手足口病患儿外周血TLR3、s100β蛋白、IL-6的表达水平及其与病情和预后的相关性

目的检测手足口病(HFMD)患儿外周血Toll样受体3(TLR3)、中枢神经特异性蛋白100β(S100β)和白细胞介素-6(IL-6)的表达水平,并探讨其与患儿病情和预后的关系。方法选择2019年1月至2023年1月郑州大学附属儿童医院、河南省儿童医院、郑州儿童医院收治的142例HFMD患儿作为观察组,依据患儿病情严重程度(轻症组与重症组)、年龄(低龄组与高龄组)和预后(良好组与不良组)进行分组,另选取87例同期健康儿童作为对照组。比较不同病情严重程度患儿与健康儿童的外周血TLR3、s100β、IL-6水平,比较观察组患儿入院不同时间、不同病情严重程度患儿入院不同时间、不同年龄患儿入院不同时间以及不同预后患儿入院不同时间的外周血TLR3、s100β、IL-6水平。结果重症组患儿的外周血TLR3、s100β、IL-6表达水平分别为1.20±0.36、(14.41±3.53)μg/L、(14.98±4.37)U/m,明显高于轻症组的0.84±0.31、(3.12±1.01)μg/L、(10.73±3.05)U/m和对照组的0.68±0.19、(1.31±0.17)μg/L、(8.95±2.36)U/m,轻症患儿的外周血TLR3、s100β、IL-6表达水平明显高于对照组,差异均有统计学意义(P<0.05);观察组患儿入院1 d的外周血TLR3、s100β、IL-6表达水平明显低于入院5 d、7 d,入院7 d的外周血TLR3、s100β、IL-6表达水平明显低于入院5d时,差异均有统计学意义(P<0.05);重症组患儿入院1d、5d、7d的外周血TLR3、s100β、IL-6表达水平明显高于轻症组,差异均有统计学意义(P<0.05);低龄组患儿入院1 d、5 d、7 d的外周血TLR3、s100β、IL-6表达水平明显高于高龄组,差异均有统计学意义(P<0.05);预后良好组患儿入院1 d、7 d的外周血TLR3表达水平分别为0.97±0.31、1.90±0.55,明显低于预后不良组的1.90±0.18、2.46±0.74,入院1 d、5 d、7 d的外周血s100β、IL-6表达水平分别为(8.17±2.31)μg/L、(32.98±10.08)μg/L、(27.27±7.66)μg/L、(12.65±4.09)U/mL、(17.41±4.06)U/mL、(14.39±4.35)U/mL,明显低于预后不良组的(13.83±1.55)μg/L、(63.53±12.31)μg/L、(49.28±10.15)μg/L、(17.03±3.51)U/mL、(22.27±2.48)U/mL、(20.35±4.58)U/mL,差异均有统计学意义(P<0.05)。结论HFMD患儿外周血TLR3、s100β、IL-6水平升高,且与病情严重程度、发病年龄以及预后有一定的关联性,临床监测以上指标变化,可提示病情严重程度、预测预后。

Oxygen-glucose deprivation of neurons transfected with toll-like receptor 3-siRNA Determination of an optimal transfection sequence

Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA （siRNA）-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.

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.

Conjugation of toll-like receptor-7 agonist to gastric cancer antigen MG7-Ag exerts antitumor effects

AIM: To investigate the effects of our tumor vaccines on reversing immune tolerance and generating therapeutic response.METHODS: Vaccines were synthesized by solid phase using an Fmoc strategy,where a small molecule toll-like receptor-7 agonist(T7) was conjugated to a monoclonal gastric cancer 7 antigen mono-epitope(T7-MG1) or tri-epitope(T7-MG3).Cytokines were measured in both mouse bone marrow dendritic cells and mouse spleen lymphocytes after exposed to the vaccines.BALB/c mice were intraperitoneally immunized with the vaccines every 2 wk for a total of three times,andthen subcutaneously challenged with Ehrlich ascites carcinoma(EAC) cells.Three weeks later,the mice were killed,and the tumors were surgically removed and weighed.Serum samples were collected from the mice,and antibody titers were determined by ELISA using an alkaline phosphate-conjugated detection antibody for total Ig G.Antibody-dependent cell-mediated cytotoxicity was detected by the lactate dehydrogenase method using natural killer cells as effectors and antibody-labeled EAC cells as targets.Cytotoxic T lymphocyte activities were also detected by the lactate dehydrogenase method using lymphocytes as effectors and EAC cells as targets.RESULTS: Vaccines were successfully synthesized and validated by analytical high performance liquid chromatography and electrospray mass spectrometry,including T7,T7-MG1,and T7-MG3.Rapid inductions of tumor necrosis factor-α and interleukin-12 in bone marrow dendritic cells and interferon γ and interleukin-12 in lymphocytes occurred in vitro after T7,T7-MG1,and T7-MG3 treatment.Immunization with T7-MG3 reduced the EAC tumor burden in BALB/c mice to 62.64% ± 5.55% compared with PBS control(P < 0.01).Six or nine weeks after the first immunization,the monoclonal gastric cancer 7 antigen antibody increased significantly in the T7-MG3 group compared with the PBS control(P < 0.01).As for antibody-dependent cell-mediated cytotoxicity,antisera obtained by immunization with T7-MG3 were able to markedly enhance cell lysis compared to PBS control(31.58% ± 2.94% vs 18.02% ± 2.26%; P < 0.01).As for cytotoxic T lymphocytes,T7-MG3 exhibited obviously greater cytotoxicity compared with PBS control(40.92% ± 4.38% vs 16.29% ± 1.90%; P < 0.01).CONCLUSION: A successful method is confirmed for the design of gastric cancer vaccines by chemical conjugation of T7 and multi-repeat-epitope of monoclonal gastric cancer 7 antigen.

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.

木犀草素通过TLR4/PI3K/AKT通路对子痫前期大鼠胎盘血管生成及细胞凋亡的影响

目的:探究木犀草素通过Toll样受体4(TLR4)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)通路对子痫前期(PE)大鼠胎盘血管生成及细胞凋亡的影响。方法:SD大鼠随机分为对照组、模型组、木犀草素(40 mg/kg)组、TLR4过表达载体组、TLR4空载组、木犀草素(40 mg/kg)+TLR4过表达载体组,每组12只,除对照组外,其余各组大鼠皮下注射L-精氨酸甲酯(LNAME)制备PE模型,以药物分组处理后,测定各组大鼠妊娠晚期平均动脉压及24 h尿蛋白含量,检测各组大鼠平均子代数目、平均子代体质量、胎盘组织CD31(血管标志物)阳性细胞比例、胎盘细胞凋亡比例,血清炎症因子IL-6、肿瘤坏死因子-α(TNF-α)、IL-17水平及胎盘组织凋亡相关蛋白、TLR4/PI3K/AKT通路蛋白表达。结果:与对照组相比,模型组大鼠平均动脉压、24 h尿蛋白含量、胎盘细胞凋亡比例、血清IL-6、TNF-α及IL-17水平、胎盘组织p-PI3K/PI3K、p-AKT/AKT、TLR4、BCL2相关X蛋白(Bax)、天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)蛋白水平显著升高(P<0.05),平均子代数目、平均子代体质量、胎盘组织CD31阳性细胞比例、B淋巴细胞瘤-2(Bcl-2)蛋白表达水平显著降低(P<0.05)。与模型组、木犀草素+TLR4过表达载体组分别相比,木犀草素组大鼠平均动脉压、24 h尿蛋白含量、胎盘细胞凋亡比例、血清IL-6、TNF-α及IL-17水平、胎盘组织p-PI3K/PI3K、p-AKT/AKT、TLR4、Bax、caspase-3蛋白水平均显著降低(P<0.05),平均子代数目、平均子代体质量、胎盘组织CD31阳性细胞比例、Bcl-2蛋白表达均显著升高(P<0.05),TLR4过表达载体组大鼠平均动脉压、24 h尿蛋白含量、胎盘细胞凋亡比例、血清IL-6、TNF-α及IL-17水平、胎盘组织p-PI3K/PI3K、p-AKT/AKT、TLR4、Bax、caspase-3蛋白水平均显著升高(P<0.05),平均子代数目、平均子代体质量、胎盘组织CD31阳性细胞比例、Bcl-2蛋白表达均显著降低(P<0.05)。与模型组相比,TLR4空载组大鼠各指标水平差异无统计学意义(P>0.05)。结论:木犀草素可通过下调TLR4/PI3K/AKT通路表达,降低PE大鼠平均动脉压,缓解妊娠后期高血压与蛋白尿症状,抑制炎症,增强胎盘血管生成,减轻胎盘组织损伤及细胞凋亡,提高妊娠大鼠子代存活率。

泥蚶TLR3基因的生物学特性及表达分析

为探究泥蚶Toll样受体3(TLR3)基因的生物学特性及其对缺氧和硫化物胁迫的响应,克隆了泥蚶TgTLR3核心片段序列,并运用实时荧光定量PCR技术检测了TgTLR3基因在血淋巴、鳃、肝胰腺、外套膜、闭壳肌、斧足等组织的分布情况以及在缺氧和硫化物胁迫下血淋巴中的表达模式。结果显示,泥蚶TgTLR3开放阅读框2634 bp,编码877个氨基酸。其中,49~593位为胞外富含亮氨酸重复序列,跨膜域为658~680位,720~865位为白细胞介素受体1结构域,蛋白质分子量为99.4 kD,等电点为7.76。氨基酸同源性比对发现,泥蚶TgTLR3氨基酸序列与毛蚶相似度高达80.05%。系统进化树显示,TgTLR3与毛蚶首先聚为一支,表明与毛蚶亲缘关系最近。TgTLR3在泥蚶各组织中均有表达,其中血淋巴表达量最高。缺氧胁迫48~96 h后,血淋巴中TgTLR3显著升高;添加硫化物后,TgTLR3在48 h显著低于缺氧组但在96 h仍显著高于对照。上述研究结果表明,TgTLR3基因在泥蚶应对缺氧和硫化物胁迫的免疫调控中可能发挥着重要作用。研究结果为探究软体动物TLR3的功能及其响应缺氧及硫化物胁迫的免疫机制提供了基础数据。

外周血BNP及TLR3、NF-κB水平与手足口病患儿病情、预后的相关性

目的探究外周血B型钠尿肽(BNP)及Toll样受体3(TLR3)、核因子κB(NF-κB)水平与手足口病患儿病情、预后的相关性。方法选取2021年8月至2023年8月邓州市疾病预防控制中心收治的102例手足口病患儿为研究对象,根据患儿患病轻重程度将其分为重症组43例,轻症组59例,对比两组患儿外周血BNP及TLR3、NF-κB水平。对出院患儿进行3个月随访,根据随访结果将预后良好患儿纳入良好组,预后不良患儿纳入不良组。多因素分析采用Logistic回归方程,受试者工作特征曲线(ROC)分析BNP、TLR3、NF-κB表达水平对手足口病患儿预后的预测价值。结果重症组患儿BNP、TLR3、NF-κB表达水平均高于轻症组,差异有统计学意义(P<0.05);不良组和良好组患儿性别、体质量指数(BMI)值、合并发热及合并皮疹相比较差异无统计学意义(P>0.05);不良组患儿年龄<3岁占比、合并肺部渗出性病变占比、合并循环障碍占比、BNP、TLR3及NF-κB水平均高于良好组,差异有统计学意义(P<0.05);经Logistic回归分析,BNP、TLR3、NF-κB表达均为影响手足口病患儿预后的独立危险因素(P<0.05);经ROC曲线分析,BNP、TLR3、NF-κB水平表达均对手足口病患儿预后不良发生均有较高的预测价值,曲线下面积(AUC)分别为0.908、0.950、0.904。结论重症手足口病患儿BNP、TLR3、NF-κB表达水平均高于轻症患儿,对评估病情严重程度具有较高参考价值,且高水平BNP、TLR3、NF-κB均为手足口病患儿预后不良的独立危险因素。

隐丹参酮调节TLR4/NF-κB/NLRP3信号通路对高糖诱导的心肌细胞焦亡的影响

目的:探讨隐丹参酮调节Toll样受体4/核转录因子κB/NOD样受体蛋白3(TLR4/NF-κB/NLRP3)信号通路对高糖诱导的心肌细胞焦亡的影响。方法:将H9c2细胞分为对照组(正常葡萄糖5.5 mmol/L)、高糖组(葡萄糖33 mmol/L)、2.5μmol/L隐丹参酮组(葡萄糖33 mmol/L+2.5μmol/L隐丹参酮)、5μmol/L隐丹参酮组(葡萄糖33 mmol/L+5μmol/L隐丹参酮)和脂多糖(LPS)组(葡萄糖33 mmol/L+5μmol/L隐丹参酮+1μg/mL TLR4/NF-κB/NLRP3信号通路激活剂LPS),各组细胞培养在相应的培养基中48 h。采用四甲基偶氮唑盐(MTT)法检测细胞存活率;2′,7′-二乙酰二氯荧光素(DCFH-DA)法检测细胞活性氧(ROS)水平;半胱氨酸天冬氨酸酶-1抑制剂(FLICA)-半胱氨酸天冬氨酸酶-1(Caspase-1)/碘化丙啶(PI)双染法检测细胞焦亡情况;酶联免疫吸附法(ELISA)检测细胞上清白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)水平;蛋白免疫印迹法(Western Blot)检测细胞GSDMD蛋白N端片段(GSDMD-N)、TLR4、NLRP3、磷酸化NF-κB p65(p-NF-κB p65)、NF-κB p65、Cleaved Caspase-1、Caspase-1蛋白表达。结果:与对照组比较,高糖组H9c2细胞存活率下降(P<0.05),细胞ROS水平、焦亡细胞比例、细胞上清IL-1β和IL-18水平及细胞TLR4、NLRP3、p-NF-κB p65/NF-κB p65、GSDMD-N、Cleaved Caspase-1/Caspase-1蛋白表达升高(P<0.05);与高糖组比较,2.5μmol/L隐丹参酮组、5μmol/L隐丹参酮组H9c2细胞存活率升高(P<0.05),细胞ROS水平、焦亡细胞比例、细胞上清IL-1β和IL-18水平及细胞TLR4、NLRP3、p-NF-κB p65/NF-κB p65、GSDMD-N、Cleaved Caspase-1/Caspase-1蛋白表达降低(P<0.05)。TLR4/NF-κB/NLRP3信号通路激活剂LPS可逆转隐丹参酮对高糖诱导细胞增殖和焦亡的抑制作用。结论:隐丹参酮可抑制TLR4/NF-κB/NLRP3信号通路减轻高糖诱导的心肌细胞焦亡。

Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

Correlation analysis of Toll-like receptor 3 content in peripheral blood with electroencephalogram parameters and neurotransmitter content in children with intractable epilepsy

Objective:To detect the content of Toll-like receptor 3 (TLR3) in peripheral blood of children with intractable epilepsy, explore the correlation between TLR3 content and EEG parameters, neurotransmitter contents.Methods:37 cases of Intractable epilepsy children in our hospital during September 2016to June 2018 were chosen as Intractable epilepsy group, 30 cases of healthy volunteers who underwent physical examination in our hospital were treated as Normal control group. The levels of TLR3, neurotransmitter [5-hydroxytryptamine (5-HT), dopamine (DA), epinephrine (E), norepinephrine (NE)] and electroencephalogram parameters [alpha, beta, delta, theta] in peripheral blood of two groups were compared. Pearson test was used to evaluate the correlation of TLR3 content in peripheral blood with EEG parameters and neurotransmitter content in children with intractable epilepsy.Results: Content of TLR3 in peripheral blood of Intractable epilepsy group was significantly higher than that of Normal control group;the alpha power and theta power of EEG parameters were lower than those of Normal control group;contents of neurotransmitters such as 5-HT, DA, E and NE were significantly lower than those of Normal control group (P<0.05). The correlation analysis showed that content of TLR3 in peripheral blood of children with intractable epilepsy was negatively correlated with levels of alpha and theta power of EEG, positively correlated with content of neurotransmitters such as 5-HT, DA, E and NE (P<0.05), but had no significant correlation was found with level of beta and delta power (P>0.05).Conclusion: The abnormal increase of TLR3 in peripheral blood of children with intractable epilepsy may be one of the direct causes of neurological impairment in children with intractable epilepsy.

TLR4/NF-κB/NLRP3通路抑制炎症反应作用的研究

Toll样受体(TLRs)是参与非特异性免疫的一类重要的蛋白质,是表达与固有免疫细胞最重要的模式识别受体之一,可识别来源于微生物的具有保守结构的分子,即病原体相关分子模式(PAMAs),从而激活机体产生免疫应答。核因子κB(NF-κB)在几乎所有类型的细胞和组织中都有表达,在调节对外部刺激的显著多样性的反应中起着关键作用,因此是多个生理和病理过程中的关键因素。而核苷酸结合寡聚化结构域样受体蛋白3(NLRP3),即NLRP3炎性小体通路在脑缺血再灌注和焦亡中具有重要意义,如NLRP3在脑缺血再灌注损伤时在小胶质细胞中被激活,随后在神经元和微血管内皮细胞中表达。由TLRs激活、NF-κB传递、NLRP3启动形成的TLR/NF-κB/NLRP3信号通路在机体各器官炎症反应中起到关键性作用,本文探讨了各类药物通过抑制该通路炎症反应,起到保护身体各大重要器官的作用。

达原饮对Hp感染模型大鼠胃组织和口周皮肤TLR4/NF-κB/NLRP3通路的影响

目的:观察达原饮对幽门螺杆菌(Hp)感染模型大鼠胃组织和口周皮肤Toll样受体4/核因子-κB/NOD样受体蛋白3(TLR4/NF-κB/NLRP3)信号通路,以及肿瘤坏死因子-α(TNF-α)与白细胞介素-1β(IL-1β)等炎症因子的影响,探讨达原饮治疗Hp感染的机制。方法:选取44只雄性SD大鼠,随机分为空白组8只及造模组36只,造模组予以Hp菌液灌胃造模,造模成功后,将造模组随机分为模型组、达原饮高剂量组、达原饮低剂量组、西药组,每组各8只,分别予以相应药物灌胃,空白组、模型组予以等量生理盐水灌胃,灌胃2周。观察大鼠造模前后一般情况改变,苏木精-伊红(HE)染色观察口周皮肤和胃组织病理学变化,酶联免疫吸附测定(ELISA)检测大鼠口周皮肤和胃组织TLR4、NF-κB、NLRP3表达以及血清中TNF-α、IL-1β表达水平。结果:给药干预后,与模型组相比,西药组、达原饮高剂量组、达原饮低剂量组大鼠皮毛柔顺光泽,饮食水量增加,大便逐渐成型,胃黏膜上皮细胞层次基本分明,排列较整齐,炎症细胞浸润减少,口周皮肤角化过度、棘层增厚、毛囊周围轻度炎症细胞浸润等情况均有不同程度改善。与模型组相比,西药组、达原饮高剂量组、达原饮低剂量组大鼠胃组织和口周皮肤TLR4、NF-κB、NLRP3蛋白表达水平均有不同程度的下调(P<0.05或P<0.01),血清炎症因子TNF-α、IL-1β表达明显下降(P<0.01)。结论:达原饮可能通过干预TLR4/NF-κB/NLRP3信号通路,抑制TLR4、NF-κB、NLRP3及下游炎症因子的表达,减轻Hp感染模型大鼠胃组织和口周皮肤炎症损伤。

维生素D_(3)辅助糖皮质激素对小儿支气管哮喘TLR4/NF-κB信号通路相关因子水平的影响

目的研究维生素D_(3)辅助糖皮质激素对小儿支气管哮喘Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路相关因子水平的影响。方法选取2019年1月—2022年1月长江大学附属荆州医院收治的120例小儿支气管哮喘患儿作为研究对象,按照随机数表法将患儿分为A组、B组和C组,每组40例。所有患儿给予常规治疗,C组在常规治疗基础上给予安慰剂,B组在常规治疗基础上给予糖皮质激素,A组在B组基础上给予维生素D_(3)。观察各组患儿喘息、胸闷、气促及咳嗽症状持续时间,分析各组患儿治疗前后TLR4和NF-κB mRNA表达,并比较3组治疗前后气道炎症指标[嗜酸性粒细胞(EOS)、白细胞介素-4(IL-4)、肿瘤坏死因子α(TNF-α)、C反应蛋白(CRP)]水平,比较各组患儿治疗前后肺功能[第1秒用力呼气容积占预计值百分比(FEV1%pred)、用力肺活量(FVC)、每秒呼气峰流速(PEF)及FEV1/FVC值]。结果A组喘息、胸闷、气促及咳嗽症状持续时间短于B组和C组(P<0.05),B组短于C组(P<0.05)。A组治疗前后TLR4和NF-κB mRNA相对表达量的差值高于B组和C组(P<0.05),B组高于C组(P<0.05)。A组治疗前后IL-4、EOS、TNF-α、CRP的差值高于B组和C组(P<0.05),B组高于C组(P<0.05)。A组治疗前后FEV1%pred、FVC、FEV1/FVC、PEF的差值高于B组和C组(P<0.05),B组高于C组(P<0.05)。Pearson相关性分析显示,IL-4、EOS、TNF-α、CRP水平与TLR4 mRNA呈正相关(r=0.501、0.574、0.462和0.474,均P<0.05),与NF-κB mRNA呈正相关(r=0.496、0.522、0.485和0.492,均P<0.05)。FEV1%pred、FVC、FEV1/FVC、PEF水平与TLR4 mRNA呈负相关(r=-0.596、-0.542、-0.513和-0.505,均P<0.05),与NF-κB mRNA呈负相关(r=-0.561、-0.505、-0.526和-0.518,均P<0.05)。结论维生素D_(3)辅助糖皮质激素治疗小儿支气管哮喘可通过调控TLR4/NF-κB信号通路,减轻患儿气道炎症反应,并有效提升肺功能,具有良好的临床应用价值。

TLR4、PTX3、PLR在评估急性缺血性脑卒中机械取栓术后患者预后中的应用

目的探讨Toll样受体4(TLR4)、正五聚蛋白3(PTX3)、血小板与淋巴细胞比值(PLR)在急性缺血性脑卒中机械取栓术后患者预后中的应用。方法回顾性分析2021年1月至2022年12月安阳市人民医院收治的160例急性缺血性脑卒中患者的病案资料,患者均接受机械取栓术治疗。用改良Rankin量表(mRS)对患者术后1、3个月的预后进行评估,术后1个月mRS≤3分为预后良好,mRS>3分为预后不良;术后3个月mRS≤2分为预后良好,mRS>2分预后不良。比较预后良好组与预后不良组TLR4、PTX3、PLR水平,分析TLR4、PTX3、PLR预测急性缺血性脑卒中机械取栓术后患者预后的价值。结果预后不良组术后1、3个月的mRS评分、TLR4、PTX3、PLR水平高于预后良好组(P<0.05),TLR4、PTX3、PLR联合预测急性缺血性脑卒中机械取栓术后1、3个月预后不良的曲线下面积(AUC)高于单一指标(P<0.05)。结论TLR4、PTX3、PLR在急性缺血性脑卒中机械取栓术后预后不良中呈高表达,对术后的预后预测有一定价值,能反映机体炎症状态及血小板活化情况,为预后预测提供重要参考依据。

桥本甲状腺炎合并甲状腺乳头状癌患者中Toll样受体3和核转录因子-κB的表达及相关性分析

目的分析桥本甲状腺炎(Hashimoto thyroiditis,HT)合并甲状腺乳头状癌(PTC)患者Toll样受体3(toll-like receptor 3,TLR3)和核转录因子-κB(nuclear transcription factor-κB,NF-κB)表达及相关性。方法收取邯郸市中心医院2020年3月~2022年3月收治的130例行手术切除的甲状腺标本,其中正常甲状腺组织标本43例,HT标本47例,HT合并PTC标本40例,分析TLR3和NF-κB在正常甲状腺组织、HT组、HT合并PTC组中的表达,分析HT合并PTC组中TLR3和NF-κB表达与临床病理参数关系,Pearson相关性分析TLR3和NF-κB的关系。结果TLR3在正常甲状腺组织、HT组、HT合并PTC组中的阳性表达率分别为0(0/43)、80.85%(38/47)、90.00%(36/40);NF-κB在以上三组中的阳性表达率分别为0(0/43)、68.09%(32/47)、85.00%(34/40)。TLR3和NF-κB在HT组、HT合并PTC组中的阳性表达率均高于正常甲状腺组织(P<0.05),TLR3和NF-κB表达与性别、年龄、HT合并PTC病理学特征、病灶类型、淋巴结转移、甲状腺包膜侵犯差异比较均无统计学意义(P均>0.05)。TLR3和NF-κB呈显著正相关(r=0.589,P<0.05)。结论TLR3和NF-κB在HT合并PTC组织中的阳性率高于正常甲状腺组织,且二者表达呈正相关。

鼻内针刺治疗病毒感染后嗅觉障碍的临床疗效及对Toll样受体3表达水平和炎症免疫反应的影响

目的探讨鼻内针刺治疗病毒感染后嗅觉障碍(post-viral olfactorydysfunction,PVOD)的临床疗效及对Toll样受体3(Toll-like receptor3,TLR3)表达水平和炎症免疫反应的影响。方法选取2020年1月—2021年10月黑龙江中医药大学附属第二医院收治的116例PVOD患者,采用随机数值表法分为对照组、鼻内针刺组各58例,分别予鼻用激素、鼻内窥镜下鼻内针刺治疗,连续治疗2周,评估两组临床疗效,记录不良反应情况;于治疗前后,进行Sniffin’Sticks主观嗅觉心理测试、嗅觉功能客观测试,检测外周血TLR3 mRNA、肿瘤坏死因子-α(tumor necrosis factor,TNF-α)、白细胞介素-6(Interleukin-6,IL-6)水平。结果治疗后,鼻内针刺组的总有效率为91.38%(53/58),明显高于对照组的77.59%(45/58)。治疗后,两组患者的Sniffin’Sticks测试结果均明显好转(P<0.05);且鼻内针刺组治疗后Sniffin’Sticks测试结果整体优于对照组(P<0.05)。鼻内针刺组治疗后的P2波潜伏期显著短于本组治疗前及对照组同期,P2波振幅显著大于本组治疗前及对照组同期(P<0.05)。鼻内针刺组治疗后的外周血TLR3 mRNA及炎性因子TNF-α、IL-6水平均显著低于本组治疗前及对照组同期(P<0.05)。治疗期间,两组均未出现严重不良反应。结论鼻内针刺治疗PVOD疗效显著,有助于改善患者的嗅觉功能,其机制可能与下调TLR3表达水平,减轻炎症免疫反应水平有关。

HPV16/18E6、TLR3蛋白和血清CRP水平在乳腺浸润性导管癌中的表达

目的探讨乳腺浸润性导管癌组织中人乳头瘤病毒16/18E6(HPV16/18E6)、Toll样受体3(TLR3)蛋白及血清C反应蛋白(CRP)水平表达之间的关系及其临床意义。方法采用免疫组化染色和电化学发光分析技术检测柳州女性50例乳腺浸润性导管癌、30例乳腺导管原位癌(DCIS)、30例乳腺导管上皮非典型增生(ADH)和30例正常乳腺对照组组织中HPV16/18E6、TLR3蛋白表达和血清CRP水平,比较分析癌组、DCIS组、ADH组及对照组中HPV16/18E6、TLR3蛋白及血清CRP表达之间的相互关系,评价癌组中HPV16/18E6、TLR3蛋白及血清CRP阳性表达与临床病理特征之间的关系及其与ER、PR、HER2、Ki-67表达的相关性。结果癌组、DCIS组、ADH组及对照组中HPV16/18E6、TLR3蛋白及血清CRP阳性表达率差异均有统计学意义(P<0.01)。癌组及DCIS组HPV16/18E6蛋白和TLR3蛋白的阳性表达率均显著高于对照组(均P<0.01),且癌组、DCIS组和ADH组中血清CRP阳性率均显著高于对照组(均P<0.01)。HPV16/18E6阳性癌组中TLR3蛋白及血清CRP的阳性率均显著高于HPV16/18E6阴性组(均P<0.05);癌组、DCIS组及ADH组中HPV16/18E6与TLR3蛋白和血清CRP表达之间均呈正相关(P<0.01),TLR3蛋白与血清CRP表达亦呈正相关(P<0.01);癌组中HPV16/18E6、TLR3蛋白和血清CRP水平均与HER2和Ki-67的表达呈正相关(P<0.001);HPV16/18E6、TLR3蛋白及血清CRP阳性表达与组织学分级、淋巴结转移和pTNM分期相关(P<0.05)。此外,HPV16/18E6和TLR3蛋白的阳性表达均与肿瘤直径大小有关(P<0.05)。结论HPV16/18感染介导的TLR3蛋白阳性表达和血清CRP高水平可能涉及了HPV16/18感染后乳腺癌的发生、发展过程,TLR3蛋白和血清CRP水平可作为临床监测、评估HPV16/18相关乳腺癌患者疾病进展和预后影响因素的指标。

Toll样受体3处理的脐带间充质干细胞与人牙髓细胞共培养对细胞矿化与抗炎修复能力的影响

目的:探究Toll样受体3(TLR3)处理的人脐带间充质干细胞(hUCMSCs)与人牙髓细胞(hDPCs)共培养后,对细胞矿化能力及抗炎修复能力的影响。方法:从健康新生儿的脐带组织中分离培养hUCMSCs,采用流式细胞术和免疫荧光染色检测细胞表面标记物CD34、CD45、CD90及CD105的表达,以对hUCMSCs进行鉴定;以TLR3激动剂Poly(I:C)(10μg/mL)处理hUCMSCs,建立hUCMSCs与h DPCs的共培养体系,测定各组细胞碱性磷酸酶(ALP)水平,茜素红染色和Von Kossa染色观察各组细胞矿化情况;利用脂多糖(LPS,10μg/mL)刺激hDPCs模拟牙髓炎症反应,MTT法检测各组细胞的增殖活性,RT-qPCR法检测各组细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)、白细胞介素10(IL-10)、牙本质涎磷蛋白(DSPP)、牙本质基质蛋白-1(DMP-1)、骨钙素(OCN)及骨桥蛋白(OPN)的mRNA表达情况。结果:分离的hUCMSCs中CD34和CD45呈阴性表达,CD90和CD105呈阳性表达,表明成功分离出hUCMSCs;与单独培养的hUCMSCs和hDPCs比较,hUCMSCs与hDPCs共培养体系内细胞ALP染色加深,ALP活性升高,有较多的矿化结节形成(P<0.05);与hUCMSCs与hDPCs共培养比较,经Poly(I:C)处理的hUCMSCs与h DPCs共培养中细胞ALP活性进一步升高,矿化结节形成更多(P<0.05)。相较于未经LPS刺激的细胞,经LPS刺激后的各组细胞增殖活性降低,细胞中TNF-α、IL-6及IL-10的mRNA相对表达量均升高,DSPP、DMP-1、OCN及OPN的mRNA相对表达量均下降(P<0.05);在LPS刺激下,相较于hUCMSCs与hDPCs共培养下,经Poly(I:C)处理的hUCMSCs与h DPCs共培养中的细胞增殖活性较高,TNF-α、IL-6的mRNA相对表达量均下降,IL-10 mRNA相对表达量进一步升高,同时,DSPP、DMP-1、OCN及OPN的mRNA相对表达量也均升高(P<0.05)。结论:TLR3激动剂Poly(I:C)处理的hUCMSCs与hDPCs共培养,可增强细胞的矿化能力,抑制LPS刺激下炎症因子水平,并促进炎症状态下牙本质形成相关基因的表达,这可能有利于牙髓炎症下的抗炎修复。

Effects of Propofol on The mRNA Expression of Toll-like Receptor 4 in BV-2 Cells During Mimic Ischemia-Reperfusion Injury In Vitro

This study investigated the effects of propofol on the mRNA expression of Toll-like receptor-4 （TLR4） in BV-2 cells during mimic ischemia-reperfusion （I/R） injury in vitro. BV-2 cells, a mouse microglia line, were cultured and divided into 4 groups at random： control group （group C）, ischemia/reperfusion group （group I/R）, low-dose propofol （25 μmol/L） intervention group （group PF25） and high-dose propofol （100 μmol/L） intervention group （group PF100）. The mRNA expression of TLR4 and NF-κB was measured by means of RT-PCR. TNF-α levels in the supernatants of BV-2 cells were detected by ELISA. The results showed that the mRNA expression of TLR4 and NF-κB was significantly higher in groups I/R, PF25 and PF100 than in group C （P〈0.01）. And the TNF-α level in the supernatants was elevated in groups I/R, PF25 and PF100 as compared with that in group C （P〈0.01）. After pre-treatment with propofol, the mRNA expressions of TLR4 and NF-κB and the TNF-α level were significantly decreased in groups PF25 and PF100 in comparison to those in group I/R （P〈0.01）. And the decrease in those indicators was more significant in group PF100 than in group PF25 （P〈0.01）. It was concluded that propofol exerted brain-protecting effects during I/R injury by suppressing the mRNA expressions of TLR4 and NF-κB and deceasing the TNF-α level.