TLR/MyD88信号通路与自身免疫性疾病

Toll样受体(Toll-like receptor,TLR)是近年来发现的一类模式识别受体,通过识别病原相关分子模式(pathogen-associated molecular pattern,PAMP),激活天然免疫.TLR信号还通过上调抗原提呈细胞(antigen presenting cells,APC)表面共刺激分子及APC分泌的炎症细胞因子调节获得性免疫.TLR/MyD88信号在自身免疫性疾病的发病过程中起重要作用.本文介绍了TLR/MYD88信号通路及其在自身免疫病如实验性自身免疫脑脊髓膜炎、类风湿性关节炎、实验性自身免疫性葡萄膜炎、实验性自身免疫性心肌炎和自身免疫性肾小球肾炎等发生发展中的作用.     

有氧运动与黑枸黄酮对抑郁症小鼠的干预及TLR4/MyD88/NF-κB通路相关因子组织差异表达

该文探讨了有氧游泳运动(aerobic swimming, SW)与黑枸黄酮(Lycium ruthenicummurrflavones,LRMF)对抑郁症小鼠的抗抑郁作用及TLR4/MyD88/NF-κB相关因子在脑和血液组织中的差异性表达。该研究构建了50只雄性KM小鼠抑郁症模型,采用游泳运动和/或黑枸黄酮(200 mg/kg)灌胃干预。通过行为学评估、Elisa酶联免疫、脑组织尼氏染色、免疫组织化学法(IHC)及qRT-PCR法研究有氧运动与黑枸黄酮干预下小鼠的行为、脑组织形态学改变及相关因子蛋白、mRNA的表达。行为学评估结果显示,建模后,与对照组比较,慢性轻度不可预见性应激(chronic unpredictable mild stress, CUMS)组行为学评估具有显著性差异(P0.01);干预后,与模型组比较,干预组行为学评估出现不同程度的改善(P0.05, P0.01)。干预组脑组织单胺类递质5-HT(5-hydroxytryptamine)、NE(norepinephrine)、DA(dopamine)含量较M组上升,黑枸黄酮联合游泳运动组最高(P0.01),其次为游泳运动组。显微观察发现,模型组脑组织神经元损伤程度最高,核固缩、深染及空泡明显,黑枸黄酮联合游泳干预效果最佳(P0.01)。IHC结果显示,干预组TLR4、MyD88、NF-κB、IL-1β蛋白表达水平较模型组降低(P0.05, P0.01),且黑枸黄酮联合游泳组蛋白表达水平最低(P0.01),游泳组优于黑枸黄酮组。qRT-PCR检测结果显示, TLR4、MyD88在脑组织中表达水平均高于血液组织(P0.05),且呈正相关。与模型组比较,黑枸黄酮联合游泳组TLR4、MyD88、NF-κB、IL-1βmRNA表达最低(P0.01),游泳组低于黑枸黄酮组(P0.05)。以上结果表明,有氧游泳运动及黑果枸杞黄酮能缓解或治疗小鼠抑郁症所导致的脑损伤,增强免疫及神经元修复,联合干预效果最佳。具体机制可能是有氧运动和LRMF抑制TLR4的活性,同时,下调MyD88的表达,从而上调抗炎症基因的表达。此外, TLR4、MyD88在脑组织和血液组织中存在中度相关关系。作为一种早期检测手段,检测血浆中相关因子循环RNA可诊断和监测抑郁症致脑损伤的情况,具有良好的临床应用前景。     

MyD88依赖的TLR4信号通路在无症状HIV阳性患者肺泡巨噬细胞中的选择性损伤

肺泡巨噬细胞（AMS）是肺部主要的效应细胞,可通过模式识别受体如Toll样受体4（TLR4）识别病原微生物,从而发挥关键的第1道防线作用。在人类免疫缺陷病毒（HIV）阳性的巨噬细胞中,     

胀果甘草多糖GiP-B1通过TLR4/MyD88/NF-κB言号通路激活巨噬细胞RAW 264.7

为研究胀果甘草多糖GiP-B1通过TLR4/MyD88/NF-κB信号通路对巨噬细胞RAW 264.7免疫功能的影响,体外以TLR4干扰RNA (TLR4-siRNA)转染巨噬细胞RAW 264.724 h,用100 μg/mL GiP-B1处理转染和未转染细胞,12 h后分别采用CCK-8和ELISA法检测GiP-B...     

布地奈德混悬液对哮喘模型小鼠肺组织TLR4/MyD88/NF-κB通路的影响

摘要 目的：探究布地奈德混悬液对哮喘模型小鼠肺组织TOLL样受体4（toll-like receptor 4,TLR4）/髓样分化因子88（myeloid differentiation factor 88,MyD88）/核因子κB（nuclear factor-κB,NF-κB）通路的影响。方法：使用4 %鸡蛋清白蛋白与2 %的Al（OH3）共同致敏小鼠,建立咳嗽变异性哮喘小鼠模型40只,将模型大鼠分别使用低、中、高剂量（0.2、1.0、2.0 g/kg）布地奈德混悬液和孟鲁司特钠进行干预,1次/日连续干预14 d,于干预14 d时采集小鼠的支气管肺泡灌注液（bronchoalveolar lavage fluid,BALF）、气管及肺组织,对各组BALF中的白细胞（white blood cell,WBC）、嗜酸性粒细胞、血清γ干扰素（interferon-γ,IFN-γ）、白细胞介素-1β（interleukin-1β,Il-1β）、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平差异开展比较,对各组小鼠肺组织黏膜上皮增生程度评分、炎症细胞浸润程度评分、病变总评分差异,以及TLR4、MyD88、p65蛋白表达差异进行分析。结果：分析显示,布地奈德混悬液能够显著降低哮喘模型小鼠BALF中白细胞及嗜酸性粒细胞数量,同时还能够改善小鼠气管和支气管黏膜上皮增生与肺组织炎症细胞浸润状态,且干预后小鼠肺组织中的TLR4、MyD88、p65蛋白表达水平出现了明显的降低。结论：布地奈德混悬液对改善小鼠哮喘效果较好,其作用机制可能与该药能够调节TLR4、MyD88、p65蛋白表达,进而影响炎症和免疫反应进程有关。     

基于TLR4/MyD88/NF-κB通路探讨美洲大蠊虫粉对脊髓损伤大鼠的保护作用

目的 探讨美洲大蠊虫粉对脊髓损伤大鼠的保护作用和可能的机制。方法 将48只SD大鼠随机分为假手术组、盐水组、美洲大蠊虫粉组、TOLL样受体4(toll-like receptors, TLR4)抑制剂组。除假手术组外其余3组均构建大鼠脊髓半横断损伤模型。术后假手术组不做治疗,盐水组与美洲大蠊虫粉组分别给予生理盐水和美洲大蠊虫粉(630 mg/kg)灌胃处理,TLR4抑制剂组给予TLR4抑制剂(3 mg/kg)腹腔注射处理。术后1、3、7、14 d运用BBB评分评估大鼠后肢运动功能,苏木素-伊红(HE)染色观察脊髓组织病理改变,免疫组化观察神经元数目变化,酶联免疫法检测炎性因子IL-1、IL-6、IL-10和TNF-α表达,Western Blot检测TLR4、髓样分化因子(myeloid differentiation factor 88,MyD88)和NF-κB p65表达。结果 与假手术组相比,盐水组BBB评分、神经元数目显著下降,而病理损伤程度、IL-1、IL-6、TNF-α、TLR4、MyD88、NF-κB p65水平显著增加(P<0.05),与盐水组相比,美洲大蠊虫粉组...     

大鼠海马神经元TLR4介导的MyD88依赖途径在神经炎症中的作用

目的:研究大鼠海马神经元是否有Toll样受体4(TLR4)介导的的髓样分化因子88(MyD88)依赖途径及该途径的激活在神经炎症中的作用。方法:采用体外培养7 d的新生大鼠海马神经元,细胞免疫荧光双标法鉴定海马神经元纯度。用TLR4配体脂多糖(LPS)或TLR4抗体预处理海马神经元,以激活或阻断TLR4的作用。实时定量PCR(RT-qPCR)方法检测海马神经元中MyD88、肿瘤坏死因子受体相关因子6(TRAF6)mRNA的表达;Westernblot方法测定海马神经元MyD88和TRAF6蛋白水平;细胞免疫荧光双标法观察海马神经元中核因子κB/P65(NF-κB/P65)的表达定位及TLR4激活或阻断后NF-κB/P65核易位情况;ELISA检测培养上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和一氧化氮(NO)的水平。结果:LPS能上调海马神经元MyD88和肿瘤坏死因子受体相关因子(TRAF6)mRNA水平;促使NF-κB/P65转位至核;增加MyD88和TRAF6蛋白的表达;增加海马神经元培养上清中TNF-α、IL-1β和NO含量;TLR4抗体预处理能减弱LPS对海马神经元NF-κB/P65核易位作用及降低培养上清中TNF-α、IL-1β和NO的水平。结论:大鼠海马神经元有TLR4介导的的MyD88依赖途径,该途径的激活能导致TNF-α、IL-lβ和NO含量的增加。海马神经元TLR4介导的MyD88依赖途径参与了神经炎症反应,神经元不是神经炎症反应中的被动者。     

胰腺癌患者循环肿瘤细胞及TLR4、TLR9、myd88的表达水平与其化疗效果及转移、复发的关系

目的:探讨胰腺癌患者循环肿瘤细胞(CTC)中Toll样受体4(TLR4)、Toll样受体9(TLR9)、髓样分化因子88(myd88)的表达水平与患者化疗效果及转移、复发的关系。方法:将我院2015年6月-2016年6月收治并确诊的48例胰腺癌患者作为试验组,收集患者循环肿瘤细胞(CTC),检测其TLR4、TLR9、myd88信号表达情况,探讨其TLR4、TLR9、myd88信号表达水平与患者化疗效果及转移、复发的关系。结果:48例胰腺癌患者检出CTC 35例,检出率为72.9%。胰腺癌死亡、转移、复发患者TLR4、TLR9、myd88表达水平分别高于其存活、未转移、未复发患者,组间具有统计学差异(P0.05)。胰腺癌化疗效果CR患者TLR4、TLR9、myd88表达水平显著低于其化疗效果PR、SD、PD患者,且四组间差异具有统计学意义(P0.05);TLR4、TLR9、myd88表达水平与被膜受侵犯、淋巴结转移、肿瘤大小、CA199水平呈正相关(P0.05)。结论:胰腺癌患者CTC中TLRs/myd88信号表达水平与患者化疗效果及转移、复发密切相关。     

Eupatilin alleviates inflammatory response after subarachnoid hemorrhage by inhibition of TLR4/MyD88/NF-κB axis

Early brain injury (EBI) is associated with the adverse prognosis of subarachnoid hemorrhage (SAH) patients. The key bioactive component of the Chinese herbal medicine Artemisia asiatica Nakai (Asteraceae) is eupatilin. Recent research reports that eupatilin suppresses inflammatory responses induced by intracranial hemorrhage. This work is performed to validate whether eupatilin can attenuate EBI and deciphers its mechanism. A SAH rat model was established by intravascular perforation in vivo. At 6 h after SAH in rats, 10 mg/kg eupatilin was injected into the rats via the caudal vein. A Sham group was set as the control. In vitro, BV2 microglia was treated with 10 μM Oxyhemoglobin (OxyHb) for 24 h, followed by 50 μM eupatilin treatment for 24 h. The SAH grade, brain water content, neurological score, and blood-brain barrier (BBB) permeability of the rats were measured 24 h later. The content of proinflammatory factors was detected via enzyme-linked immunosorbent assay. Western blot analysis was conducted to analyze the expression levels of TLR4/MyD88/NF-κB pathway-associated proteins. In vivo, eupatilin administration alleviated neurological injury, and decreased brain edema and BBB injury after SAH in rats. Eupatilin markedly reduced the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), and suppressed the expression levels of MyD88, TLR4, and p-NF-κB p65 in the SAH rats' cerebral tissues. Eupatilin treatment also reduced the levels of IL-1β, IL-6, and TNF-α, and repressed the expression levels of MyD88, TLR4, and p-NF-κB p65 in OxyHb-induced BV2 microglia. Additionally, pyrrolidine dithiocarbamate or resatorvid enhanced the suppressive effects of eupatilin on OxyHb-induced inflammatory responses in BV2 microglia. Eupatilin ameliorates SAH-induced EBI via modulating the TLR4/MyD88/NF-κB pathway in rat model.     

基于TLR4MyD88信号通路观察粪菌移植对DSS诱导小鼠溃疡性结肠炎干预作用机制

摘要 目的：探讨粪菌移植（fecal microbiota transplantation,FMT）对葡聚糖硫酸钠（dextran sodium sulfate, DSS）诱导小鼠溃疡性结肠炎（ulcerative colitis,UC）干预作用及其炎症相关的分子机制。方法：30只昆明小鼠采用DSS喂养建立结肠炎小鼠模型并随机分为模型组、FMT低剂量组和FMT高剂量组,另取10只作为对照组。FMT低剂量组、FMT高剂量组自造模后第1 d开始分别给予8 g/kg、15 g/kg粪菌剂量灌肠处理,灌肠体积0.2 mL。对照组和模型组给予等体积生理盐水灌肠处理。分别在实验第1、7、14及21 d称量小鼠体重。实验结束后处死小鼠,取出结直肠,观察各组小鼠结直肠形态变化并通过HE染色观察病变程度。ELISA检测小鼠结直肠组织匀浆液上清中肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、白细胞介素-6（interleukin 6, IL-6）、白细胞介素-4（interleukin 4, IL-4）和白细胞介素-10（interleukin 10, IL-10）的表达变化;免疫组化法和RT-qPCR分别检测结直肠组织中核因子κB-P65亚基 (nuclear factor-κB P65, NF-κB P65)蛋白和mRNA的表达情况;Western blotting检测结直肠组织中Toll 样受体4 (Toll-like receptor 4,TLR4)、髓样分化因子 88(myeloid differentiation factor 88,MyD88)的蛋白表达情况。结果：对照组相比,造模小鼠在第6 d起至第14 d体重明显减轻,差异具有统计学意义（P<0.05）;与模型组相比,FMT低、高剂量组小鼠第10 d起至第14 d体重明显减轻,FMT高剂量组小鼠体重升高更加明显,差异具有统计学意义（P<0.05）;与对照组相比,模型组小鼠结直肠组织中TNF-α、IL-6、NF-κB P65 mRNA和蛋白阳性表达、TLR4、MyD88 蛋白表达均显著升高(P<0.05),IL-4、IL-10表达显著下降（P<0.05）;FMT高剂量组IL-4、IL-10较模型组显著升高,其余指标均显著下降（P<0.05）。结论：FMT可通过抑制TLR4/MyD88/NF-κB信号通路缓解炎症反应,发挥对UC的治疗作用。     

黄芩苷对实验性结肠炎小鼠TLRs/MyD88通路的作用研究

本文研究黄芩苷对葡聚糖硫酸钠(DSS)诱导的结肠炎小鼠模型的疗效,并从TLRs/MyD88通路探讨其作用机制。C57BL/6小鼠24只,分为空白对照组、模型组和黄芩苷组,用3.5%DSS诱导结肠炎模型,黄芩苷(30 mg/kg)干预7天,记录小鼠的疾病活动指数(DAI)评分,HE染色观察病理改变并评分,检测结肠髓过氧化物酶(MPO)活性,ELISA法检测肿瘤坏死因子(TNF)-α和白介素(IL)-6浓度,Real-time PCR法检测TLR2、TLR4和MyD88 mRNA表达水平。结果显示黄芩苷能有效抑制结肠炎小鼠DAI评分、结肠组织病理学评分和MPO活性,降低IL-6和TNF-α表达,降低TLR2、TLR4和MyD88 mRNA的表达。表明黄芩苷能有效缓解DSS诱导的结肠炎小鼠模型的症状,降低炎症反应,其作用机制可能是与TLRs/MyD88通路相关。     

基于TLR4/MyD88/NF-κB信号通路探讨安肠汤联合艾灸治疗肝郁脾虚证腹泻型肠易激综合征的疗效及其机制

摘要 目的：基于Toll样受体4（TLR4）/髓样分化因子88（MyD88）/核因子-κB（NF-κB）信号通路探讨安肠汤联合艾灸治疗肝郁脾虚证腹泻型肠易激综合征（IBS）的疗效及其机制。方法：采用随机数字表法,将广州中医药大学第一附属医院在2019年4月~2022年12月期间收治的108例腹泻型IBS患者分为对照组（常规药物联合艾灸治疗,n=54）和研究组（对照组基础上接受安肠汤治疗,n=54）。对比两组疗效、中医证候总积分、IBS症状严重程度问卷（IBS-SSS）评分、肠屏障功能指标、TLR4/MyD88/NF-κB信号通路相关信使核糖核酸（mRNA）表达水平。结果：研究组的临床总有效率高于对照组（P<0.05）。两组治疗后中医证候总积分、IBS-SSS评分下降,且研究组低于对照组（P<0.05）。两组治疗后肠脂肪酸结合蛋白（IFABP）、D-乳酸及二胺氧化酶（DAO）下降,且研究组低于对照组（P<0.05）。两组治疗后TLR4、MyD88、NF-κB mRNA表达下降,且研究组低于对照组（P<0.05）。结论：安肠汤联合艾灸治疗肝郁脾虚证腹泻型IBS患者,可有效改善临床症状和肠屏障功能,疗效较好,可能与调节TLR4/MyD88/NF-κB信号通路有关。     

Repeated stimulation by LPS promotes the senescence of DPSCs via TLR4/MyD88-NF-κB-p53/p21 signaling

Dental pulp stem cells (DPSCs), one type of mesenchymal stem cells, are considered to be a type of tool cells for regenerative medicine and tissue engineering. Our previous studies found that the stimulation with lipopolysaccharide (LPS) might introduce senescence of DPSCs, and this senescence would have a positive correlation with the concentration of LPS. The β-galactosidase (SA-β-gal) staining was used to evaluate the senescence of DPSCs and immunofluorescence to show the morphology of DPSCs. Our findings suggested that the activity of SA-β-gal has increased after repeated stimulation with LPS and the morphology of DPSCs has changed with the stimulation with LPS. We also found that LPS bound to the Toll-like receptor 4 (TLR4)/myeloid differentiation factor (MyD) 88 signaling pathway. Protein and mRNA expression of TLR4, MyD88 were enhanced in DPSCs with LPS stimulation, resulting in the activation of nuclear factor-κB (NF-κB) signaling, which exhibited the expression of p65 improved in the nucleus while the decreasing of IκB-α. Simultaneously, the expression of p53 and p21, the downstream proteins of the NF-κB signaling, has increased. In summary, DPSCs tend to undergo senescence after repeated stimulation in an inflammatory microenvironment. Ultimately, these findings may lead to a new direction for cell-based therapy in oral diseases and other regenerative medicines.     

Xiao-Chai-Hu-Tang ameliorates tumor growth in cancer comorbid depressive symptoms via modulating gut microbiota-mediated TLR4/MyD88/NF-κB signaling pathway

BackgroundDepressive symptoms are thought to promote cancer development and depressive remission has been reported to be effective for defeating cancer. The herbal formula Xiao-Chai-Hu-Tang (XCHT), that has an anti-depressive efficacy, has been widely utilized in China. However, its anti-cancer effect and underlying mechanisms remain unclear.PurposeThe present study aims to investigate the effects of XCHT on the depression-associated tumor and its potential mechanisms.MethodsA placebo-controlled trial was conducted in cancer patients comorbid with depressive symptoms to evaluate the effects of XCHT on depressive scales, tumor-related immune indicators, and gut microbial composition. A xenografted colorectal cancer (CRC) mouse model exposure to chronic restraint stress (CRS) was established to examine XCHT effects on tumorigenesis in vivo. Further, by manipulating gut bacteria with fecal microbial transplantation (FMT) or antibiotics-induced bacterial elimination in CRS-associated xenografted model, gut microbiota-mediated anti-tumor mechanism was explored.ResultsIn cancer patients comorbid with depressive symptoms, XCHT showed substantial effects on improvement of depressive scales, system inflammatory levels and gut dysbiosis. In vivo, XCHT inhibited tumor growth and prolonged survival time in addition to showing anti-depressive effect. Similarly, in our clinical trial, XCHT partially reversed gut dysbiosis, particularly through reducing abundances of Parabacteroides, Blautia and Ruminococcaceae bacterium. Manipulation of gut bacteria in CRS-associated xenografted model further proved that the inhibition of XCHT on tumor progression was mediated by gut microbiota and that the underlying mechanism involves in downregulation of TLR4/MyD88/NF-κB signaling.ConclusionsWe demonstrated that gut microbiota mediates the anti-tumor action of the formula XCHT in cancer patients and models that were comorbid with depressive symptoms. This study implies a novel clinical significance of anti-depressive herbal medicine in the cancer treatment and clarifies the important role of gut microbiota in treating cancer accompanied by depressive symptoms.     

FoxO1 regulates TLR4/MyD88/MD2-NF-κB inflammatory signalling in mucosal barrier injury of inflammatory bowel disease

In this study, FoxO1 transgenic mice (transgenic, FoxO1-Tg) and C57BL/6 wild-type (wild-type, FoxO1-WT) mice were used to establish chronic colitis by drinking water containing dextran sulphate sodium (DSS). Afterwards, we observed the life changes in mice and assessed the pathological changes by H&E tissue staining. In addition, the TLR4/MyD88/MD2-NF-κB inflammatory signals were detected. As a result, under DSS treatment, the activation level of TLR4/MyD88/MD2-NF-κB inflammatory signal was higher in FoxO1-Tg mice than that in FoxO1-WT mice. Meanwhile, the intestinal mucosal tissue damage was more severe, the down-regulation of tight junction protein level was more significant and the life quality was decreased to a higher degree in FoxO1-Tg mice compared with those in FoxO1-WT mice. Caco-2 cells were used to mimic the intestinal mucosal barrier model for in vitro assays. In addition, lentiviral packaging FoxO1 overexpressing plasmid was transfected into Caco-2 cells for FoxO1 overexpression. TNF-α intervention was performed for intestinal mucosal inflammatory response model. Consequently, the down-regulation of FoxO1 inhibited the activation of TLR4/MyD88/MD2-NF-κB inflammatory signal, decreased the mucosal barrier permeability and up-regulated the expression of tight junction protein. By contrast, the overexpression of FoxO1 increased the mucosal barrier permeability and down-regulated the level of tight junction protein.     

奥利亚罗非鱼髓样分化因子(MyD88)的克隆及其在组织中的表达

髓样分化因子（myeloid differentiation factor 88,MyD88）是TLR（toll-like receptor）信号通路的关键接头蛋白,在先天性免疫中具有重要作用。通过RACE-RCR技术克隆了奥利亚罗非鱼（Oreochromis aureus）MyD88基因cDNA全长序列（GenBank登录号：JN032017）。序列分析表明,奥利亚罗非鱼MyD88 基因全长为1 611 bp,其中包括155 bp的5’非编码区,589 bp的3’非编码区和867 bp的编码区,编码288个氨基酸残基。MyD88蛋白N端具有死亡结构域,C端具有TIR结构域。同源性分析表明,奥利亚罗非鱼MyD88氨基酸序列与鳜鱼（Siniperca chuats）相似性最高,为85.8%,与其他鱼类相似性为70%~82%,与哺乳动物相似性为63%~66%;系统进化树分析表明,奥利亚罗非鱼MyD88与同属鲈形目的鳜鱼、大黄鱼（Larimichthys crocea）聚在一起。采用实时定量PCR方法检测MyD88在奥利亚罗非鱼各组织中的表达情况。结果显示,MyD88在所有被测组织中都有表达,其中表达量最高的是卵巢,其次在小肠、脾、肝、肾、鳃和血液中有较高的表达量,肌肉、精巢组织中表达量最低。本研究可为进一步探讨MyD88在奥利亚罗非鱼TLR信号通路中的作用奠定一定的基础。     

基于TLR4/MYD88信号通路探讨大黄牡丹汤对急性胰腺炎大鼠模型的保护作用

目的基于TLR4/MYD88信号通路研究大黄牡丹汤对逆行性胰胆管注射法制作急性胰腺炎(acute pancreatitis,AP)大鼠模型的保护作用。方法 SPF级Wistar大鼠96只,按照随机数字表法分为:假手术组、AP模型观察组、奥曲肽阳性对照组、大黄牡丹汤高、中、低剂量组,除假手术组经胰胆管逆行注射生理盐水,其余各组均采用经胰胆管逆行注射5%牛磺胆酸钠溶液造模,给与药物干预6 d。观察大鼠一般生存状况;测定血清淀粉酶(AMS)、谷丙转氨酶(ALT)、谷草转氨酶(AST)含量; HE观察胰腺病理组织改变; RT-PCR、Westen Blot、IHC法检测胰腺组织TLR4、MYD88、IRAK-2、IRAK-4基因蛋白表达水平; ELISA法检测胰腺组织中炎性因子IL-2、iNOS、IFN-γ含量。结果 (1)与假手术组比较,AP模型观察组大鼠一般生存状况相对较差,血清淀粉酶(AMS)、谷丙转氨酶(ALT)、谷草转氨酶(AST)均显著升高,镜下可见胰腺组织结构散乱,坏死、充血严重,胰腺组织中TLR4、MYD88、IRAK-2、IRAK-4基因蛋白表达水平均显著升高,胰腺组织匀浆液中IL-2、iNOS、IFN-γ含量均显著升高,差异具有统计学意义(P0.05);(2)干预后,各治疗组大鼠一般生存状况有不同程度改善,血清淀粉酶(AMS)、谷丙转氨酶(ALT)、谷草转氨酶(AST)均下降,镜下间质性水肿以及坏死灶明显改善,胰腺组织中TLR4、MYD88、IRAK-2、IRAK-4基因蛋白表达水平均下降,胰腺组织匀浆液中IL-2、iNOS、IFN-γ含量均下降,其中尤以大黄牡丹汤高剂量组显著,差异具有统计学意义(P0.05)。结论大黄牡丹汤改善急性胰腺炎大鼠胰腺损伤,其作用机制可能与抑制TLR4/MYD88信号通路有关。     

TLR9的特性及其介导的TLR9/CpG-DNA免疫信号通路

TLRs在天然免疫应答中发挥着重要作用,构成了机体抗感染的第一道防线,是沟通天然免疫和获得性免疫的桥梁。其中TLR9已经被证实能够识别细菌DNA中免疫刺激序列CpG,从而激活哺乳动物细胞的天然免疫机制,这一发现具有重要的生物学意义与应用价值。它不仅进一步推进了外源DNA激活哺乳动物抗感染天然免疫的进一步研究,更为CpG-DNA应用于抗感染、肿瘤和免疫缺陷疾病等的治疗提供新的思路,为改进DNA疫苗效果提供非常有益的帮助。     

TLR4/P38/JNK信号通路在海马神经元凋亡中的作用

目的：探讨Toll样受体4（TLR4）/P38/JNK信号通路在海马神经元凋亡中的作用及其机制,为神经退行性疾病（ND）的发病机制与防治研究提供新的实验依据。方法：采用体外培养7 d的新生大鼠海马神经元,免疫荧光双标法鉴定海马神经元纯度。用TLR4配体脂多糖（LPS）或TLR4抗体预处理海马神经元,以激活或阻断TLR4的作用。实验1设正常对照组、LPS组及TLR4抗体+ LPS组;免疫荧光法检测P-P38,P-JNK的表达。实验2分为6组：正常对照组,LPS组,TLR4抗体+ LPS组,SB202190（抑制P38） + LPS组,SP600125（抑制JNK） + LPS组,PD98059（抑制ERK） + LPS组;分别用TLR4抗体、P38、JNK及ERK的抑制剂预处理海马神经元后再给以LPS刺激24 h,Western blot法检测Bcl-2,Bax,Active-caspase-3的表达变化;流式细胞术检测海马神经元凋亡率。结果：LPS组海马神经元P-P38、P-JNK的表达明显高于正常对照组（P < 0. 01）,TLR4抗体+ LPS组P-P38,P-JNK表达显著低于LPS组（P <0.01）。与正常对照组相比,LPS组海马神经元Bcl-2/Bax表达减少、Active-caspase-3表达增加,海马神经元凋亡率增加（P < 0.01）。而TLR4抗体+ LPS组、SB202190 + LPS组、SP600125 + LPS组Bcl-2/Bax显著高于LPS组、Active cas-pase-3显著低于LPS组（P < 0.01）,海马神经元凋亡率显著低于LPS组（P < 0. 05,P < 0. 01）。PD98059 + LPS组与LPS组海马神经元凋亡率无明显差异。结论：①海马神经元中有TLR4介导的P38/JNK信号通路。②海马神经元TLR4激活后,P-P38、P-JNK表达增加,使Bcl-2/Bax的比例降低和Active-caspase-3表达增加,从而促进海马神经元的凋亡。海马神经元凋亡过程中有TLR4介导的P38/JNK信号通路的参与。