广谱模式识别分子Toll-like receptor 2的研究进展

TLR-2（Toll-like receptor 2,TLR-2）是哺乳动物TLRs（Toll-like receptors,TLRs）家族的一员,作为细胞表面的天然受体蛋白,主要参与病原微生物产物的识别及炎症信号传导,介导天然抗感染兔疫;最近又发现其参与机体对非感染因子所致炎性组织损伤的识别。通过对TLR-2参与的识别和细胞内信号传导机制的研究,可为深入探讨抵御微生物感染的机制、对自身正常与非正常组织的识别提供新的思路。

人Toll-like receptor 2配基模拟肽的初步研究

TLR-2(Toll-likereceptor2)是介导天然免疫的重要模式识别分子,可参与识别多种病原体及其产物.为探索被TLR-2所识别配基的结构共性,以真核细胞表达的人TLR-2胞外段蛋白(A26￣T588)为钓饵筛选噬菌体12肽库,获得一高度保守的阳性噬菌体克隆P12-1,实验发现P12-1可与不同形式的TLR-2胞外段结合,并且可刺激细胞分泌TNFα,提示P12-1可能模拟TLR-2配基的结构与生物学活性.

人Toll-like receptor 2胞外段的克隆和表达

目的 克隆、表达人Toll-like receptor2(TLR2)胞外段(A26-T588)基因,获得人TLR2胞外段蛋白。方法 RT-PCR扩增TLR2胞外段基因,以pcDNA3.1+质粒为载体在HEK293细胞中表达TLR2胞外段蛋白,同时以pcDNA3.1+/TLR2(A26-T588)重组质粒免疫昆明鼠,制备抗TLR2胞外段蛋白多抗。结果 PCR扩增及重组质粒测序结果表明成功地构建了pcDNA3.1+/TLR2(A26-T588)真核表达质粒,SDS-PAGE分析纯化产物在M_r为68000处出现明显蛋白条带。重组质粒DNA免疫小鼠3次后,血清抗体滴度可达1∶250。TLR2胞外段蛋白可与LPS结合,并在一定的质量浓度范围内呈剂量依赖性。结论 构建的pcDNA3.1+/TLR2(A26-T588)真核表达质粒可在哺乳动物细胞中表达TLR2胞外段蛋白,并与重组质粒DNA免疫小鼠抗血清及TLR2单克隆抗体TL2.1特异性反应,由此证明其表达正确。

Protection against hepatic ischemia/reperfusion injury via downregulation of toll-like receptor 2 expression by inhibition of Kupffer cell function

AIM： To elucidate the mechanism of liver protection by inhibition of Kupffer cells （KCs） function.METHODS： All the animals were randomly divided into three groups. Blockade group （gadolinium chloride solution （GdCl3） injection plus ischemia/reperfusion （I/R） injury）：GdCl3 solution was injected once every 24 h for 2 d via the tail vein before I/R injury. Non-blockade group （saline solution injection plus I/R injury）： saline instead of GdCl3 as a control was injected as in the blockade group. Sham group： saline was injected without I/R injury. Liver samples were collected 4 h after blood inflow restoration. The blockade of the function of KCs was verified by immunostaining with an anti-CD68 mAb. Toll-like receptor2 （TLR2） was immunostained with a goat antimouse polydonal anti-TLR2 antibody. Membrane proteins were extracted from the liver samples and TLR2 protein was analyzed by Western blot. Portal vein serum and plasma were taken respectively at the same time point for further detection of the levels of tumor necrosis factor-α （TNF-α） and alanine aminotransferase （ALT）, an indicator of liver function.I/R injury via downregulation of theexpression of TLR2.RESULTS： Compared to non-blockade group, CD^68＋ cells significantly reduced in blockade group （OPTDI, optical density integral）： 32.97±10.55 vs 185.65±21.88, P〈0.01） and the liver function impairment was relieved partially （level of ALT： 435.89±178.37 U/L vs 890.21±272.91 U/L,P〈0.01）. The expression of TLR2 protein in blockade group significantly decreased compared to that in non- group（method of immunohistochemistry, OPDTI： 75.74±17.44 vs 170.58±25.14, P〈0.01; method of Western blot, A value： 125.89±15.49 vs 433.91±35.53, P〈0.02）. The latter correlated with the variation of CD68 staining （r = 0.745,P〈0.05）. Also the level of portal vein TNF-α decreased in blockade group compared to that in non-blockade group （84.45±14.73 ng/L vs 112.32±17.56 ng/L, P〈0.05）, butwas still higher than that in sham group （84.45±14.73 ng/L vs 6.07±5.33 ng/L, P〈0.01）.CONCLUSION： Inhibition of the function of KCs may protect liver against I/R injury via downregulation of the expression of TLR2.

Science Letters:A synthetic Toll-like receptor 2 ligand decreases allergic immune responses in a mouse rhinitis model sensitized to mite allergen

It has been proposed that activation of Toll-like receptors （TLRs） plays crucial roles in the polarization of adaptive immune responses. A synthetic Toll-like receptor 2 （TLR2） ligand, Pam3CSK4, has been reported to modulate the balance of Th1/Th2 responses. We evaluated the modulation effect of Pam3CSK4 on allergic immune response in a mouse rhinitis model sensitized to house dust mite allergen （HDM）. Mice were sensitized and challenged with Dermatophagoidesfarinae allergen （Der f）, and then the allergic mice were treated by Pam3CSK4. Nasal allergic symptoms and eosinophils were scored. Der f-specific cytokine responses were examined in the splenocytes and bronchoalveolar lavage fluid （BALF）. Serum level of total IgE was also detected. After establishing a mouse allergic rhinitis model with HDM, we have showed that Pam3CSK4 treatment not only ameliorated the nasal allergic symptoms remarkably but also decreased the eosinophils and total inflammation cells in BALF significantly. Analysis of cytokine profile found that IFN-7 released from either BALF or stimulated splenocytes increased markedly in Pam3CSK4-treated mice, while IL-13 decreased significantly. Moreover, serum level of total IgE was significantly lower in Pam3CSK4-treated mice than in the untreated. Thus, in an allergic rhinitis mouse model developed with HDM, Pam3CSK4 was shown to exhibit an antiallergic effect, indicating its potential application in allergic diseases.

Over-expression of Toll-like receptor 2 up-regulates heme oxygenase-1 expression and decreases oxidative injury in dairy goats

Background： Mastitis, an infection caused by Gram-positive bacteria, produces udder inflammation and oxidative injury in milk-producing mammals. Toll-like receptor 2（TLR2） is important for host recognition of invading Grampositive microbes. Over-expression of TLR2 in transgenic dairy goats is a useful model for studying various aspects of infection with Gram-positive bacteria, in vivo.Methods： We over-expressed TLR2 in transgenic dairy goats. Pam3CSK4, a component of Gram-positive bacteria,triggered the TLR2 signal pathway by stimulating the monocytes-macrophages from the TLR2-positive transgenic goats, and induced over-expression of activator protein-1（AP-1）, phosphatidylinositol 3-kinase（PI3K） and transcription factor nuclear factor kappa B（NF-κB） and inflammation factors downstream of the signal pathway.Results： Compared with wild-type controls, measurements of various oxidative stress-related molecules showed that TLR2, when over-expressed in transgenic goat monocytes-macrophages, resulted in weak lipid damage, high level expression of anti-oxidative stress proteins, and significantly increased m RNA levels of transcription factor NF-E2-related factor-2（Nrf2） and the downstream gene, heme oxygenase-1（HO-1）. When Pam3CSK4 was used to stimulate ear tissue in vivo the HO-1 protein of the transgenic goats had a relatively high expression level.Conclusions： The results indicate that the oxidative injury in goats over-expressing TLR2 was reduced following Pam3CSK4 stimulation. The underlying mechanism for this reduction was increased expression of the anti-oxidation gene HO-1 by activation of the Nrf2 signal pathway.

Toll-like receptor 2及其信号传导通路在阿片类物质诱导细胞凋亡中的作用

目的:成瘾物质(Addictive drugs)导致的中毒性脑病的中枢神经系统损害以神经元凋亡为突出表现,具体机制仍有待进一步的研究探讨。方法:Toll-like receptor 2(TLR2)不仅是激活机体自身免疫防御和炎症反应的重要受体,而且广泛参与多种细胞功能,包括细胞凋亡。我们使用体外培养的HEK293和高表达TLR2的HEK2932种细胞,以及体外培养小鼠皮层原代神经元细胞,研究经过吗啡处理后细胞存活率和凋亡的变化,以探讨TLR2信号通路在吗啡诱导的细胞凋亡中的作用。结果:TLR2高表达导致吗啡诱导的细胞存活率下降和细胞凋亡的显著增加。使用MyD88抑制体竞争性抑制MyD88并阻断TLR2信号通路后,吗啡诱导的TLR2高表达细胞的凋亡也被明显抑制。长期的吗啡处理导致正常神经元的TLR2在信使RNA(mRNA)水平和蛋白水平表达明显增高,而且在TLR2功能缺失的原代神经元细胞中,吗啡诱导的caspase-3活性增高被阻断,吗啡诱导的神经元凋亡也被明显抑制。结论:TLR2信号通路参与吗啡诱导的神经元凋亡。

N-acetylcysteine inhibits activation of toll-like receptor 2 and 4 gene expression in the liver and lung after partial hepatic ischemia-reperfusion injury in mice

BACKGROUND: Toll-like receptor 2 and 4 (TLR2/4) may play important roles in ischemia-reperfusion (I/R) injury, and N-acetylcysteine (NAC) can prevent the generation of reactive oxygen species (ROS) induced by I/R injury. This study aimed to investigate the changes in TLR2/4 gene expression in the liver and lung after I/R injury with or without NAC pretreatment. METHODS: BALB/c mice were used in a model of partial hepatic I/R injury and randomly assigned to a sham-operated control group (SH), a hepatic ischemia/reperfusion group (I/R) or a NAC pretreated, hepatic I/R group (I/R-NAC). The levels of TNF-alpha in the portal vein and plasma alanine aminotransferase (ALT) were measured at 1 and 3 hours after reperfusion. The lung wet-to-dry ratio was measured, and the expression of TLR2/4 mRNA and protein in the liver and lung were assessed with RT-PCR and Western blotting at the same time points. RESULTS: Compared with the I/R group, the expression of TLR2/4 mRNA and protein in the liver and lung in the I/R-NAC group was decreased at the same time point (P<0.05). The levels of portal vein TNF-a and plasma ALT increased continuously in the l/R group at I and 3 hours of reperfusion compared with the SH group; however, they declined significantly in the group pretreated with NAC (P<0.05). The extent of lung edema was relieved in the I/R-NAC group compared with the I/R group (P<0.05). CONCLUSIONS: TLR2/4 was activated in the liver and lung in the process of partial hepatic I/R injury. NAC inhibited the activation of TLR2/4 and the induction of TNF-alpha resulting from I/R injury via modulating the redox state, thus it may mitigate liver and lung injury following partial hepatic I/R in mice.

Short-Term Inhalation of Nitric Oxide Inhibits Activations of Toll-Like Receptor 2 and 4 in the Lung after Ischemia-Reperfusion Injury in Mice

In order to investigate the effects of different terms of inhaled nitric oxide （NO） preconditioning with low concentration on the activations of Toll-like receptor 2 and 4 （TLR2/4） in the lung ischemia-reperfusion （IR） injury in mice, we divided the male C57BL mice into five groups： sham （S） group, IR group, NO 1-min preconditioning group （15 ppm NO inhalation for 1 min before ischemia, NO 1-min）, NO 10-min preconditioning group （15 ppm NO inhalation for 10 min before ischemia, NO 10-min）, NO 60-min preconditioning group （15 ppm NO inhalation for 60 min before ischemia, NO 60-min）. The changes of partial pressure of oxygen in artery （PaO2）, left lung wet-to-dry weight ratio （W/D）, and myeloperoxidase （MPO） in the injured lung were measured in every group at 6th h of reperfusion after 60 min of left lung ischemia. The changes of TLR2/4 activations and plasma TNF-a were measured in this procedure in additional mice. As compared with IR group, PaO2 increased, MPO and W/D decreased evidently after reperfusion in NO 10-min group. The changes in NO 60-min group were similar to those in NO 10-rain group. There was no difference between NO 1-min and IR group. In NO inhalation group, the expressions levels of TLR2/4 mRNA and proteins were diminished, TNF-~t concentrations were decreased, and the lung injuries were ameliorated effectively. We concluded that short term inhalation of NO protected lung IR injury. But the protective effect of NO was not increased with extension of inhaled NO. Inhaled NO could inhibit the activations of TLR2/4 in the lung after IR injury. TLR signal pathway might contribute to the effect of protection with NO in this model.

Expression and significance of toll-like receptor 2,4 in peripheral blood mononuclear cells in patients with systemic inflammatory response syndrome

To explore changes of toll-like receptor (TLR) 2,4 in peripheral blood mononuclear cells (PBMC) in acute abdomen patients with systemic inflammatory response syndrome (SIRS) and their significance.Methods A clinical study was done on 103 patients of which 65 were with SIRS.The mRNA expression of TLR2,4 were detected by RT-PCR;the expression of TNF-α and IL-6 were observed by ELISA;the correlation between TLR2,4 mRNA,the level of TNF-α and IL-6,and the clinical course was evaluated.Results TLR2 mRNA ,TNF-α and IL-6 were upregulated markedly on the first day of hospitalization,then decreased gradually;TLR2 mRNA maintained on high level till the 5th day.The expression of TLR2,4 mRNA was positive correlated with the level of TNF-α and IL-6,and the length of stay.TLR2,4 mRNA expression increased in patients with multiple organ failure.Conclusion In actue abdomen patients with SIRS,the expression of TLR2,4 of PBMC increased markedly,indicating its improtant role in the pathogenesis of SIRS.4 refs,2 figs,2 tabs.

Faecal and mucosal microbiota in patients with functional gastrointestinal disorders: Correlation with toll-like receptor 2/toll-like receptor 4 expression

AIM To investigate the intestinal luminal microbiota(LM)and mucosa-associated microbiota(MAM)in Chinese patients with functional gastrointestinal disorders(FGIDs)and examine the association between these communities and the expression of toll-like receptor(TLR)2 and TLR4. METHODS Thirty-two Chinese subjects who suffered from symptoms of FGIDs,as confirmed by gastroenterologists,were enrolled in this study.Fresh faecal samples and descending colonic mucosal biopsies were collected from the subjects before(faecal)and during(mucosal) flexible colonoscopy.For analysis of the samples,we performed high-throughput sequencing of the V3-V4region of the 16S rR NA gene and reverse transcription(RT)-PCR to detect the expression of colonic TLR2 and TLR4.Differences in the stool and mucosal microbiota were examined and a correlation network analysis was performed. RESULTS The microbiota of faecal samples was significantly more diverse and richer than that of the mucosal samples,and the LM and MAM populations differed significantly.TLR2 expression showed a significant positive correlation with TLR4 expression.In the MAM samples,the genera Faecalibacterium and Ruminococcus,which belong to the family Ruminococcaceae,were inversely correlated with TLR4 expression(r=-0.45817,P=0.0083 and r=-0.5306,P=0.0018,respectively).Granulicatella,which belongs to Carnobacteriaceae,and Streptococcus,which belongs to Streptococcaceae,were inversely correlated with TLR2 expression(r=-0.5573,P=0.0010 and r=-0.5435,P=0.0013,respectively).In the LM samples,examination at phylum,class,or order level revealed no correlation with TLR4 expression.Faecalibacterium,which belongs to Ruminococcaceae,and Streptococcus,which belongs to Streptococcaceae,were inversely correlated with TLR2 expression(r=-0.5743,P=0.0058 and r=-0.3905,P=0.0271,respectively). CONCLUSION Microbial compositions of LM and MAM in Chinese patients with FGIDs are different.Expression of TLRs may be affected by the type of bacteria that are present in the gut.

Role of nitric oxide in Toll-like receptor 2 and 4 mRNA expression in liver of acute hemorrhagic necrotizing pancreatitis rats

AIM： To investigate the role of nitric oxide （NO） in Tolllike receptor 2 （TLR2）/4mRNA expression in livers of acute hemorrhagic necrotizing pancreatitis （AHNP） rats. METHODS： One hundred and ten SD male rats were randomly divided into sham-operated group （n = 10）, AHNP group （n = 30）, chloroquine （CQ）-treated group （n = 30） and L-Arg-treated group （n = 40）. TLR2/4mRNA expression in the liver of AHNP rats was measured by RT-PCR. RESULTS： Expression of TLR2/4mRNA could be detected in the liver of AHNP rats in sham-operated group （0.155E-5±0.230E-6 and 0.115E-2±0.545E-4）, but was markedly increased at 3 h in AHNP group （0.197E-2±0.114E-3 and 0.175±0.349E-2） peaking at 12 h （0.294E-2 ± 0.998E-4 and 2.673 ± 2.795E-2, P〈 0.01）. Hepatic injuries were aggravated, TNF-α concentration in the liver was increased and NO concentration was decreased （P〈 0.05 or P〈 0.01）. When TLR2/4mRNA expression was inhibited by CQ （3 h： 1.037E-4±3.299E-6 and 0.026±3.462E-3; 6 h： 1.884E-4±4.679E-6 and 0.108±6.115E-3; 12 h： 2.443E-4±7.714E-6 and 0.348±6.807E-3; P 〈 0.01）, hepatic injuries were relieved, NO concentration in the liver was increased and TNF-α concentration was decreased （P〈0.05 or P〈0.01）. When rats with AHNP were treated with L-Arg, TLR2/4mRNA expression in the liver could be effectively inhibited （50 mg-T： 0.232E-2±0.532E-4 and 0.230±6.883E-3; 100 mg-T： 0.210E-2± 1.691E-4 and 0.187±0.849E-2; 200 mg-T： 0.163E-2±0.404E-4 and 0.107±0.195E-2; 400 mg-T： 0.100E-2±0.317E-4 and 0.084±0.552E-2; P〈0.01） and hepatic injuries were relieved. At the same time, NO concentration in the liver was markedly increased and TNF-α concentration was decreased （P〈0.05 or P〈O.OI）, CONCLUSION： The expression of TLR2/4mRNA is increased and hepatic injuries are aggravated in the liver of AHNP rats. TLR2/4mRNA gene expression in the liver of AHNP rats can be markedly inhibited by NO, leading to the relief of hepatic injuries.

Effect of nitric oxide on toll-like receptor 2 and 4 gene expression in rats with acute lung injury complicated by acute hemorrhage necrotizing pancreatitis

BACKGROUND: Toll-like receptor (TLR) 2/4 might play important roles in mediating proinflammatory cytokine synthesis and release. And nitric oxide (NO) has been used to treat acute respiratory distress syndrome (ARDS). This study aimed to investigate the changes in TLR2/4 gene expression in the lungs of rats with acute lung injury (ALI) complicated by acute hemorrhage necrotizing pancreatitis (AHNP) and the effect of NO on the TLR2/4 gene expression. METHODS: One hundred and ten SD male rats were randomly divided into sham-operated group ( n = 10) , AHNP group (n = 30) , chloroquine-treated group ( n = 30) , and L-Arg-treated group (n =40). The lungs were dissected for lung histological scoring, and bronchoalveolar lavages were harvested for lung injury indexing. TLR2/4 mRNA expression in the lungs was measured by RT-PCR. RESULTS: TLR2/4mRNA was detected in the lungs with low values in the sham-operated group (0.016±0. 210E-2, 0.112 ±0.750E-2) , but it was markedly increased at 3 hours in the AHNP group (0.787±0.751E-2, 1.512 ±1.794E-2) , peaking at 12 hours (1.113 ±6.141E-2, 2.957±2.620E-2; P <0.05 or P <0.01). When lung injuries were aggravated, TNF-α concentrations in the lungs were increased, but NO concentrations were decreased ( P < 0.05 or P < 0.01 ) . When TLR2/4mRNA was inhibited by CQ (3h: 0.313 ± 5.491E-2, 0.005 ±1.419E-3 ; 6h: 0.488 ±7.442E-2, 0.010 ± 1.518E-3; 12h: 0.883 ± 8.911E-2, 0.024 ± 2.760E-3; P< 0.05 or P <0.01) , lung injuries were relieved. NO concentrations in the lungs were increased but TNF-α concentrations were decreased (P <0. 05 or P <0.01). When the rats with AHNP were treated with L-Arg, TLR2/4mRNA expression in the lungs could be effectively inhibited (50mg-T: 0.656 ±3. 977E-2, 1. 501 ±6.111E-2; 100mg-T: 0.260± 0.891E-2, 0.732 ±5.135E-2; 200mg-T: 0.126 ±0.914E-2, 0.414 ± 1.678E-2; 400mg-T: 0.091 ±0.399E-2, 0.287 ± 0.176E-2; P <0.05 or P <0. 01) and lung injuries were relieved. At the same time, NO concentrations in the lungs were markedly increased, but TNF-α concentrations were decreased (P <0.05 or P <0.01). CONCLUSIONS: The expression of TLR2/4mRNA is increased in the lungs in rats with AHNP and lung injuries are aggravated. TLR2/4mRNA gene expression of the lungs of rats with AHNP could be markedly inhibited by NO, leading to the relief of lung injuries.

The Expression of Toll-like Receptor 2 and 4 mRNA in Local Tissues of Model of Oropharyngeal Candidiasis in Mice

Summary: To investigate the expression of Toll-like receptor (TLR) 2 and 4 mRNA in local tissues of model of oropharyngeal candidiasis in mice and to explore the potential role of TLR2 and TLR4 in earlier period of immune response, a murine model of oropharyngeal candidiasis inoculated by cotton wool balls saturated with candida albicans was established. Mice were sacrificed at the indicated time points and the oropharyngeal tissues were excised. The expression of TLR2 and TLR4 mRNA was detected by RT-PCR. The results showed that low level of TLR2/4 mRNA could be detected in oropharyngeal tissues, but they were markedly up-regulated 6 h after inoculation, peaking after 12-24 h. Tissue TLR4 mRNA was gradually down-regulated 24-48 h, while TLR2 mRNA levels remained high up to the 72nd h. These data suggested that oropharyngeal infection of Candida albicans could result in up-regulation of TLR2/4 mRNA expression in local tissues, which might play important roles in earlier period of immune response.

Expression and Significance of Toll-like Receptor 2,4 of Peripheral Blood Mononuclear Cells in Acute Abdomen Patients Associated with Systemic Inflammatory Response Syndrome

The changes of Toll-like receptor （TLR） 2, 4 of peripheral blood mononuclear cells （PBMCs） in the acute abdomen patients associated with systemic inflammatory response syndrome （SIRS） and their potential significance were explored. A clinical study was performed on 103 acute abdomen patients in whom 65 were associated with SIRS. Forty healthy individuals served as normal controls. The mRNA expression of TLR2, 4 was detected by RT-PCR, and the expression of TNF-α and IL-6 by ELISA. The level of plasma endotoxin, hospital stay and mortality were measured. It was found that the endotoxin level was increased to varying degrees in all the acute abdomen patients, and the endotoxin level was and hospital stay longer in SIRS group than in non-SIRS group （P〈0.01）. TLR2 mRNA, TLR4 mRNA, IL-6 and TNF-ct could be detected with low value in normal controls, but they were up-regulated markedly on the 1 st day after admission. Then TLR4 mRNA, IL-6 and TNF-α were decreased gradually, but TLR2 mRNA maintained at a high level till the 5th day. These indexes above in SIRS group were higher than those in non-SIRS group （P〈0.01）. The results of correlation analysis revealed the expression of TLR2, 4 mRNA was positively correlated with the levels of TNF-α and IL-6, and the hospital stay, The results of Logistic regression demonstrated that overexpression of TLR2, 4 mRNA might result in higher risk of multiple organ dysfunction syndrome （MODS）. It was concluded that in the acute abdomen patients associated with SIRS, the expression of TLR2, 4 in PBMCs was increased markedly, suggesting that TLR might play an important role in the pathogenesis of acute abdomen associated with SIRS.

The expression of toll-like receptor 2, 4 of livers in mice with systemic inflammatory response syndrome

BACKGROUND: The complicated pathogenesis of systemic inflammatory response syndrome (SIRS) is a hot topic in critical care medicine. In this study we explored the expression of toll-like receptor (TLR) 2, 4 of livers in SIRS mice and evaluated the role of TLR2, 4 in the pathogenesis of SIRS. METHODS: Forty BABL/C mice were randomly divided into 2 groups: control (n=20) and SIRS (n=20). SIRS model was induced by severe acute pancreatitis. Blood routine, blood amylase, glutamic pyruvic transaminase and temperature were measured. Histological changes of pancreases and livers were observed microscopically. The mRNA expressions of TLR2, 4 were detected by real-time polymerase chain reaction (PCR). The protein expressions of TLR2, 4 were examined by Western blot. RESULTS: Marked edema, inflammatory cell infiltration, hemorrhage, and necrosis were observed in the pancreases and livers of SIRS mice. The concentrations of amylase and glutamic pyruvic transaminase were increased significantly. Body temperature and white blood cell count were decreased. The mRNA and protein expressions of TLR2, 4 increased markedly in SIRS mice. Significant difference was observed between SIRS and control mice (P【0.01). CONCLUSION: The expressions of TLR2, 4 of livers were increased markedly in SIRS mice, indicating that TLR might play an important role in the pathogenesis of SIRS.

Expression of Toll-like Receptor 2/4 on Alveolar Macrophage in the Model of Total Hepatic Ischemia/Reperfusion Injury in Mice

Objective： To explore the expression and meaning of Toll-like receptor 2/4 in alveolar macrophage during the process of total hepatic ischemia in mice. Methods： BALB/c mice were used in a model of total hepatic ischemia/reperfusion. Alveolar Macrophage were collected at the time point of lh, 6h and 12h by the means of bronchoalveolar lavage （BAL）, and its TLR2/4 mRNA and protein were detected with Flow Cytometry and Real-time PCR. The level of TNF in BAL fluid were measured. The concentration of MPO, the ratio of wet/dry and lung histological scores were used to assess the degrees of lung injuries. Results： At the three time points of hepatic ischemia reperfusion, the expression of TLR2/4 protein of and mRNA were up-regulated and the level of TLR2 was on the rise continually. TLR4 at the time of 6 h reached the peak value （P〈0.01）. The level of TNF-2 in BAL fluid reached the highest point at the time of 6 h （P〈0.01）. The ratio of wet/dry rose continually during hepatic ischemia reperfusion. After 1 h, the level of MPO increased rapidly. Then it reached the peak value during the period of 6 h to 12 h. Conclusion： TLR2/4 on the mice of alveolar macrophage were activated in the process of hepatic ischemia/reperfusion and involved in the injury of lung.

Toll-like receptors 2 polymorphism is associated with psoriasis: A case-control study in the northern Chinese population

Background:Psoriasis is a disease caused by genetics and immune system dysfunction,affecting the skin and joints.Toll-like receptors(TLRs)play an important role in triggering the innate immune response and controlling adaptive immunity.The role of TLR2 in the progression of psoriasis is not well understood.Methods:A case-control study was conducted on a northern Chinese Han population,consisting of psoriasis patients and healthy control subjects.Genotyping was performed using the tetra-primer amplification refractory mutation system-polymerase chain reaction(ARMS-PCR),and allele and genotype frequencies of four SNPs in TLR2 were analyzed in 270 psoriasis patients and 246 healthy controls.Results:Four TLR2 SNPs(rs11938228,rs4696480,rs3804099,rs5743699)were genotyped and found to be in linkage disequilibrium.The genotype distributions of rs11938228 and rs4696480 in two groups were in Hardy-Weinberg equilibrium and statistically significant except for the overdominance model.The haplotypes ATTC and ATCC were found to be protective against psoriasis.Conclusion:Our study found a correlation between TLR2 genetic variations and the likelihood of psoriasis in northern China.

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.

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.