Acacetin protects against cerebral ischemia-reperfusion injury via the NLRP3 signaling pathway

Acacetin(5,7-dihydroxy-4′-methoxyflavone), a potential neuroprotective agent, has an inhibitory effect on lipopolysaccharide-induced neuroinflammatory reactions. However, whether acacetin has an effect on inflammatory corpuscle 3(NLRP3) after cerebral ischemia-reperfusion injury has not been fully determined. This study used an improved suture method to establish a cerebral ischemia-reperfusion injury model in C57BL/6 mice. After ischemia with middle cerebral artery occlusion for 1 hour, reperfusion with intraperitoneal injection of 25 mg/kg of acacetin(acacetin group) or an equal volume of saline(0.1 mL/10 g, middle cerebral artery occlusion group) was used to investigate the effect of acacetin on cerebral ischemia-reperfusion injury. Infarct volume and neurological function scores were determined by 2,3,5-triphenyltetrazolium chloride staining and the Zea-Longa scoring method. Compared with the middle cerebral artery occlusion group, neurological function scores and cerebral infarction volumes were significantly reduced in the acacetin group. To understand the effect of acacetin on microglia-mediated inflammatory response after cerebral ischemia-reperfusion injury, immunohistochemistry for the microglia marker calcium adapter protein ionized calcium-binding adaptor molecule 1(Iba1) was examined in the hippocampus of ischemic brain tissue. In addition, tumor necrosis factor-α, interleukin-1β, and interleukin-6 expression in ischemic brain tissue of mice was quantified by enzyme-linked immunosorbent assay. Expression of Iba1, tumor necrosis factor-α, interleukin-1β and interleukin-6 was significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Western blot assay results showed that expression of Toll-like receptor 4, nuclear factor kappa B, NLRP3, procaspase-1, caspase-1, pro-interleukin-1β, and interleukin-1β were significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Our findings indicate that acacetin has a protective effect on cerebral ischemia-reperfusion injury, and its mechanism of action is associated with inhibition of microglia-mediated inflammation and the NLRP3 signaling pathway.

Neuroprotection mediated by the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage

The Wnt/Frizzled signaling pathway participates in many inflammation-linked diseases. However, the inflammatory response mediated by the Wnt/Frizzled signaling pathway in experimental subarachnoid hemorrhage has not been thoroughly investigated. Consequently, in this study, we examined the potential role of the Wnt/Frizzled signaling pathway in early brain injury in rat models of subarachnoid hemorrhage.Simultaneously, possible neuroprotective mechanisms were also investigated. Experimental subarachnoid hemorrhage rat models were induced by injecting autologous blood into the prechiasmatic cistern. Experiment 1 was designed to examine expression of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. In total, 42 adult rats were divided into sham(injection of equivalent volume of saline), 6-, 12-, 24-, 48-, 72-hour, and 1-week subarachnoid hemorrhage groups. Experiment 2 was designed to examine neuroprotective mechanisms of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. Rats were treated with recombinant human Wnt1(rhwnt1), small interfering Wnt1(siwnt1) RNA, and monoclonal antibody of Frizzled1(anti-Frizzled1) at 48 hours after subarachnoid hemorrhage. Expression levels of Wnt1, Frizzled1, β-catenin, peroxisome proliferator-activated receptor-γ, CD36, and active nuclear factor-κB were examined by western blot assay and immunofluorescence staining. Microglia type conversion and inflammatory cytokine levels in brain tissue were examined by immunofluorescence staining and enzyme-linked immunosorbent assay. Our results show that compared with the sham group, expression levels of Wnt1, Frizzled1, and β-catenin were low and reduced to a minimum at 48 hours, gradually returning to baseline at 1 week after subarachnoid hemorrhage. rhwnt1 treatment markedly increased Wnt1 expression and alleviated subarachnoid hemorrhage-induced early brain injury(within 72 hours), including cortical cell apoptosis, brain edema, and neurobehavioral deficits, accompanied by increasing protein levels of β-catenin, CD36, and peroxisome proliferator-activated receptor-γ and decreasing protein levels of nuclear factor-κB. Of note, rhwnt1 promoted M2-type microglia conversion and inhibited release of inflammatory cytokines(interleukin-1β, interleukin-6, and tumor necrosis factor-α). In contrast, siwnt1 RNA and anti-Frizzled1 treatment both resulted in an opposite effect. In conclusion, the Wnt/Frizzled1 signaling pathway may participate in subarachnoid hemorrhage-induced early brain injury via inhibiting the inflammatory response, including regulating microglia type conversion and decreasing inflammatory cytokine release. The study was approved by the Animal Ethics Committee of Anhui Medical University and First Affiliated Hospital of USTC,Division of Life Sciences and Medicine, University of Science and Technology of China(approval No. LLSC-20180202) in May 2017.

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。

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。

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。

泼尼松联合阿奇霉素序贯疗法治疗儿童重症肺炎支原体肺炎的疗效及对血清TLR4/MyD88/NF-κB信号通路相关蛋白和下游炎性因子水平的影响

目的探讨泼尼松联合阿奇霉素序贯疗法治疗儿童重症肺炎支原体肺炎的疗效及对血清Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核因子κB(NF-κB)信号通路相关蛋白和下游炎性因子水平的影响。方法招募2019年1月至2022年6月唐山市妇幼保健院收治的儿童重症肺炎支原体肺炎患者120例,采用随机数字表法将其分为对照组(采用阿奇霉素治疗,60例)和观察组(采用泼尼松联合阿奇霉素治疗,60例)。治疗4周后,比较两组治疗效果、临床肺部感染量表(CIPS)评分、TLR4/MyD88/NF-κB信号通路相关蛋白以及下游炎性因子的水平。结果观察组患者的体温恢复时间、咳嗽缓解时间、咳痰缓解时间、喘息缓解时间、肺部啰音消失时间较对照组更快,住院时间更短,差异有统计学意义(P<0.05)。治疗后,两组的体温、白细胞计数、气道分泌物、氧合情况、胸部X射线、痰培养等CIPS项目评分以及总分均较治疗前降低,且观察组评分较对照组更低,差异有统计学意义(P<0.05)。治疗后,两组TLR4、MyD88、NF-κB、C-反应蛋白(CRP)、白细胞介素-6(IL-6)、降钙素原(PCT)水平均降低,且观察组水平较对照组更低,差异有统计学意义(P<0.05)。结论泼尼松联合阿奇霉素序贯疗法对人体血清TLR4/MyD88/NF-κB信号通路相关蛋白及下游炎性因子水平有显著下调作用,可提高儿童重症肺炎支原体肺炎患者的临床疗效。

柚皮素对过敏性鼻炎模型大鼠鼻黏膜组织TLR4/NF-κB/TNF-α信号通路的影响

目的探究柚皮素对过敏性鼻炎(AR)模型大鼠鼻黏膜组织Toll样受体4(TLR4)/核因子κB(NF-κB)/肿瘤坏死因子α(TNF-α)信号通路的影响。方法建立AR大鼠模型,48只大鼠以随机数字表法平均分为4组:模型组、柚皮素低(100 mg/kg)剂量组、柚皮素高(200 mg/kg)剂量组、柚皮素高(200 mg/kg)剂量+脂多糖(LPS)(TLR4通路激活剂,0.4 mg/kg)组,另取12只SD大鼠设为对照组。以药物分组干预治疗后,观察大鼠鼻炎症状并进行评分;苏木精-伊红(HE)染色检测大鼠鼻黏膜组织病理形态改变;试剂盒测定大鼠血清IgE及炎性因子白细胞介素(IL)-17、IL-18水平;免疫组织化学染色检测大鼠鼻黏膜组织CD19、CD23表达;免疫印迹法检测大鼠鼻黏膜组织TLR4/NF-κB/TNF-α通路蛋白表达。结果与对照组相比,模型组大鼠鼻黏膜组织发生严重病理损伤,鼻炎症状评分、血清IgE、IL-17及IL-18水平、鼻黏膜组织CD19、CD23阳性细胞比例、鼻黏膜组织TLR4、核内NF-κB p65、TNF-α蛋白表达水平显著升高(P<0.05);与模型组相比,柚皮素干预组大鼠鼻黏膜组织病理损伤均减轻,鼻炎症状评分、血清IgE、IL-17及IL-18水平、鼻黏膜组织CD19、CD23阳性细胞比例、鼻黏膜组织TLR4、核内NF-κB p65、TNF-α蛋白表达水平均降低,且柚皮素高剂量组大鼠各指标改变程度更强;与柚皮素高剂量组相比,柚皮素高剂量+LPS组大鼠鼻黏膜组织病理损伤加重,鼻炎症状评分、血清IgE、IL-17及IL-18水平、鼻黏膜组织CD19、CD23阳性细胞比例、鼻黏膜组织TLR4、核内NF-κB p65、TNF-α蛋白表达水平显著升高(P<0.05)。结论柚皮素可通过抑制TLR4/NF-κB/TNF-α通路表达而减轻超敏反应及炎症,从而缓解鼻黏膜组织损伤,改善AR大鼠症状。

蛛网膜下腔出血患者TLR4/NF-κB信号通路变化及意义

目的探究蛛网膜下腔出血患者Toll样受体4(Toll like receptor 4,TLR4)/核因子κB(nuclear factor kappa B,NF-κB)信号通路变化及意义。方法选取作者医院2018-05/2021-06月收治的60例蛛网膜下腔出血患者作为观察组,另选取同期体检健康者60例作为对照组。比较两组TLR4/NF-κB信号通路相关因子表达,分析蛛网膜下腔出血患者TLR4/NF-κB信号通路相关因子与神经功能损害美国国立卫生院卒中量表(national institutes of health stroke scale,NIHSS)评分的关系。比较不同预后患者发病第1、3、7、14天外周血TLR4/NF-κB信号通路相关因子,绘制受试者工作特征(receiver operating characteristic,ROC)曲线,评价TLR4/NF-κB信号通路相关因子对蛛网膜下腔出血患者预后的预测价值,分析TLR4/NF-κB信号通路相关因子与蛛网膜下腔出血患者预后不良风险的关系。结果观察组TLR4 mRNA、NF-κB mRNA表达均高于对照组,组间比较差异有统计学意义(P均<0.05)。蛛网膜下腔出血患者入院当天NIHSS评分为(11.24±3.12)分,根据Pearson相关性模型分析可知,蛛网膜下腔出血患者TLR4 mRNA、NF-κB mRNA表达均与NIHSS评分呈正相关关系(P<0.05)。不同组别间患者TLR4 mRNA、NF-κB mRNA相对表达量差异有统计学意义(F组间=8.037、19.348,P均<0.001),不同时间点患者TLR4 mRNA相对表达量差异有统计学意义(F时间=5.128、16.207,P均<0.001),且时间与组间存在交互效应(F交互=6.497、18.032,P=0.015、<0.001)。预后不良组患者发病第1天TLR4 mRNA、NF-κB mRNA相对表达量较预后良好组患者差异无统计学意义(P>0.05)。预后不良组患者发病第3天、7天、14天TLR4 mRNA、NF-κB mRNA相对表达量均高于预后良好组患者(P均<0.05)。预后不良组患者、预后良好组患者发病第3天TLR4 mRNA、NF-κB mRNA相对表达量升至最高(P均<0.05);发病第7天、14天TLR4 mRNA、NF-κB mRNA相对表达量均较发病第3天明显下降,且发病第14天降至最低(P均<0.05)。以预后不良作为阳性样本,预后良好作为阴性样本,绘制ROC曲线,结果显示,发病第3、7、14天TLR4 mRNA、NF-κB mRNA联合预测蛛网膜下腔出血患者预后的AUC分别为0.854、0.894、0.915,较各指标单独诊断价值明显提高。Logistic回归分析显示,TLR4 mRNA、NF-κB mRNA仍与蛛网膜下腔出血患者预后不良有关(P均<0.05)。结论蛛网膜下腔出血后TLR4/NF-κB信号通路被激活,TLR4 mRNA、NF-κB mRNA表达明显上调,且TLR4/NF-κB信号通路与蛛网膜下腔出血患者早期脑损伤有关,为临床治疗提供了新思路,有助于改善患者预后。

乌司他丁抑制TLR4/NF-κB信号通路对心肺复苏大鼠心功能的改善作用

目的旨在探究乌司他丁对心肺复苏大鼠的影响。方法通过窒息构建心脏骤停大鼠模型,窒息9.5 min后,对大鼠静脉注射肾上腺素,并对大鼠胸部进行按压以实施心肺复苏。Kaplan-Meier存活曲线分析大鼠存活率,神经系统缺损评分(NDS)评估大鼠神经系统缺损,转棒疲劳实验评估大鼠运动协调及平衡能力。ELISA法检测血清炎性因子白细胞介素-1β(IL-1β)、肿瘤坏死因子(TNF-α)、IL-6水平,western blotting检测大脑皮层TLR4/NF-κB信号通路的表达。结果乌司他丁治疗提高大鼠存活率,改善心脏骤停后的心功能及大鼠神经认知功能、运动协调能力。乌司他丁降低心脏骤停/心肺复苏后促炎因子TNF-α、IL-1β、IL-6水平,降低TLR4蛋白及p-p65/p65水平。结论乌司他丁抑制TLR4/NF-κB通路活性,对心肺复苏大鼠心功能及运动协调能力具有改善作用。

Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway

Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae （Gegen）, has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment （intravenous injection） re- duced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-a in brain tissue. Pretreatment with puerarin （intravenous injection） attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 （JAK2） and signal transducers and activators of transcription 3 （STAT3） activation and nuclear factor kappa B （NF-KB） inhibition. These observa- tions were inhibited by the alpha7 nicotinic acetylcholine receptor （a7nAchR） antagonist a-bungarotoxin （a-BGT）. In addition, puerarin pretreatment increased the expression of a7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory re- sponse. Our results also indicated that the anti-inflammatory effect of puerarin may partly be medi- ated through the activation of the cholinergic anti-inflammatory pathway.

Electroacupuncture Delays Cartilage Degeneration by Modulating Nuclear Factor-κB Signaling Pathway

Objective: To illustrate the molecular mechanisms underlying the therapeutic effects of electroacupuncture(EA) on knee osteoarthritis(OA). Methods: Twenty-seven six-month-old New Zealand white rabbits were allocated into three groups in accordance with a random number table: normal group(no surgery-induced OA;without treatment), model group(surgery-induced OA;without treatment) and EA group [surgery-induced OA;received treatment with EA at acupoints Dubi(ST 35) and Neixiyan(EX-LE 5), 30 min twice a day]. After eight consecutive weeks of treatment, the histopathological alterations in cartilage were observed using optical microscopy and transmission electron microscopy, cartilage degeneration was evaluated by modified Mankin’s score principles, the synovial fluid concentration of interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α) and matrix metalloproteinase-3(MMP-3) were evaluated by enzyme-linked immunosorbent assay, and the protein expression levels of IL-1β, IL-6, TNF-α, MMP-3, IκB kinase-β(IKK-β), nuclear factor of α light polypeptide gene enhancer in B-cells inhibitor α(IκB-α) and nuclear factor-κB(NF-κB) p65 were quantified by Western blot analysis. Results: EA treatment significantly improved cartilage structure arrangement and reduced cellular degeneration. The IL-1β, IL-6, TNF-α and MMP-3 of synovial fluid in the EA-treated group were significantly decreased compared with the model group(all P<0.01). Compared with the model group, the IL-1β, IL-6, TNF-α, MMP-3, IKK-β and NF-κB p65 protein expressions in cartilage of EA-treated group were significantly decreased(all P<0.01), whereas IκB-α expression was significantly up-regulated(P<0.01). Conclusion: EA treatment may delay cartilage degeneration by down-regulating inflammatory factors through NF-κB signaling pathway, which may, in part, explain its clinical efficacy in the treatment of knee OA.

Influence of Silencing TRAF6 with shRNA on LPS/TLR4 Signaling in vitro

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide(LPS)/toll-like receptor(TLR)-4 signaling pathway in vitro.Four plasmids(pGCsi-TRAF6-shRNA1,2,3,4) containing different shRNA sequences were designed and synthesized.The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay.Inflammatory cellular models were established by LPS stimulation.Levels of TNF-α,IL-1β and TGF-β1 in the supernatants,mRNA expressions of TRAF6,IL-6 and COX-2,protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA,real-time quantitative PCR and Western blotting,respectively.The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h.The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1,2 groups than in the TRAF6-shRNA3,4 groups.Therefore,pGCsi-TRAF6-shRNA1,2 were selected for the subsequent experiments.Our results still showed that pGCsi-TRAF6-shRNA1,2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-α,IL-1β,IL-6 and COX-2,and inhibit NF-κB nuclear translocation.Moreover,pGCsi-TRAF6-shRNA1,2 could suppress the release of TGF-β1 at the protein level.It was concluded that the recombinant plasmid pTRAF6-shRNA can,to some extent,inhibit inflammatory response stimulated by LPS at the initial phase.TRAF6 may become the potential therapeutic target of many inflammation-related diseases.

Maraviroc promotes recovery from traumatic brain injury in mice by suppression of neuroinflammation and activation of neurotoxic reactive astrocytes

Neuroinflammation and the NACHT,LRR,and PYD domains-containing protein 3 inflammasome play crucial roles in secondary tissue damage following an initial insult in patients with traumatic brain injury(TBI).Maraviroc,a C-C chemokine receptor type 5 antagonist,has been viewed as a new therapeutic strategy for many neuroinflammatory diseases.We studied the effect of maraviroc on TBI-induced neuroinflammation.A moderate-TBI mouse model was subjected to a controlled cortical impact device.Maraviroc or vehicle was injected intraperitoneally 1 hour after TBI and then once per day for 3 consecutive days.Western blot,immunohistochemistry,and TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)analyses were performed to evaluate the molecular mechanisms of maraviroc at 3 days post-TBI.Our results suggest that maraviroc administration reduced NACHT,LRR,and PYD domains-containing protein 3 inflammasome activation,modulated microglial polarization from M1 to M2,decreased neutrophil and macrophage infiltration,and inhibited the release of inflammatory factors after TBI.Moreover,maraviroc treatment decreased the activation of neurotoxic reactive astrocytes,which,in turn,exacerbated neuronal cell death.Additionally,we confirmed the neuroprotective effect of maraviroc using the modified neurological severity score,rotarod test,Morris water maze test,and lesion volume measurements.In summary,our findings indicate that maraviroc might be a desirable pharmacotherapeutic strategy for TBI,and C-C chemokine receptor type 5 might be a promising pharmacotherapeutic target to improve recovery after TBI.

Toll样受体4信号转导研究进展

Toll样受体(Toll-like-receptors,TLRs)是一个主要分布于炎症细胞的识别病源分子的受体超家族,其中TLR4主要识别革兰阴性细菌细胞壁成分脂多糖(lipopolysaccharide,LPS)。LPS与TLR4结合后活化髓样分化因子88(myeloid differentiation factor 88,MyD88)依赖性和非依赖性两条信号途径;前者活化丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)和核因子-κB(nuclear factor kappa B,NF-κB)信号通路,后者活化NF-κB和干扰素调节因子-3(IFN-regulated factor-3,IRF3)信号通路。通过这些信号途径TLR4诱导炎症细胞释放炎症因子介导炎症反应;同时TLR4通过活化树突状细胞促进抗原递呈,介导先天性免疫向获得性免疫的转化。此外,TLR4能诱导磷脂酰肌醇-3激酶-蛋白激酶B(PI3K-AKT)的信号转导,LPS介导的细胞存活和增殖与TLR4活化PI3K-AKT途径有关。

黄芩素通过TLR4/NF-κB信号通路对小鼠结肠炎的干预作用

目的:探讨黄芩素通过TLR4/NF-κB信号通路对葡聚糖硫酸钠(DSS)诱导的实验性结肠炎的干预作用。方法:将C57BL/6雄性小鼠随机分为5组,即对照组、结肠炎组、黄芩素低剂量(Ba-L)治疗组[10 mg/(kg·d)]、黄芩素高剂量(Ba-H)治疗组[25 mg/(kg·d)]和5-氨基水杨酸(5-ASA)治疗组,每组各12只。对照组小鼠饮用清水,其余各组小鼠均连续7 d饮用3.5%的DSS溶液构建实验性结肠炎模型。在DSS造模开始前7 d和造模开始后给予不同剂量黄芩素或5-ASA灌胃治疗,共14 d。比较各组小鼠之间的平均体质量、疾病活动指数(DAI)以及结肠组织学活动程度评分(HAI);采用实时荧光定量PCR法测定各组小鼠的结肠组织中IL-1β、TNF-α、IFN-γ、TLR4、NF-κB p65、MyD88的mRNA水平;采用免疫荧光技术测定肠上皮细胞中NF-κB p65的入核率。结果:结肠炎组小鼠与对照组小鼠相比,出现严重的结肠炎,表现为小鼠体质量降低、全结肠长度缩短、DAI和HAI大幅度升高(均P<0.01)。与结肠炎组小鼠相比,Ba-L治疗组、Ba-H治疗组和5-ASA治疗组小鼠的结肠炎有明显改善(均P<0.05)。结肠炎组小鼠的结肠组织中IFN-γ、TNF-α、IL-1β、My D88、NF-κB p65、TLR4的mRNA表达水平均明显高于对照组小鼠(均P<0.01);而Ba-L治疗组、Ba-H治疗组及5-ASA治疗组中上述mRNA表达水平则均明显低于结肠炎组小鼠(均P<0.05)。结肠炎组小鼠的肠上皮细胞中NF-κB p65的入核率明显高于对照组小鼠(P<0.001),但Ba-L治疗组、Ba-H治疗组以及5-ASA治疗组的肠上皮细胞中NF-κB p65的入核率则明显低于结肠炎组(均P<0.001)。结论:黄芩素可能通过TLR4/NF-κB信号通路抑制DSS诱导的结肠炎症。

CD14-TLR4-NF-κB信号传导通路在脂多糖诱导ALI/ARDS中的作用及机制研究

目的探讨白细胞分化抗原14(CD14)-Toll样受体4(TLR4)-核因子激活的B细胞的κ-轻链增强(NF-κB)信号传导通路在脂多糖(LPS)诱导急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)中作用及机制。方法于2020年1—12月,选取购自北京宝元兴业科技有限公司的清洁级健康雄性SD大鼠90只,分为A、B、C三组,每组各30只,C组为正常大鼠模型组,B组为ALI/ARDS模型组大鼠,A组为ALI/ARDS+CD14、TLR4以及NF-κB模拟物模型组大鼠,比较三组大鼠相关指标的差异。结果造模12 h后,A、B、C三组大鼠的呼吸频率(RR)分别是(127.92±17.04)次/分、(87.51±8.42)次/分、(55.28±3.73)次/分,A组>B组>C组,动脉血氧分压(PaO2)分别是(7.63±1.16)mmHg、(10.58±1.65)mmHg、(13.46±2.05)mmHg,A组<B组<C组,均差异有统计学意义(P<0.05)。A、B、C三组大鼠的CD14蛋白表达水平分别是(0.94±0.23)、(0.69±0.18)、(0.48±0.13),TLR4蛋白表达水平分别是(0.97±0.26)、(0.65±0.13)、(0.43±0.11),NF-κB P65蛋白表达水平分别是(1.03±0.28)、(0.67±0.17)、(0.47±0.12),脂多糖蛋白表达水平分别是(0.98±0.27)、(0.66±0.16)、(0.45±0.12),均A组>B组>C组,均差异有统计学意义(P<0.05)。Pearson相关性分析显示,A组大鼠的脂多糖蛋白分别与CD14蛋白、TLR4蛋白以及NF-κB P65蛋白均呈正相关(P<0.05)。结论CD14-TLR4-NF-κB信号传导通路能够增强脂多糖表达并促进机体炎性活动,参与ALI/ARDS的发生发展。

多囊卵巢综合征伴胰岛素抵抗相关信号通路的研究进展

多囊卵巢综合征(PCOS)是一种好发于青春期及育龄期女性的涉及诸多因素的内分泌代谢性疾病,临床主要表现为闭经、体胖、多毛痤疮以及妊娠率显著降低,但目前其病因病机尚不清楚。胰岛素抵抗(IR)与多种慢性非传染性疾病(高血压、糖尿病、心脑血管疾病、PCOS等)相关,目前已知磷脂酰肌醇-3-激酶/蛋白激酶B、肝激酶B1/AMP活化的蛋白激酶、沉默信息调节因子1/叉头框转录因子O1、Toll样因子4/核因子κB信号通路参与了PCOS伴IR(PCOS-IR)的作用机制,因此充分认识PCOS-IR的发病机制在疾病预防中有重要临床意义。

TSNⅡA对大鼠IRI肾脏TLR4/NF-κB信号通路的影响

目的探讨丹参酮ⅡA(TSNⅡA)对大鼠肾脏缺血再灌注损伤(IRI)肾脏Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号通路的影响及对炎症因子水平的下调作用,并分析其对肾脏IRI保护作用机制。方法选取雄性Wistar大鼠建立大鼠肾IRI模型,采用随机数字表表法随机分为五组,每组12只,其中A组大鼠为假手术组;B组为IRI模型组;C组为ST2825干预组,注射ST2825(100 mg/kg);D组为Bay-7082干预组,注射Bay-7082(50μg/kg);E组为TSNⅡA干预组:实验前3 d开始TSNⅡA干预组股静脉注射丹参酮ⅡA注射液(5 mg/kg),每天1次。18 h后,取大鼠下腔静脉血液2 ml,检测血清肿瘤坏死因子(TNF-α)、白细胞介素1(IL-1)、白细胞介素6(IL-6)浓度,并处死大鼠,应用免疫组化法检测肾脏TLR4和NF-κB阳性表达率。结果 B、C、D和E组TLR4和NF-κB阳性率均高于A组,C、D和E组TLR4和NF-κB阳性率均低于B组,E组TLR4和NF-κB阳性率分别为(38.27±2.87)%和(40.76±4.20)%,均高于C和D组,差异均具有统计学意义(P<0.05)。B、C、D和E组血清TNF-α、IL-1和IL-6水平均高于A组,C、D和E组血清TNF-α、IL-1和IL-6水平均低于B组,E组血清TNF-α、IL-1和IL-6水平分别为(76.02±4.28)pg/L、(86.41±6.32)ng/L和(80.28±5.60)ng/L,均高于C和D组,差异均具有统计学意义(P<0.05)。结论TSNⅡA能够下调大鼠肾脏TLR4和NF-κB水平,抑制TLR4/NF-κB信号通路,降低炎症因子水平,实现对肾脏IRI的改善与保护作用。

TLR-NF-κB信号通路与病毒性心肌炎的研究进展

Toll样受体是一类主要表达于炎性细胞与微生物识别有关的受体家族,在天然免疫反应中具有重要的作用。研究发现Toll样受体-核转录因子κB(TLR-NF-κB)信号转导通路的激活能够诱导炎性细胞释放炎性因子,介导炎性反应,同时介导先天性免疫向获得性免疫的转化,从而参与病毒性心肌炎(VMC)的病理、生理进程。因此,探讨VMC时TLR-NF-κB信号转导通路的作用,将为进一步揭示VMC的发病机制和寻求有效的防治措施提供重要的理论依据。

白果内酯对脓毒症急性肺损伤大鼠TLR4NFκB信号通路及Th1Th2细胞的影响

目的探讨白果内酯(BB)对脓毒症致急性肺损伤(ALI)大鼠Toll样受体-4(TLR4)/核因子-κB(NF-κB)信号通路及辅助性T细胞1/2(Th1/Th2)的影响。方法50只SD大鼠随机分为假手术组(Sham组)、模型组(ALI组)、BB低(2.5 mg/kg)剂量组、BB高(10 mg/kg)剂量组、地塞米松阳性对照组(0.45 mg/kg),每组各10只,除Sham组外,其余各组采用盲肠结扎穿孔法复制脓毒症ALI模型,术后6 h各药物组经尾静脉注射相应剂量药物,Sham组、ALI组经尾静脉注射生理盐水,均3次/d,共3 d。末次给药1 h后处死大鼠,取肺组织,以苏木精-伊红染色(HE)检测各组大鼠肺组织病理变化;以酶联免疫吸附(ELISA)试剂盒检测大鼠肺组织氧化物酶(MPO)活性、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平;凝胶电泳迁移率转变分析(EMSA)法检测肺组织NF-κB活性;以蛋白免疫印迹法检测肺组织中TLR4通路蛋白表达;采用流式细胞仪(FACS)检测肺脏细胞中辅助性T细胞1/2(Th1/Th2)比值。结果与Sham组相比,ALI组大鼠肺组织可见水肿、炎性细胞浸润等病理损伤,肺组织MPO、IL-6、TNF-α含量、TLR4蛋白表达、NF-κB活性均明显升高(P<0.05),Th1/Th2、TNF-α/IL-6均明显降低(P<0.05)。与ALI组相比,BB低、高剂量组及地塞米松阳性组肺组织水肿、炎性细胞浸润等病理损伤减轻,肺组织MPO、IL-6、TNF-α含量、TLR4蛋白表达、NF-κB活性均明显降低(P<0.05),Th1/Th2、TNF-α/IL-6均明显升高(P<0.05)。与BB低剂量组相比,BB高剂量组及地塞米松阳性组肺组织水肿、炎性细胞浸润等病理损伤减轻,MPO、IL-6、TNF-α含量、TLR4蛋白表达、NF-κB活性均明显降低(P<0.05),Th1/Th2、TNF-α/IL-6均明显升高(P<0.05)。结论BB可抑制TLR4/NF-κB信号通路活化,调节Th1/Th2平衡,减轻炎性介质释放,改善脓毒症ALI大鼠肺组织损伤。