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.

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.

Receptor activator of nuclear factorκB ligand/osteoprotegerin axis and vascular calcifications in patients with chronic kidney disease

Vascular calcifications are commonly observed in patients with chronic kidney disease （CKD） and contri-bute to the excessive cardiovascular morbidity and mortality rates observed in these patients populations. Although the pathogenetic mechanisms are not yet fully elucidated, recent evidence suggests a link between bone metabolism and the development and progression of vascular calcifications. Moreover, accumulating data indicate that receptor activator of nuclear factor κB ligand/osteoprotegerin axis which plays essential roles in the regulation of bone metabolism is also involved in extra-osseous bone formation. Further studies are required to establish the prognostic significance of the above biomarkers as predictors of the presence and severity of vascular calcifications in CKD patients and of cardiovascular morbidity and mortality. Moreover, randomized clinical trials are needed to clarify whether inhibition of osteoclast activity will protect from vascular calcifcations.

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.

Apigenin ameliorates imiquimod-induced psoriasis in C57BL/6J mice by inactivating STAT3 and NF-κB

Psoriasis is a chronic autoimmune disease featured by patches on the skin.It is caused by malfunction of immune cells and keratinocytes with inflammation as one of its key features.Apigenin(API)is a natural flavonoid with anti-inflammatory and immunoregulatory properties.Therefore,we speculated that API can ameliorate psoriasis,and determined its effect on the development of psoriasis by using imiquimod(IMQ)-induced psoriasis mouse model.Our results showed that API attenuated IMQ-induced phenotypic changes,such as erythema,scaling and epidermal thickening,and improved splenic hyperplasia.Abnormal differentiation of immune cells was restored in API-treated mice.Mechanistically,we revealed that API is a key regulator of signal transducer activator of transcription 3(STAT3).API regulated immune responses by reducing interleukin-23(IL-23)/STAT3/IL-17A axis.Moreover,it suppressed IMQ-caused cell hyperproliferation by inactivating STAT3 through regulation of extracellular signal-regulated kinase 1/2 and nuclear factor-κB(NF-κB)pathway.Furthermore,API reduced expression of inflammatory cytokines through inactivation of NF-κB.Taken together,our study demonstrates that API can ameliorate psoriasis and may be considered as a strategy for psoriasis treatment.

Glycine Attenuates Myocardial Fibrosis in Myocardial Infarction in Rats Partly through Modulating Signal Transducer and Activator of Transcription 3/Nuclear Factor-κB/Transforming Growth Factor-β axis

Objective: Inflammation and fibrosis are strongly associated with each other. Glycine is present in various traditional Chinese medicines and exhibits anti-inflammatory activity. However, the effects of glycine on myocardial fibrosis(MF) in rats with myocardial infarction(MI) have not been reported. The purpose of this study is to investigate the effects of glycine therapy on MF and comprehend its underlying mechanisms. Materials and Methods: Left anterior descending artery ligation-induced MI in Sprague Dawley rats was leveraged to assess the therapeutic effects of Glycine. Rats received either normal saline or glycine(0.5 mg/g bodyweight) for 7 days. Results: Glycine upregulated cardiac ejection fraction and fractional shortening to improve cardiac function, as evaluated by echocardiography. Histological and immunohistochemical analyses demonstrated that glycine could decrease inflammatory cell infiltration and alleviate collagen deposition. Western blotting revealed that nuclear factor-κB(NF-κB)-mediated inflammatory signaling was also downregulated by glycine treatment. The expression of signal transducer and activator of transcription 3(STAT3), tumor necrosis factor-α, and transforming growth factor-β(TGF-β) was decreased significantly in the glycine-treated group compared to the model group. Thus, glycine plays a protective role against myocardial ischemia and subsequent MF. Conclusion: The protective effects of glycine were achieved partly through STAT3/NF-κB/TGF-β signaling pathway.

辐射对RANKL诱导RAW264.7细胞向破骨细胞分化中CBFα1表达水平的影响

为了研究辐射对于破骨细胞中Notch通路的影响,进一步了解辐射诱发骨损伤的机理,本研究采用核因子κB受体活化因子配体(Receptor activator of nuclear Factor-B Ligand,RANKL)诱导RAW264.7细胞系生成的破骨细胞,经2 Gy 137Csγ射线照射后应用实时定量聚合酶链反应(Real-time polymerase chain reaction,Real-time PCR)方法,检测其Notch通路重要靶位启动子C结合因子α1(C Promoter-Binding Factorα1,CBFα1)表达的变化情况。实验结果表明,在2 Gy 137Csγ射线照射后,CBFα1在RANKL作用下生成的破骨细胞中表达明显增高。辐射诱发的Notch通路中CBFα1表达增高,可能是由于辐射增强了Notch通路对破骨细胞分化的促进作用,增加了破骨细胞的数量,增强了其活性,从而导致骨损伤、骨质疏松、甚至骨折的发病率增高。

PTHrP促进RANKL诱导巨噬细胞分化为破骨细胞参与中耳胆脂瘤骨破坏

目的:骨质进行性吸收破坏是中耳胆脂瘤最重要的临床特征之一,可导致一系列颅内外并发症,而目前中耳胆脂瘤骨破坏的机制尚未明确。本研究旨在探究甲状旁腺激素相关蛋白(parathyroid hormone-related protein,PTHrP)参与中耳胆脂瘤骨破坏的机制。方法:收集后天性中耳胆脂瘤患者的25例胆脂瘤标本和13例外耳道正常皮肤组织标本。采用免疫组织化学染色方法检测PTHrP、核因子κB受体活化因子配体(receptor activator for nuclear factor-kappa B ligand,RANKL)和骨保护素(osteoprotegerin,OPG)在中耳胆脂瘤和外耳道正常皮肤组织中的表达,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法检测中耳胆脂瘤和外耳道正常皮肤组织中是否存在TRAP阳性多核巨噬细胞。选取小鼠单核巨噬细胞RAW264.7细胞进行干预,分为RANKL干预组和PTHrP+RANKL共同干预组,采用TRAP染色法检测2组破骨细胞的生成情况,实时聚合酶链反应(real-time polymerase chain reaction,real-time PCR)检测干预后2组破骨细胞相关基因TRAP、组织蛋白酶K(cathepsin K,CTSK)和活化T细胞核因子1(nuclear factor of activated T cell cytoplasmic 1,NFATc1)的mRNA表达水平,骨吸收陷窝实验检测2组破骨细胞的骨吸收功能。结果:免疫组织化学染色结果显示,PTHrP和RANKL在中耳胆脂瘤组织中的表达均显著增高,OPG表达降低(均P<0.05),且PTHrP的表达与RANKL、RANKL/OPG比值均呈显著正相关,与OPG表达呈显著负相关(分别r=0.385、r=0.417、r=-0.316,均P<0.05)。同时,PTHrP、RANKL的表达水平与中耳胆脂瘤的骨破坏程度均呈显著正相关(分别r=0.413、r=0.505,均P<0.05)。TRAP染色结果显示中耳胆脂瘤上皮周围基质中有大量TRAP阳性细胞,并存在细胞核数量为3个或3个以上的TRAP阳性破骨细胞。RANKL或PTHrP+RANKL联合干预5 d后,与RANKL干预组相比,PTHrP+RANKL联合干预组的破骨细胞数量显著增加(P<0.05),且破骨细胞相关基因TRAP、CTSK和NFATc1的mRNA表达水平均升高(均P<0.05)。骨吸收陷窝扫描电镜结果显示RANKL干预组、PTHrP+RANKL联合干预组的骨片表面均形成骨吸收陷窝;与RANKL干预组相比,PTHrP+RANKL联合干预组的骨片表面骨吸收陷窝数量显著增加(P<0.05),面积也更大。结论:PTHrP可能通过促进RANKL诱导胆脂瘤组织周围基质中的巨噬细胞分化为破骨细胞,参与中耳胆脂瘤骨破坏。

抗MCV抗体与RANKL、OPG、TRACP-5b及RA疾病活动度的相关性研究

目的探索类风湿关节炎(rheumatoid arthritis,RA)患者抗突变型瓜氨酸波形蛋白(mutant citrulline vimentin,MCV)抗体与骨代谢标志物及疾病活动度之间的关系。方法收集119例RA患者临床资料和血清,检测抗MCV抗体及RANKL、OPG和TRACP-5b等骨代谢标志物,分析抗MCV抗体与骨代谢标志物之间的相关性,并探索抗MCV抗体与疾病活动是否有关联。结果RA患者抗MCV抗体滴度与RANKL、TRACP-5b水平及RANKL/OPG均无显著相关性(P>0.05),与OPG呈正相关但相关系数低(r=0.183,P<0.05)。②抗MCV抗体与DAS28(r=0.376,P<0.01)、ESR(r=0.440,P<0.01)、RF-IgM(r=0.376,P<0.01)呈正相关。结论抗MCV抗体与血清中骨代谢相关标志物TRACP-5b、RANKL、OPG、RANKL/OPG均无显著相关性,提示抗MCV抗体可能不直接通过影响骨代谢参与RA骨侵蚀进展。抗MCV抗体与疾病活动度呈正相关,提示它对RA病情评估可能有一定的价值。

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 .

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.

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.

Regulation of bone destruction in rheumatoid arthritis through RANKL-RANK pathways

Recent studies have demonstrated that osteoclasts, the primary cells responsible for bone resorption, are mainly involved in bone and joint destruction in rheumatoid arthritis(RA) patients. Recent progress in bone cell biology has revealed the molecular mechanism of osteoclast differentiation and bone resorption by mature osteoclasts. We highlight here the potential role of the receptor activator of nuclear factor κB ligand(RANKL)-RANK pathways in bone destruction in RA and review recent clinical trials treating RA by targeting RANKL.

Mindin is upregulated during colitis and may activate NF-κB in a TLR-9 mediated manner

AIM:To investigate the regulation of mindin expression and the signaling pathway involved during inflammation.METHODS:C57BL/6 mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 6 d to induce acute colitis,and then the colon was harvested for histological analysis or for RNA isolation.mRNA expression of mindin and nuclear factor (NF)-κB p65 was analyzed by quantitative real time polymerase chain reaction (RT-PCR) and mindin expression construct was conf irmed by Western blotting.Mouse macrophage and intestinal epithelial lineage cells were stimulated with different cytokines and toll-like receptor (TLR) ligands,before pNF-κB-luciferase activity was assessed using the Dual-Luciferase reporter assay system.RESULTS:mRNA expression of mindin was upregulated 4.7 ± 1.1 fold compared with the baseline during DSS-induced intestinal inflammation in the mice.Stimulation with CpG-ODN (a known TLR-9 ligand) induced 4.2 ± 0.3 fold upregulation of mindin expression in RAW 264.7 cells.Full-length of mindin was cloned from cDNA of mouse mesenteric lymph node,then the pCMV-Mindin-Flag expression vector was established and the protein expression level was confi rmed.Transfection of the mindin construct and stimulation with CpG-ODN signifi cantly increased the NF-κB-luciferase activity by 2.5 ± 0.3 and 4.5 ± 0.5 fold in RAW264.7 and CMT93 cells,respectively (P < 0.01).CONCLUSION:Mindin expression is upregulated during intestinal inflammation and may induce NF-κB promoter activation in a TLR-9 mediated manner.

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.

Promising Effects of Zerumbone on the Regulation of Tumor-promoting Cytokines Induced by TNF-α-activated Fibroblasts

Inflammation plays an important role in the development of several cancers.Inflammatory cytokines,including tumor necrosis factor-α(TNF-α),are associated with the induction of inflammation.Chronic inflammation contributes to the progression of cancer through several mechanisms,including increased cytokine production and activation of transcription factors,such as nuclear factor-κB(NF-κB).Zerumbone(ZER),a component of subtropical ginger(Zingiber zerumbet Smith),seems to have anti-inflammatory,anti-cancer,and antioxidant activities.In this study,we aimed to explore the protective function and mechanisms of ZER against TNF-α-induced cancer-promoting cytokines.We found that the viability of stimulated human fibroblast cell lines was reduced after treatment with ZER(IC50=18µmol/L),compared to un-stimulated fibroblasts(IC50=40µmol/L).Besides,ZER inhibited mRNA expression and protein secretion of transforming growth factor-β(TGF-β),interleukin-33(IL-33),monocyte chemoattractant protein-1(MCP-1),and stromal cell-derived factor 1(SDF-1),which were produced by TNF-α-induced fibroblasts,as measured by quantitative real time-PCR(qRT-PCR)and ELISA assays.The mRNA expression levels of TGF-β,IL-33,SDF-1,and MCP-1 showed 8,5,2.5,and 4-fold reductions,respectively.Moreover,secretion of TGF-β,IL-33,SDF-1,and MCP-1 was reduced to 3.65±0.34 ng/mL,6.3±0.26,1703.6±295.2,and 5.02±0.18 pg/mL,respectively,compared to the untreated group.In addition,the conditioned media(CM)of TNF-α-stimulated fibroblasts increased the NF-κB expression in colorectal cancer cell lines(HCT-116 and Sw48),while in the vicinity of ZER,the expression of NF-κB was reversed.Considering the significant effects of ZER,this component can be used as an appropriate alternative herbal treatment for cancer-related chronic inflammation.

CARMA3: A novel scaffold protein in regulation of NF-κB activation and diseases

CARD recruited membrane associated protein 3 (CARMA3) is a novel scaffold protein. It belongs to the CARMA protein family, and is known to activate nuclear factor (NF)- κB. However, it is still unknown which receptor functions upstream of CARMA3 to trigger NF-κB activation. Recently, several studies have demonstrated that CARMA3 serves as an indispensable adaptor protein in NF-κB signaling under some G protein-coupled receptors (GP- CRs), such as lysophosphatidic acid (LPA) receptor and angiotensin (Ang) Ⅱ receptor. Mechanistically, CARMA3 recruits its essential downstream molecules Bcl10 and MALT1 to form the CBM (CARMA3-Bcl10-MALT1) signalosome whereby it triggers NF-κB activation. GPCRs and NF-κB play pivotal roles in the regulation of various cellular functions, therefore, aberrant regulation of the GPCR/NF-κB signaling axis leads to the development of many types of diseases, such as cancer and atherogenesis. Recently, the GPCR/CARMA3/NF-κB signaling axis has been confirmed in these specific diseases and it plays crucial roles in the pathogenesis of disease progression. In ovarian cancer cell lines, knockdown of CARMA3 abolishes LPA receptor-induced NF-κB activation, and reduces LPA-induced ovarian cancer invasion. In vascular smooth cells, downregulation of CARMA3 substantially impairs Ang-Ⅱ-receptor-induced NF-κB activation, and in vivo studies have confirmed that Bcl10- deficient mice are protected from developing Ang-Ⅱ-receptor-induced atherosclerosis and aortic aneurysms. In this review, we summarize the biology of CARMA3, describe the role of the GPCR/CARMA3/NF-κB signaling axis in ovarian cancer and atherogenesis, and speculate about the potential roles of this signaling axis in other types of cancer and diseases. With a significant increase in the identification of LPA- and Ang-Ⅱ-like ligands, such as endothelin-1, which also activates NF-κB via CARMA3 and contributes to the development of many diseases, CARMA3 is emerging as a novel therapeutic target for various types of cancer and other diseases.

Down-regulation of peroxisome proliferator-activated receptor γ coactivator-1α expression in fatty acid-induced pancreatic betacell apoptosis involves nuclear factor-κB pathway

Background Pancreatic beta-cell apoptosis induced by lipotoxicity, to a large extent, contributes to the progression of type 2 diabetes. To investigate the mechanism of free fatty acid induced apoptosis, we aimed to study the effects of palmitic acid （PA） on the apoptosis and peroxisome proliferator-activated receptor y coactivator-1α （PGC-1α） expression in βTC3 cells as well as the possible role of nuclear factor-KB （NF-KB） in this process. Methods Hoechst 33258 was used to detect βTC3 cell apoptosis, which was induced by PA stimulation for 12 hours. PGC-1α expression was analyzed by reverse transcription polymerase chain reaction, IκB kinase β （IKKβ）, IκBα NF-KB-inducing kinase （NIK） and ReI-B expressions were analyzed by Western blotting. MGβ2 was employed to block the endogenous IκBαdegradation before PA administration, and then its effect on PA-inducing cell apoptosis and PGC-1α mRNA expression was analyzed. Results Significant increased cell apoptosis was found at the concentration of 0.5 mmol/L and 1.0 mmol/L PA administration. PA （0.5 mmol/L） could extensively reduced the expression of PGC-1α mRNA. After exposing βTC3 cells to 0.5 mmol/L PA for different time periods （0, 4, 6, 8, 10 and 12 hours）, IKKβ protein expression increased while IκBα NIK and ReI-B protein expression declined in a time-dependent manner. Pretreatment with MGβ2 to inhibit the degradation of IκBα partially prevented the down-regulation of PGC-1α mRNA expression after 12-hour PA treatment in accordance with the decrease of PA induced apoptosis. Conclusions NF-KB canonical pathway was activated in PA-mediated βTC3 cell apoptosis, whereas non-canonical pathway was inhibited. Reduced PGC-1α expression by PA in βTC3 cells could involve the activation of canonical NF-KB pathway, so as to deteriorate the PA induced apoptosis.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.