Efficacy of Jiangzhi Xiaoban tablet(降脂消斑片)on toll-like receptor 4/nuclear factor-kappa B/nod-like receptor protein 3 signaling pathway in mice with atherosclerosis induced by high-fat diet

OBJECTIVE:To study the effect of Jiangzhi Xiaoban tablet(降脂消斑片,JZXB)on toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/Nod-like receptor protein 3(NLRP3)signaling pathway expression in atherosclerosis(AS)mice by establishing a mouse model of AS,and to explore its mechanism of prevention and treatment of AS.METHODS:Sixty-four male C57BL/6J mice were randomly divided into two groups,12 in the normal control group and 52 in the model group(MOD).Seven weeks later,two mice in each of the above two groups were randomly sacrificed,and the whole aortic tissue of the mice was taken out for hematoxylin-eosin staining.After successful modeling,50 mice in the modeling group were randomly divided into 5 groups:MOD,atorvastatin group(ATO),low-dose group of JZXB(JZXB-L),middle-dose group of JZXB(JZXB-M),and high-dose group of JZXB(JZXB-H),10 mice in each group.The mice in each group were killed after 6 weeks of preventive administration.HE staining was used to observe the pathological changes of aorta in AS mice.The levels of serum triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)were detected by automatic biochemical analyzer.The levels of inflammatory factor interleukin-1β(IL-1β)were detected by enzyme linked immunosorbent assay.The expression of TLR4,NF-κB and NLRP3 proteins in aortic tissue was detected by immunohistochemistry.RESULTS:Compared with the MOD,the levels of serum TC,TG and LDL-C in the JZXB-H and ATO were significantly decreased,while the level of HDL-C was significantly increased.The levels of serum TG,LDL-C in the JZXB-M were significantly decreased,and the level of HDL-C was significantly increased.Compared with the MOD,the levels of IL-1βwere significantly decreased,aortic lesions were significantly improved,and the expression of TLR4,NF-κB,and NLRP3 proteins in the aortic tissue was significantly decreased in the JZXB-H,JZXB-M,and ATO.CONCLUSION:JZXB has inhibitory effect on atherosclerosis in mice,and its mechanism may be through regulating the TLR4/NF-κB/NLRP3 signaling pathway and reducing the inflammatory response,so as to play a role in inhibiting atherosclerosis.

Toll-like receptor 4/nuclear factor-kappa B signaling detected in brain after early subarachnoid hemorrhage

Background Inflammation and immunity play a vital role in the pathogenesis of early brain injury after subarachnoid hemorrhage （SAH）. Nuclear factor-kappa B （NF-κB） regulates many genes essential for inflammation and immunity and is activated by toll-like receptor （TLR）. This study aimed to detect the expression of the toll-like receptor 4/nuclear factor-kappa B （TLR4/NF-κB） signaling in the rat brain after early SAH. Methods The rats were decapitated and their brains were removed at 0, 2, 4, 6, 12, 24 and 48 hours after a single injection of blood into the prechiasmatic cistern, mRNA expression of TLR4 was measured by Taqman real-time RT-PCR, and protein expression by immunohistochemistry and Western blotting. NF-κB activity and concentrations of tumor necrosis factor-alpha （TNF-α）, interleukin-lbeta （IL-1β） and interleukin-6 （IL-6） were measured by enzyme-linked immunosorbent assay （ELISA）. Results TaqMan real-time RT-PCR and Western blotting identified a biphasic change in TLR4 expression in both mRNA and protein： an initial peak （2-6 hours） and a sustained elevation （12-48 hours）. Immunohistochemical staining showed the inducible expression of TLR4-like immunoreactions predominantly in glial cells and vascular endothelium. A similar biphasic change in the activation of NF-κB subunit p65 as well as the production of NF-κB-regulated proinflammatory cytokines （TNF-α, IL-1β and IL-6） were detected by ELISA. Conclusions These data suggest that experimental SAH induces significant up-regulation of TLR4 expression and the NF-κB signaling in early brain injury. Activation of the TLR4/NF-κB signaling may regulate the inflammatory responses after SAH.

Endogenous danger signals trigger hepatic ischemia/reperfusion injury through toll-like receptor 4/nuclear factor-kappa B pathway

Background Restoration of blood flow to the ischemic liver lobes may paradoxically exacerbate tissue injury, which is called hepatic ischemia/reperfusion injury （IRI）. Toll-like receptor 4 （TLR4）, expressed on several liver cell types, and the nuclear factor-kappa B （NF-KB） signaling pathway are crucial to mediating hepatic inflammatory response. Because IRI is essentially a kind of profound acute inflammatory reaction evoked by many kinds of danger signals, we investigated TLR4/NF-KB signaling pathway activation in a murine model of partial hepatic IRI. Methods Wild-type mice （WT, C3H/HeN） or TLR4 mutant mice （C3H/HeJ） were subjected to 45 minutes of partial hepatic ischemia followed by 1 hour, 3 hours of reperfusion. Sham group accepted the same procedure without the obstruction of blood supply. At the end of reperfusion, the compromise of liver function and the histological change of liver sections were measured as the severity of liver injury. The level of endotoxin in the portal vein was measured by limulus assay. NF-KB activation was determined by electrophoretic mobility shift assay （EMSA）. The levels of tumor necrosis factor-a （TNF-a） and intedeukin-1β （IL-1β） in systemic blood after hepatic IRI were assessed by enzyme-linked immunosorbent assay （ELISA）. Results The compromise of liver function and the morphological injuries in mutant mice were relieved more markedly than those in WT mice after partial hepatic IRI. NF-KB activation in WT mice was stronger than that in TLR4 mutant mice, and both were stronger than those in the sham operated mice （P〈0.01）. Endotoxin in each group was undetectable. The levels of TNF-α and IL-1β in systemic blood were elevated in both strains, but lower in the sham operated group. These mediators were significantly decreased in TLR4 mutant mice compared with those in WT mice （P〈0.01）. Conclusions The TLR4/NF-KB signaling pathway may mediate hepatic IRI triggered by endogenous danger signals. Inhibition of the TLR4/NF-KB pathway may be a potential therapeutic target for attenuating ischemia/reperfusion-induced tissue damage in some clinical settings.

Pharmacological Inhibition of Macrophage Toll-like Receptor 4/Nuclear Factor-kappa B Alleviates Rhabdomyolysis-induced Acute Kidney Injury

Background：Acute kidney injury （AKI） is the most common and life-threatening systemic complication ofrhabdomyolysis.Inflammation plays an important role in the development of rhabdomyolysis-induced AKI.This study aimed to investigate the kidney model of AKI caused by rhabdomyolysis to verify the role ofmacrophage Toll-like receptor 4/nuclear factor-kappa B （TLR4/NF-κB） signaling pathway.Methods：C57BL/6 mice were injected with a 50% glycerin solution at bilateral back limbs to induce rhabdomyolysis,and CLI-095 or pyrrolidine dithiocarbamate （PDTC） was intraperitoneally injected at 0.5 h before molding.Serum creatinine levels,creatine kinase,the expression of tumor necrosis factor （TNF）-c,interleukin （IL）-1β and I L-6,and hematoxylin and eosin stainings of kidney tissues were tested.The infiltration of macrophage,mRNA levels,and protein expression of TLR4 and NF-κB were investigated by immunofluorescence double-staining techniques,reverse transcriptase-quantitative polymerase chain reaction,and Western blotting,respectively.In vitro,macrophage RAW264.7 was stimulated by ferrous myoglobin;the cytokines,TLR4 and NF-κB expressions were also detected.Results：In an in vivo study,using CLI-095 or PDTC to block TLR4/NF-κB,functional and histologic results showed that the inhibition of TLR4 or NF-κB alleviated glycerol-induced renal damages （P 〈 0.0 1）.CLI-095 or PDTC administration suppressed proinflammatory cytokine （TNF-c,IL-6,and IL-1 β） production and macrophage infiltration into the kidney （P 〈 0.01）.Moreover,in an in vitro study,CLI-095 or PDTC suppressed myoglobin-induced expression ofTLR4,NF-κB,and proinflammatory cytokine levels in macrophage RAW264.7 cells （P 〈 0.01）.Conclusion：The pharmacological inhibition of TLR4/NF-κB exhibited protective effects on rhabdomyolysis-induced AKI by the regulation of proinflammatory cytokine production and macrophage infiltration.

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.

Protective effect of modified Huangqi Chifeng decoction(加味黄芪赤风汤)on immunoglobulin A nephropathy through toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B signaling pathway

OBJECTIVE:To examine the nephroprotective mechanism of modified Huangqi Chifeng decoction(加味黄芪赤风汤,MHCD)in immunoglobulin A nephropathy(IgAN)rats.METHODS:To establish the IgAN rat model,the bovine serum albumin,lipopolysaccharide,and carbon tetrachloride 4 method was employed.The rats were then randomly assigned to the control,model,telmisartan,and high-,medium-,and low-dose MHCD groups,and were administered the respective treatments via intragastric administration for 8 weeks.The levels of 24-h urinary protein,serum creatinine(CRE),and blood urea nitrogen(BUN)were measured in each group.Pathological alterations were detected.IgA deposition was visualized through the use of immunofluorescence staining.The ultrastructure of the kidney was observed using a transmission electron microscope.The expression levels of interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1),and transforming growth factor-β1(TGF-β1)were examined by immunohistochemistry and quantitative polymerase chain reaction.Levels of toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),and nuclear factor-kappa B(NF-κB)P65,were examined by immunohistochemistry,Western blotting,and quantitative polymerase chain reaction.RESULTS:The 24-h urine protein level in each group increased significantly at week 6,and worsen from then on.But this process can be reversed by treatments of telmisartan,and high-,medium-,and low-dose of MHCD,and these treatments did not affect renal function.Telmisartan,and high-,and medium-dose of MHCD reduced IgA deposition.Renal histopathology demonstrated the protective effect of high-,medium-,and low-dose of MHCD against kidney injury.The expression levels of MCP-1,IL-6,and TGF-β1 in kidney tissues were downregulated by low,medium and high doses of MHCD treatment.Additionally,treatment of low,medium and high doses of MHCD decreased the protein and mRNA levels of TLR4,MyD88,and NF-κB.CONCLUSIONS:MHCD exerted nephroprotective effects on IgAN rats,and MHCD regulated the expressions of key targets in TLR4/MyD88/NF-κB signaling pathway,thereby alleviating renal inflammation by inhibiting MCP-1,IL-6 expressions,and ameliorating renal fibrosis by inhibiting TGF-β1 expression.

五味子乙素通过TLR4/NF-κB信号通路对急性胰腺炎大鼠肺部损伤的影响

目的:探讨五味子乙素通过Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号通路对急性胰腺炎(AP)大鼠肺部损伤的影响。方法:取SD大鼠,通过胆胰管内逆行注射5%牛磺胆酸钠方法诱导建立AP肺损伤模型,经随机数表法分为模型组、五味子乙素组、TLR4过表达载体组、TLR4空载组、五味子乙素+TLR4过表达载体组,每组12只大鼠,再取12只SD大鼠仅翻动肠管不注射5%牛磺胆酸钠,作为假手术组。以药物分别干预大鼠后,检测各组大鼠肺功能及各组大鼠腹水量与肺组织湿重/干重(W/D);HE染色检测各组大鼠肺组织病理形态并评分;检测各组大鼠动脉血气;全自动生化分析仪检测大鼠血清淀粉酶,ELISA检测炎症细胞因子IL-6、IL-18水平;蛋白免疫印迹法检测肺组织TLR4/NF-κB通路蛋白表达;免疫组织化学染色检测肺组织TLR4蛋白表达。结果:与假手术组相比,模型组大鼠肺组织出现病理损伤改变,模型组大鼠MV、PEF、PaO_(2)、OI显著降低(P<0.05),Ri、腹水量与W/D、PaCO_(2)、Holfbauer评分、血清淀粉酶、IL-6与IL-18水平、肺组织TLR4阳性细胞比例、TLR4与MYD88蛋白表达、p-NF-κB p65/NF-κB p65水平显著升高(P<0.05)。与模型组、五味子乙素+TLR4过表达载体组分别相比,五味子乙素组大鼠肺组织病理损伤改变程度均减轻,MV、PEF、PaO_(2)、OI均升高(P<0.05),Ri、腹水量与W/D、PaCO_(2)、Holfbauer评分、血清淀粉酶、IL-6与IL-18水平、肺组织TLR4阳性细胞比例、TLR4与MYD88蛋白表达、p-NF-κB p65/NF-κB p65水平均降低(P<0.05);TLR4过表达载体组大鼠肺组织病理损伤改变程度均加重,MV、PEF、PaO_(2)、OI均降低(P<0.05),Ri、腹水量与W/D、PaCO_(2)、Holfbauer评分、血清淀粉酶、IL-6与IL-18水平、肺组织TLR4阳性细胞比例、TLR4与MYD88蛋白表达、p-NF-κB p65/NF-κB p65水平均升高(P<0.05)。与模型组相比,TLR4空载组大鼠各指标差异无统计学意义(P>0.05)。结论:五味子乙素可通过下调TLR4/NF-κB信号通路,抑制炎症,减轻AP大鼠肺部损伤,修复肺功能。

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.

Methylprednisolone inhibits activated CD4^+ T cell survival promoted by toll-like receptor ligands

BACKGROUND: Methylprednisolone (MP) can affect the survival of CD4(+) T lymphocytes and plays an important role in adaptive immune responses; however, its mechanism of action is not clear. Recent studies have shown that toll-like receptors (TLRs) on CD4(+) T cells can directly modulate adaptive immune responses by affecting the survival and proliferation of activated CD4(+) T cells. This study aimed to investigate the relationship between MP, TLRs and activated CD4(+) T cells. METHODS: We separated and purified CD4(+) T cells from mice, activated them in vitro, and co-cultured them with TLR ligands, MP or inhibitors of nuclear factor-kappa B (NF-kappa B) and activator protein 1 (AP-1). We then assessed CD4(+) T cell survival and proliferation and the expression of NF-kappa B and AP-1. RESULTS: Activated CD4(+) T cells showed increased TLR-3 and TLR-9 mRNA expression, but polyinosinic-polycytidylic acid (poly I:C) and MP had no effect on the expression of these mRNAs. Still, poly I:C and CpG oligodeoxynucleotides (CpG DNA) increased the survival of activated CD4(+) T cells, whereas MP reduced the survival of activated CD4(+) T cells and could inhibit the survival effects of poly I:C and CpG DNA. The NF-kappa B essential modifier-binding domain (NBD) inhibited the survival of activated CD4(+) T cells induced by poly I:C and CpG DNA, but the AP-1 inhibitor crucumin did not have the same effect. The increased expression of NF-kappa B induced by poly I:C and CpG DNA in activated CD4(+) T cells could be inhibited by MP, but the same was not true for the increased expression of AP-1 induced by poly I:C and CpG DNA. Finally, the proliferation of activated CD4(+) T cells was not affected by poly I:C or MP. CONCLUSION: The survival of activated CD4(+) T cells is promoted by TLR ligands, but this effect is inhibited by MP.

脂氧素A4抑制TLR4/MyD88/NF-κB通路减缓脓毒症性急性肾损伤

目的探讨脂氧素A4(LXA4)通过抑制TLR4/MyD88/NF-κB通路减缓脓毒症性急性肾损伤(SAKI)。方法将40只无特定病原体级雄性C57BL/6J小鼠随机分为SAKI组、SAKI+LXA4组、假手术组、假手术+LXA4组,每组10只。采用盲肠结扎穿孔术进行SAKI造模,SAKI+LXA4组、假手术+LXA4组在术后30 min腹腔注射LXA4(40 ng/kg)。各组小鼠在造模术后24 h收集血清、尿液、肾组织。酶联免疫吸附试验(ELISA)测定各组小鼠血肌酐(Scr)、血尿素氮(Bun)、白细胞介素-1β(IL-1β)、IL-6、肿瘤坏死因子-α(TNF-α),尿液中性粒细胞明胶酶相关性脂质运载蛋白(NGAL)及肾损伤分子1(KIM-1);HE及PAS染色观察小鼠肾脏损伤情况;实时荧光定量PCR检测各组小鼠肾脏Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核因子-κB p65(NF-κB p65)mRNA水平;免疫组化法、蛋白免疫印迹实验检测各组小鼠TLR4、MyD88、NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)的表达。结果ELISA实验提示SAKI组Scr、Bun、IL-1β、IL-6、TNF-α、NGAL、KIM-1水平均高于SAKI+LXA4组(P<0.05),假手术组及假手术+LXA4组Scr、Bun、IL-1β、IL-6、TNF-α、NGAL、KIM-1无明显上升;HE及PAS染色提示SAKI组肾损伤程度明显高于SAKI+LXA4组(P<0.05),假手术组及假手术+LXA4组无明显肾损伤;实时荧光定量PCR提示SAKI组较SAKI+LXA4组TLR4、MyD88、NF-κB p65 mRNA升高(P<0.05),假手术组与假手术+LXA4组TLR4、MyD88、NF-κB p65 mRNA均低于SAKI组及SAKI+LXA4组(P<0.05);免疫组化法、蛋白免疫印迹实验结果提示SAKI组较SAKI+LXA4组TLR4、MyD88、NF-κB p65、p-NF-κB p65表达升高(P<0.05),假手术组与假手术+LXA4组TLR4、MyD88、NF-κB p65、p-NF-κB p65表达均低于SAKI组及SAKI+LXA4组(P<0.05)。结论TLR4/MyD88/NF-κB通路在SAKI发生发展中起重要作用,LXA4可能通过抑制TLR4/MyD88/NF-κB信号通路减缓SAKI。

Nr4a1激动剂胞孢子酮B挽救小鼠噪声性听力损失

目的:探究核受体亚家族4A组成员1(nuclear receptor subfamily 4 group A member 1,Nr4a1)Nr4a1激动剂胞孢子酮B(cytosporone B,Csn-B)对小鼠噪声暴露后听力损失的治疗作用。方法:采用双氧水刺激HEI-OC1毛细胞系的方法构建氧化应激细胞模型;通过实时荧光定量PCR(quantitative real-time PCR,q PCR)检测细胞中Nr4a1的mRNA表达水平;分别通过细胞计数试剂盒(cell counting kit-8,CCK8)及流式细胞术的方法检测细胞活力和细胞凋亡水平以评估Csn-B预处理后经双氧水刺激的细胞状态。构建小鼠噪声性听力损失模型,运用qPCR和免疫荧光技术检测噪声暴露后Nr4a1在小鼠耳蜗中的表达;通过检测听性脑干反应(auditory brainstem response,ABR)评估噪声暴露后以及Csn-B连续治疗13 d后小鼠听力情况。结果:双氧水刺激后HEI-OC1毛细胞中Nr4a1表达上升,细胞活力显著下降,凋亡水平显著升高;Csn-B预处理HEI-OC1毛细胞经双氧水刺激,细胞活力显著高于对照组而凋亡水平则显著低于对照组。在体研究结果显示,噪声暴露后小鼠听力显著降低,Nr4a1在小鼠耳蜗中的表达水平显著升高。噪声暴露后经Csn-B治疗小鼠听力得到改善,主要表现为Click-ABR以及Tone Burst-ABR(4000、8000Hz处)阈值下降。结论:Nr4a1激动剂Csn-B增强内耳毛细胞对氧化应激损伤的抵御能力,部分改善噪声暴露后的小鼠听力。

Yemazhui(Herba Eupatorii Lindleyani)ameliorates lipopolysaccharide-induced acute lung injury via modulation of the toll-like receptor 4/nuclear factor kappa-B/nod-like receptor family pyrin domain-containing 3 protein signaling pathway and intestinal flor

OBJECTIVE:To investigate the impact of Yemazhui(Herba Eupatorii Lindleyani,HEL)against lipopolysaccharide(LPS)-induced acute lung injury(ALI)and explore its underlying mechanism in vivo.METHODS:The chemical constituents of HEL were analyzed by ultra-high performance liquid chromatographyquadrupole time-of-flight mass spectrometry method.Then,HEL was found to suppress LPS-induced ALI in vivo.Six-week-old male Sprague-Dawley rats were randomly divided into 6 groups:control,LPS,Dexamethasone(Dex),HEL low dose 6 g/kg(HEL-L),HEL medium dose 18 g/kg(HEL-M)and HEL high dose 54 g/kg(HEL-H)groups.The model rats were intratracheally injected with 3 mg/kg LPS to establish an ALI model.Leukocyte counts,lung wet/dry weight ratio,as well as myeloperoxidase(MPO)activity were determined followed by the detection with hematoxylin and eosin staining,enzyme linked immunosorbent assay,quantitative real time polymerase chain reaction,western blotting,immunohistochemistry,and immunofluorescence.Besides,to explore the effect of HEL on ALI-mediated intestinal flora,we performed 16s rRNA sequencing analysis of intestinal contents.RESULTS:HEL attenuated LPS-induced inflammation in lung tissue and intestinal flora disturbance.Mechanism study indicated that HEL suppressed the lung coefficient and wet/dry weight ratio of LPS-induced ALI in rats,inhibited leukocytes exudation and MPO activity,and improved the pathological injury of lung tissue.In addition,HEL reduced the expression of tumor necrosis factoralpha,interleukin-1beta(IL-1β)and interleukin-6(IL-6)in bronchoalveolar lavage fluid and serum,and inhibited nuclear displacement of nuclear factor kappa-B p65(NF-κBp65).And 18 g/kg HEL also reduced the expression levels of toll-like receptor 4(TLR4),myeloid differentiation factor 88,NF-κBp65,phosphorylated inhibitor kappa B alpha(phospho-IκBα),nod-like receptor family pyrin domain-containing 3 protein(NLRP3),IL-1β,and interleukin-18(IL-18)in lung tissue,and regulated intestinal flora disturbance.CONCLUSIONS:In summary,our findings revealed that HEL has a protective effect on LPS-induced ALI in rats,and its mechanism may be related to inhibiting TLR4/NF-κB/NLRP3 signaling pathway and improving intestinal flora disturbance.

Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons

Lipopolysaccharide stimulates Toll-like receptor 4 on immune cells to produce immune mediators. Toll-like receptor 4 is also expressed by non-immune cells, which can be stimulated by lipopolysaccharide. However, whether Toll-like receptor 4 is expressed by primary cultured hippocampal neurons and its specific role in lipopolysaccharide-induced neuroinflammation is currently undefined, in this study, Toll-like receptor 4 antibody blocking was used to analyze the Toll-like receptor 4 signaling pathway and changes in inflammation of lipopolysaccharide stimulated hippocampal neurons. Immunofluorescence showed that Toll-like receptor 4 protein was mainly located in the membrane of hippocampal neurons. Quantitative reverse transcription-PCR and western blot assay showed that after stimulation of lipopolysaccharide, the mRNA and protein levels of Toll-like receptor 4 and the mRNA levels of interleukin-ll3 and tumor necrosis factor-（] were significantly increased. In addition, there was increased phosphorylation and degradation of kappa B a inhibitor in the cytosol and increased nuclear factor-KB p65 expression in the nuclei. Pretreatment with Toll-like receptor 4 antibody could almost completely block this increase. These experimental findings indicate that lipopolysaccharide participates in neuroinflammation by stimulating Toll-like receptor 4/nuclear factor-KB pathway in hippocampal neurons, which may be both ＂passive victims＂ and ＂activators＂ of neuroinflammation.

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。

基于TLR4/NF-κB信号通路及血清CXCL16、ACA评价依达拉奉右莰醇对ACI神经功能的保护作用

目的基于Toll样受体(TLR)4/核因子κB(NF-κB)信号通路及血清CXC型趋化因子配体16(CXCL16)、抗心磷脂抗体(ACA)评价依达拉奉右莰醇治疗急性脑梗死(ACI)的疗效及神经功能保护作用。方法将102例ACI患者根据是否使用依达拉奉右莰醇分为两组:观察组(n=54)均在常规治疗基础上接受依达拉奉右莰醇治疗,对照组(n=48)未用依达拉奉治疗。对比两组治疗前及治疗7d、14d、30d后神经功能缺损量表(NIHSS),治疗前及治疗14d检测血清TLR4、NF-κB、白介素1β(IL-1β)、白介素6(IL-6)、肿瘤坏死因子α(TNF-α)、CXCL16、ACA,随访3个月采用改良Rankin量表(mRS)评价预后。结果治疗后7d、14d、30d,两组NIHSS评分均下降,而观察组明显低于对照组(P<0.05)。治疗14d后,两组血清TLR4、NF-κB、IL-1β、IL-6、TNF-α、CXCL16、ACA水平均下降,且观察组明显低于对照组(P<0.05)。随访3个月,观察组mRS均分低于对照组(P<0.05),但两组预后差异无统计学意义(79.63%vs 68.75%,P>0.05)。结论依达拉奉右莰醇用于ACI的治疗可保护神经功能,其机制可能与调节TLR4/NF-κB信号通路及血清CXCL16、ACA表达有关。

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。

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。

基于TLR4/MyD88/NF-kB信号通路探讨追风透骨胶囊减缓兔膝骨关节炎模型软骨退变的作用机制

目的 观察追风透骨胶囊对兔膝骨关节炎(knee osteoarthritis, KOA)模型关节软骨退变的干预作用,并基于Toll样受体4(Toll like receptor 4, TLR4)/髓细胞分化初级反应蛋白88(myeloid differentiation primary response protein 88, MyD88)/核因子kappa-B(nuclear factor kappa-B, NF-κB)信号通路探讨追风透骨胶囊的可能作用机制。方法 从50只6月龄雄性新西兰兔中随机选取10只作为正常组,其余40只为KOA造模组,用改良Videman造模法制备兔KOA模型。模型验证后,从正常组中随机选取6只作为空白组(A组),KOA造模组抽取30只随机分为模型组(B组)、硫酸氨基葡萄糖组(C组)、追风透骨胶囊低剂量组(D组)、追风透骨胶囊中剂量组(E组)、追风透骨胶囊高剂量组(F组),每组6只。A、B组予蒸馏水灌胃,C、D、E、F组予相应药物灌胃,疗程均为6周。给药结束后,取造模侧膝关节软骨,予大体及HE染色观察,并用Pelletier评分及Mankin评分评估;以Western blot、RT-PCR法检测软骨中TLR4、My D88、NF-κB蛋白及其mRNA的表达;免疫组化法检测白细胞介素-1β(interleukin-1β, IL-1β)、白细胞介素-6(interleukin-6, IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)的含量。结果 与A组比较,B组膝关节软骨破坏明显,Pelletier评分及Mankin评分均升高(P<0.01),软骨中TLR4、MyD88、NF-κB蛋白及其mRNA表达水平均上调(P<0.01),IL-1β、IL-6、TNF-α含量均升高(P<0.01)。与B组比较,C、D、E、F组的软骨损伤程度轻,软骨Mankin评分均降低(P<0.01),软骨中TLR4、MyD88、NF-κB蛋白及m RNA表达水平均下调(P<0.01),IL-1β、IL-6、TNF-α含量均降低(P<0.01),此外,F组软骨Pelletier评分较B组降低明显(P<0.05)。结论 追风透骨胶囊能延缓改良Videman造模法诱导的兔KOA模型关节软骨退变,其作用机制可能与下调软骨中TLR4、MyD88、NF-κB蛋白及m RNA的表达,抑制TLR4/MyD88/NF-κB信号通路的活化,降低IL-1β、IL-6、TNF-α在软骨中的含量,从而减轻炎症反应相关。

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。

基于TLR4/NF-κB信号通路沉默miR-21对糖尿病肾病大鼠的干预效果

目的探究基于Toll样受体4(Toll-like receptor 4,TLR4)/核转录因子-κB(nuclear factor kappa B,NF-κB)信号通路沉默微小RNA-21(MicroRNA-21,miR-21)对糖尿病肾病大鼠的干预效果。方法选取45只大鼠,10只作为空白组,其余35只建立糖尿病肾病模型,最终有30只建模成功,将最终建模成功的30只大鼠分为模型组、上调组和下调组,每组10只。做miR-21转染,将10μl miR-21过表达慢病毒悬液、miR-21沉默慢病毒悬液分别注射于上调组、下调组大鼠胃组织中,空白组、模型组注射同剂量的蒸馏水灌胃,注射24 h后观察大鼠变化。结果与空白相比,模型组、上调组、下调组白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1(interleukin-1,IL-1)、C-C基序趋化因子2(CC chemokine ligand 2,CCL2)、内皮素-1(endothelin 1,ET-1)、结缔组织生长因子(connective tissue growth factor,CTGF)、转化生长因子-β1(transforming the growth factor-β1,TGF-β1)水平、TLR4、NF-κB表达量上升,一氧化氮(nitric oxide,NO)水平下降(P<0.05);与模型组相比,上调组IL-6、IL-1、CCL2、ET-1、CTGF、TGF-β1水平、TLR4、NF-κB表达量上升(P<0.05),NO水平下降(P<0.05),下调组IL-6、IL-1、CCL2、ET-1、CTGF、TGF-β1水平、TLR4、NF-κB表达量下降(P<0.05),NO水平上升(P<0.05);与上调组相比,下调组IL-6、IL-1、CCL2、ET-1、CTGF、TGF-β1水平、TLR4、NF-κB表达量下降(P<0.05),NO水平上升(P<0.05)。结论沉默miR-21可缓解糖尿病肾病大鼠炎性反应、肾纤维化,改善内皮功能,其机制可能与TLR4/NF-κB信号通路被抑制有关。