MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4

BACKGROUND Diabetic kidney disease(DKD)is a major complication of diabetes mellitus.Renal tubular epithelial cell(TEC)damage,which is strongly associated with the inflammatory response and mesenchymal trans-differentiation,plays a significant role in DKD;However,the precise molecular mechanism is unknown.The recently identified microRNA-630(miR-630)has been hypothesized to be closely associated with cell migration,apoptosis,and autophagy.However,the association between miR-630 and DKD and the underlying mechanism remain unknown.AIM To investigate how miR-630 affects TEC injury and the inflammatory response in DKD rats.METHODS Streptozotocin was administered to six-week-old male rats to create a hypergly cemic diabetic model.In the second week of modeling,the rats were divided into control,DKD,negative control of lentivirus,and miR-630 overexpression groups.After 8 wk,urine and blood samples were collected for the kidney injury assays,and renal tissues were removed for further molecular assays.The target gene for miR-630 was predicted using bioinformatics,and the association between miR-630 and toll-like receptor 4(TLR4)was confirmed using in vitro investigations and double luciferase reporter gene assays.Overexpression of miR-630 in DKD rats led to changes in body weight,renal weight index,basic blood parameters and histopathological changes.RESULTS The expression level of miR-630 was reduced in the kidney tissue of rats with DKD(P<0.05).The miR-630 and TLR4 expressions in rat renal TECs(NRK-52E)were measured using quantitative reverse transcription polymerase chain reaction.The mRNA expression level of miR-630 was significantly lower in the high-glucose(HG)and HG+mimic negative control(NC)groups than in the normal glucose(NG)group(P<0.05).In contrast,the mRNA expression level of TLR4 was significantly higher in these groups(P<0.05).However,miR-630 mRNA expression increased and TLR4 mRNA expression significantly decreased in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Furthermore,the levels of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β),and IL-6 were significantly higher in the HG and HG+mimic NC groups than in NG group(P<0.05).However,the levels of these cytokines were significantly lower in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Notably,changes in protein expression were observed.The HG and HG+mimic NC groups showed a significant decrease in E-cadherin protein expression,whereas TLR4,α-smooth muscle actin(SMA),and collagen IV protein expression increased(P<0.05).Conversely,the HG+miR-630 mimic group exhibited a significant increase in E-cadherin protein expression and a notable decrease in TLR4,α-SMA,and collagen IV protein expression than in the HG+mimic NC group(P<0.05).The miR-630 targets TLR4 gene expression.In vivo experiments demonstrated that DKD rats treated with miR-630 agomir exhibited significantly higher miR-630 mRNA expression than DKD rats injected with agomir NC.Additionally,rats treated with miR-630 agomir showed significant reductions in urinary albumin,blood glucose,TLR4,and proinflammatory markers(TNF-α,IL-1β,and IL-6)expression levels(P<0.05).Moreover,these rats exhibited fewer kidney lesions and reduced infiltration of inflammatory cells.CONCLUSION MiR-630 may inhibit the inflammatory reaction of DKD by targeting TLR4,and has a protective effect on DKD.

Divergent expression of bacterial wall sensing toll-like receptors 2 and 4 in colorectal cancer

To characterize the expression of toll-like receptors (TLR) 2 and 4 in colorectal cancer (CRC) and in normal colorectal mucosa. METHODSWe analysed tissue samples from a prospective series of 118 unselected surgically treated patients with CRC. Sections from formalin fixed, paraffin embedded specimens were analysed for TLR2 and TLR4 expression by immunohistochemistry. Two independent assessors evaluated separately expression at the normal mucosa, at the invasive front and the bulk of the carcinoma, and in the lymph node metastases when present. Expression levels in different locations were compared and their associations with clinicopathological features including TNM-stage and the grade of the tumour and 5-year follow-up observations were analysed. RESULTSNormal colorectal epithelium showed a gradient of expression of both TLR2 and TLR4 with low levels in the crypt bases and high levels in the surface. In CRC, expression of both TLRs was present in all cases and in the major proportion of tumour cells. Compared to normal epithelium, TLR4 expression was significantly weaker but TLR2 expression stronger in carcinoma cells. Weak TLR4 expression in the invasive front was associated with distant metastases and worse cancer-specific survival at 5 years. In tumours of the proximal colon the cancer-specific survival at 5 years was 36.9% better with strong TLR4 expression as compared with those with weak expression (P = 0.044). In contrast, TLR2 expression levels were not associated with prognosis. Tumour cells in the lymph node metastases showed higher TLR4 expression and lower TLR2 expression than cells in primary tumours. CONCLUSIONTumour cells in CRC show downregulation of TLR4 and upregulation of TLR2. Low expression of TLR4 in the invasive front predicts poor prognosis and metastatic disease.

Relationship of Toll-Like Receptors 2 and 4 Gene Polymorphisms with Essential Hypertension in Chinese Han Population

Objective: There are numerous studies suggesting that genetic polymor-phisms of inflammation factors Toll-like receptors 2 and 4 (TLR2, TLR4) might play a role in the pathophysiological process of hypertension. In this study, we evaluated the association in a sample of members of the Chinese Han population. Method: We selected four single nucleotide polymor-phisms (SNP) of TLR2 (rs3804099, rs3804100, rs7656411) and TLR4 (rs1927906) genes, and measured the distributions of genotypic and allelic frequencies in 1063 participants, including 391 essential hypertension pa-tients and 672 controls. Result: No significant differences in the genotypic and allelic frequencies of the four SNPs were detected between cases and controls. However, three haplotypes, CCG, TTG and TTT of TLR2, were significantly associated with a decrease in the risk of essential hyperten-sion (OR: 0.512, 95% CI: 0.397 - 0.660, P P = 0.0038;OR: 0.797, 95% CI: 0.667 - 0.952, P = 0.0122, respectively). Inversely, the risk of essential hypertension increased sig-nificantly in patients with the CTG, TCG or TCT haplotypes (OR: 2.924, 95% CI: 2.157 - 3.963, P P P Conclusion: Our study suggested that haplotypes (CCG, TTG, TTT, CTG, TCG and TCT) of TLR2 might have profound effects on the development of essential hypertension in the Chinese Han population.

Betaine inhibits Toll-like receptor 4 expression in rats with ethanol-induced liver injury

AIM:To test whether ethanol feeding could induce Toll-like receptor 4(TLR4)responses,assess the hepatoprotective effect of betaine and its inhibitive effect on TLR4 in animal models of alcoholic liver injury.METHODS:Forty-eight female Sprague-Dawley rats were randomly divided into four groups as control,model,low and high dose betaine groups.Except control group,all rats were fed with high fat-containing diet plus ethanol and fish oil gavages for 8 wk.Betaine was administered intragastrically after exposure of ethanol for 4 wk.The changes of liver histology were examined.The expression of TLR4 mRNA and protein was detected by RT-PCR and Western blotting,respectively.The serum aminotransferase activity alanine transarninase(ALT),aspartate aminotransferase(AST),serum endotoxin,and liver inflammatory factors tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin-18(IL-18)were also assayed.RESULTS:Compared with control group,rats of model group developed marked liver injury,accompanied by an increase of ALT(159.41±7.74 U/L vs 59.47± 2.34 U/L,P<0.0001),AST(248.25±1.40 U/L vs 116.89±3.48 U/L,P<0.0001),endotoxin(135.37± 30.17 ng/L vs 44.15±7.54 ng/L,P<0.0001),TNF-α(20.81±8.58 pg/mL vs 9.34±2.57 pg/mL,P=0.0003),IFN-γ(30.18±7.60 pg/mL vs 16.86±9.49 pg/mL,P= 0.0039)and IL-18(40.99±8.25 pg/mL vs 19.73±9.31 pg/mL,P=0.0001).At the same time,the expression of TLR4 mRNA and protein was markedly induced in the liver after chronic ethanol consumption(1.45±0.07 vs 0.44±0.04,P<0.0001;1.83±0.13 vs 0.56±0.08,P<0.0001).Compared with model group,betaine feeding resulted in significant decreases of ALT(64.93 ±6.06 U/L vs 159.41±7.74 U/L,P<0.0001),AST(188.73±1.11 U/L vs 248.25±1.40 U/L,P<0.0001),endotoxin(61.80±12.56 ng/L vs 135.37±30.17 ng/L,P<0.0001),TNF-α(9.79±1.32 pg/mL vs 20.81± 8.58 pg/mL,P=0.0003),IFN-γ(18.02±5.96 pg/mL vs 30.18±7.60 pg/mL,P=0.0008)and IL-18(18.23±7.01 pg/mL vs 40.99±8.25 pg/mL,P<0.0001).Betaine also improved liver steatosis.The expression levels of TLR4 mRNA or protein in liver tissues were significantly lowered(0.62±0.04 vs 1.45±0.07,P<0.0001;and 0.65±0.06 vs 1.83±0.13,P<0.0001).There was a statistical difference of TLR4 mRNA and protein expression between high-and low-dose betaine groups(0.62±0.04 vs 0.73±0.05,P<0.0001,and 0.65±0.06 vs 0.81±0.09,P<0.0001).CONCLUSION:Betaine can prevent the alcoholinduced liver injury effectively and improve the liver function.The expression of TLR4 increases significantly in ethanol-fed rats and betaine administration can inhibit TLR4 expression.

Association of Toll-like receptor 4 Signaling Pathway with Steroid-induced Femoral Head Osteonecrosis in Rats

Osteonecrosis of the femoral head is frequently observed in patients treated with excessive corticosteroids. However, the pathogenesis of corticosteroid-induced osteonecrosis remains unclear. The purpose of this study was to investigate the role of Toil-like receptor 4 （TLR4） signaling pathway in steroid-induced femoral head osteonecrosis in rats. Male Sprague-Dawley rats were injected intramus- cularly with 20 mg/kg methylprednisolone （MP） for 8 weeks, twice per week. The animals were sacri- ficed at 2, 4 and 8 weeks after the last MP injection, respectively, and then allocated to the 2-, 4- and 8-week model groups （n=24 each）. Rats in the control group （n=12） were not given any treatment. Histopathological analysis was performed and the concentration of tartrate-resistant acid phosphatase （TRAP） in plasma was determined. The activation of osteoclasts in the femoral head was assessed by TRAP staining. The expression of TLR4, MyD88, TRAF6 and NF-Id3 p65 that are involved in TLR4 signaling, and MCP-1 production were detected by using real-time PCR （RT-PCR） and Western blotting. The results showed that the osteonecrosis in the femoral head was clearly observed and the concentra- tion of TRAP in the plasma was increased in the model rats. The femoral head tissues in MP-treated rats were positive for TRAP and the intensity of TRAP staining was greater in MP-treated rats than in con- trol rats. As compared with the control group, the mRNA expression of TLR4 signaling-related factors was enhanced significantly at 4 and 8 weeks, and the protein levels of these factors increased signifi- cantly with time. It was concluded that MP could induce the femoral head osteonecrosis in rats, which was associated with osteoclast activation via the TLR4 signaling pathway. These findings suggest that TLR4 signaling pathway plays a pivotal role in the pathogenesis of steroid-induced osteonecrosis.

Starring role of toll-like receptor-4 activation in the gutliver axis

Since the introduction of the term "gut-liver axis", many studies have focused on the functional links of intestinal microbiota, barrier function and immune responses to liver physiology. Intestinal and extra-intestinal diseases alter microbiota composition and lead to dysbiosis, which aggravates impaired intestinal barrier function via increased lipopolysaccharide translocation. The subsequent increased passage of gut-derived product from the intestinal lumen to the organ wall and bloodstream affects gut motility and liver biology. The activation of the toll-like receptor 4(TLR-4) likely plays a key role in both cases. This review analyzed the most recent literature on the gut-liver axis, with a particular focus on the role of TLR-4 activation. Findings that linked liver disease with dysbiosis are evaluated, and links between dysbiosis and alterations of intestinal permeability and motility are discussed. We also examine the mechanisms of translocated gut bacteria and/or the bacterial product activation of liver inflammation and fibrogenesis via activity on different hepatic cell types.

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis

AIM To determine the effects of ω-3 fatty acids(ω-3FA) on the toll-like receptor 4(TLR4)/nuclear factor κB p56(NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis(SAP).METHODS A total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay. RESULTS The expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group(P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h(P < 0.05).CONCLUSION During the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

Expression and Immune Effect of Toll-Like Receptor 4 in Human Trophoblast Cells

This study investigated the expression and immune effect of TLR4 in human trophoblast cells. The expression level of TLR4 mRNA in normal and LPS-stimulated human term trophoblast cells （1 mg/L LPS, 12 h） was detected by RT-PCR. In LPS-stimulated human term trophoblast cells of TLR4-blocked group and non-TLR4-blocked group, and normal term trophoblast cells of blank control group, apoptosis rate was measured by flow cytometry （FCM）, and the level of TNF-α determined by using enzyme linked immunosorbent assay （ELISA） respectively. RT-PCR results showed that the expression level of TLR4 mRNA in LPS-stimulated human trophoblast cells was significantly higher than that in normal cells （P〈0.01）. FCM revealed that there was significant difference in apoptosis rate of LPS-stimulated human term trophoblast cells between TLR4-blocked group and non-TLR4-blocked group （P〈0.05）, or between TLR4 antibody-blocked group and blank control group. ELISA indicated that the level of TNF-α in LPS-stimulated human trophoblast cells also had statistical differences between TLR4 antibody-blocked group and non-TLR4 antibody-blocked group （P〈0.05）. Our results suggest that TLR4 plays an important role in the immunological mechanism of apoptosis and secretion of TNF-α of human term trophoblast cells stimulated by LPS.

A 5'-flanking region polymorphism in toll-like receptor 4 is associated with gastric cancer in a Chinese population

Objective： Inflammation induced by H.pylori colonization in the stomach is related to the development of gastric cancer and the genetic variations of the genes involved in the immune responses modify the host response to the infection. The aim of this study was to evaluate whether polymorphisms in the toll-like receptor 4 （TLR4） gene, a key regulator of both innate and adaptive immunity, were related to the susceptibility to

Production of interleukin-1β related to mammalian target of rapamycin/Toll-like receptor 4 signaling pathway during Aspergillus fumigatus infection of the mouse cornea

AIM：To elucidate the effect of rapamycin on regulating the production of interleukin（IL）-1β in Aspergillus fumigatus（A.fumigatus）-induced keratitis and to verify whether the expression of IL-1β in A.fumigatus keratitis is associated with the mammalian target of rapamycin（mT OR）/Toll-like receptor 4（TLR4） signaling pathway.METHODS：Fungal keratitis mouse models of susceptible C57 BL/6 mice were established using A.fumigatus.The mice were subsequently treated with rapamycin.The protein levels of p-mT OR,TLR4,and IL-1β in normal and infected corneal tissue were measured by Western blot.The TLR4 and IL-1β m RNA levels were determined by real-time polymerase chain reaction（PCR）.RESULTS：In C57 BL/6 mice,rapamycin treatment decreased the clinical scores and production of the pro-inflammatory cytokine,IL-1β.The expression of TLR4,stimulated by A.fumigatus,was reduced as well when the mT OR signaling pathway was suppressed by rapamycin.CONCLUSION：Rapamycin is beneficial for the outcome of fungal keratitis and has an inhibitory effect expression of the inflammatory cytokine IL-1β.The inhibitory effect on IL-1β expression can be associated with the mT OR/TLR4 signaling pathway in A.fumigatus infection in mice.

Mechanisms of Increased Expression of Toll-Like Receptor-4 in Human Monocyte/Macrophage-derived Foam Cells

Summary： The mechanisms of increased expression of toll-like receptor 4 （TLR-4） during the formation of foam cells were explored. Low density lipoprotein （LDL） was prepared by density gradient ultracentrifugation and oxidized by incubation with CuClz. The human monocytic leukemia cell line （THP-1） was cultured in RPMI1640. The differentiation of THP-1 cells into macrophages （MPs） was induced by using myristate acetate （PMA） for 48 h. The macrophages were then incubated with oxidized LDL （ox-LDL） to generate foam cells （FCs）. The mRNA and protein expression levels of human TLR-4 were detected by immunocytochemistry, Western blotting and reverse transcription polymerase chain reaction （RT-PCR）. The results showed that the TLR-4 mRNA and the protein expression levels were significantly increased during the differentiation of monocytes into macrophages （P〈0. 05） as well as during the formation of lipid-laden foam cells （P〈0.05）. It was concluded that the upregulation of human TLR-4 gene expression during the differentiation of monocytes into macrophages and the differentiation of macrophages into foam cells could increase TLR-4 protein synthesis dramatically, which may enhance the ability of foam cells inflammation reaction in atherosclerosis.

Impact of Probiotics on Toll-like Receptor 4 Expression in an Experimental Model of Ulcerative Colitis

Toll-like receptors （TLRs） are key components of the innate immune system which trigger antimicrobial host defense responses. This study aimed to investigate the impact of probiotics （Lactoba- cillus, Bifiidobacterium） on the expression of TLR4 and tumor necrosis factor-alpha （TNF-α in the co- lon mucosa of rat experimental ulcerative colitis model induced by trinitrobenzene sulfonic acid （TNBS）/ethanol and immune complexes. The gross and histological changes of the colonic mucosa were observed and assessed by the means-standard deviation and independent samples t-test. The pro- tein expression levels of TLR4 and TNF-α were detected by using immunohistochemistry and Westem blotting, respectively. It was revealed that there was visible infiltration of inflammatory cells, formation of crypt abscess, and the reduction of goblet cells in the colon tissue of experimental models. As com- pared with the control group, the levels of TLR4 and TNF-α protein were significantly increased in the model group （P〈0.01 for both）. No significant difference was found in the expression of TLR4 and TNF-α between the two-week probiotics treatment group and the model group （P〉0.05）, whereas sig- nificant reductions were shown in rats which were treated with probiotics for four weeks as compared with the model group （P〈0.01）. There was no significant difference between two probiotics-treated groups. Our results implied that probiotics were likely to play a key role in protecting ulcerative colitis by reducing the inflammatory factor TNF-α expression through inhibiting the TLR4 expression in the colon tissue of experimental models.

Toll-like receptor 4-mediated signaling participates in apoptosis of hippocampal neurons

The phosphatidylinositol 3 kinase （PI3K）/protein kinase B （AKT） signaling pathway is considered important for cell survival and has been shown to mediate various anti-apoptotic biological effects. This study explored the role of the Toll-like receptor 4 （TLR4）-mediated PI3K/AKT-glycogen syn-thase kinase 3β （GSK-3β） signaling pathways in lipopolysaccharide-induced apoptosis in a primary culture of hippocampal neurons. Results demonstrated that the apoptotic ratio of hippocampal neurons stimulated by lipopolysaccharide was significantly higher compared with the control group. Both the expression of P-AKTser473 and P-GSK-3βSserg in hippocampal neurons stimulated by lipopolysaccharide decreased compared with the control, while the level of active Caspase-3 and the ratio of Bax/Bcl-2 were significantly increased. The level of active Caspase-3 and the ratio of Bax/Bcl-2 in hippocampal neurons treated with TLR4 antibody or the GSK-3β inhibitor, LiCl, de-creased before intervention with lipopolysaccharide, but increased after treatment with the AKT in-hibitor, LY294002. These findings suggest that the TLR4-PI3K/AKT-GSKβ signaling pathway may be involved in lipopolysaccharide-induced apoptosis of hippocampal neurons.

Toll-like receptor 4 polymorphisms and bacterial infections in patients with cirrhosis and ascites

AIM To assess the relationship between the presence of toll-like receptor 4(TLR4) polymorphisms and bacterial infections in cirrhotic patients with ascites. METHODS We prospectively included consecutive patients with cirrhosis and ascites hospitalized during a 6-year period. Patients with human immunodeficiency virus(HIV) infection or any other immunodeficiency, patients with advanced hepatocellular carcinoma(beyond Milan's criteria) or any other condition determining poor short-term prognosis, and patients with a permanent urinary catheter were excluded. The presence of D299 G and/or T399 I TLR4 polymorphisms was determined by sequencing and related to the incidence and probability of bacterial infections, other complications of cirrhosis, hepatocellular carcinoma, and mortality during follow-up. A multivariate analysis to identify predictive variables of mortality in the whole series was performed. RESULTS We included 258 patients: 28(10.8%) were carriers of D299G and/or T399I TLR4 polymorphisms(polymorphism group) and 230 patients were not(wildtype group). The probability of developing any bacterial infection at one-year follow-up was 78% in the polymorphism group and 69% in the wild-type group(P = 0.54). The one-year probability of presenting infections caused by gram-negative bacilli(51% vs 44%, P = 0.68), infections caused by gram-positive cocci(49% vs 40%, P = 0.53), and spontaneous bacterial peritonitis(29% vs 34%, respectively, P = 0.99) did not differ between the two groups. The oneyear probability of transplant-free survival was 55% in the polymorphism group and 66% in the wild-type group(P = 0.15). Multivariate analysis confirmed that age, Child-Pugh score, active alcohol intake, previous hepatic encephalopathy, hepatocellular carcinoma and serum creatinine were associated with a higher risk of death during follow-up. CONCLUSION Genetic polymorphisms D299 G and/or T399 I of TLR4 do not seem to play a relevant role in the predisposition of cirrhotic patients with ascites to bacterial infections.

Lactobacilli inhibit interleukin-8 production induced by Helicobacter pylori lipopolysaccharide-activated Toll-like receptor 4

AIM： To investigate the effect of Lactobacillus bulgaricus （LBG） on the Toll-like receptor 4 （TLR4） pathway and interleukin-8 （IL-8） production in SGC-7901 cells treated with Helicobacter pyloriSydney strain 1 lipopolysaccharide （HpyloriSS1-LPS）. METHODS： SGC-7901 cells were treated with HpyloriSS1-LPS in the presence or absence of pretreatment for 1 h with viable LBG or supernatant recovered from LBG culture MRS broth （LBG-s）. Cellular lysates were prepared for Western blot with anti-TLR4, anti-transforming growth factor β-activated kinase 1 （TAK1）, anti-phospho-TAK1, anti-nuclear factor κB （NF-κB）, anti-p38 mitogen-activated protein kinase （p38MAPK）, and anti-phospho-p38MAPK antibodies. The amount of IL-8 in cell culture medium was measured by ELISA. RESULTS： H pyloriSS1-LPS up-regulated the expression of TLR4, stimulated the phosphorylation of TAKI, subsequently enhanced the activation of NF- κB and the phosphorylation of p38MAPK in a time- dependent manner, leading to augmentation of IL-8 production in SGC-7901 cells. Viable LBG or LBG-s pretreatment attenuated the expression of TLR4, inhibited the phosphorylation of TAK1 and p38MAPK, prevented the activation of NF-κB, and consequently blocked IL-8 production.CONCLUSION： H py/oriSS1-LPS induces IL-8 production through activating TLR4 signaling in SGC-7901 cells and viable LBG or LBG-s prevents H pyloriSS1-LPS-mediated IL-8 production via inhibition of the TLR4 pathway.

Impact of Toll-like Receptor 4 Deficiency on Cerebrocardiac Syndrome

In order to investigate the role of Toll-like receptor 4 （TLR4） in cerebrocardiac syndrome （CCS）, the partial cerebral ischemia/reperfusion （I/R） models in mice with different TLR4 genotypes were established in the present study. TLR4 wild-type （C3H/HeN） and mutant （C3H/HeJ） mice of 6-8 weeks of age were divided into 4 groups at random： C3H/HeN sham group （n=10）, C3H/HeJ sham group （n=10）, C3H/HeN model group （n=10） and C3H/HeJ model group （n=10）. Partial cerebral I/R was caused by the middle cerebral artery occlusion （MCAO） to duplicate CCS models in mice. After the operation, the electrocardiogram （ECG）, the level of tumor necrosis factor-alpha （TNF-c0 in myocardial tissue and the cardiac pathological changes were observed in each group. It was shown that the brain infarct volume in C3H/HeN model group was larger than that in C3H/HeJ model group （P〈0.01）. The ST segment change and T wave inversion occurred frequently in model groups. Moreover, the TNF-ct level in C3H/HeN model group was higher than that in C3H/HeJ model group （P〈0.01）. The myocar- dial injury was aggravated in C3H/HeN group as compared with C3H/HeJ group. It was concluded that TLR4 was implicated in the development of CCS.

Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons

Lipopolysaccharide stimulates Toll-like receptor 4 on immune cells to produce immune mediators. Toll-like receptor 4 is also expressed by non-immune cells, which can be stimulated by lipopolysaccharide. However, whether Toll-like receptor 4 is expressed by primary cultured hippocampal neurons and its specific role in lipopolysaccharide-induced neuroinflammation is currently undefined, in this study, Toll-like receptor 4 antibody blocking was used to analyze the Toll-like receptor 4 signaling pathway and changes in inflammation of lipopolysaccharide stimulated hippocampal neurons. Immunofluorescence showed that Toll-like receptor 4 protein was mainly located in the membrane of hippocampal neurons. Quantitative reverse transcription-PCR and western blot assay showed that after stimulation of lipopolysaccharide, the mRNA and protein levels of Toll-like receptor 4 and the mRNA levels of interleukin-ll3 and tumor necrosis factor-（] were significantly increased. In addition, there was increased phosphorylation and degradation of kappa B a inhibitor in the cytosol and increased nuclear factor-KB p65 expression in the nuclei. Pretreatment with Toll-like receptor 4 antibody could almost completely block this increase. These experimental findings indicate that lipopolysaccharide participates in neuroinflammation by stimulating Toll-like receptor 4/nuclear factor-KB pathway in hippocampal neurons, which may be both ＂passive victims＂ and ＂activators＂ of neuroinflammation.

Association between polymorphisms in the Toll-like receptor 4,CD14,and CARD15/NOD2and inflammatory bowel disease in the Greek population

AIM: Crohn's disease(CD)and ulcerative colitis(UC)are multifactorial diseases with a significant genetic background.Apart from CARD15/NOD2 gene, evidence is accumulating that molecules related to the innate immune response such as CD14 or Toll-like receptor 4 (TLR4), are involved in their pathogenesis. In further exploring the genetic background of these diseases, we investigated the variations in the CARD15/NOD2 gene (Arg702Trp,Gly908Arg and Leu1007fsinsC), and polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) as well as in the promoter of the CD14 gene (T/C at position -159) in Greek patients with CD and UC.METHODS: DNA was obtained from 120 patients with CD,85 with UC and 100 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis.RESULTS: The 299Gly allele frequency of the TLR4 gene and the T allele and TT genotype frequendes of the CD14 promoter were significantly higher in CD patients only compared to healthy individuals (P = 0.026＜0.05; P = 0.0048＜0.01 and P= 0.047＜0.05 respectively). Concerning the NOD2/CARD15mutations the overall presence in CD patients was significantly higher than that in UC patients or in controls.Additionally, 51.67% of the CD patients were carriers of a TLR4 and/or CD14 polymorphic allele and at least one variant of the NOD2/CARD15, compared to 27% of the UC patients. It should be pointed out that both frequencies significantly increased as compared with the 10% frequency of multiple carriers found in healthy controls. A possible interaction of the NOD2/CARD15 with TLR4 and especially CD14, increased the risk of developing inflammatory bowel disease (IBD).CONCLUSION: Our results indicate that co-existence of a mutation in either the TLR4 or CD14 gene, and in NOD2/CARD15is associated with an increased susceptibility to developing CD compared to UC, and to developing either CD or UC compared to healthy individuals.

Effect of ω-3 Polyunsaturated Fatty Acid on Toll-like Receptors in Patients with Severe Multiple Trauma

This study examined the effects of ω-3 polyunsaturated fatty acid（ω-3PUFA） on the expression of toll-like receptor 2（TLR2）,toll-like receptor 4（TLR4） and some related inflammatory factors in peripheral blood mononuclear cells（PBMCs） of patients with early-stage severe multiple trauma.Thirty-two patients who were admitted to the Department of Traumatic Surgery,Tongji Hospital（Wuhan,China） between May 2010 and November 2010,and diagnosed as having severe multiple trauma with a injury severity score（ISS） no less than 16,were enrolled in the study and divided into two groups at random（n=16 in each）：ω-3PUFA group and control group in which routine parenteral nutrition supplemented with ω-3PUFA or not was administered to the patients in two groups for consecutive 7 days.Peripheral blood from these patients was collected within 2 h of admission（day 0）,and 1,3,5 and 7 days after the nutritional support.PBMCs were isolated and used for detection of the mRNA and protein expression of TLR2 and TLR4 by using real-time PCR and flow cytometry respectively,the levels of NF-κB by quantum dots-based immunofluorescence assay,the levels of TNF-α,IL-2,IL-6 and COX-2 by ELISA,respectively.The results showed that the mRNA and protein expression of TLR2 and TLR4 in PBMCs was significantly lower in ω-3PUFA group than in control group 5 and 7 days after nutrition support（both P0.05）.The levels of TNF-α,IL-2,IL-6 and COX-2 were found to be substantially decreased in PBMCs in ω-3PUFA group as compared with control group at 5th and 7th day（P0.05 for all）.It was concluded that ω-3PUFA can remarkably decrease the expression of TLR2,TLR4 and some related inflammatory factors in NF-κB signaling pathway in PBMCs of patients with severe multiple trauma,which suggests that ω-3PUFA may suppress the excessive inflammatory response meditated by the TLRs/NF-κB signaling pathway.

Expression of Toll-like Receptor 4 in Neonatal Cord Blood Mononuclear Cells in Patients with Preeclampsia

The expression of Toll-like receptor 4 (TLR4) in neonatal cord blood mononuclear cells (MNCs) and serum TNF-α were investigated in order to explore the roles of TLR4 in the pathogenesis of preeclampsia.The study enrolled 27 patients suffering from preeclampsia (experimental group) and 21 normal pregnancy patients (control group).After MNCs were separated, the expression of TLR4 mRNA and protein was detected by using real-time quantitative PCR and Western blotting respectively, and the expression of TNF-α by using ELISA.The results showed the TLR4 mRNA level in cord blood MNCs (2-CT:0.07±0.17), TLR4 protein expression level (absorbance ratio:0.81%±0.15%) and TNF-α level (9.5±1.73 pg/mL) were all increased in experimental group as compared with control group with the differences being statistically significant (P【0.05).There was a positive correlation between the expression of TLR4 mRNA and TNF-α in both experimental group and control group (r=0.54 and 0.53, respectively, P【0.05).It was concluded that TLR4 expression in the experimental group of cord blood MNCs was increased and there was a positive correlation between the expression of TLR4 mRNA and TNF-α in both groups.TLR4-mediated release of inflammatory cytokines may be one of the important reasons leading to preeclampsia.