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.

儿童肺炎支原体肺炎肺泡灌洗液CARDS TX、HMGB1、TLR2表达及临床意义

目的研究肺炎支原体肺炎(MPP)患儿肺泡灌洗液中社区获得性呼吸窘迫综合征毒素(CARDS TX)、高迁移率族蛋白1(HMGB1)、Toll样受体2(TLR2)表达,探讨其在MPP发病中的临床意义。方法回顾性分析55例MPP病例组及20例对照组临床资料,同时检测两组支气管肺泡灌洗液(BALF)中CARDS TX、HMGB1、TLR2、TLR4、晚期糖基化终末产物受体(RAGE)及髓样分化因子88(MyD88)表达,体外检测不同浓度CARDS TX刺激下HMGB1、TLR2的表达并进行比较。结果与对照组相比,MPP组LYM绝对值计数、PA、CD3^(+)、CD3^(+)CD4^(+)、CD3^(+)CD8^(+)、CD3-CD19^(+)、NK cell、CD19^(+)CD23^(+)明显下降,CRP、LDH、D-二聚体、IgA、IgG、IgM、CARDS TX、HMGB1、TLR2、MyD88均升高,差异有统计学意义(P<0.05或0.001)。CARDS TX刺激后HMGB1、TLR2的表达升高,且呈正相关,差异有统计学意义(P<0.001)。结论CARDS TX可能通过HMGB1-TLR2-MyD88途径参与肺炎支原体(MP)的致病。

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.

清渣汤对玫瑰痤疮模型小鼠皮损组织VEGF、TLR2、KLK5表达的影响

目的:观察清渣汤口服对玫瑰痤疮模型小鼠皮损组织血管内皮生长因子(VEGF)、Toll样受体2(TLR2)、激肽释放酶5(KLK5)表达的影响。方法:7周龄健康雄性小鼠36只,用随机数字表法将其分为6组:清渣汤高剂组、中剂组、低剂组、阳性对照组(多西环素片)、模型对照组、正常对照组,每组6只。除正常组外,其余各组均进行玫瑰痤疮造模。造模24 h后给药,模型组给予0.2 mL生理盐水灌胃,清渣汤3剂组分别给予25 mg/kg(低剂量组)、50 mg/kg(中剂量组)和100 mg/kg(高剂量组)清渣汤灌胃,阳性对照组给予30 mg/kg盐酸多西环素溶液灌胃,每日1次,共给药4周。免疫组化法检测皮损部位皮肤组织VEGF、TLR2、KLK5蛋白表达水平。结果:模型组小鼠皮损组织VEGF、TLR2、KLK5蛋白表达较正常组明显增强(P<0.01);与模型组比较,阳性对照组和清渣汤高、中、低剂量组小鼠皮损组织VEGF、TLR2、KLK5蛋白表达减弱(P<0.05)。结论:清渣汤能够抑制玫瑰痤疮小鼠模型皮损部VEGF、TLR2、KLK5蛋白表达水平,从而减轻炎症反应。

Toll样受体2(TLR2)与结核分枝杆菌感染的相关研究进展

Toll样受体2(TLR2)是白细胞表面表达的一种模式识别受体,已发现多种配体可以激动或抑制TLR2的活化,进而调控免疫细胞的炎症和凋亡状态。结核分枝杆菌(MTB)通常寄生在巨噬细胞中,但MTB感染机体后,包括巨噬细胞在内的多种免疫细胞表面的TLR2都可与其相互作用,释放细胞因子,影响MTB在体内的状态和增殖。在分子层面,TLR2的多态性有待更多探索,目前研究的大部分TLR2的多态性被认为与MTB的易感性无关。文中总结了TLR2与配体、感染后免疫调节活动、多态性与MTB易感性等方面的研究进展。

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。

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。

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。

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.

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.

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.

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.

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.

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样受体2对儿童肺炎支原体肺炎肺部炎症表现和肿瘤坏死因子-α的调节作用

目的 探讨Toll样受体2(TLR2)对肺炎支原体肺炎(MPP)病儿肺部炎症表现和肿瘤坏死因子-α(TNF-α)的调节作用。方法 选取2021年1月至2022年1月郑州大学附属儿童医院诊治的MPP病儿27例,其中难治性肺炎支原体肺炎(RMPP)病儿12例、MPP病儿15例。选取非感染病儿12例作为对照(NC)组。检测并比较不同病儿外周血TLR和TNF-α的表达,培养A549细胞并比较其与中性粒细胞肺炎支原体(MP)刺激的TNF-α表达,以及TLR2敲除后MP刺激的TNF-α表达。结果 MPP病儿血清TNF-α水平高于NC组,且RMPP组高于MPP组(P<0.05)。RMPP组外周血中性粒细胞TLR1和TLR2mRNA高于MPP组(P<0.05)。MPP组TLR2 mRNA高于NC组(P<0.05)。MP刺激组的TNF-α[A549细胞:(19.50±1.30)ng/L,中性粒细胞:(505.60±92.40)ng/L]和TLR2(A549细胞:3 760.17±772.06,中性粒细胞:4 224.00±711.12)表达显著高于NC组(P<0.05)。TLR2敲除后A549细胞后,与对照组相比,TLR2敲除的细胞MP刺激诱导的TNF-α表达受到显著抑制。蛋白质印迹法显示,MP刺激A549细胞中MyD88和NF-κB蛋白表达均明显增加(P<0.05)。阻断Myd88或NF-κB可明显减弱MP诱导的A549细胞TNF-α表达(P<0.05)。结论 TLR2可调节MPP通过TLR2-MyD88-NF-κB信号通路介导的肺部炎症和TNF-α释放。

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.

室旁核TLR4阻断改善2型糖尿病大鼠糖代谢及机制

目的 聚焦于下丘脑室旁核(PVN)区域,研究中枢Toll样受体(TLR)4阻断对2型糖尿病(T2DM)大鼠糖代谢的影响及探讨相应影响机制。方法 将SD大鼠随机分为:正常对照(NC+PVN vehicle)组、正常干预(NC+PVN TAK-242)组、糖尿病(DM+PVN vehicle)组、糖尿病干预(DM+PVN TAK-242)组。高糖高脂联合小剂量链脲佐菌素(STZ)方法建立T2DM大鼠模型,皮下植入室旁核微型渗透泵(28 d,输注速率0.11 L/h)给予TAK-242(一种TLR4特异性抑制剂)和人工脑脊液;各组大鼠监测体重、空腹血糖(FBG)、葡萄糖耐量试验(GTT)、胰岛素耐量试验(ITT)和丙酮酸耐量试验(PTT)评估体内糖代谢情况;肾交感神经放电检测交感神经活性;Western印迹检测还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶(NOX)4、Cu-Zn超氧化物歧化酶(SOD)、NF-κB通路激活标志物(TH)、谷氨酸脱羧酶(GAD)67蛋白表达;二氢乙淀(DHE)检测PVN中活性氧簇(ROS)水平;免疫荧光检测PVN中TLR4、TH阳性神经元表达数量;免疫组化检测PVN中神经元激活标志物(c-FOS)、NF-κB通路激活标志物(p-IKKβ)阳性神经元表达数量。结果 与正常组相比,T2DM组大鼠体重显著降低,FBG显著升高,体内存在胰岛素抵抗和糖异生紊乱,肾交感神经放电显著增加,PVN内TLR4、TH、ROS、c-FOS、p-IKKβ阳性神经元表达数量显著增多,PVN内NOX4、TH蛋白表达显著升高,Cu-Zn SOD、GAD67蛋白表达显著降低(P<0.05)。经TAK-242治疗的T2DM大鼠体重下降速度显著降低,FBG不断升高趋势被控制,体内胰岛素抵抗和糖异生紊乱显著改善,肾交感神经放电显著降低,PVN内TLR4、TH、ROS、c-FOS、p-IKKβ阳性神经元表达数量显著减少,PVN内NOX4、TH蛋白表达降低,Cu-Zn SOD、GAD67蛋白表达升高(P<0.05)。结论 PVN慢性输注TAK-242可有效阻断下丘脑PVN TLR4的表达,改善PVN内氧化应激和神经元凋亡,改善PVN内兴奋性神经递质和抑制性神经递质失平衡,进而降低交感神经活性,改善T2DM大鼠体内糖代谢。

TLR2 mAb和TLR4 mAb对急性期溃疡性结肠炎小鼠结肠黏膜炎症因子IFN-γ、IL-4及IL-17表达的影响

目的探讨TLR2单克隆抗体(toll-like receptor 2 monoclonal antibodies,TLR2 mAb)和TLR4单克隆抗体(toll-like receptor 4 monoclonal antibodies,TLR4 mAb)对葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导的急性期溃疡性结肠炎(ulcerative colitis,UC)小鼠结肠黏膜的炎症因子IFN-γ、IL-4及IL-17表达的影响。方法50只雄性BALB/c小鼠分为5组(每组10只):对照组(A)、模型组(B)及TLR2 mAb干预组(C)、TLR4mAb干预组(D)、TLR2 mAb和TLR4 mAb联合干预组(E)。A组小鼠饮用蒸馏水7 d;B-E组小鼠仅饮用5%DSS水溶液7 d以产生急性期溃疡性结肠炎模型。造模开始的同时,每隔48 h分别给予C、D、E干预组小鼠以TLR2 mAb(10μg)、TLR4 mAb(10μg)、TLR2 mAb(10μg)+TLR4 mAb(10μg)腹腔内注射,A、B组给予腹腔内注射生理盐水,以观察TLR2 mAb和TLR4 mAb的干预作用。观察指标包括疾病活动指数(disease activityindex,DAI)、结肠组织病理学评分(histopathological score,HS)。造模及干预7 d后处死小鼠,Real-ti me PCR法检测各组结肠黏膜TLR2、TLR4、IFN-γ、IL-4及IL-17的mRNA表达。结果(1)与A组相比,B组小鼠肠道DAI及HS明显增高(P<0.01)。与B组相比,E组小鼠肠道DAI(P<0.05)和HS(P<0.01)均明显降低。与D组相比,E组小鼠肠道HS明显降低(P<0.05)。(2)与A组相比,B组小鼠结肠黏膜TLR2、TLR4表达明显增高(P<0.01,P<0.05)。与B组相比,C、D、E组小鼠结肠黏膜TLR2、TLR4表达均有不同程度的降低。(3)与A组相比,B组小鼠结肠黏膜IFN-γ、IL-4及IL-17的mRNA表达明显增高(P<0.01,P<0.05,P<0.01)。与B组相比,C、D、E组小鼠结肠黏膜IFN-γ、IL-4及IL-17的mRNA表达均有不同程度的降低。结论TLR2 mAb和TLR4 mAb可能通过下调小鼠结肠黏膜的炎症因子IFN-γ、IL-4、IL-17的mRNA表达而发挥其干预作用。

TLR2基因单核苷酸多态性与重症社区获得性肺炎易感性和预后的关联研究

目的探讨Toll样受体2（toll—like receptor2，TLR2）基因单核苷酸多态性（single nucleotide polymorphism，SNP）与中国汉族人群重症社区获得性肺炎的易感性和预后的相关性。方法收集2005—05—2008—04中山医院急诊科收治的360例社区获得性肺炎患者的病史资料和血液样本，其中包括重症肺炎和非重症肺炎患者各180例。根据Hapmap中国人群数据库选择SNPs和标签SNPs（Tag SNPs），利用Primer3软件设计引物；采用DNA抽提试剂盒提取外周血的DNA，PCR-直接测序法进行SNPs分型；利用Haploview 4．1和SPSS15．0软件进行数据分析。结果TLR2基因2个TagSNPs即rs1898830和rs3804099在研究样本中符合Hardy—Weinberg平衡，2个SNPs等位基因频率和基因型频率在重症肺炎和非重症肺炎组问比较差异无统计学意义，存活组与死亡组比较差异也无统计学意义（P〉0．05）。结论TLR2基因单核苷酸多态性与中国汉族人群重症社区获得性肺炎的易感性和预后无明显的相关性。

Toll样受体2(TLR2)激动剂Pam3Csk4预处理对耐甲氧西林金黄色葡萄球菌攻击小鼠的保护作用

目的耐甲氧西林金黄色葡萄球菌(MRSA)感染日趋严重,免疫防治被寄予厚望。本研究旨在探索Toll样受体2(TLR2)激动剂Pam3Csk4预处理小鼠对MRSA攻击的保护作用。方法每只昆明小鼠用(25、50、100)μg Pam3Csk4尾静脉预处理12、24 h,小鼠尾静脉注射7×1010集落形成单位(CFU)/kg的MRSA(ATCC43300)。小鼠生存率按前24 h每2 h观察1次,后续每6 h记录小鼠存活情况,共观察1周。感染MRSA(3×108CFU/只)6 h后,菌落计数法观察肝、脾和肾等器官对MRSA的清除能力;3×108CFU/只MRSA攻击6、12、24 h后,ELISA检测小鼠血清中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、γ干扰素(IFN-γ)和IL-10含量,实时荧光定量PCR(q PCR)检测MRSA攻击后小鼠脾脏TNF-α、IL-6、IFN-γ和IL-10的mRNA水平,以及Pam3Csk4预处理小鼠24 h后,CXC趋化因子配体1(CXCL1)和Fcγ受体Ⅲ(FcγRⅢ)表达量。结果 100μg Pam3Csk4预处理能显著提高MRSA攻击小鼠的生存率,与Pam3Csk4预处理时间呈时间依赖关系;MRSA攻击小鼠的生存时间与Pam3Csk4预处理剂量相关,50μg以上剂量Pam3Csk4预处理能显著提高MRSA攻击小鼠生存率至70%以上;与对照组比较,Pam3Csk4预处理小鼠血清中TNF-α在6 h和12 h均显著降低,24 h后2组间无显著差别;IL-6在处理6 h内显著降低,12 h和24 h后无显著差异,IFN-γ在24 h内均显著降低,而IL-10则无显著变化;Pam3Csk4预处理24 h后,脾脏中CXCL1和FcγRⅢ表达量均显著高于生理盐水组。结论 Pam3Csk4预处理对MRSA攻击小鼠具有保护作用。