Toll-like receptors is a super-family of pathogen recognition-receptors discovered in recent years.During the process of ischemia-reperfusion, the Toll-like receptor 4 (TLR4) combines with lipopolysaccharide and many endogenous ligands such as high mobility group protein B1, heparan sulfate, and fibrinogen. Through the myeloid differentiation protein 88 -dependent and -independent signaling pathways, the products induce the release of inflammatory cytokine-mediated inflammatory response, leading to injuries. ω-3 polyunsaturated fatty acids, by inhibiting the signal pathway activation and target gene expression of TLR4, can influence the function of many immune cells and regulate the body's inflammatory response and immune function. This article reviews the function of TLR4 during ischemia-reperfusion injury and the possible interventional role of ω-3 polyunsaturated fatty acids.

As the largest protective barrier of the body,gastrointestinal tract helps the organism resist the invasion and attacks of harmful substances from the outside world.There are three kinds of protective barriers:mechanical barrier,biotical barrier,and immune barrier.In recent years,increasing researches indicate the importance of gastrointestinal immune barrier and the important immunologic mechanism of enteral nutrition in improving the clinical outcome.This article reviews the effect of gut immunity in the improvement of clinical outcome by enteral nutrition.

OBJECTIVETo investigate the role of high mobility group box 1 (HMGB1) in the signaling pathway of mouse intestinal ischemia-reperfusion injury.

METHODSTwenty-four Specific Pathogen free male C57BL / 6 mice were randomly divided into three groups (n = 8) : the sham operation group (sham), the control group(control) and the HMGB1 antibody group (anti-HMGB1). The vehicle alone or anti-HMGB1 antibody(1 mg/kg, 0. 025%) was injected respectively via the caudal vein 30 min prior to ischemia in the control group or the anti-HMGB1 group. All mice were anesthetized,opened abdominal wall and exposed arteria mesenterica superior. The control group and the anti-HMGBl group underwent 60 min of mesenteric ischemia and 60 min of reperfusion and the sham group were merely opened abdominal wall for 120 min without ischemia-reperfusion. The levels of NF-κB p65, IL-6 and TNF-α in plasma and the activity of MPO in lung and liver and the morphological changes of lung and intestinal tissue were measured. The mRNA levels of HMGB1 and NF-κB were evaluated using real-time quantitative PCR and the protein levels of HMGB1 and NF-KB were evaluated using Western blot. The experimental data was analyzed using one-way analysis of variance.

RESULTSThe levels of IL-6, TNF-α and NF-κB p65 in plasma was significantly higher in the control group and the anti-HMGB1 group compared with the sham group (the sham group vs. the control group vs. the anti-HMGB1 group, NF-κB p65, 104. 64 ± 11. 89: 228. 53 ± 24. 85: 145. 00 ± 33. 63, F = 38. 036, P <0. 05; IL-6,50. 02 ± 6. 33:104. 91 ± 31. 18:62. 28 ± 6. 73, F = 49. 763, P < 0. 05; TNF-α, 43. 79 ± 4. 18: 70. 81 ± 6. 97: 52. 76 ± 5. 71, F = 34. 571, P < 0. 05). The increasing degree in the anti- HMGB1 group was significantly reduced compared with the control group (P <0. 05). The activity of MPO of liver and lung in the control group and the anti-HMGB1 group was significantly higher than those in the sham group (P <0. 05). Compared with the sham group, the degree of tissue injury in jejunum, ileum and lung was serious in the control group, and that in the anti-HMGB1 group was significantly lower than the control group. The expression of HMGB1 mRNA and NF-κB mRNA in the lung and the ileum in the sham group and the control group were all higher than the sham group (HMGB1 mRNA in lung: sham group 1. 04 ± 0. 19 vs. control group 2. 25 ± 0. 18 vs. anti-HMGB1 group 1. 89 0. 18, F = 66. 203, P < 0. 05; in ileum: 1. 14 ± 0. 54 vs. 6. 26 ± 0. 60 vs. 4. 93 0. 55, F = 133. 427, P < 0. 05; NF-κB mRNA in lung: 1. 03 ± 0. 21 vs. 2. 04 ± 0. 29 vs. 1. 42 ± 0. 23, F =26. 229, P < 0. 05; ileum: 1. 03 ± 0. 23 vs. 3. 71 ± 0. 53 vs. 2. 23 ± 0. 55, F = 50. 477, P <0. 05). Subjected to intestinal ischemia-reperfusion injury, the protein expression of HMGB1 and NF-κB in the lung, jejunum and ileum in the control group and the anti-HMGB1 group increased compared with the sham group(P <0. 05), but that was significantly lower in the anti-HMGB1 group than the control group (P <0. 05).

CONCLUSIONThe administration of anti-HMGB1 antibodies may reduce the damage caused by ischemia-reperfusion effectively.

Animals ; HMGB1 Protein ; metabolism ; Interleukin-6 ; Intestines ; metabolism ; Liver ; Lung ; Male ; Mesenteric Artery, Superior ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; RNA, Messenger ; Reperfusion Injury ; metabolism ; Signal Transduction ; Transcription Factor RelA ; Tumor Necrosis Factor-alpha

Objective To investigate the expression of high mobility group box 1 (HMGBI) of TLR4 endogenous ligand and distant organ tissue injury after intestine ischemia/reperfusion and drainage of lymph fluid in rats. Methods Twenty-four Sprague-Dawley (SD) male rats (SPF grade) were evenly divided into 3 groups:Sham surgery group,intestine ischemia-reperfusion (I/R) group,and intestine ischemia-reperfusion with drainage of intestine lymph fluid (IR + drainage) group.The injury of distant organs such as lungs,liver,kidney was evaluated;The expression of high mobility group box 1 (HMGBI) of TLR4 endogenous ligand in intestine,lung and liver after the ischemia-reperfusion injury was measured by immunohistochemistry.Result HE stained sections,as well as HMGB1 immunohistochemistry results showed that the injury of ischemia/reperfusion (I/R) group and ischemia/reperfusion (I/R) + drainage group were more severe than that in the sham group.A large number of cells stained in I/R group,indicating that HMGB1 expression increased.The injury in I/R + drainage group was significantly less severe than I/R group.Western blot tests showed that the expression of HMGB1 in jejunum,ileum,liver,lung increased significantly in I/R group after L/R injury.Gray-scale values of HMGB1/β-actin were 0.3145 ± 0.0549、 1.7352 ± 0.3280、1.4443 ± 0.0926、3.1382 ± 0.4202.Lymph drainage significantly alleviated the damage,the expression of HMGB1 were significantly lower (P ＜0.05).Gray-scale values of HMGB1/β-actin were 0.1745 ± 0.0327、 1.1083 ± 0.2098、 1.1862 ± 0.1221、2.1095 ± 0.1993. Conclusion Increased expression of HMGB1 of TLR4 endogenous ligand is associated with intestinal and distant tissue injury during intestinal ischemia-reperfusion injury.Drainage of lymph fluid can block the gutlymph pathway and thus reduce the source of HMGB1 from the intestinal as well as the injury of distant tissue.

Objective To investigate intestinal mucosal injury and the change of free amino acid levels in plasma with intestinal ischemia/reperfusion.Methods Twenty-four Sprague-Dawley (SD) male rats (SPF grade) were randomly divided into 3 groups with 8 rats in each group:Blank group,Sham group and ischemia/reperfusion (I/R) group.The rats in I/R group were subjected to 60 min ischemia by clamping the superior mesenteric artery (SMA),followed by 120 min repeffusion.All rats were sacrificed with blood withdraw through inferior vena cava.The plasma was precipitated with Sulfosalicylic acid and the supernatant free amino acid levels were measured and the intestinal mucosal thickness and villus length were also assayed.Results In the I/R group the total free amino acids,essential amino acids (EAA),glutamine and branched-chain amino acids (BCCA) were remarkably lower [the total free amino acids:I/R vs Blank vs Sham:(4585.1 326.1) vs (5661.5 ±581.9) vs (5337.9±998.7) μmol/L (F=5.075,P=0.016); EAA:I/Rvs Blank vs Sham:(1401.3 ±183.4) vs (2147.6 ± 265.1) vs (1796.2 ± 440.8) μmol/L (F =1 1.216,P =0.000) ; glutamine:I/R vs Blank vs Sham:(646.1 ± 34.7) vs (895.7 ± 258.8) vs (839.1 ± 163.7) μmol/L (F =4.326,P =0.027) ; BCCA:I/R vs Blank vs Sham:(507.8 ± 119.0) vs (912.2 ± 165.8) vs (671.9 ± 79.8) μmol/L (F =10.662,P =0.001)]and the jejunum and ileum mucosal thickness and villus height were decreased compared to Blank and Sham groups [jejunum mucosal thickness:I/R vs Blank vs Sham:(401.50 ± 117.79) vs (529.22 ±54.73) vs (499.54 ±64.48) μm (F=31.869,P =0.000) ; jejunum villus height:I/R vs Blank vs Sham:(271.37 ± 84.29) vs (365.26 ± 46.98) vs (349.67 ± 56.11) μm (F =30.472,P =0.000) ; ileum mucosal thickness:I/R vs Blank vs Sham:(254.20 ± 43.56) vs (324.70 ± 30.56) vs (298.26 ± 58.46) μm (F =30.442,P =0.000) ; ileum villus height:I/R vs Blank vs Sham:(169.37 ± 37.25) vs (221.62 ± 37.26) vs (193.25 ± 38.39) μm (F =24.145,P =0.000)],and The EAA and BCAA in the I/R group were lower than the Sham group (respectively,P ＜0.05).There was no significant difference in aromatic amino acids (AAA) among the three groups [I/R vs Blank vs Sham:(273.2 ± 37.4) vs (296.8 ± 55.6) vs (281.9 ± 7.3) μmol/L (F =0.578,P =0.570)].The ratio BCAA/AAA in the Sham and I/R groups were significantly lower than the Blank group [(I/R vs Blank vs Sham:(2.4 ±0.6) vs.(1.9 ±0.4) vs (3.1 ±0.7) (F =5.215,P =0.014)],while the I/R group was decreased slightly compared to the Sham group,but the difference was not significant (P ＞ 0.05).The ethanolamine phosphate,taurine,citrulline,cystine,phosphoserine levels were reduced in the Sham and I/R groups compared to the Blank group [ethanolamine phosphate:I/R vs Blank vs Sham:(11.4 ± 1.9) vs (14.3 ± 3.4) vs (10.1±1.7) μmol/L(F=5.897,P=0.009);taurine:I/R vs BlankvsSham:(341.1±36.3) vs(533.2±90.8) vs (439.2±105.4) μmol/L (F=10.702,P=0.001); citrulline:I/R vs Blank vs Sham:(57.7±3.2) vs (73.1 ±16.2) vs (58.1 ±3.8) μmol/L (F=6.360,P =0.007); cystine:I/R vs Blank vs Sham:(20.0 ± 3.6) vs (60.6 ± 24.6) vs (36.3 ± 5.8) μmol/L (F =15.344,P =0.000) ; phosphoserine:I/R vs BlankvsSham:(10.2±1.1) vs (15.8±5.4) vs (11.7 ±3.4) μmol/L (F=4.878,P=0.018)],and the taurine and cystine in I/R groups were significantly decreased than the Sham group (respectively,P ＜ 0.05).The ornithine and arginine were comparatively reduced in I/R in contrast to the Blank and Sham groups [ornithine:I/R vs Blank vs Sham:(81.5 ± 19.0) vs (125.5 ±42.3) vs (114.9 ± 19.5) μmol/L (F =4.961,P =0.017) ;arginine:I/R vs Blank vs Sham:(199.2 ± 8.0) vs (258.9 ± 14.6) vs (248.7 ± 38.4) μmol/L (F =13.940,P =0.000)].The tryptophan and glutamic acid concentrations were increased in the Sham and I/R groups [tryptophan:L/R vs Blank vs Sham:(125.9 ± 12.1) vs (103.1 ± 29.9) vs (128.9 ± 18.5) μmol/L (F =5.429,P =0.031) ; glutamic acid:I/R vs Blank vs Sham:(188.6 ± 29.8) vs (93.6 ± 29.4) vs (125.4 ± 43.8) μmol/L (F =15.241,P =0.000)] and it was lower in the Sham group than the I/R group (P ＜ 0.05).Conclusion Intestinal ischemia-reperfusion can cause intestinal mucosal injury and the change of free amino acid levels in plasma and intestinal barrier damage may be related to the decline glutamine concentration and the increase of protein catabolism.

Objective To study several measuring methods of the intestinal mucosa barrier and evaluate the correlation between different methods for determining the intestinal damage.Methods Sixteen specific pathogen free (SPF) male Sprague-Dawley rats were randomly divided into two groups:the Control group (n =8) and the ischemia/reperfusion (I/R) group (n =8).After adaptive feeding for 5 days,I/R group was put into ischemia model for 60 min and the Control group was merely opened on its abdominal wall but without ischemia for 60 min.After having been fed for another day,all rats were killed and specimens were collected.The plasma diamine oxidase (DAO),D-lactate (D-LAC),endotoxin,and glutamine (Gln) levels were detected,and the intestinal mucosal morphology was observed.The intestinal permeability (L/M) was detected 1 day before and after the surgery.Results The plasma DAO,D-LAC,and endotoxin levels were significantly higher in I/R group compared with the Control group (DAO:(0.498 ±0.032) vs (0.247 ±0.051) U/ml,t=-11.790,P=0.000; D-LAC:(5.47±1.55) vs (3.83±0.63) mg/L,t=-2.757,P=0.022; endotoxin:(0.0395±0.002 8) vs (0.025 6 ±0.004 5) EU/ml,t =-7.377,P =0.000).The plasma Gln concentration was significantly lower than that in the Control group [(646.12 ± 34.75) vs (839.13 ± 163.76) μmol/L,t =3.261,P =0.012).The L/M value on the 1 st postoperative day was significantly higher than that in the I/R group [(3.63 ±2.09) vs (1.22 ±0.66),t =-3.118,P =0.013)].The jejunum mucosal thickness,jejunum villus height,ileal mucosal thickness,and ileal villus height were significantly lower in I/R group compared with the Control group after operation [(329.80 ±64.68) vs (512.82 ±38.41) μm,t=6.881,P=0.000; (253.06±69.33) vs (386.79±56.39) μm,t=4.232,P=0.001; (205.89± 18.71) vs (335.29±27.71) μm,t=10.945,P=0.000; (135.61 ±22.30) vs (253.18±31.02) μm,t =8.705,P =0.000].After intestinal ischemia/reperfusion,DAO,D-LAC,endotoxin and L/M were all increased and positively correlated with each other.The plasma concentration of Gln and the morphological changes of jejunum and ileum were negatively correlated with DAO,D-LAC,endotoxin,and L/M,respectively.Conclusions After intestinal ischemia/rcperfusion,the levels of all examination indicators obviously change and correlate with each other.The DAO,D-LAC,endotoxin,and L/M are positively related to each other and negatively correlated with the intestinal barrier function.Gln is positively correlated with small intestinal mucosal morphology and negatively correlated with others,respectively.

Objective To study the clinical effect of low molecular weight heparin on fetal growth restriction (FGR) prevention in early pregnancy.Methods 100 pregnant women had been employed in our study,they were divided into two groups,observation( n =60) and control( n =40) group.Both groups were given the natural vitamin E 0.1g,and folie acid 0.4mg daily oral administration,continuing to 12 weeks of gestation.5000 u of subcutaneous dalteparin sodium was added to pregnant women with notmal level of D-dimer in the observation group until 12 weeks of pregnancy,while the dose of Fragmin was adjusted in pregnant women with abnormal level of D-dimer until retuned to normal level.The pregnacy outcome of two groups were compared.Results The gestational age,birth weight,placental weight of the obseruation group was signiticantly higher than thal of the control group ( t =4.55,2.79,11.91,all P ＜ 0.05 ),while FGR,oligohydramnies,fetal distress and the incidence of hypertensive disorters of the obseruation group were significantly lower than that of control group ( x2 =6.50,20.55,7.87,3.76,all P ＜ 0.05 ) ; Compared to the control group,the observation group didn't have higher incidence of intrauterine fetal death,neonatal asphyxia and perinatal death ( P ＞ 0.05).Conclusion Low molecular weight heparin using in early pregnancy was effective in preventing FGR in pregnant women with FGR risk factors.

Objective To measure intestinal permeability of postoperative fasting patients and evaluate their intestinal barrier function.Methods Twenty patients after moderate or minor operation ,10 male and 10 female,were included in the study .After 3 to 5 days of fasting,when their bowel functon returned to normal,10 ml solution of lactulose(2g) with mannitol (1g) were given orally,urine of 6 hours was collected.As a control group,20 healthy volunteers were also included.There were no statistical differences in sex and age between two groups.Results The L/M of the patients and healthy volunteers were 0.1989?0.1186 and 0.4398? 0.2165 respectively,the difference was significant.Conclusions The intestinal permenbility of postperative fasting patients was elevated when compared with that of volunteers.

TLR4 mediates I/R injury involving endogenous ligands.Interaction of TLR4 with endogenous ligands provides a critical link between tissue damage and activation of the innate immune response.In the early phase of liver,kidney,heart,or lung I/R injury,endogenous ligands are secreted from several kinds of cells,they are recognized by TLR4.Interaction of TLR4 with endogenous ligands,such as HMGB1,seems to be the most important trigger of inflammation and initiates signaling cascades leading to inflammatory and immune responses.Blocking the interaction of TLR4 with endogenous ligands may be useful in clinical management of inflammation and cellular necrosis caused by ischemic insults.