Role of osteoprotegerin/receptor activator of nuclear factor kappa B/receptor activator of nuclear factor kappa B ligand axis in nonalcoholic fatty liver disease

Concomitantly with the increase in the prevalences of overweight/obesity, nonalcoholic fatty liver disease(NAFLD) has worldwide become the main cause of chronic liver disease in both adults and children. Patients with fatty liver display features of metabolic syndrome(Met S), like insulin resistance(IR), glucose intolerance, hypertension and dyslipidemia. Recently, epidemiological studies have linked obesity, Met S, and NAFLD to decreased bone mineral density and osteoporosis, highlighting an intricate interplay among bone, adipose tissue, and liver. Osteoprotegerin(OPG), an important symbol of the receptor activator of nuclear factor-B ligand/receptor activator of nuclear factor kappa B/OPG system activation, typically considered for its role in bone metabolism, may also play critical roles in the initiation and perpetuation of obesityrelated comorbidities. Clinical data have indicated that OPG concentrations are associated with hypertension, left ventricular hypertrophy, vascular calcification, endothelial dysfunction, and severity of liver damage in chronic hepatitis C. Nonetheless, the relationship between circulating OPG and IR as a key feature of Met S as well as between OPG and NAFLD remains uncertain. Thus, the aims of the present review are to provide the existent knowledge on these associations and to discuss briefly the underlying mechanisms linking OPG and NAFLD.

Effect of Triptolide on Expression of Receptor Activator of Nuclear Factor-κB Ligand in Rat Adjuvant Induced Arthritis

The effect of triptolide （TP） on the expression of receptor activator of nuclear factor-κB ligand （RANKL） and osteoprotegerin （OPG） was explored in rat adjuvant induced arthritis （AA）. AA was induced in Wistar rats. Arthritis rats were treated with TP and methotrexate （MTX） at the onset （day 9） of arthritis. On the peak of arthritis （day 24）, the expression of RANKL and OPG protein in the joints and RANKL mRNA in peripheral blood mononuclear cells （PBMC） was detected. TNF-α and IL-1β levels in peripheral blood were determined. Bone erosion scores were also evaluated. The results showed that bone erosion scores in TP and MTX groups were lower than in AA group （.P〈0.01） ; The expression levels of RANKL in the synovium （P〈0.01） and bone （P〈0.05）, and OPG level in synovium （P〈0.05） were lower in TP group than in AA group （P〈0.05）. In TP group, the expression levels of RANKL mRNA and TNF-α, IL-1β in PBMC were lower than in AA group （all P〈0.01）. It was concluded that TP could inhibit rat adjuvant arthritis bone erosion by suppressing the expression of RANKL.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.

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.

TLR4 upregulates CBS expression through NF-κB activation in a rat model of irritable bowel syndrome with chronic visceral hypersensitivity

AIM:To investigate the roles of toll-like receptor 4(TLR4) and nuclear factor(NF)-κB on cystathionine βsynthetase(CBS) expression and visceral hypersensitivity in rats.METHODS:This study used 1-7-wk-old male SpragueDawley rats.Western blot analysis was employed to measure the expression of TLR4,NF-kB and the endogenous hydrogen sulfide-producing enzyme CBS in colon dorsal root ganglia(DRG) from control and "irritable bowel syndrome" rats induced by neonatal colonic inflammation(NCI).Colon-specific DRG neurons were labeled with Dil and acutely dissociated to measure excitability with patch-clamp techniques.Immunofluorescence was employed to determine the co-expression of TLR4,NF-kB and CBS in Dil-labeled DRG neurons.RESULTS:NCI significantly upregulated the expression of TLR4 in colon-related DRGs(0.34 ± 0.12 vs 0.72 ±0.02 for the control and NCI groups,respectively,P <0.05).Intrathecal administration of the TLR4-selective inhibitor CLI-095 significantly enhanced the colorectal distention threshold of NCI rats.CLI-095 treatment also markedly reversed the hyperexcitability of colonspecific DRG neurons and reduced the expression of CBS(1.7 ± 0.1 vs 1.1 ± 0.04,p < 0.05) and of the NF-kB subunit p65(0.8 ± 0.1 vs 0.5 ± 0.1,P< 0.05).Furthermore,the NF-KB-selective inhibitor pyrrolidine dithiocarbamate(PDTC) significantly reduced the upregulation of CBS(1.0 ± 0.1 vs 0.6 ± 0.1,P< 0.05)and attenuated visceral hypersensitivity in the NCI rats.In vitro,incubation of cultured DRG neurons with the TLR4 agonist lipopolysaccharide significantly enhanced the expression of p65(control vs 8 h:0.9 ± 0.1 vs1.3 ± 0.1;control vs 12 h:0.9 ± 0.1 vs 1.3 ± 0.1,P< 0.05;control vs 24 h:0.9 ± 0.1 vs 1.6 ± 0.1,P <0.01) and CBS(control vs 12 h:1.0 ± 0.1 vs 2.2 ±0.4;control vs 24 h:1.0 ± 0.1 vs 2.6 ± 0.1,P< 0.05),whereas the inhibition of p65 via pre-incubation with PDTC significantly reversed the upregulation of CBS expression(1.2 ± 0.1 vs 0.6 ± 0.0,P< 0.01).CONCLUSION:Our results suggest that the activation of TLR4 by NCI upregulates CBS expression,which is mediated by the NF-kB signaling pathway,thus contributing to visceral hypersensitivity.

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.

核因子κB受体活化因子信号转导机制与破骨细胞的活化

背景:破骨细胞是目前已知的唯一一种骨吸收细胞,其生命活动对骨骼的正常发育和骨骼损伤修复至关重要。在绝大多数骨病中,破骨细胞均显示出异常增殖分化和骨吸收活性增加,而核因子κB受体活化因子信号是调控破骨细胞生命过程的关键信号通路。目的:总结对破骨细胞核因子κB受体活化因子信号下游靶点及DNA转录因子的最新研究进展,为相关疾病的研究和治疗提供依据。方法:文献检索数据库包括中国知网、万方、维普数据库、PubMed、Embase及Web of Science数据库,中文检索词为“破骨细胞,破骨前体细胞,骨质疏松症,骨代谢,发病机制,表观遗传学,信号通路,信号传导,转录因子,组织工程”,英文检索词为“osteoclasts,osteoclast precursor cells,osteoporosis,bone metabolism,pathogenesis,epigenetics,signaling pathway,signal transduction,transcription factors,tissue engineering”,时间设置为2017-2021年,根据纳入和排除标准共筛选52篇文献。结果与结论:核因子κB受体活化因子的特殊结构决定了其信号传导需要与肿瘤坏死因子受体激活因子6结合来募集多种蛋白、活性酶以及细胞因子,形成具有内在酶活性的核因子κB受体活化因子复合物;该复合物通过激活核因子κB、丝裂原活化蛋白激酶等信号通路的传导,最终调控破骨细胞分化、增殖、骨吸收等生命过程。

RANKL,a necessary chance for clinical application to osteoporosis and cancer-related bone diseases

Osteoporosis is a common bone disease characterized by reduced bone and increased risk of fracture. In postmenopausal women, osteoporosis results from bone loss attributable to estrogen deficiency. Osteoclast differentiation and activation is mediated by receptor activator of nuclear factor-κB ligand(RANKL), its receptor receptor activator of nuclear factor-κB(RANK), and a decoy receptor for RANKL, osteoprotegerin(OPG). The OPG/RANKL/RANK system plays a pivotal role in osteoclast biology. Currently, a fully human antiRANKL monoclonal antibody named denosumab is being clinically used for the treatment of osteoporosis and cancer-related bone disorders. This review describes recent advances in RANKL-related research, a story from bench to bedside. First, the discovery of the key factors, OPG/RANKL/RANK, revealed the molecular mechanism of osteoclastogenesis. Second, we established three animal models:(1) a novel and rapid bone loss model by administration of glutathione-S transferase-RANKL fusion protein to mice;(2) a novel mouse model of hypercalcemia with anorexia by overexpression of soluble RANKL using an adenovirus vector; and(3) a novel mouse model of osteopetrosis by administration of a denosumab-like anti-mouse RANKL neutralizing monoclonal antibody. Lastly, anti-human RANKL monoclonal antibody has been successfully applied to the treatment of osteoporosis and cancer-related bone disorders in many countries. This is a real example of applying basic science to clinical practice.

Receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression in chronic apical periodontitis：possible association with inflammatory cells

Background Receptor activator of nuclear factor kappa B （NF-κB） ligand （RANKL） and osteoprotegerin （OPG） have been recently shown to play important roles in bone resorption. The aim of this study was to investigate the possible association between the expression of bone resorption regulators （RANKL and OPG） and inflammatory cell infiltration in chronic apical periodontitis.Methods The samples of chronic periapical lesions （n=40） and healthy periapical tissues （n=10） were examined for immunohistochemical analysis of RANKL and OPG. Lesion samples were further analyzed for the inflammatory infiltration condition. The inflammatory cell infiltration was scored in relation to immunohistochemical reactivity for CD3, CD20 and CD68.Results The number of RANKL-positive cells and the ratio of RANKL/OPG in chronic apical periodontitis were significantly higher than those in healthy periapical tissues （P＜0.001）. The number of RANKL-positive cells was higher in lesions with severe inflammatory infiltration than in those with light inflammatory infiltration （P＜0.05）. Significantly increased RANKL expression was found with T lymphocytes （CD3＋）, macrophages （CD68＋） and B lymphocytes （CD20＋）infiltration （P＜0.05）. No association was found between the ratio of RANKL/OPG and inflammatory cell infiltration.Conclusions RANKL expression was increased with T, B lymphocytes and macrophages infiltration, respectively in chronic periapical lesions. RANKL appears to be closely related to periapical inflammatory infiltrates. The relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption.

Magnolol protects against acute gastrointestinal injury in sepsis by down-regulating regulated on activation,normal T-cell expressed and secreted

BACKGROUND Sepsis is a major medical challenge.Magnolol is an active constituent of Houpu that improves tissue function and exerts strong anti-endotoxin and anti-inflammatory effects,but the mechanism by which it reduces intestinal inflammation in sepsis is yet unclear.AIM To assess the protective effect of magnolol on intestinal mucosal epithelial cells in sepsis and elucidate the underlying mechanisms.METHODS Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,and regulated on activation,normal T-cell expressed and secreted(RANTES)levels in serum and ileal tissue in animal studies.The histopathological changes of the ileal mucosa in different groups were observed under a microscope.Cell Counting Kit-8 and cell permeability assays were used to determine the concentration of drug-containing serum that did not affect the activity of Caco2 cells but inhibited lipopolysaccharide(LPS)-induced decrease in permeability.Immunofluorescence and Western blot assays were used to detect the levels of RANTES,inhibitor of nuclear factor kappa-B kinaseβ(IKKβ),phosphorylated IKKβ(p-IKKβ),inhibitor of nuclear factor kappa-B kinaseα(IκBα),p65,and p-p65 proteins in different groups in vitro.RESULTS In rats treated with LPS by intravenous tail injection in the presence or absence of magnolol,magnolol inhibited the expression of proinflammatory cytokines,IL-1β,IL-6,and TNF-αin a dose-dependent manner.In addition,magnolol suppressed the production of RANTES in LPS-stimulated sepsis rats.Moreover,in vitro studies suggested that magnolol inhibited the increase of p65 nucleation,thereby markedly downregulating the production of the phosphorylated form of IKKβin LPS-treated Caco2 cells.Specifically,magnolol inhibited the translocation of the transcription factor nuclear factor-kappa B(NF-κB)from the cytosol into the nucleus and down-regulated the expression level of the chemokine RANTES in LPS-stimulated Caco2 cells.CONCLUSION Magnolol down-regulates RANTES levels by inhibiting the LPS/NF-κB signaling pathways,thereby suppressing IL-1β,IL-6,and TNF-αexpression to alleviate the mucosal barrier dysfunction in sepsis.

Notch信号促进核因子κB受体活化因子配体诱导的破骨细胞分化的体外研究

目的探讨Notch信号抑制对核因子κB受体活化因子配体(RANKL)诱导的小鼠RAW264.7细胞向破骨细胞分化的影响。方法建立RANKL诱导的小鼠RAW264.7细胞体外分化模型,实时定量聚合酶链反应(real-time PCR)检测Notch信号成分(Notch1、Notch2、Delta1、Jagged1)、下游靶基因Hes1以及破骨细胞标志基因抗酒石酸酸性磷酸酶(TRAP)和Cathepsin K在诱导前后mRNA的表达。在诱导体系中加入不同浓度的γ分泌酶抑制剂(GSI),抑制Notch受体的表达,TRAP染色检测破骨细胞分化的变化情况。结果 50 ng·m L-1 RANKL诱导小鼠RAW264.7细胞3 d,Notch1、Notch2、Delta1、Jagged1及Hes1的mRNA表达均有不同程度的提高,其中以Notch2、Jagged1增高最明显;破骨细胞标志基因表达显著增高。在RANKL诱导的同时加入不同浓度GSI,抑制Notch的表达,可致Notch下游靶基因Hes1表达下降,同时TRAP阳性细胞计数显著减少,且呈剂量依赖性。结论 Notch信号可促进RANKL诱导的RAW264.7细胞向破骨细胞分化。

Anluohuaxianwan Alleviates Carbon Tetrachloride-Induced Hepatic Fibrosis in Rats through Upregulation of Peroxisome Proliferator-Activated Receptor-Gamma and Downregulation of Nuclear Factor-Kappa B/IκBα Signaling Pathway

Objective:The aim of this study was to investigate the effects of traditional Chinese medicine Anluohuaxianwan(ALHXW)on peroxisome proliferator-activated receptor-gamma(PPARγ)and nuclear factor-kappa B(NF-κB)signaling pathways using a rat model of carbon model groups were gavaged with saline for 6 weeks.Liver function was measured,and liver fibrosis and necroinflammation were assessed.Protein and messenger RNA expression levels of PPARγ,NF-κB,and Inhibitorαof NF-κB(IκBα)were analyzed by Western blot and reverse transcription–quantitative polymerase chain reaction.Results:ALHXW markedly alleviated liver injury compared with the model group,as indicated by the improvements in disease status,the morphology of liver and spleen,the liver and spleen indexes,and liver function.The extent of liver fibrosis was improved,hepatic stellate cell activation was inhibited,the expression of PPARγand IκBαwas significantly higher,and the expression of NF-κB was significantly lower in the treatment group as compared with the model group.Conclusions:ALHXW treatment can alleviate CCl4-induced liver fibrosis in rats, and the potential antifibrogenic mechanisms may occur through the upregulation of PPARγ expression and downregulation of NF-κB/IκBα signaling pathway.

Different effects of antisense IRAK-2 oligonucleotide on IL-1 and TNF-stimulated NF-kappa B activation

Objective: To investigate the role of interleukin-1 (IL-1) receptor associated kinase-2 (IRAK-2) in IL1 and TNF-stimulated nuclear factor-kappa B (NF-kappa B ) activation. Metbods: The 293 cell was trans fectedwith antisense IRAK-2 oligonucleotide (IRAK-2 ODN) followed by stimulating the cell with IL-1 or tumor necrosis factor (TNF), and then the levels of NF-kappa B activation was analyzed by sandwich enzyme-linked immunosorbent assay (ELISA ). Result: Pre-transfecting with antisense IRAK-2 ODN could remarkably decreasethe levels of NF-kappa B activation stimulated by IL-1 in time- and concentration-dependent manner, it can not attenuate the one stimulated by TNF. Conclusion: The responses of IL-1 and TNF-stimulated NF-kappa B activation to antisense IRAK-2 oligonucleotids were different. IRAK-2 plays a key role in the IL-1 signaling events leading to NF kappa B activation.

核因子κB受体活化因子配体抑制剂地舒单抗在肺癌骨转移中的应用

目前,随着基因靶向治疗和免疫治疗在肺癌领域中取得巨大突破,晚期肺癌患者总生存期(OS)显著延长,但发生远处骨转移及骨相关事件(SRE),如骨痛、病理性骨折、脊髓压迫、高钙血症等风险也随之增大,严重影响了患者的生活质量。因此,在全身治疗基础上应积极预防和治疗骨转移及SRE治疗。临床研究表明,核因子κB受体活化因子(RANK)配体(RANKL)/RANK/骨保护素信号通路在肿瘤骨转移中发挥着重要作用,阻断该通路能有效预防和治疗SRE。RANKL抑制剂地舒单抗(商品名:安加维)是一种人免疫球蛋白G2单克隆抗体,可特异性结合RANKL而阻断破骨细胞参与的RANKL/RANK/骨保护素信号通路激活,最终发挥预防骨转移及SRE的作用。与双磷酸盐类药物比较,地舒单抗疗效显著,能明显延长患者OS和SRE的发生时间。同时,地舒单抗还具有抗肿瘤作用。该文就地舒单抗的作用机制、临床研究及安全性等方面的最新研究进行了阐述,以期为临床医生提供参考。

龈沟液RANKL、音猬因子水平对牙缺失Nobel种植体早期稳定性评估价值

目的探讨龈沟液核因子-κB受体活化因子配体(RANKL)、音猬因子水平对牙缺失Nobel种植体早期稳定性评估价值。方法选取2020年6月至2021年12月邯郸市第一医院牙缺失患者128例作为观察组,均为Nobel种植体修复,选取同期体检健康者64例作为对照组。对比两组及观察组术后即刻、术后3个月龈沟液RANKL、音猬因子水平、种植体稳定系数(ISQ)值,多重线性回归模型分析Nobel种植体早期稳定性影响因素,并分析龈沟液RANKL、音猬因子水平对早期稳定性评估价值。结果观察组术后3个月龈沟液RANKL、音猬因子水平低于术后即刻,差异有统计学意义(P<0.05);观察组术后3个月ISQ值高于术后即刻,差异有统计学意义(P<0.05)。术后即刻龈沟液RANKL、音猬因子水平与术后3个月ISQ值呈负相关(P<0.05)。术后即刻及术后3个月龈沟液RANKL、音猬因子水平对应的线性系数差异有统计学意义(P<0.05);建立多重线性回归模型:术后3个月ISQ值=100.968-4.607×术后咬(牙合)痛-2.886×常食坚果-0.254×术后即刻龈沟液RANKL-0.280×术后即刻龈沟液音猬因子,回归模型具有统计学意义(P<0.05),自变量可解释术后3个月ISQ值74.59%的变异量。结论牙缺失患者进行Nobel种植后龈沟液RANKL、音猬因子呈低表达,术后通过检测二者水平可有效判断种植体早期稳定性。

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.

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.

Imbalance of osteoprotegerin/receptor activator of nuclear factor-κB ligand and oxidative stress in patients with obstructive sleep apnea-hypopnea syndrome

Background:Obstructive sleep apnea-hypopnea syndrome (OSAHS) is associated with a higher prevalence of osteoporosis.However,the underlying mechanisms linking OSAHS with bone loss are still unclear.The aim of this study was to investigate the changes of receptor activator of nuclear factor-κB ligand (RANKL,an osteoclastogenesis-promoting factor) and osteoprotegerin (OPG,the decoy receptor for RANKL),oxidative stress and bone metabolism markers in OSAHS,in order to understand the potential mechanisms underlying bone loss in OSAHS patients.Methods:Forty-eight male patients with OSAHS,confirmed by polysomnography (PSG) study,were enrolled.Twenty male subjects who were confirmed as not having OSAHS served as the controls.The subjects’bone mineral density (BMD) was assessed in lumbar spine and femoral neck using dual-energy X-ray absorptiometry (DXA).Blood samples were collected from all subjects for measurement of RANKL,OPG,the bone formation marker bone-specific alkaline phosphatase (BAP),the bone resorption marker tartrate-resistant acid phosphatase 5b (TRAP-5b),and total antioxidant capacity (TAOC).Results:The BMD and the T-score of the femoral neck and the lumbar spine were significantly lower in OSAHS patients as compared to the control group (P< 0.05).The serum level of BAP was significantly decreased in the OSAHS group (15.62 ± 5.20 μg/L) as compared to the control group (18.83 ± 5.50 μg/L,t= -2.235,P< 0.05),while the levels of TRAP-5b did not differ between the two groups (t= -1.447,P> 0.05).The serum level of OPG and the OPG/RANKL ratio were lower in the OSAHS group compared to the control group (bothP< 0.05).TAOC level was also decreased significantly in the OSAHS group (P< 0.05).Correlation analysis showed that the TAOC level was positively correlated with BAP in the OSAHS group (r= 0.248,P= 0.04),but there were no correlations between TAOC and the BMD or the T-scores.The correlations between the level of OPG (or the OPG/RANKL ratio) and BMD or TAOC did not reach significance.Conclusion:In OSAHS patients,lower levels of TAOC were associated with decreased bone formation,suggesting a role of oxidative stress in bone loss,while the role of OPG/RANKL imbalance in bone metabolism in OSAHS needs further evaluation .

Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

Background The cannabinoid receptor-2 （CB2） is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist （AM630） on receptor activator of nuclear factor kappa B （RANK） ligand （RANKL）induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase （TRAP） staining using a commercial kit. Total ribonucleic acid （RNA）was isolated and real-time reverse transcriptase-polymerase chain reaction （RT-PCR） was done to examine the expression of RANK, cathepsin K （CPK） and nuclear factor kappa B （NF-κB）. The extracellular signal-regulated kinase （ERK）,phosphorylation of ERK （P-ERK） and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazoliumbromide （MTT） assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of 〉100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

Effect of Wenhua Juanbi Recipe(温化蠲痹方) on Expression of Receptor Activator of Nuclear Factor Kappa B Ligand,Osteoprotegerin,and Tumor Necrosis Factor Receptor Superfamily Member 14 in Rats with Collagen-Induced Arthritis

Objective： To study the effect of Wenhua Juanbi Recipe（温化蠲痹方, WJR） on expression of receptor activator of nuclear factor kappa B ligand（RANKL）, osteoprotegerin（OPG）, and tumor necrosis factor receptor superfamily member 14（TNFRSF14, also known as LIGHT） in rats with collagen-induced arthritis（CIA）. Methods： CIA rats were generated by subcutaneous injection of bovine collagen type-Ⅱ at the tail base. Sixty CIA rats were randomly assigned（10 animals/group） to： model, methotrexate（MTX）-treated（0.78 mg/kg body weight）, and WJR-treated（22.9 g/kg） groups. Healthy normal rats（n=10） were used as the normal control. Treatments or saline were administered once daily by oral gavage. Rats were sacrificed at day 28 post-treatment and knee synovium and peripheral blood serum were collected. Toe swelling degree and expression of RANKL, OPG, and LIGHT were determined by Western blot and immunohistochemistry. Results： Compared with the normal group, toe swelling degree was significantly increased in the model group（P〈0.01）. After treatment, toe swelling degree decreased significantly in the WJR and MTX groups compared with the model group（P〈0.01）. Compared with the normal group, expression of RANKL and LIGHT were significantly increased and OPG significantly decreased in peripheral blood and synovium of the model group（P〈0.01）. Conversely, RANKL and LIGHT expression were significantly reduced and OPG increased in the WJR and MTX groups compared with the model group（P〈0.01）. No statistically significant difference existed between WJR and MTX groups. Conclusion： WJR likely acts by reducing RANKL expression and increasing OPG expression, thus inhibiting RANKL/RANK interaction and reducing LIGHT expression, thereby inhibiting osteoclast formation/activation to block bone erosion.