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.

Fecal microbiota transplantation alleviates experimental colitis through the Toll-like receptor 4 signaling pathway

BACKGROUND Fecal microbiota transplantation(FMT)has shown promising therapeutic effects on mice with experimental colitis and patients with ulcerative colitis(UC).FMT modulates the Toll-like receptor 4(TLR4)signaling pathway to treat some other diseases.However,it remains unknown whether this modulation is also involved in the treatment of UC.AIM To clarify the necessity of TLR4 signaling pathway in FMT on dextran sodium sulphate(DSS)-induced mice and explain the mechanism of FMT on UC,through association analysis of gut microbiota with colon transcriptome in mice.METHODS A mouse colitis model was constructed with wild-type(WT)and TLR4-knockout(KO)mice.Fecal microbiota was transplanted by gavage.Colon inflammation severity was measured by disease activity index(DAI)scoring and hematoxylin and eosin staining.Gut microbiota structure was analyzed through 16S ribosomal RNA sequencing.Gene expression in the mouse colon was obtained by transcriptome sequencing.RESULTS The KO(DSS+Water)and KO(DSS+FMT)groups displayed indistinguishable body weight loss,colon length,DAI score,and histology score,which showed that FMT could not inhibit the disease in KO mice.In mice treated with FMT,the relative abundance of Akkermansia decreased,and Lactobacillus became dominant.In particular,compared with those in WT mice,the scores of DAI and colon histology were clearly decreased in the KO-DSS group.Microbiota structure showed a significant difference between KO and WT mice.Akkermansia were the dominant genus in healthy KO mice.The ineffectiveness of FMT in KO mice was related to the decreased abundance of Akkermansia.Gene Ontology enrichment analysis showed that differentially expressed genes between each group were mainly involved in cytoplasmic translation and cellular response to DNA damage stimulus.The top nine genes correlating with Akkermansia included Aqp4,Clca4a,Dpm3,Fau,Mcrip1,Meis3,Nupr1 L,Pank3,and Rps13(|R|>0.9,P<0.01).CONCLUSION FMT may ameliorate DSS-induced colitis by regulating the TLR4 signaling pathway.TLR4 modulates the composition of gut microbiota and the expression of related genes to ameliorate colitis and maintain the stability of the intestinal environment.Akkermansia bear great therapeutic potential for colitis.

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.

HMGN1激活TLR4/MyD88/NF-κB p65/IKK-β信号通路诱导小鼠BV2小胶质细胞活化上调其促炎介质表达

目的 探究高迁移率族核小体结合蛋白1(HMGN1)对小鼠BV2细胞炎症反应的影响并探究其可能的机制。方法使用(0、 100、 200、 500、 1000、 2000)ng/mL的重组HMGN1孵育BV2细胞6 h。用显微镜观察细胞形态变化,实时定量PCR检测细胞中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、 IL-1β、单核细胞趋化蛋白1(MCP-1) mRNA水平。随后把小胶质细胞随机分成对照组、模型组、抑制剂组和拮抗剂组。模型组用500 ng/mL的HMGN1处理BV2细胞,拮抗剂组在模型组的基础上加入瑞沙托维(resatorvid)/TAK-242进行干预,使用实时定量PCR和免疫荧光细胞化学染色检测细胞中的M1/M2型标志物的表达情况,Western blot法检测诱导型一氧化氮合酶(iNOS)、 Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核因子κB p65(NF-κB p65)和NF-κB抑制物激酶β(IKK-β)的蛋白表达。结果 HMGN1处理后,BV2细胞形态发生明显变化,呈阿米巴样;与0 ng/mL HMGN1组相比,TNF-α、 IL-6、 IL-1β、 MCP-1的mRNA水平随HMGN1剂量的增加而升高;M1型标志物iNOS的mRNA水平随HMGN1剂量的增加而升高,M2型标志物CD206的水平随HMGN1剂量的升高而降低。与模型组相比,拮抗剂组M1型标志物iNOS的mRNA水平显著降低,M2型标志物CD206的水平显著升高。免疫荧光细胞化学染色结果也显示MI型标志物iNOS在拮抗剂组中的表达降低。Western blot的结果提示,拮抗剂组iNOS、 TLR4、 MyD88、 NF-κB p65和IKK-β的蛋白表达显著降低。结论 HMGN1可能通过激活TLR4/MyD88/NF-κB p65/IKK-β信号通路诱导BV2小胶质细胞活化来上调其促炎介质的水平。

沙棘黄酮通过调控TLR4/NF-κB信号通路改善大鼠多囊卵巢综合征的作用

目的:探究沙棘黄酮改善大鼠多囊卵巢综合征(polycystic ovarian syndrome,PCOS)的作用及机制,为PCOS的防治提供新的思路。方法:采用高脂饲料联合灌胃来曲唑法复制PCOS模型,并随机分为模型组、低、高剂量沙棘黄酮组(200、400 mg/kg)及二甲双胍组(100 mg/kg),灌胃21 d后检测相关指标变化。结果:与模型组比较,低、高剂量沙棘黄酮组大鼠卵巢指数、发情间期时像百分率、空腹血糖(FBG)、空腹胰岛素(FINS)含量、胰岛素抵抗指数(HOMA-IR)、血清促黄体生成素(LH)活性及睾酮(T)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、C反应蛋白(CRP)含量显著降低(P<0.05,P<0.01),促卵泡刺激素(FSH)活性极显著增加(P<0.01);低、高剂量沙棘黄酮组大鼠卵巢组织病理形态有所改善,Toll样受体4(TLR4)、髓样分化因子88(My D88)、核转录因子κBp65(NF-κBp65)mRNA及TLR4、MyD88、p-NF-κBp65蛋白表达显著降低(P<0.05,P<0.01)。结论:沙棘黄酮可以改善PCOS大鼠症状,减轻胰岛素抵抗,调节性激素水平,修复卵巢组织病理改变,其机制与抑制TLR4/NF-κB信号通路介导的炎症反应有关。

苦豆碱通过抑制TLR4/NF-κB/NLRP3通路改善香烟烟雾诱导的人支气管上皮细胞损伤

目的探究苦豆碱(Alo)对香烟烟雾诱导的人支气管上皮细胞损伤的作用及其可能的作用机制。方法16HBE人支气管上皮细胞经100 mL/L香烟烟雾提取物(CSE)和(50、100、200)μmol/L Alo共处理后,CCK-8法检测细胞活力,试剂盒检测乳酸脱氢酶(LDH)活性;原位末端转移酶标记技术(TUNEL)、Western blot法检测细胞凋亡,ELISA检测炎性因子水平;2′,7′-二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针和相关试剂盒检测氧化应激水平;Western blot法检测Toll样受体4(TLR4)/核因子κB(NF-κB)/含pyrin结构域核苷酸结合寡聚结构域样受体家族蛋白3(NLRP3)通路相关蛋白表达水平。16HBE细胞经100 mL/L CSE和200μmol/L Alo共处理后,采用上述方法检测过表达TLR4对TLR4/NF-κB/NLRP3通路、细胞LDH活性、凋亡、炎症反应及氧化应激的影响。结果CSE暴露可降低16HBE细胞活力,增加LDH释放和细胞凋亡,增强炎症反应和氧化应激水平,且激活TLR4/NF-κB/NLRP3通路;经Alo处理后,细胞活性升高,LDH释放减少、凋亡降低、炎症减轻、氧化应激水平下降,且TLR4/NF-κB/NLRP3通路失活;TLR4过表达可逆转Alo处理对CSE诱导的16HBE细胞损伤的保护作用。结论Alo可通过抑制TLR4/NF-κB/NLRP3通路减轻CSE诱导的人支气管上皮细胞损伤。

银杏叶提取物基于TLR4/NF-κB通路对骨质疏松大鼠的干预效果及作用机制

目的分析银杏叶提取物基于TLR4/NF-κB通路对骨质疏松大鼠的干预效果及作用机制。方法选取周龄在4~6周的SPF健康雌性SD大鼠40只,随机分对照组(A组)、骨质疏松组(B组)、阳性药物己烯雌酚组(C组)和银杏叶提取物(D组),适应性饲养1周后,第2周进行建模,饲养3个月后起给予药物干预,C组大鼠每日给予己烯雌酚0.05 mg·kg^(-1)·d^(-1),D组大鼠给予银杏叶提取物,以200 mg·kg^(-1)·d^(-1)的剂量给药,给药方式均采用灌胃给药。C组、D组进行药物干预6周,与此同时,A组、B组给予10 mL·kg^(-1)·d^(-1)0.9%氯化钠溶液灌胃。给药周期满6周后,麻醉大鼠成功,处死大鼠,取出股骨组织,检测并观察各组大鼠碱性磷酸酶(alkaline phosphatase,ALP)、骨保护素(osteoprotegerin,OPG)、25-羟基维生素D 3(25-hydroxyvitamin D_(3),25OHD)、抗酒石酸盐酸性磷酸酶(Tartrate-resistant acid phosphatase,TRACP)、钙离子(calcium ion,Ca^(2+))、镁离子(magnesian ion,Mg^(2+))、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、过氧化氢酶(catalase,CAT)水平及toll样受体4(toll-like receptoR4,TLR4)、核因子κB(nucleaRfactoRkappa-B,NF-κB)蛋白表达。结果与A组比较,B组、C组、D组ALP、TRACP、MDA、TLR4、NF-κB水平升高,OPG、25OHD、Ca^(2+)、Mg^(2+)、SOD、GSH-Px、CAT水平降低,差异有统计学意义(P<0.05);与B组比较,C组、D组ALP、TRACP、MDA、TLR4、NF-κB水平降低,OPG、25OHD、Ca^(2+)、Mg^(2+)、SOD、GSH-Px、CAT水平升高,差异有统计学意义(P<0.05);与C组比较,D组ALP、TRACP、MDA、TLR4、NF-κB水平降低,OPG、25OHD、Ca^(2+)、Mg^(2+)、SOD、GSH-Px、CAT水平升高,差异有统计学意义(P<0.05)。结论银杏叶提取可能会通过抑制TLR4/NF-κB通路活化来改善骨质疏松大鼠骨代谢水平及氧化应激反应,影响破骨细胞吸收,同时会调节机体Ca^(2+)、Mg^(2+)水平,未来对临床骨质疏松的治疗有重要意义。

拉考沙胺联合奥卡西平治疗局灶性癫痫的疗效及对脑电图频段功率、血清TLR-4、NF-κB水平影响

目的探讨拉考沙胺联合奥卡西平治疗局灶性癫痫的疗效及对患者脑电图频段功率、血清Toll样受体-4(TLR-4)、核转录因子-κB(NF-κB)水平影响。方法选取2022年1月至2023年2月本院收治的局灶性癫痫患者102例,根据随机数字表法分为基线组和试验组,每组51例。基线组采用奥卡西平治疗,试验组采用拉考沙胺联合奥卡西平治疗。两组均治疗6个月后评估疗效。比较两组患者治疗前、治疗6个月癫痫发作频率、脑电图频段功率,检测血清TLR-4、NF-κB水平。分析两组患者治疗6个月TLR-4、NF-κB水平与疗效的关系。统计两组患者治疗期间不良反应发生率。结果试验组治疗总有效率高于基线组(P<0.05)。治疗6个月,两组患者癫痫发作频率降低,且试验组低于基线组(P<0.05);两组α、β频段功率升高,δ、θ频段功率降低,且试验组变化幅度大于基线组(P<0.05)。治疗6个月,试验组、基线组TLR-4(r=-0.820、-0.666,P均<0.001)、NF-κB(r=-0.744、-0.643,P均<0.001)水平与疗效均呈负相关。治疗期间试验组不良反应发生率与基线组比较,差异无统计学意义(P>0.05)。结论拉考沙胺联合奥卡西平治疗局灶性癫痫疗效显著,可有效改善患者脑电图频段功率,减少发作频率,且安全性有保障。

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.

天麻素对缺血缺氧诱导的小胶质细胞中TLR4表达的影响

目的:探究天麻素(GAS)对缺血缺氧性损伤(HIBD)后小胶质细胞toll样受体4(TLR4)表达的影响。方法:体内创建新生大鼠HIBD模型,将30只3 d龄SD大鼠随机组成3组:假手术组(sham)、HIBD模型组、HIBD模型+GAS干预组(HIBD+G);体外创建BV2细胞氧糖剥夺(OGD)模型,实验随机设置对照组(control)、OGD组、OGD+GAS干预组(OGD+G)。Western Blot和免疫荧光双标染色技术均检测体外各组细胞及体内大鼠模型左侧大脑胼胝体区TLR4的表达。结果:OGD诱导的小胶质细胞中TLR4表达显著增加;GAS干预后TLR4表达显著减少(P<0.05)。结论:GAS激活的小胶质细胞TLR4的表达具有抑制效应,从而对HIBD发挥神经保护作用。

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。

苍附导痰汤对肥胖型PCOS-IR模型大鼠卵巢TLR4/NF-κB p65信号通路的影响

目的 探讨苍附导痰汤对肥胖型PCOS-IR (polycystic ovarian syndrome-insulin resistance, PCOS-IR)模型大鼠卵巢Toll受体4(TLR4)/核转录因子κB p65(NF-κB p65)信号通路的调控作用。方法 48只♀大鼠随机分为正常组8只和模型组40只。来曲唑(1 mg·kg^(-1))联合高脂饮食建立肥胖型PCOS-IR大鼠模型,快速革兰染色法观察动情周期,挑选24只模型大鼠随机分为:模型组、阳性药(二甲双胍135 mg·kg^(-1))组、苍附导痰汤高、低剂量(57.96、14.49 g·kg^(-1))组,每组各6只,药物干预21 d。观察动情周期、卵巢和子宫指数变化;血液生化仪测定空腹血糖(FBG)和血脂(甘油三酯TG和总胆固醇TC)变化;酶联免疫吸附(ELISA)法测定空腹胰岛素(FINS)水平;免疫组化法和荧光定量PCR法检测卵巢中TLR4和NF-κB p65蛋白及基因的表达。结果 与正常组比较,模型组大鼠动情周期紊乱,卵巢多囊性改变明显,FBG、TG、TC含量和FINS、HOMA-IR水平上调,卵巢中TLR4和NF-κB p65蛋白及mRNA表达均增加(P<0.05);与模型组比较,苍附导痰汤高剂量组大鼠排卵周期得到改善,卵巢多囊性改变减轻,上述指标出现明显逆转(P<0.05)。结论 苍附导痰汤能有效改善肥胖型PCOS-IR大鼠卵巢排卵和糖脂代谢功能,作用机制可能与调控卵巢TLR4/NF-κB p65信号通路有关。

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。

有氧运动对CAP大鼠前列腺组织miR-155表达、TLR4/NF-κB信号通路的影响

目的探讨有氧运动对慢性非细菌性前列腺炎(CAP)大鼠前列腺组织miR-155表达、Toll样受体(TLR)4/核转录因子(NF)-κB信号通路的影响。方法雄性健康SD大鼠随机分为正常对照组(正常组)、CAP模型对照组(模型组)、游泳运动观察组(运动组),每组10只,注射消痔灵进行大鼠造模,模型形成期7 d,后运动组进行50 min/(次·d)的游泳运动,每周运动6 d,持续28 d,正常组和模型组不进行任何运动,自由活动取食;末次运动结束后2 h处死大鼠,采用ELISA检测前列腺组织白细胞介素(IL)-8、IL-6、肿瘤坏死因子(TNF)-α表达,Western印迹检测TLR4、NF-κB P65蛋白相对表达,实时荧光定量-聚合酶链反应(RT-PCR)检测miR-155表达,观察各组前列腺组织病理学改变。结果28 d游泳运动结束后,与正常组比较,模型组和运动组前列腺组织内IL-8、IL-6、TNF-α、TLR4、NF-κB P65和miR-155表达均显著升高(P<0.05);与模型组比较,运动组上述各项观察指标均显著降低(P<0.05);相关分析显示,TLR4、NF-κB P65与IL-8、IL-6、TNF-α表达呈正相关,miR-155与TLR4、NF-κB P65、IL-8、IL-6、TNF-α表达呈正相关(均P<0.01);病理观察显示,正常组前列腺组织学观察未见异常改变,模型组前列腺组织内可见腺体上皮细胞及纤维组织明显增生,间质中淋巴细胞等慢性炎症细胞浸润;与模型组比较,运动组前列腺腺体上皮细胞轻度增生,间质轻度纤维化,慢性炎症细胞浸润现象明显好转。结论有氧运动能够对CAP大鼠炎症起到缓解作用,其作用机制可能与miR-155调控TLR4/NF-κB信号通路有关。

Betaine inhibits Toll-like receptor 4 expression in rats with ethanol-induced liver injury

AIM:To test whether ethanol feeding could induce Toll-like receptor 4(TLR4)responses,assess the hepatoprotective effect of betaine and its inhibitive effect on TLR4 in animal models of alcoholic liver injury.METHODS:Forty-eight female Sprague-Dawley rats were randomly divided into four groups as control,model,low and high dose betaine groups.Except control group,all rats were fed with high fat-containing diet plus ethanol and fish oil gavages for 8 wk.Betaine was administered intragastrically after exposure of ethanol for 4 wk.The changes of liver histology were examined.The expression of TLR4 mRNA and protein was detected by RT-PCR and Western blotting,respectively.The serum aminotransferase activity alanine transarninase(ALT),aspartate aminotransferase(AST),serum endotoxin,and liver inflammatory factors tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin-18(IL-18)were also assayed.RESULTS:Compared with control group,rats of model group developed marked liver injury,accompanied by an increase of ALT(159.41±7.74 U/L vs 59.47± 2.34 U/L,P<0.0001),AST(248.25±1.40 U/L vs 116.89±3.48 U/L,P<0.0001),endotoxin(135.37± 30.17 ng/L vs 44.15±7.54 ng/L,P<0.0001),TNF-α(20.81±8.58 pg/mL vs 9.34±2.57 pg/mL,P=0.0003),IFN-γ(30.18±7.60 pg/mL vs 16.86±9.49 pg/mL,P= 0.0039)and IL-18(40.99±8.25 pg/mL vs 19.73±9.31 pg/mL,P=0.0001).At the same time,the expression of TLR4 mRNA and protein was markedly induced in the liver after chronic ethanol consumption(1.45±0.07 vs 0.44±0.04,P<0.0001;1.83±0.13 vs 0.56±0.08,P<0.0001).Compared with model group,betaine feeding resulted in significant decreases of ALT(64.93 ±6.06 U/L vs 159.41±7.74 U/L,P<0.0001),AST(188.73±1.11 U/L vs 248.25±1.40 U/L,P<0.0001),endotoxin(61.80±12.56 ng/L vs 135.37±30.17 ng/L,P<0.0001),TNF-α(9.79±1.32 pg/mL vs 20.81± 8.58 pg/mL,P=0.0003),IFN-γ(18.02±5.96 pg/mL vs 30.18±7.60 pg/mL,P=0.0008)and IL-18(18.23±7.01 pg/mL vs 40.99±8.25 pg/mL,P<0.0001).Betaine also improved liver steatosis.The expression levels of TLR4 mRNA or protein in liver tissues were significantly lowered(0.62±0.04 vs 1.45±0.07,P<0.0001;and 0.65±0.06 vs 1.83±0.13,P<0.0001).There was a statistical difference of TLR4 mRNA and protein expression between high-and low-dose betaine groups(0.62±0.04 vs 0.73±0.05,P<0.0001,and 0.65±0.06 vs 0.81±0.09,P<0.0001).CONCLUSION:Betaine can prevent the alcoholinduced liver injury effectively and improve the liver function.The expression of TLR4 increases significantly in ethanol-fed rats and betaine administration can inhibit TLR4 expression.

双氢青蒿素抑制TLR4/NF-κB信号通路对高糖刺激的足细胞功能障碍的改善作用

目的探究双氢青蒿素通过调控Toll样受体4(TLR4)/核因子-κB(NF-κB)信号通路对D-葡萄糖诱导的人肾小球足细胞(HGPC)炎症、增殖、迁移和侵袭的影响。方法体外培养HGPC细胞,将HGPC细胞分为对照组(5 mmol·L^(-1)D-葡萄糖)、高糖组(30 mmol·L^(-1)D-葡萄糖)和不同浓度双氢青蒿素组(30 mmol·L^(-1)D-葡萄糖+5、10、20、40μmol·L^(-1)双氢青蒿素),筛选出合适的双氢青蒿素作用浓度;随后将细胞分为对照组、高糖组、双氢青蒿素组(30 mmol·L^(-1)D-葡萄糖+20μmol·L^(-1)双氢青蒿素)、抑制剂组(30 mmol·L^(-1)D-葡萄糖+5μmol·L^(-1)NF-κB通路抑制剂BAY 11-7082)、双氢青蒿素+抑制剂组(30 mmol·L^(-1)D-葡萄糖+20μmol·L^(-1)双氢青蒿素+5μmol·L^(-1)BAY 11-7082)和双氢青蒿素+激活剂组(30 mmol·L^(-1)D-葡萄糖+20μmol·L^(-1)双氢青蒿素+1μmol·L^(-1)NF-κB通路激活剂Prostratin),干预24 h。细胞计数试剂盒细胞8(CCK-8)法检测细胞活力;酶联免疫吸附试验(ELISA)法检测炎症因子白细胞介素(IL)-1β和IL-8的表达水平;5-乙炔基-2’脱氧尿嘧啶核苷(EdU)法检测细胞的增殖能力;Transwell小室法检测细胞迁移和侵袭能力;蛋白免疫印迹(Western Blot)法检测细胞周期蛋白D1(CyclinD1)、基质金属蛋白酶9(MMP-9)和TLR4/NF-κB通路相关蛋白表达水平。结果CCK-8法检测结果显示20μmol·L^(-1)双氢青蒿素对D-葡萄糖HGPC细胞活力恢复效果最好,因此选择20μmol·L^(-1)双氢青蒿素用于后续实验。与对照组比较,高糖组细胞增殖率和CyclinD1蛋白相对表达量明显降低(P<0.05),炎症因子(IL-1β和IL-8)、迁移数、侵袭数、MMP-9、TLR4和磷酸化(p)-NF-κB p65蛋白相对表达量明显升高(P<0.05);双氢青蒿素组、抑制剂组、双氢青蒿素+抑制剂组及双氢青蒿素+激活剂组中双氢青蒿素和BAY11-7082明显抑制了D-葡萄糖对HGPC细胞的上述作用(P<0.05);与双氢青蒿素组比较,双氢青蒿素+抑制剂组中BAY11-7082增强了双氢青蒿素对D-葡萄糖诱导的HGPC细胞的作用(P<0.05),双氢青蒿素+激活剂组中Prostratin则削弱了双氢青蒿素对D-葡萄糖诱导的HGPC细胞的作用(P<0.05)。结论双氢青蒿素能抑制D-葡萄糖诱导的HGPC细胞迁移和侵袭,并促进其增殖,能有效改善D-葡萄糖刺激对HGPC细胞的炎症损伤,其作用机制可能与阻滞TLR4/NF-κB信号通路信号转导有关。

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.

Lactobacilli inhibit interleukin-8 production induced by Helicobacter pylori lipopolysaccharide-activated Toll-like receptor 4

AIM： To investigate the effect of Lactobacillus bulgaricus （LBG） on the Toll-like receptor 4 （TLR4） pathway and interleukin-8 （IL-8） production in SGC-7901 cells treated with Helicobacter pyloriSydney strain 1 lipopolysaccharide （HpyloriSS1-LPS）. METHODS： SGC-7901 cells were treated with HpyloriSS1-LPS in the presence or absence of pretreatment for 1 h with viable LBG or supernatant recovered from LBG culture MRS broth （LBG-s）. Cellular lysates were prepared for Western blot with anti-TLR4, anti-transforming growth factor β-activated kinase 1 （TAK1）, anti-phospho-TAK1, anti-nuclear factor κB （NF-κB）, anti-p38 mitogen-activated protein kinase （p38MAPK）, and anti-phospho-p38MAPK antibodies. The amount of IL-8 in cell culture medium was measured by ELISA. RESULTS： H pyloriSS1-LPS up-regulated the expression of TLR4, stimulated the phosphorylation of TAKI, subsequently enhanced the activation of NF- κB and the phosphorylation of p38MAPK in a time- dependent manner, leading to augmentation of IL-8 production in SGC-7901 cells. Viable LBG or LBG-s pretreatment attenuated the expression of TLR4, inhibited the phosphorylation of TAK1 and p38MAPK, prevented the activation of NF-κB, and consequently blocked IL-8 production.CONCLUSION： H py/oriSS1-LPS induces IL-8 production through activating TLR4 signaling in SGC-7901 cells and viable LBG or LBG-s prevents H pyloriSS1-LPS-mediated IL-8 production via inhibition of the TLR4 pathway.

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.

Association of Toll-like receptor 4 Signaling Pathway with Steroid-induced Femoral Head Osteonecrosis in Rats

Osteonecrosis of the femoral head is frequently observed in patients treated with excessive corticosteroids. However, the pathogenesis of corticosteroid-induced osteonecrosis remains unclear. The purpose of this study was to investigate the role of Toil-like receptor 4 （TLR4） signaling pathway in steroid-induced femoral head osteonecrosis in rats. Male Sprague-Dawley rats were injected intramus- cularly with 20 mg/kg methylprednisolone （MP） for 8 weeks, twice per week. The animals were sacri- ficed at 2, 4 and 8 weeks after the last MP injection, respectively, and then allocated to the 2-, 4- and 8-week model groups （n=24 each）. Rats in the control group （n=12） were not given any treatment. Histopathological analysis was performed and the concentration of tartrate-resistant acid phosphatase （TRAP） in plasma was determined. The activation of osteoclasts in the femoral head was assessed by TRAP staining. The expression of TLR4, MyD88, TRAF6 and NF-Id3 p65 that are involved in TLR4 signaling, and MCP-1 production were detected by using real-time PCR （RT-PCR） and Western blotting. The results showed that the osteonecrosis in the femoral head was clearly observed and the concentra- tion of TRAP in the plasma was increased in the model rats. The femoral head tissues in MP-treated rats were positive for TRAP and the intensity of TRAP staining was greater in MP-treated rats than in con- trol rats. As compared with the control group, the mRNA expression of TLR4 signaling-related factors was enhanced significantly at 4 and 8 weeks, and the protein levels of these factors increased signifi- cantly with time. It was concluded that MP could induce the femoral head osteonecrosis in rats, which was associated with osteoclast activation via the TLR4 signaling pathway. These findings suggest that TLR4 signaling pathway plays a pivotal role in the pathogenesis of steroid-induced osteonecrosis.