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.

Mycophenolate mofetil affects monocyte Toll-like receptor 4 signaling during mouse renal ischemia/reperfusion injury

Background Mycophenolate mofetil （MMF） has been used to prevent transplant rejection for many years and has been shown to have protective effects against renal failure. The objective was to investigate the effect of MMF on monocyte Toll-like receptor 4 （TLR4） signaling in the early stages of renal ischemia/reperfusion injury （IRI） of mice. Methods Sixty BALB/C mice were randomly divided into two groups： an IRI group, in which renal IRI was induced by clamping the renal pedicles for 45 minutes, and an MMF group, in which MMF was given （40 mg.kg-l.d-1, intraperitoneally） from 2 days before renal IRI. The plasma creatinine level and renal tissue damage of each group mice were observed 6, 12, 24, and 48 hours after reperfusion. The concentration of plasma high-mobility group box 1 （HMGB-1） （TLR4 ligand）, interleukin 6 （IL-6）, monocyte chemoattractant protein-1 （MCP-1）, and tumor necrosis factor a （TNF-cc） and the expression of TLR-4 on monocytes were determined. Results The plasma creatinine concentration in the MMF group was lower compared to the IRI group （after reperfusion of 6, 12, 24, or 48 hours, P 〈0.05）. Pathological analysis showed that the renal damage was slighter, TLR-4 expression was reduced （after reperfusion of 6, 12, 24, or 48 hours, P 〈0.05）, and the concentration of cytokines in the plasma was lower （P 〈0.05） in the MMF group. No differences in the concentrations of HMGB-1 were observed （P 〉0.05）. Conclusion Monocyte TLR4 signaling is important in the early stage of kidney IRI, but MMF can inhibit it and improve renal function.

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.

Study on the Relationship between Heat Shock Protein 70 and Toll-like Receptor-4 of Monocytes

Summary: To explore the relation between human heat shock protein 70 (hsp70) and TLR4 in human monocytes in vitro, human monocytes were stimulated with various concentrations of HSP70, and TNF-α production in supernatants was measured by ELISA. Pre-incubated with or without anti-TLR4 mAb, and stimulated with hsp70 (5.0 μg/ml), NF-κB p65 of human monocytes in different time points were detected by immunohistochemistry and monocyte surface expression of TLR4 was measured by flow cytometry. After the human monocytes were pre-incubated with various concentrations of anti-TLR4 and stimulated with hsp70 (5.0 μg/ml), TNF-α production in supernatants was measured. The results showed that hsp70 enhanced NF-κB activation, which was clearly inhibited by anti-TLR4, with the positive cell ratios being 67.44 %, 39.17 %, 31.56 % and 28.05 %, respectively. TLR4 was rapidly down-regulated in the presence of hsp70. MFI of TLR4 on monocytes in different time points were 87.77±5.38, 78.16±6.01 and 45.17±4.97 (P<0.05), 26.98±5.83 (P<0.01), respectively. Moreover, hsp70-induced TNF-α production by human monocytes was inhibited by anti-TLR4. It is suggested that TLR4 is involved in the hsp70-mediated activation of innate immunity.

Fecal microbiota transplantation alleviates experimental colitis through the Toll-like receptor 4 signaling pathway

BACKGROUND Fecal microbiota transplantation(FMT)has shown promising therapeutic effects on mice with experimental colitis and patients with ulcerative colitis(UC).FMT modulates the Toll-like receptor 4(TLR4)signaling pathway to treat some other diseases.However,it remains unknown whether this modulation is also involved in the treatment of UC.AIM To clarify the necessity of TLR4 signaling pathway in FMT on dextran sodium sulphate(DSS)-induced mice and explain the mechanism of FMT on UC,through association analysis of gut microbiota with colon transcriptome in mice.METHODS A mouse colitis model was constructed with wild-type(WT)and TLR4-knockout(KO)mice.Fecal microbiota was transplanted by gavage.Colon inflammation severity was measured by disease activity index(DAI)scoring and hematoxylin and eosin staining.Gut microbiota structure was analyzed through 16S ribosomal RNA sequencing.Gene expression in the mouse colon was obtained by transcriptome sequencing.RESULTS The KO(DSS+Water)and KO(DSS+FMT)groups displayed indistinguishable body weight loss,colon length,DAI score,and histology score,which showed that FMT could not inhibit the disease in KO mice.In mice treated with FMT,the relative abundance of Akkermansia decreased,and Lactobacillus became dominant.In particular,compared with those in WT mice,the scores of DAI and colon histology were clearly decreased in the KO-DSS group.Microbiota structure showed a significant difference between KO and WT mice.Akkermansia were the dominant genus in healthy KO mice.The ineffectiveness of FMT in KO mice was related to the decreased abundance of Akkermansia.Gene Ontology enrichment analysis showed that differentially expressed genes between each group were mainly involved in cytoplasmic translation and cellular response to DNA damage stimulus.The top nine genes correlating with Akkermansia included Aqp4,Clca4a,Dpm3,Fau,Mcrip1,Meis3,Nupr1 L,Pank3,and Rps13(|R|>0.9,P<0.01).CONCLUSION FMT may ameliorate DSS-induced colitis by regulating the TLR4 signaling pathway.TLR4 modulates the composition of gut microbiota and the expression of related genes to ameliorate colitis and maintain the stability of the intestinal environment.Akkermansia bear great therapeutic potential for colitis.

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.

Distinct toll-like receptor expression in monocytes and T cells in chronic HCV infection

AIM： Hepatitis C virus often establishes chronic infections. Recent studies suggest that viral and bacterial infections are more common in HCV-infected patients compared to controls. Pathogens are recognized by Toll-like receptors （TLRs） to shape adaptive and innate immune responses. METHODS： In this study, to infected host to recognize assess the ability of HCV-infected host to recognize invading pathogens, we investigated Toll-like receptor expression in innate （monocytes） and adaptive （T cells） immune cells by realtime PCR. RESULTS： We determined that RNA levels for TLRs 2, 6. 7, 8, 9 and 10 mRNA levels were upregulated in both monocytes and T cells in HCV-infected patients compared to controls. TLR4 was only upregulated in T lymphocytes, while TLR5 was selectively increased in monocytes of HCV-infected patients. MD-2, a TLR4 coreceptor, was increased in patients＇ monocytes and T cells while CD14 and MyD88 were increased only in monocytes. CONCLUSION： Our data reveal novel details on TLR expression that likely relates to innate recognition of pathogens and immune defense in HCV-infected individuals.

Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

The noble gas argon has the potential to protect neuronal cells from cell death.So far,this effect has been studied in treatment after acute damage.Preconditioning using argon has not yet been investigated.In this study,human neuroblastoma SH-SY5Y cells were treated with different concentrations of argon(25%,50%,and 74%;21%O_(2),5%CO_(2),balance nitrogen)at different time intervals before inflicting damage with rotenone(20μM,4 hours).Apoptosis was determined by flow cytometry after annexin V and propidium iodide staining.Surface expressions of Toll-like receptors 2 and 4 were also examined.Cells were also processed for analysis by western blot and qPCR to determine the expression of apoptotic and inflammatory proteins,such as extracellular-signal regulated kinase(ERK1/2),nuclear transcription factor-κB(NF-κB),protein kinase B(Akt),caspase-3,Bax,Bcl-2,interleukin-8,and heat shock proteins.Immunohistochemical staining was performed for TLR2 and 4 and interleukin-8.Cells were also pretreated with OxPAPC,an antagonist of TLR2 and 4 to elucidate the molecular mechanism.Results showed that argon preconditioning before rotenone application caused a dose-dependent but not a time-dependent reduction in the number of apoptotic cells.Preconditioning with 74%argon for 2 hours was used for further experiments showing the most promising results.Argon decreased the surface expression of TLR2 and 4,whereas OxPAPC treatment partially abolished the protective effect of argon.Argon increased phosphorylation of ERK1/2 but decreased NF-κB and Akt.Preconditioning inhibited mitochondrial apoptosis and the heat shock response.Argon also suppressed the expression of the pro-inflammatory cytokine interleukin-8.Immunohistochemistry confirmed the alteration of TLRs and interleukin-8.OxPAPC reversed the argon effect on ERK1/2,Bax,Bcl-2,caspase-3,and interleukin-8 expression,but not on NF-κB and the heat shock proteins.Taken together,argon preconditioning protects against apoptosis of neuronal cells and mediates its action via Toll-like receptors.Argon may represent a promising therapeutic alternative in various clinical settings,such as the treatment of stroke.

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.

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.

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.

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.

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.

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.

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.

Clinical significance of NOD2/CARD15 and Toll-like receptor 4 gene single nucleotide polymorphisms in inflammatory bowel disease

AIM： To evaluate the role of genetic factors in the pathogenesis of Crohn＇s disease （CD） and ulcerative colitis （UC）, we investigated the single nucleotide polymorphisms （SNPs） of NOD2/CARD15 （R702W, Gg08R and L1007finsC）, and Toll-like receptor 4 （TLR4） genes （D299G and T399I） in a selected inflammatory bowel disease （IBD） population coming from Southern Italy. METHODS： Allele and genotype frequencies of NOD2/ CARD15 （R702W, Gg08R and L1007finsC） and TLR4 （D299G and T399I） SNPs were examined in 133 CD patients, in 45 UC patients, and in 103 healthy controls. A genotype-phenotype correlation was performed. RESULTS： NOD2/CARD15 R702W mutation was significantly more frequent in CD （9.8%） than in controls （2.4%, P = 0.001） and in UC （2.3%, P = 0.03）. No significant difference was found between UC patients and control group （P 〉 0.05）. In CD and UC patients, no significant association with G908R variant was found. L1007finsC SNP showed an association with CD （9.8%） compared with controls （2.9%, P = 0.002） and UC patients （2.3%, P = 0.01）. Moreover, in CD patients, G908R and L1007finsC mutations were significantly associated with different phenotypes compared to CD wild-type patients. No association of IBD with the TLR4 SNPs was found in either cohort （allele frequencies： D299G-controls 3.9%, CD 3.7%, UC 3.4%, P 〉 0.05; T399I-controls 2.9%, CD 3.0%, UC 3.4%, P 〉 0.05）. CONCLUSION： These findings confirm that, in our IBD patients selected from Southern Italy, the NOD2/ CARD15, but not TLR4 SNPs, are associated with increased risk of CD.

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 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.