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.

Silencing of Jumonji domain-containing 1C inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells via nuclear factor-κB signaling

BACKGROUND Osteoporosis is a common metabolic bone disorder induced by an imbalance between osteoclastic activity and osteogenic activity.During osteoporosis,bone mesenchymal stem cells(BMSCs)exhibit an increased ability to differentiate into adipocytes and a decreased ability to differentiate into osteoblasts,resulting in bone loss.Jumonji domain-containing 1C(JMJD1C)has been demonstrated to suppress osteoclastogenesis.AIM To examine the effect of JMJD1C on the osteogenesis of BMSCs and the potential underlying mechanism.METHODS BMSCs were isolated from mouse bone marrow tissues.Oil Red O staining,Alizarin red staining,alkaline phosphatase staining and the expression of adipo-genic and osteogenic-associated genes were assessed to determine the differen-tiation of BMSCs.Bone marrow-derived macrophages(BMMs)were incubated with receptor activator of nuclear factor-kappaΒligand to induce osteoclast differentiation,and osteoclast differen-tiation was confirmed by tartrate-resistant acid phosphatase staining.Other related genes were measured via reverse transcription coupled to the quantitative polymerase chain reaction and western blotting.Enzyme-linked immunosorbent assays were used to measure the levels of inflammatory cytokines,including tumor necrosis factor alpha,interleukin-6 and interleukin-1 beta.RESULTS The osteogenic and adipogenic differentiation potential of BMSCs isolated from mouse bone marrow samples was evaluated.JMJD1C mRNA and protein expression was upregulated in BMSCs after osteoblast induction,while p-nuclear factor-κB(NF-κB)and inflammatory cytokines were not significantly altered.Knockdown of JMJD1C repressed osteogenic differentiation and enhanced NF-κB activation and inflammatory cytokine release in BMSCs.Moreover,JMJD1C expression decreased during BMM osteoclast differentiation.CONCLUSION The JMJD1C/NF-κB signaling pathway is potentially involved in BMSC osteogenic differentiation and may play vital roles in the pathogenesis of osteoporosis.

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 .

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.

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.

Jianpi Qingchang decoction alleviates ulcerative colitis by inhibiting nuclear factor-κB activation

To investigate the therapeutic effect of Jianpi Qingchang decoction (JPQCD) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice.METHODSC57BL/c mice were injected intragastrically with 5% DSS instead of drinking water for 7 d, and their body weight, diarrhea severity and fecal bleeding were monitored, while the mice in the control group were treated with standard drinking water, without DSS. After 7 d, the DSS drinking water was changed to normal water and the DSS group continued with DSS water. The control and DSS groups were given normal saline by intragastric injection. The 5-aminosalicylic acid (5-ASA) group was treated orally with 5-ASA at a dose of 100 mg/kg daily. The JPQCD group was treated orally with JPQCD at a dose of 17.1 g/kg daily. On day 14, the colon length was measured, the colorectal histopathological damage score was assessed, and protein levels of interleukin (IL)-1β, IL-8 and tumor necrosis factor-alpha (TNF-α) in colon supernatants were measured by enzyme-linked immunosorbent assay. mRNA expression of IL-1β, IL-8, TNF-α and nuclear factor-kappa B (NF-κB) was detected by real-time quantitative polymerase chain reaction. Western blotting was used to detect the protein expression of NF-κB and inhibitor of kappa B.RESULTSAcute inflammation occurred in the mice administered DSS, including the symptoms of losing body weight, loose feces/watery diarrhea and presence of fecal blood; all these symptoms worsened at 7 d. The colons of mice treated with DSS were assessed by histological examination, and the results confirmed that acute inflammation had occurred, as evidenced by loss of colonic mucosa and chronic inflammatory cell infiltration, and these features extended into the deeper layer of the colon walls. The expression levels of IL-1β, IL-8 and TNF-α in the DSS group were higher than those in the control group (P < 0.05), and the expression levels of IL-1β, IL-8 and TNF-α in the JPQCD and 5-ASA groups were lower than those in the DSS group after treating with JPQCD and 5-ASA. Comparing with the DSS group, the mRNA level of IL-1β, IL-8, TNF-α and NF-κB was significantly reduced by 5-ASA and JPQCD. The difference between JPQCD and 5-ASA groups was not statistically significant (P > 0.05). Comparing with the DSS group, due to using JPQCD and 5-ASA, significant suppression of activation in DSS-induced NF-κB and increased phosphorylation of IκB in mice with experimental colitis occurred (P < 0.05). The difference between the JPQCD group and the 5-ASA group was not statistically significant (P > 0.05).CONCLUSIONActivation of the NF-κB signaling pathway is inhibited by JPQCD, which shows the potential mechanism by which JPQCD treats UC.

Propofol induces apoptosis and increases gemcitabine sensitivity in pancreatic cancer cells in vitro by inhibition of nuclear factor-κ B activity

AIM:To investigate the effect of propofol on human pancreatic cells and the molecular mechanism of propofol action.METHODS:We used the human pancreatic cancer cell line MIAPaCa-2 for in vitro studies measuring growth inhibition and degree of apoptotic cell death induced by propofol alone,gemcitabine alone,or propofol followed by gemcitabine.All experiments were conducted in triplicate and carried out on three or more separate occasions.Data were means of the three or more independent experiments±SE.Statistically significant differences were determined by two-tailed unpaired Student’s t test and defined as P<0.05.RESULTS:Pretreatment of cells with propofol for 24 h followed by gemcitabine resulted in 24%-75% growth inhibition compared with 6%-18%when gemcitabine was used alone.Overall growth inhibition was directly correlated with apoptotic cell death.We also showed that propofol potentiated gemcitabine-induced killing by downregulation of nuclear factor-κB(NF-κB).In contrast,NF-κB was upregulated when pancreatic cancer cells were exposed to gemcitabine alone,suggesting a potential mechanism of acquired chemoresistance.CONCLUSION:Inactivation of the NF-κB signaling pathway by propofol might abrogate gemcitabineinduced activation of NF-κB,resulting in chemosensitization of pancreatic tumors to gemcitabine.

Effects of Ginsenoside Rg1 on nuclear factor-kappa B activity in beta amyloid protein-treated neural cells

BACKGROUND： Modern pharmacological studies have shown that Ginsenoside Rgl is one of the active components of ginseng that promote intelligence in the nervous system. Ginsenoside Rgl can improve memory and learning in mouse models of β-amyloid protein （Aβ）-induced dementia. OBJECTIVE： To investigate whether effects of Ginsenoside Rgl against Aβ are associated with activity of nuclear factor-kappa B （NF-κB）. DESIGN, TIME AND SETTING： The randomized performed at the DME Center, Institute of Clinica controlled, cell biological experiment was Pharmacology, Guangzhou University of Chinese Medicine, China from July 2005 to May 2006. MATERIALS： Beta-amyloid fragment 25-35 （Aβ25-35） was supplied by the Neural Biochemical Laboratory, Xuanwu Hospital, Capital Medical University, China. Ginsenoside Rgl was obtained from National Institute for the Control of Pharmaceutical and Biological Products, China. Rabbit anti-rat NF-κB p65 antibody was purchased from Santa Cruz Biotechnology, USA. METHODS： Hippocampal neurons and cortical astrocytes of neonatal Sprague Dawley rats were harvested and treated with various concentrations （0, 5, 10, 20, and 40 μmol/L） of Aβ for 6, 12, and 24 hours to establish cellular models of Alzheimer＇s disease. Cellular models were pretreated with various concentrations of Ginsenoside Rgl （1,2, 4, 8, and 16 μmol/L）. According to cell morphology and activity, the following conditions were selected： 40 μmol/L Aβ for 24 hours, as well as 2, 4, and 8 μmol/L Ginsenoside Rg1. NF-κB activity was observed using immunofluorescence and cytochemical staining. MAIN OUTCOME MEASURES： Morphology and viability of hippocampal neurons and cortical astrocytes, and activities of NF-κB were measured. RESULTS： Hippocampal neuron activity was significantly greater in the normal and 2 and 4 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.05）. Astrocyte activity was significantly greater in the normal, 1,2, 4, 8, and 16 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.05）. NF-κB activity of hippocampal neurons was significantly greater in the normal, 2, 4, and 8 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.01）. NF-κB activity of astrocytes was significantly less in the normal, 2, 4, and 8 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.01 or P 〈 0.05）. No significant difference in NF-κB activity was determined between the 2 μmol/L Ginsenoside Rgl and normal groups （P 〉 0.05）. CONCLUSION： Ginsenoside Rgl protected neural cells by upregulating NF-κB activity in neurons and downregulating NF-κB activity in astrocytes. Ginsenoside Rgl （2 μmol/L） maintained cell activity and NF-κB activity at normal levels.

Methanol extract of Codium fragile inhibits tumor necrosis factor-ɑ-induced matrix metalloproteinase-9 and invasiveness of MDA-MB-231 cells by suppressing nuclear factor-κB activation

Objective:To evaluate whether the methanol extract of Codium fragile(MECF) regulates tumor necrosis factor-α(TNF-α)-induced invasion of human breast cancer MDA-MB-231 cells by suppressing matrix metalloproteinase-9(MMP-9).Methods:Reverse transcriptionpolymerase chain reaction(RT-PCR) and western blot analysis were performed to analyze the expression of MMP-9 and nuclear factor-κB(NF-κB) subunits,p65 and p50,and IκB in MDA-MB-231 cells.3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT) assay was used for cell viability.MMP-9 activity and invasion were measured by gelatin zymography and a matrigel invasion assay,respectively.NF- κB activity was measured by an electrophoretic mobility shift assay and luciferase activity.Results:MECF had no effects on cell viability up to a concentration of 100 μg/mL in human breast cancer MDA-MB-231 cells regardless of the presence of TNF-α.MDA-MB-231 cells that were stimulated with TNF-α showed a marked increase of invasion compared to the untreated control,whereas pretreatment with MECF downregulated the TNF-α-induced invasion of MDA-MB-231 cells.Additionally,zymography,western blot analysis,and reverse transcriptase-polymerase chain reaction(RT-PCR) confirmed that MECF decreased TNF-α-induced MMP-9 expression and activity which is a key regulator for cancer invasion.According to an electrophoretic morbidity shift assay,pretreatment with MECF in MDA-MB-231 cells significantly decreased the TNF-α-induced DNA-binding activity of nuclear factor- κB(NF- κB),which is an important transcription factor for regulating cancer invasion-related genes such as MMP-9.Furthermore,treatment with MECF sustained the expression of p65 and p50 in response to TNF-α in the cytosolic compartment.The luciferase assay demonstrated that MECF attenuated TNF-α-induced NF- κB luciferase activity.Conclusion:MECF exhibited its antiinvasive capability by downregulating TNF-α-induced MMP-9 expression,resulting from the suppression of NF- κB activity in the human breast cancer cell line MDA-MB-231.

Autophagy plays a protective role in advanced glycation end products-induced apoptosis of chondrocytes via regulation of tumor necrosis factor-α,nuclear factor-κ B and reactive oxygen species

Objective: To study the adverse effects of advanced glycation end products(AGEs) on chondrocytes and the role of autophagy in this process. Methods: Chondrocytes were harvested from the human articular cartilage tissues in surgery. AGEs were administered during chondrocytes culture. The rapamycin was used to induce autophagy. The cell viability was determined by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay.The expression of tumor necrosis factor-α(TNF-α) and nuclear factor-κ B(NF-κ B) was detected by quantitative real-time polymerase chain reaction. The reactive oxygen species(ROS) production and apoptosis of the chondrocytes were determined by fluorescent probe and flow cytometer, respectively. Results: The chondrocytes viability was significantly reduced after 12 h incubation with AGEs(P<0.01)). In contrast, rapamycin pretreatment increased the chondrocytes viability through autophagy. AGEs increased TNF-α and NF-κ B mRNA expression of chondrocytes and autophagy receded or proceeded the change. AGEs increased intracellular ROS accumulation and autophagy reversed the change. AGEs accelerated chondrocytes apoptosis and autophagy suspended apoptosis. Conclusions: Accumulation of AGEs may have an adverse role for chondrocytes by increasing TNF-α and NF-κB expression, ROS accumulation and apoptosis; meanwhile, autophagy ameliorates the AGEsinduced adverse effects.

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.

Salvianic acid A inhibits induction of inflammatory mediators by blocking Nuclear Factor-κB activation in macrophages

Objective To investigate the anti-inflammation effect and possible mechanism of Salvianic acid A(SAA)in mouse peritoneal macrophages.Methods Peritoneal macrophages were obtained from BALB/c mice.LPS induced nitric oxide(NO),tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)in supernatant,protein expression of inducible nitric oxide synthase(iNOS),matrix metalloproteinase-9(MMP-9)and activation of nuclear factor-kappa B(NF-κB)in the extract were measured.Results SAA strongly inhibited the excessive production of NO,TNF-α and IL-6 in LPS-induced peritoneal macrophages in a concentration-dependent manner and blocked the expression of iNOS and MMP-9.Treatment with LPS alone increased the translocation of NF-κB(p65)from cytosol to the nucleus,but the SAA inhibited the translocation of NF-κB(p65).Conclusions The results showed that SAA had strong anti-inflammatory effects in LPS-stimulated peritoneal macrophages.The important mechanism is due to its inhibition of NF-κB activation.

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.

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.

Blocking NF-kB nuclear translocation leads to p53-related autophagy activation and cell apoptosis

AIM: To investigate the anti-tumor effects of nuclear factor-κB (NF-κB) inhibitor SN50 and related mechanisms of SGC7901 human gastric carcinoma cells. METHODS: MTT assay was used to determine the cytotoxic effects of SN50 in gastric cancer cell line SGC7901. Hoechst 33258 staining was used to detect apoptosis morphological changes after SN50 treatment. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after SN50 treatment.Immunofluorescence staining was used to detect the expression of light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Western blotting analysis were used to determine the expression of proteins involved in apoptosis and autophagy including p53, p53 upregulated modulator of apoptosis (PUMA), damage-regulated autophagy modulator (DRAM), LC3 and Beclin 1. We detected the effects of p53-mediated autophagy activation on the apoptosis of SGC7901 cells with the p53 inhibitor pifithrin-α. RESULTS: The viability of SGC7901 cells was inhibited after SN50 treatment. Inductions in the expression of apoptotic protein p53 and PUMA as well as autophagic protein DRAM, LC3 and Beclin 1 were detected with Western blotting analysis. SN50-treated cells exhibited punctuate microtubule-associated protein 1 LC3 in immunoreactivity and MDC-labeled vesicles increased after treatment of SN50 by MDC staining. Collapse of mitochondrial membrane potential Δψ were detected for 6 to 24 h after SN50 treatment. SN50-induced increases in PUMA, DRAM, LC3 and Beclin 1 and cell death were blocked by the p53 specific inhibitor pifithrin-α. CONCLUSION: The anti-tumor activity of NF-κB inhibitors is associated with p53-mediated activation of autophagy.

Helicobacter pylori tumor necrosis factor-α inducing protein promotes cytokine expression via nuclear factor-κB

AIM:To study the effects of Helicobacter pylori(H. pylori)tumor necrosis factor-α(TNF)inducing protein (Tip-α)on cytokine expression and its mechanism. METHODS:We cloned Tip-αfrom the H.pylori strain 26695,transformed Escherichia coli with an expression plasmid,and then confirmed the expression product by Western blotting.Using different concentrations of Tip-αthat affected SGC7901 and GES-1 cells at different times,we assessed cytokine levels using enzyme-linked immunosorbent assay.We blocked SGC7901 cells with pyrrolidine dithiocarbamate(PDTC),a specific inhibitor of nuclear factorκB(NF-κB).We then detected interleukin(IL)-1βand TNF-αlevels in SGC7901 cells. RESULTS:Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein,which indicated that recombinant Tip-αprotein was recombined successfully.The levels of IL-1β,IL-8 and TNF-αwere sig-nificantly higher following Tip-αinterference,whether GES-1 cells or SGC-7901 cells were used(P<0.05).However,the levels of cytokines(including IL-1β,IL-8 and TNF-α)secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-αat the same concentration and for the same duration(P<0.05).After blocking NF-κB with PDTC, the cells(GES-1 cells and SGC-7901 cells)underwent interference with Tip-α.We found that IL-1βand TNF-αlevels were significantly decreased compared to cells that only underwent Tip-αinterference(P<0.05). CONCLUSION:Tip-αplays an important role in cyto-kine expression through NF-κB.

Total polysaccharides of the Sijunzi decoction attenuate tumor necrosis factor-α-induced damage to the barrier function of a Caco-2 cell monolayer via the nuclear factor-κB-myosin light chain kinase-myosin light chain pathway

AIM To explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction(TPSJ) on the epithelial barriers in vitro. METHODS Caco-2 cell monolayers were treated with or without TPSJ in the presence or absence of TNF-α, and paracellular permeability and transepithelial electrical resistance(TEER) were measured to evaluate the epithelial barrier function. Immunofluorescence and western blotting were respecti-vely used to evaluate the distribution and expression of the tight junction proteins claudin 1, claudin 2, zo3, and occludin in Caco-2 cells. western blotting was also used to evaluate the cellular expression of myosin light chain(MLC), phosphorylated MLC(pM LC), MLC kinase(MLCK), and nuclear factor(NF)-κB p65. RESULTS TPSJ promoted the proliferation of Caco-2 cells and inhibited TNF-α-induced secretion of pro-inflammatory cyto-kines. Furthermore, TPSJ significantly ameliorated both the reduction of TEER and the increased paracellular permeability observed in tumor necrosis factor(TNF)-α-damaged Caco-2 monolayers. Furthermore, TPSJ remarkably attenuated TNF-α-induced morphological changes, downregulated the expression of claudin 1, claudin 2, zo3, and occludin, and markedly suppressed TNF-α-mediated upregulation of p-MLC and MLCK expression. Finally, TPSJ inhibited the activation and expression of NF-κB p65. CONCLUSION Our results demonstrate that TPSJ alleviates the TNF-α-induced impairment of the intestinal epithelial cell barrier function by suppressing NF-κB p65-mediated phosphorylation of MLCK and MLC.

Clinicopathologic significance of expression of nuclear factor-kB RelA and its target gene products in gastric cancer patients

AIM:To assess the prognostic significance of nuclear factor-kB (NF-kB) and its target genes in gastric cancer. METHODS:The tumor tissues of 115 patients with gastric cancer were immunohistochemically evaluated using monoclonal antibodies against NF-kB RelA. Preoperative serum levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) were assessed via enzyme-linked immuno-sorbent assay. C-reactive protein (CRP) and serum amyloid A (SAA) were measured via immunotrubidimetry. RESULTS:Positive rate of NF-kB RelA was 42.6%. NF-kB RelA expression in tumor tissues was also related to serum levels of IL-6 (P = 0.044) and CRP (P = 0.010). IL-6, SAA, CRP were related to depth of invasion, VEGF and SAA were correlated with lymph node metastasis. IL-6, VEGF, SAA and CRP were related to the stage. Univariate analysis demonstrated that immunostaining of NF-kB RelA, levels of IL-6, VEGF, SAA were significantly related with both disease free survival and over-all survival (OS). Multivariate analysis verified that NF-kB RelA [hazard ratio (HR): 3.40, P = 0.024] and SAA (HR: 3.39, P = 0.045) were independently associated with OS. CONCLUSION: Increased expression of NF-kB RelA and high levels of serum SAA were associated with poor OS in gastric cancer patients.

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.