)and a microspectrofluorometry interfaced with an inverted microscope for 50min.Washed cells by m-HBSS(10%FBS),and added m-HBSS(10%FBS)1ml.â‘¡Intracellular calcium([Ca²⁺]_i)of PMφ:The changes of PMφ's fluorescent calcium indicator was measured by fluorescence imaging system,Charge Coupled Device(CCD)and MetaFluor Ratio Imaging Software.Measured changes of F340 and F380,and token[Ca²⁺]_i by F340/F380.Given different levels of([Ca²⁺]o),to observe the changes of[Ca²⁺]_i levels in PMφinduced by LPS,and the effection of glycine on them.6.Effect of glycine on PMφ[Ca²⁺]_i stimulated by LPSIn the condition of[Ca²⁺]o=1.25mmol/L:LPS stimulated PMφ[Ca²⁺]_i increasing in a dose-dependent manner.And Gly had significantly inhibition on PMφ[Ca2+]I stimulated by LPS in a dose-dependent mannerIn the condition of[Ca²⁺]o=0,The peak value of PMφ[Ca²⁺]_i stimulated by LPS was 44%than which in the condition of[Ca²⁺]o=1.25mmol/L.And Gly didn't had significantly inhibition on PMφ[Ca²⁺]_i stimulated by LPS.Results1.Changes of LPS in different group:LPS level in every group was begin raising from 3h,and declined at 24h.For A reached peak value at 3h,B at 6h.C,D both reached peak value at 12h,and 24h was lower than 6h in B(P＜0.05).The level of LPS in B was obviously higher than each other group at each time point(P＜0.05), and C was not significantly dfferent from D(P＞0.05).2.Changes of TNF-αin different groups:TNF-αof A was begin raising after burned,and reached peak value at 24h.The plasma level of TNF-αin B,C,and D decreased gradually from 3h to 24h(P＜0.05).B increased obviously than A at every time points(P＜0.05),and higher than C or D at 12h,24h(P＜0.05).At 6h, 12h,24h C was obviously lower than D(P＜0.05).3.Changes of IL-10 in different groups:In group B,the plasma levels of 3h and 6h were higher than 12h and 24h(P＞0.05).The level of IL-10 in group D was higher than group C at 6h,12h,24h(P＜0.05).C and D were obviously higher than A and B(P＜0.05).4.Changes of TNF-α/IL-10 in different groups:B was higher than other groups at each time point,and rising to peak value at 3h.TNF-α/IL-10 of A,C,D were changed around the base line.5.In the condition of[Ca²⁺]o =1.25mmol/L,different level of LPS could induced the increasing of[Ca²⁺],in a dose-dependent manner.And Gly can also blunt the [Ca²⁺]_i increasing induced by LPS in a dose-dependent manner.6.In the condition of[Ca²⁺]o =0,[Ca²⁺]_i induced by LPS is 44%of in the condition of[Ca²⁺]o =1.25mmol/L.Gly had no effect on[Ca²⁺]_i induced by LPS.Conclusions1.Made the animal model of endotoxin after 20%TBSA superficial second degree burn injury successfully.The level of LPS in LPS group was obviously higher than Burn group at each time point(P＜0.05),2.Gly could resist inflammation and sepsis,as blunt the increasing of TNF-αand LPS.Gly also has the effect on IL-10 release.3.Compared withGln which has certain effect on sepsis resisting,Gly has stronger effect on LPS resisting and IL-10 inducing,but weaker effect on TNF-αblunting.4.The protect mechanism of Gly:Gly could inhibit enteric endotoxin translocation, and blunt the increasing of LPS.5.The increasing of[Ca²⁺]_i induced by LPS was because of the release of[Ca²⁺]_i at the beginning,But hold out with the calcium influx from extracellular space later.Glycine can blunt the transient elevation in[Ca²⁺]_i in a dose-dependent-manner,induced by LPS by inhibiting the calcium influx from extracellular space.The inhibitory effect of glycine on[Ca²⁺]_i plays a pivotal role in the inhibitory efect of glycine on TNF-αproduction in PMφ.