Protective Effects And Mechanism Of Combined Application Of Glutamine And Probiotics On Rat Intestines After Burn Injury

Severe burn induces intestinal inflammation and oxidative stress,which leads to intestinal damage and results in impairment of intestinal mechanical barrier and intestinal disorders.Previous studies have discovered that Glutamine(Gln)or probiotics treatment could reduce inflammatory response and intestinal damage.Studies reported that Gln inhibited oxidative stress and inflammatory response in intestines after burn injury by nutritional supplements and that probiotics protected intestines and inhibits inflammatory response through modifying intestinal micro-ecology,but the detailed mechanism still remained unknown.Whether the combined application of Gln and probiotics could reduce intestinal inflammation,impair oxidative stress and protect intestines more effectively still needs further study.NO expression increased significantly in rat burn models,indicating that it plays a crucial role in intestinal inflammation and oxidative stress after burn injury.NO is an important endogenous physiological active substance that maintains basic physiological functions under normal conditions.If the expression of NO increased aberrantly,it often leads to oxidative stress and inflammatory response.Considering that Gln and probiotics inhibit oxidative stress and intestinal inflammation and intestines produce large amounts of NO after burn injury,we speculate that Gln and probiotics might function by inhibiting the aberrant expression of NO and the combined application of Gln and probiotics have a better protective effect via the inhibition of oxidative stress and inflammatory response.As a result,rat burn models were employed to estimate the protective effect of the combined application of Gln and probiotics.Whether the combined application of Gln and probiotics inhibited oxidative stress and inflammatory response through impairing the expression of NO and the underlying mechanism were further investigated.Our results discovered that oxidative stress and inflammatory response increased significantly with an elevation of NO expression after severe burn resulting in the damage of intestines;oxidative stress happened earlier than inflammatory response and the inhibition of oxidative stress led to less release of inflammatory factors;induced nitric oxide synthase(i NOS)activated inflammatory response through NF-?B pathway and the inhibition of i NOS formation significantly impaired inflammatory response;burn injury impaired the the methylation of the coding gene of iNOS and the combined application of Gln and probiotics inhibited the formation and release of NO through promoting the methylation of i NOS gene,giving rise to the inhibition of oxidative stress and inflammatory response after burn injuries.Consequently,i NOS might play a crucial role in intestinal oxidative stress and inflammatory response after burn injuries and its regulation through DNA methylation by the combined application of Gln and probiotics resulted in protective effects from injuries due to attenuated oxidative stress and inflammatory response.IntroductionHuge amounts of body fluids exuding in early stage of severe burn and redistribution of blood caused by stress response lead to significant decline in intestinal blood flow.As a result,hypoxia and energy deficiency caused by intestinal ischemia will damage intestines through oxidative stress and inflammatory response.Necrosis and apoptosis of enterocytes due to intestinal injuries destroy the intercellular tight junctions,leading to increased intestinal permeability.Oxidative stress and inflammatory response are further enhanced because of the translocation of bacteria and macromolecular substance,and finally giving rise to severe systematic inflammatory reactions.Therefore,the protection of intestines is a critical part of treatment of severe burn.Gln,the main source of energy of enterocyte,significantly decreases after severe burn.Previous findings suggested that Gln supplements had a strong protective effect on intestinal injuries after burn through the significant inhibition of intestinal inflammation and the protection of intestinal mechanical barrier.Probiotics were also reported to protect intestinal mechanical barrier through modulating intestinal flora,inhibiting the proliferation of harmful bacteria and reversing intestinal disorders.The combined application of Gln and probiotics might be more effective in protecting the injured intestines from burn injury.As mentioned above that the aberrant formation and release of NO in the intestines of rat burn models,indicating that NO might be an important molecular related to stress response damaging the rat intestines.The combined application of Gln and probiotics might protect intestines after burn injury through the inhibition of aberrant NO release.NO is an endogenous physiological active substance widely distributed in the gastrointestinal tract.It is involved in the movement of the gastrointestinal tract and the protection of the gastrointestinal mucosa under physiological condition.However,NO is also involved in or aggravates inflammatory responses during burn injury and intestinal inflammation.The synthesis of NO is catalyzed by nitric oxide synthase(NOS)from L-arginine.There are three types of NOS currently known including neuronal NOS(nNOS),endothelial NOS(eNOS)and inducible NOS(i NOS),the first two of which are known as constructional NOS(c NOS).cNOS is Ca2+-calmodulin dependent,which produces small quantities of NO to maintain normal physiological functions.However,i NOS is Ca2+-calmodulin independent with harmful effects due to the overproduction of NO.Therefore,the key process of NO formation is the expression of i NOS.Our study demonstrated that burn injuries stimulated the body to increase the expression level of NO,ROS and MDA,and the level of inflammatory factors like IL-6,TNF-? and IL-8;Moreover,the level of IL-4 was decreased;the combined application of Gln and probiotics significantly impaired oxidative stress and the release of inflammatory factors with a protective effect of intestinal mechanical barrier.The inhibition of NO formation by using L-NAME(NOS inhibitor)was employed in rat burn models to discover the role of NO in intestinal inflammation induced by burn injury.We demonstrated that the elevation of TNF-? and IL-6 came later than ROS,NO and MDA,indicating that oxidative stress happened earlier than inflammatory response.L-NAME not only inhibited the expression of i NOS in intestines and the formation of NO,but decreased the levels of ROS,MDA,TNF-?,IL-6 and increased the level of SOD as well,resulted in attenuated oxidative stress and inflammatory response.The combined application of Gln and probiotics achieved similar effect with L-NAME,indicating that the combined application impaired oxidative stress and inflammatory response through the formation of NO and that i NOS is the key meditator of NO formation and release.Interestingly,we further discovered the regulatory effect of iNOS on NF-?B;the methylation level of the gene of i NOS had a significant role in iNOS expression.We also discovered that the combined application of Gln and probiotics inhibited the formation of NO and promoted the expression of PPAR? through the inhibition of iNOS expression via the promotion of the methylation level of the gene of iNOS resulting in impaired intestinal oxidative stress and inflammatory response.In conclusion,our results not only demonstrated the important role of i NOS in intestinal burn-induced inflammation and oxidative stress,but explored the mechanism of i NOS downregulation and attenuated inflammatory damage by the combined application of Gln and probiotics as well.Objectives1.To discover the mechanism of intestinal and the body's oxidative stress and inflammatory response after severe burn and the role of iNOS in this process.2.To explore the effect and mechanism of the treatment of intestinal damage after severe burn by combined application of Gln and probiotics.3.To illustrate the mechanism of the overexpression of iNOS in intestines after burn injury and the epigenetic regulation of i NOS with the combined application of Gln and probiotics.Materials and Methods?.The establishment of burn models and main materials1.110 male Wistar rats(8 weeks old,140g-185g)were purchased from Laboratory Animal Center of the Third Military Medical University(Chongqing,China).Rats were divided into some groups(Rats were divided into two groups according to our experimental design):(1)control group,burn model group,Gln treatment group,probiotics treatment group and Gln+probiotics treatment group(2)control group,burn model group and L-NAME(inhibitor of nitric oxide synthase)group.Burn model of 20% body surface area of full-thickness skin was made by dipping the hair removal area on the back of the rats with 100? boiling water for 15 s under anesthesia.Rats in control group were treated with 25? water for 15 s under anesthesia.2.Part ?: Rats in control group were lavaged with 1ml saline after injury.Rats in burn model group were lavaged with 1ml saline after injury.Rats in Gln treatment group were lavaged with Gln(1.5g/kg)+ 1ml saline after injury.Rats in probiotics treatment group were lavaged with probiotics(300mg/kg)+ 1ml saline after injury.Rats in Gln + probiotics treatment group were lavaged with Gln(1.5g/kg)+probiotics(300mg/kg)+1ml saline after injury.Rats in different groups were treated with different methods once a day for 7 days continuously.Intestine and blood samples were collected 7 days after injury under anesthesia.3.Part ?: Rats in control group and burn model group were treated with 1ml saline(tail intravenous injection)immediately after injury.Rats in L-NAME group were treated with L-NAME HCL(100mg/kg,tail intravenous injection)immediately after injury.Blood samples of rats in control group,burn model group and L-NAME group were collected at 0,1,6,24 h after injury,respectively.Intestine specimens were collected at 24 h after injury under anesthesia.?.Biochemical index1.IL-6,IL-4,IL-8 and TNF-? were measured by ELISA and fluorescent assays.2.ROS,MDA and SOD were measured by chemiluminescent assay.3.NO was measured using NO detection kit.?.q RT-PCR(quantitative real-time polymerase chain reaction)analysis.Total RNA was prepared from rat intestines and then reverse-transcription was performed.The mRNA expression of iNOS,e NOS,n NOS,NF-?B,Bax,EGF,IL-6,TNF-?,sICAM-1,s VCAM-1,PPAR?,DNMT-1,Tet1 and GADPH was detected using qRT-PCR analysis.?.TUNNEL analysisTUNNEL kit was employed to detect the apoptosis of intestinal cells and the number apoptotic cells in 100?m2 was estimated.?.HE stainingHE staining was performed on tissue sections prepared from rat intestines with different treatments to observe the injury condition.?.Western blotting(WB)The proteins were separated using SDS-PAGE and transferred onto polyvinylidene fluoride(PVDF)membranes.The membranes were blocked with evaporated milk and subsequently incubated with anti-human NF-?B,EGF,Bax,IKKB,PERK,ERK,P100,P52,DNMT-1,Tet1,i NOS,e NOS,nNOS,PPAR? and GAPDH primary antibodies.The membranes were then incubated with appropriate HRP-conjugated secondary antibodies and chemiluminescence was detected using SuperSignal West Femto Maximum Sensitivity Substrate(ECL,Pierce-Thermo Scientific,USA).?.Methylation-Specific PCRGenomic DNA was prepared and then treated with bisulphite.Methylated DNA of e NOS and i NOS was amplified using DNA methylation detection kit.?.Statistical analysesData are presented as the mean ± SD.Statistical analysis was performed using an SPSS 22.0 software.one-way ANOVA was used to compare the difference among groups.P < 0.05 was considered significant.Results?.Burn injury led to oxidative stress and inflammatory response and the combined application of Gln and probiotics significantly reduced oxidative stress and inflammatory response.Chemiluminescent assay was used to detect oxidative stress markers in blood samples from burned rats and our results showed that NO,ROS and MDA were at a high level after burn injury and the activity of SOD was severely impaired.The expression in blood samples of IL-6,TNF-? and IL-8 were upregulated and IL-4 downregulated according to ELISA analysis.The combined application of Gln and probiotics inhibited the expression of ROS,MDA,NO,TNF-?,IL-6 and IL-8 in blood and improved the level of IL-8 and the activity of SOD.Results of intestinal oxidative stress and inflammatory markers showed that IL-4 had no significant change and that other indicators shared the same expression changes.These findings indicated that burn injury led to intestinal and body's oxidative stress and inflammatory response and the combined application of Gln and probiotics significantly reduced them.?.Intestinal and body's oxidative stress and inflammatory response after burn injury were influenced by the expression of NO;and iNOS modulated the level of intestinal inflammation through the regulation of NF-?B.The effect and mechanism of NO on oxidative stress and inflammatory response after L-NAME treatment in burn rats were detected using q RT-PCR,Western blotting,ELISA and chemiluminescence.Our results showed that the changes of SOD,MDA,ROS and NO came earlier than IL-6 and TNF-? in blood samples,indicating that oxidative stress happened earlier than inflammation in burn injury.The intestinal levels of NO,ROS,MDA,TNF-? and IL-6 were significantly increased and SOD decreased 24 hours after burn injury.The intestinal levels of NO,ROS,MDA,TNF-? and IL-6 were significantly decreased and SOD increased in L-NAME group.With the inhibition of NO formation by using L-NAME(NOS inhibitor),we discovered that the oxidative stress and inflammatory markers were also decreased with the impaired formation of NO.Furthermore,the expression of P65(NF-?B)was downregulated with the inhibition of iNOS expression,indicating that i NOS might modulate inflammation through the regulation of NF-?B.?.The protective effect and the mechanism of combined application of Gln and probiotics on intestines after burn injury.Histological analysis showed that the combined application of Gln and probiotics significantly repaired intestinal damages and recovered intestinal mechanical barrier.The combined application reduced intestinal damage and promoted intestinal reparation through inhibiting the apoptosis of enterocytes(results from TUNNEL analysis)and attenuating the expression of an apoptosis-related protein BAX and promoting the expression of a reparation-related cytokine EGF(results from Western blotting and q RT-PCR).Western blotting and q RT-PCR analyses discovered that the combined application impaired the expression of iNOS in intestines resulting in inflammatory meditation and protection of intestines,and this might be realized through classical NF-?B pathway;and that the combined application inhibited the function of NF-?B through promoting the expression of PPAR?.Consequently,the combined application of Gln and probiotics plays an important role in the modulation of i NOS expression and intestinal protection.?.The combined application of Gln and probiotics regulates the expression of i NOS by modulating methylation level of the coding gene of iNOS.MS-PCR analyses showed that the methylation level of the coding gene of i NOS decreased after burn injury and it could be increased by the combined application;and that the methylation level of the coding gene of eNOS didn't show significant difference in burn injury and the combined application.Western blotting and q RT-PCR analyses showed that the expression of methylation transferase(DNMT-1)was obviously decreased and demethylase(Tet1)increased after burn injury.However,the combined application of Gln and probiotics after burn injury significantly improved the activity and expression of DNMT-1 while decreasing the activity and expression of Tet1.This indicated that the combined application regulated the expression of iNOS via the modulation of methylation level of the coding gene of iNOS.ConclusionsThe combined application of Gln and probiotics inhibited the expression of i NOS through the promotion of methylation level of the coding gene of iNOS,resulting in the downregulation of NO,the inhibition of oxidative stress and inflammatory response,the apoptosis of enterocytes,the promotion of intestinal reparation and the protective effect of intestinal barrier function.The combined application of Gln and probiotics promoted the expression of PPAR? due to the inhibition of NF-?B,resulting in the reduced intestinal inflammation.We postulate that this might be one of the mechanism that the combined application of Gln and probiotics function in the protection of intestines after burn injury.In conclusion,our study discovered for the first time that the aberrant expression of i NOS played an important role in oxidative stress and inflammatory response in intestines after burn injury;and the combined application of Gln and probiotics protected intestines after burn injury through the inhibition of i NOS expression.