Study On Anti-inflammatory Effect Of Gamma-glutamylcysteine In Mouse Models Of Sepsis

Sepsis,a syndrome of physiologic,pathologic,and biochemical abnormalities induced by infection,is a major public health concern.It is characterized by redox imbalance and severe oxidative stress.Glutathione(GSH)serves several vital functions including scavenging free radicals,and plays a major role in maintaining intracellular redox balance.Low glutathione concentrations are often seen in adult septic patients.While GSH in circulating blood are unable to be taken in to the majority of human cells directly,limiting the officinal of exogenous GSH.GSH is a tripeptide composed of glutamate,cysteine and glycine.N-acetyl-l-cysteine(NAC),a cysteine prodrug,is easily transported into cells and increase intracellular GSH levels Research has shown that NAC has anti-inflammatory and antioxidant effects,and it has therapeutic effect on sepsis combined with other drugs.After the deacetylation of NAC,there are two reactions for GSH synthesis catalyzed by glutamate cysteine ligase(GCL)and glutathione synthetase(GSS).While y-glutamylcysteine(?-GC),an intermediate dipeptide with sulfhydryl group in GSH synthesis pathway,need one step catalyzed by GSS adding glycine to ?-GC to generate GSH.And it was reported that ?-GC restores intracellular reduced GSH stores after hydrogen peroxide treatment in vitro.This study demonstrated the therapeutic effect of ?-GC on sepsis.The concrete results are as following:The sepsis models in BALB/c mice established by cecal ligation and puncture(CLP)or intraperitoneal injection with lipopolysaccharide(LPS)were used to test the therapeutic effect of ?-GC.We find that oral administration of ?-GC reduced lethality in both mouse sepsis models and ?-GC has a better effect than NAC,but reduced GSH barely functioned.ELISA assay showed that the release of early pro-inflammatory cytokines both in serum and tissues of septic mice,including tumor necrosis factor-?(TNF-?),Interleukin-1 beta(IL-1?),were inhibited by ?-GC.Tissue biopsies and H&E staining showed that ?-GC obviously alleviated the pathological injury of septic mice in lung and renal.MTT detection proved the low cytotoxity of?-GC to various cell lines.Studies revealed that ?-GC inhibited LPS-stimulated productions of TNF-?,1L-1?,high mobility group box 1(HMGB1)in RAW264.7 cells,suggesting inhibitory effect of ?-GC on early and late proinflammatory cytokines release in macrophages.Inducible Nitric oxide synthases(iNOS)and prostaglandin H2 synthase(COX-2)are two inflammatory indicators.?-GC inhibited the expression of iNOS and the following formation of NO(nitric oxide)induced by LPS,and it had no effect on COX-2In inflammation responses,excessive reactive oxygen species(ROS)formation can induce oxidative stress.?-GC suppressed LPS-induced ROS accumulation,which suggested ?-GC could alleviate oxidative stress caused by inflammatory stimulation.Thiol-reduced GSH is the predominant antioxidant form existing in most cells Therefore,we measured the intracellular GSH level after LPS and ?-GC treatment Intracellular GSH level was increased slightly when cell treated with only ?-GC,while the ratio of reduced GSH:oxidized GSH was dramatically enlarged by ?-GC when exposed to LPS.NAC also prevented the declines of total GSH and ratio of reduced GSH:oxidized GSH,but it did not have a significant effect like y-GC.To probe into the mechanism of LPS on GSH synthesis efficiency from ?-GC,we examined the expression of GSS which is the y-GC catalyzing enzyme.Our results showed that LPS,through Nrf2 pathway and NF-?B pathway,elevated the GSS expression at both protein and mRNA levels.And the suppression of Nrf2 pathway and NF-?B pathway impact the GSH synthetic efficiency from ?-GC.In conclusion,exogenous ?-GC can enter the body through digestive tract,and ameliorated pathological injury in septic mice;?-GC can play an anti-inflammatory effect by improving intracellular reduced GSH level,removing LPS-induced ROS accumulation and reducing the release of inflammatory mediators.Inflammatory stimulus such as LPS,through Nrf2 pathway and NF-?B pathway,up-regulate the GSS expression,and thus promote the efficiency of GSH synthesis from ?-GC.In vivo and in vitro experiments demonstrated that ?-GC has a better therapeutic effect than NAC against inflammation.The present study suggested that ?-GC may act as a potential therapeutic reagent for sepsis.