| BackgroundSepsis is life threatening organ dysfunction caused by a dysregulated host response to infection,and continues to be the leading cause of mortality in the intensive care unit in developed countries.Accumulating evidence showed that abnormal host immune responses,inflammatory cytokines trigger of a "cytokine storm" resulting in subsequent systemic inflammatory response syndrome(SIRS),septic shock,and multiple organ dysfunction syndrome(MODS)and death.Significant advances have been made in understanding the pathogenesis of sepsis,development of new therapeutic agent toll-like receptor 4 antagonists that have been evaluated in clinical trials,however,there are only few successful results.Patients with severe sepsis usually have severe injury in their gastrointestinal system.Therefore,it is essential to study the underlying mechanisms of sepsis-induced gastrointestinal injury and develop novel therapeutic strategies to decrease the morbidity and mortality in septic patients.The gastrointestinal tract has long been hypothesized to play an integral role in the pathophysiology of sepsis,by acting as a motor that both drives and perpetuates multiple organ dysfunction.The gastrointestinal tract,a highly specialized intrinsic immune system,possesses the highest concentration of immune cells in the human body to maintain homeostasis and protect the body from incoming pathogens.In the past decades,numerous studies have reported that macrophages and mast cells(MCs)were implicated in the mediation of sepsis by the modulation of inflammatory and immune responses in a mouse cecal ligation puncture(CLP)model.For example,previous studies demonstrated that macrophages increased acute lung injury(ALI)through increased expression of macrophage inhibitory factor(MIF)in a sepsis-induced ALI rat model.MCs increases the recruitment of neutrophils through release of several inflammatory mediators that includes tumor necrosis factor(TNF),histamine and leukotrienes,and reduced animal survival in lipopolysaccharide(LPS)-induced sepsis rodent model.However,the exact role of macrophages,remain unclear in sepsis.The human intestinal microbiota,composed of 1013 to 1014 microorganisms that play an important role in epithelial barrier and gut immune system.Among the intestinal microbiota,probiotics that includes Bacillus subtilis,Bifidobacterium,Lactobacillus and Enterococcus are living microorganisms that have beneficial effects on the host.A growing body of experimental and clinical evidence has shown that probiotics exert their protective roles through inhibition of pathogen adhesion to intestinal surface,thereby improving function of gut epithelial barrier,and modulates immune function through regulation of secretion of various inflammatory mediators in several inflammatory bowel,infectious,gastrointestinal diseases.Therefore,probiotics could be used as a potential agent for the treatment of sepsis-induced gastrointestinal diseases,due to the barrier integrity and permeability of intestine.The expression of the tight junction(TJ)proteins decreased during weaning,and as a result,barrier integrity was impaired.This facilitated pathogen penetration and permits bacteriotoxins to enter the body.The consumption of probiotic bacteria contributes to intestinal function by maintaining paracellular permeability,enhancing the physical mucous layer,stimulating the immune system,and modulating resident microbiota composition and activity.Recently,Chen et al.have reported that probiotic pre-administration could significantly reduce the mortality rate in a mouse model of CLP-induced sepsis.However,the underlying mechanisms of probiotics in regulating sepsis-induced gastrointestinal injury have not been elucidated.Objectives1.To investigate the protective effect of bacillus subtilis and enterococcus faecalis(LCBE)on cecal ligation and perforation(CLP)-induced sepsis mice.2.To study the effect of probiotics on the pathological damage of ileum tissue in septic mice.To explore its anti-inflammatory and protective effects.3.To study the effect of probiotics on the activation and transformation of macrophages and MCs degranulation in septic mice.4.To investigate the role of probiotics in the activation of Akt signaling pathway in septic mice.Material and Methods1.One week before the establishment of the mouse cecal ligation perforation(CLP)model,mice were given probiotics capsules or saline at a dose of 200 1/day(400 mg/kg).The mice were then randomly divided into three groups:sham group,CLP control group and clp-probiotics group for CLP surgery.In the sham group,only the cecum was separated,but no ligation and puncture was performed.After treatment,the mice were treated with anti-infective therapy for 2 days,and the 7-day survival rate of CLP mice was observed to verify the effect of probiotics on the mortality of clp-induced sepsis mice.2.At 72 h after CLP,mice were sacrificed,blood and peritoneal lavage were collected,and terminal ileum tissues were taken out.The histopathological score of ileum was calculated according to the corresponding scoring criteria after HE staining.WB was used to detect the expression of TJ protein(zo-1,claudin-1 and occludin),so as to indicate whether probiotics could alleviate the pathological injury of the ileal tissue of clp-induced sepsis mice.3.The serum levels of TNF-a,L-6 and IL-10 were determined by ELISA according to the instructions.The ileal tissues were homogenized with PBS,ultrasonically treated and centrifuged,and the supernatant was collected to determine the levels of TNF-a,L-6 and IL-10 by ELISA.Total ileal tissue protein was extracted and the expression of TGF-β1 was detected by Western blot to analyze the influence of probiotics on the expression of inflammatory factors and anti-inflammatory factors in the blood and ileal tissues of clp-induced sepsis mice.4.The expressions of CDllc(marker of Ml-type cells)and CD206(marker of M2-type cells)in ileal specimens CD68 and ileal tissue were detected by IHC and WB,respectively,to study the effect of probiotics on the activation and transformation of macrophages in sepsis mice.5.Histamine concentrations in serum and peritoneal lavage fluid were determined by ELISA.After the ileum sections were stained with toluidine blue,MCs were observed under an optical microscope,and the inhibitory effect of probiotics on the degranulation of MCs was studied.6.WB was used to detect the expression of p-Akt and Akt in ileum samples to study the role of probiotics in the activation of Akt signaling pathway in sepsis mice.7.Statistical analysis:SPSS 18.0 Software(IBM SPSS,USA)and GraphPad Prism 5.0 Software(GraphPad Software,Inc.,USA)were used for statistical analysis.Depending on the distribution and type of the data,the data is expressed as the mean mean standard error(SEM)or median quartile.Significant differences were analyzed using unpaired two-tailed student t-tests,or ANOVA and Bonferroni methods,or kruskal-wallis tests and subsequent Dunn multiple comparisons.P<0.05 was considered statistically significant.Results1.Effect of probiotics(LCBE enteric-coated capsules)on the survival rate of sepsis mice:the 7-day survival rate of LCBE enteric-coated capsules pretreated mice was significantly higher than that of the control group of sepsis mice,indicating that probiotics/LCBE enteric-coated capsules could improve the survival rate of clp-induced sepsis mice.2.Effect of probiotics(LCBE enteric-coated capsules)on pathological damage of ileal tissue in septic mice:compared with sham surgery,CLP surgery resulted in significantly lower villi length and significantly increased Chiu’s score.However,compared with the CLP control group,the length of villi was significantly increased in the probiotic/LCBE enteric-coated capsule pretreatment group,while the Chiu’s score was significantly decreased.Meanwhile,the pretreatment of probiotics/LCBE enteric-coated capsules could restore the expression of TJ protein.These results suggest that LCBE enteric-coated capsules can improve the pathologic damage of the ileum in clp-induced sepsis mice.3.Effect of probiotics(LCBE enteric-coated capsules)on the expression of inflammatory cytokines and anti-inflammatory cytokines in the blood and ileum of clp-induced sepsis mice:compared with the CLP control group,the levels of pro inflammatory cytokines IL-6 and TNF-a in the serum and ileum of the clp-probiotics group were significantly decreased.However,the anti-inflammatory factors IL-10 and TGF-β1 did not differ significantly between the CLP control group and the CLP-probiotics group.This indicated that LCBE could inhibit the expression of proinflammatory factors IL-6 and TNF-a,but had no significant effect on the expression of anti-inflammatory factors TGF-β1 and IL-10.This means that LCBE enteric capsules provide a protective effect by reducing the inflammatory components of clp-induced sepsis in mice.4.Regulation of activation and transformation of macrophages by probiotics(LCBE enteric-coated capsules):IHC staining analysis showed that CD68 was positively stained in the ileal sections of mice from the clp-control group,while CD68 was significantly weaker in the clp-probiotics group,indicating that LCBE enteric-coated capsules could inhibit the activation of macrophages.In addition,CD11c expression was significantly reduced in the clp-probiotics group compared with the CLP control group.However,the expression of CD206 did not change significantly in the sham group,the clp-control group and the clp-probiotics group.These results suggest that probiotics can play a protective role by inhibiting macrophage activation and transformation.5.Effects of probiotics(LCBE enteric-coated capsules)enteric capsules on MC degranulation and histamine secretion:toluidine blue staining results showed that degranulation of MCs was significant in the CLP control group,but not in the clp-probiotics group.The histamine levels in serum and peritoneal lavage fluid of CLP control group were significantly higher than those of sham group.However,the histamine levels in serum and peritoneal lavage fluid of mice in the clp-probiotics group were significantly lower than those in the clp-probiotics group:These results suggest that probiotics can play a protective role by inhibiting MCs degranulation.6.Effects of probiotics(LCBE enteric-coated capsules)on Akt signaling pathway:compared with the clp-control group,the p-akt level in the clp-probiotics group was increased,indicating that the Akt signaling pathway could be activated by pretreatment with probiotics.conclusionProbiotics can alleviate clp-induced sepsis in mice by reducing intestinal inflammatory responses,changing macrophage activation,polarization and MCs degranulation,and regulating the Akt signaling pathway. |
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