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.

Prognostic role of the stromal cell derived factor-1 in patients with hepatitis B virus-related acute-on-chronic liver failure

BACKGROUND Stromal cell derived factor-1(SDF-1)plays a pivotal role in the recruitment of stem cells to injured livers.However,the changes of SDF-l in patients with hepatitis B virus(HBV)-related acute-on-chronic liver failure(ACLF)have yet to be elucidated.AIM To study the SDF-1 changes in patients with HBV-related ACLF.METHODS 30 patients with HBV-related ACLF,27 patients with chronic hepatitis B and 20 healthy individuals are involved in our study.The SDF-l mRNA expression in liver tissue was detected by quantitative real-time polymerase chain reaction.Immunohistochemical staining was performed to illustrate the expression of SDFl,CXC receptor 4(CXCR4)and Ki67.The serum SDF-l concentrations were also detected by enzyme-linked immunosorbent assays.RESULTS The expression of SDF-1 mRNA from ACLF patients was remarkably higher than that from other patients(both P<0.05).The expression of SDF-l,CXCR4 and Ki67 from ACLF were the highest among the three groups(all P<0.01).The serum SDF-l levels in ACLF patients were significantly lower than that in other patients(both P<0.01).Moreover,in ACLF patients,the serum SDF-1 Levels were positively correlated with serum total bilirubin and international normalized ratio.In addition,the serum SDF-l levels in survival were significantly lower compared with the non-survivals(P<0.05).The area under the curve for the serum SDF-1 level in predicting 28-d mortality was 0.722(P<0.05).CONCLUSION This study provides the SDF-1 changes in patients with HBV-related ACLF.The SDF-1 Level at admission may serve as a promising prognostic marker for predicting short-term prognosis.

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.

N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats

AIM:To observe the effects of N-acetylserotonin(NAS)administration on retinal ischemia-reperfusion(RIR)injury in rats and explore the underlying mechanisms involving the high mobility group box 1(HMGB1)/receptor for advanced glycation end-products(RAGE)/nuclear factor-kappa B(NF-κB)signaling pathway.METHODS:A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye.Eighty male Sprague Dawley were randomly divided into five groups:sham group(n=8),RIR group(n=28),RIR+NAS group(n=28),RIR+FPS-ZM1 group(n=8)and RIR+NAS+FPS-ZM1 group(n=8).The therapeutic effects of NAS were examined by hematoxylin-eosin(H&E)staining,and retinal ganglion cells(RGCs)counting.The expression of interleukin 1 beta(IL-1β),HMGB1,RAGE,and nod-like receptor 3(NLRP3)proteins and the phosphorylation of nuclear factorkappa B(p-NF-κB)were analyzed by immunohistochemistry staining and Western blot analysis.The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay(ELISA).RESULTS:H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats.With NAS therapy,the HMGB1 and RAGE expression decreased significantly,and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression.Additionally,NAS exhibited an anti-inflammatory effect by reducing IL-1βexpression.The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression,so as to the IL-1βexpression and retinal edema,accompanied by an increase of RGCs in RIR rats.CONCLUSION:NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway,which may be a useful therapeutic target for retinal disease.

桥本甲状腺炎合并甲状腺乳头状癌患者中Toll样受体3和核转录因子-κB的表达及相关性分析

目的分析桥本甲状腺炎(Hashimoto thyroiditis,HT)合并甲状腺乳头状癌(PTC)患者Toll样受体3(toll-like receptor 3,TLR3)和核转录因子-κB(nuclear transcription factor-κB,NF-κB)表达及相关性。方法收取邯郸市中心医院2020年3月~2022年3月收治的130例行手术切除的甲状腺标本,其中正常甲状腺组织标本43例,HT标本47例,HT合并PTC标本40例,分析TLR3和NF-κB在正常甲状腺组织、HT组、HT合并PTC组中的表达,分析HT合并PTC组中TLR3和NF-κB表达与临床病理参数关系,Pearson相关性分析TLR3和NF-κB的关系。结果TLR3在正常甲状腺组织、HT组、HT合并PTC组中的阳性表达率分别为0(0/43)、80.85%(38/47)、90.00%(36/40);NF-κB在以上三组中的阳性表达率分别为0(0/43)、68.09%(32/47)、85.00%(34/40)。TLR3和NF-κB在HT组、HT合并PTC组中的阳性表达率均高于正常甲状腺组织(P<0.05),TLR3和NF-κB表达与性别、年龄、HT合并PTC病理学特征、病灶类型、淋巴结转移、甲状腺包膜侵犯差异比较均无统计学意义(P均>0.05)。TLR3和NF-κB呈显著正相关(r=0.589,P<0.05)。结论TLR3和NF-κB在HT合并PTC组织中的阳性率高于正常甲状腺组织,且二者表达呈正相关。

Hepatitis B virus X protein up-regulates tumor necrosis factor-α expression in cultured mesangial cells via ERKs and NF-κB pathways

Objective:To investigate the effects of hepatitis B virus(HBV)X protein(HBx)on the expression of tumor necrosis factor-α(TNF-α)in glomerular mesangial cells(GMCs)and the underlying intracellular signal pathways.Methods:The plasmid pCI-neo-X that carries the X gene of hepatitis B virus was transfected into cultured GMCs.HBx expression in the transfected GMCs was assessed by Western-blot.TNF-αprotein and mRNA were assessed by ELISA and semi-quantitative RT-PCR,respectively.Three kinase inhibitors-U0126,an inhibitor of extracellular signal-regulated kinases(ERKs);lactacvstin,an inhibitor of nuclear factor-κB(NF-κB);and SB203580,a selective inhibitor of p38 MAP kinase(p38 MAPK)were used to determine which intracellular signal pathways may underlie the action of HBx on TNF-αexpression in transfected GMCs.Results:A significant increase in HBx expression in pCI-neo-X transfected GMCs was detected at 36 h and 48 h,which was not affected by any of those kinase inhibitors mentioned above.A similar increase in the expression of both TNF-αprotein and mRNA was also observed at 36 h and 48 h,which was significantly decreased in the presence of U0126 or lactacytin,but not SB203580.Conclusions:HBx upregulates TNF-αexpression in cultured GMCs,possibly through ERKs and NF-κB pathway,but not p38 MAPK pathway.

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.

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.

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.

CYLD deletion triggers nuclear factor-κB-signaling and increases cell death resistance in murine hepatocytes

AIM:To analyze the role of CYLD for receptor-mediated cell death of murine hepatocytes in acute liver injury models.METHODS:Hepatocyte cell death in CYLD knockout mice(CYLD-/-)was analyzed by application of liver injury models for CD95-(Jo2)and tumor necrosis factor(TNF)-α-[D-Gal N/lipopolysaccharide(LPS)]induced apoptosis.Liver injury was assessed by measurement of serum transaminases and histological analysis.Apoptosis induction was quantified by cleaved PARP staining and Western blotting of activated caspases.Nuclear factor(NF)-κB,ERK,Akt and jun amino-terminal kinases signaling were assessed.Primary Hepatocytes were isolated by two step-collagenase perfusion and treated with recombinant TNF-αand with the CD95-ligand Jo2.Cell viability was analyzed by MTT-assay.RESULTS:Livers of CYLD-/-mice showed increased anti-apoptotic NF-κB signaling.In both applied liver injury models CYLD-/-mice showed a significantly reduced apoptosis sensitivity.After D-Gal N/LPS treatment CYLD-/-mice exhibited significantly lower levels of alanine aminotransferase(ALT)(295 U/L vs 859 U/L,P<0.05)and aspartate aminotransferase(AST)(560 U/L vs 1025 U/L,P<0.01).After Jo injection CYLD-/-mice showed 2-fold lower ALT(50 U/L vs 110 U/L,P<0.01)and lower AST(250 U/L vs 435 U/L,P<0.01)serumlevels compared to WT mice.In addition,isolated CYLD-/-primary murine hepatocytes(PMH)were less sensitive towards death receptor-mediated apoptosis and showed increased levels of Bcl-2,XIAP,c IAP1/2,survivin and c-FLIP expression upon TNF-and CD95-receptor triggering,respectively.Inhibition of NF-κB activation by the inhibitor of NF-κB phosphorylation inhibitor BAY 11-7085 inhibited the expression of antiapoptotic proteins and re-sensitized CYLD-/-PMH towards TNF-and CD95-receptor mediated cell death.CONCLUSION:CYLD is a central regulator of apoptotic cell death in murine hepatocytes by controlling NF-κB dependent anti-apoptotic signaling.

Phosphoinositide 3-Kinase/Akt and Nuclear Factor-κB Are Involved in Staphylococcus Aureus-induced Apoptosis in U937 Cells

Objective To explore the mechanisms involved in Staphylococcus aureus （S. aureus） invading human monocytic U937 cells. Methods S. aureus were added to U937 cells at multiplicity of infections （MOI） of 20：1 for 0, 15, 30, 60, and 90 minutes, respectively. Cell apoptosis was analyzed with Hoechst 33258 staining and Annexin V-fluorescein isothiocyanate （FITC）/propidium iodide （PI） flow cytometry analysis. Akt and nuclear factor-κB （NF-κB） activities were detected by Western blotting. Results Infection of U937 cells with S. aureus induced rapid cell death in a time-dependent manner, and the cells displayed characteristic features of apoptosis. S. aureus-induced apoptosis was associated with a prominent downregulation of activated （phosphorylated） Akt and NF-κB. The inhibition of phosphorylated Akt by LY294002 led to the inhibition of NF-κB in a dose-dependent manner. Inhibition of Akt with LY294002 caused further increase in apoptosis of U937 cells. Conclusions S. aureus can stimulate the apoptosis of U937 ceils. S. aureus induces apoptosis of U937 cells by inhibiting Akt-regulated NF-κB.

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.

Lysophosphatidic acid induced nuclear translocation of nuclear factor-κB in Panc-1 cells by mobilizing cytosolic free calcium

AIM: To clarify whether Lysophosphatidic acid (LPA) activates the nuclear translocation of nuclear factor-κB (NF-κB) in pancreatic cancer. METHODS: Panc-1, a human pancreatic cancer cell line, was used throughout the study. The expression of LPA receptors was confirmed by reverse-transcript polymerase chain reaction (RT-PCR). Cytosolic free calcium was measured by fluorescent calcium indicator fura-2, and the localization of NF-κB was visualized by immunofluorescent method with or without various agents, which effect cell signaling. RESULTS: Panc-1 expressed LPA receptors, LPA1, LPA2 and LPA3. LPA caused the elevation of cytosolic free calcium dose-dependently. LPA also caused the nuclear translocation of NF-κB. Cytosolic free calcium was attenuated by pertussis toxin (PTX) and U73122, an inhibitor of phospholipase C. The translocation of NF-κB was similarly attenuated by PTX and U73122, but phorbol ester, an activator of protein kinase C, alone did not translocate NF-κB. Furthermore, the translocation of NF-κB was completely blocked by Ca2+ chelator BAPTA-AM. Thapsigargin, an endoplasmic- reticulum Ca2+-ATPase pump inhibitor, also promoted the translocation of NF-κB. Staurosporine, a proteinkinase C inhibitor, attenuated translocation of NF-κB induced by LPA. CONCLUSION: These findings suggest that protein kinase C is activated endogenously in Panc-1, and protein kinase C is essential for activating NF-κB with cytosolic calcium and that LPA induces the nuclear translocation of NF-κB in Panc-1 by mobilizing cytosolic free calcium.

Effect of Ganoderma lucidum spore on expression of insulin-like growth factor-1, nuclear factor-kappa B, and neuronal apoptosis in the epileptic rat brain

BACKGROUND：It has been reported that Ganoderma lucidum spore powder, a very well known Chinese traditional medicine, can affect immunoregulation, free radical scavenging, and anti-hypoxia responses. OBJECTIVE： To investigate the effect of Ganoderma lucidum spore powder on expression of insulin-like growth factor-1 （IGF-1）, nuclear factor-κB （NF-κB） and neuronal apoptosis in rats with pentylenetetrazol （PTZ）-induced epilepsy. DESIGN, TIME AND SETTING： A cellular and molecular biology experiment with randomized controlled study design was performed at the Central Laboratory of Basic Medical College of Jiamusi University from June to August 2005. MATERIALS： Thirty healthy, adult, male, Wistar rats were selected and randomly divided into 3 groups （10 rats per group）： control, epilepsy model, and Ganoderma lucidum spore powder. A sub-eclampsia PTZ dose （35 mg/kg） was intraperitoneally injected to induce epilepsy in the latter two groups. Wild Ganoderma lucidum spore powder （30 g/L） was provided by the wild Ganoderma lucidum plant nursery at Jiamusi, China. Immunohistochemical detection and terminal deoxynucleotidyl transferase-mediate dUTP nick end-labeling （TUNEL） kits were purchased from Wuhan Boster Biological Technology Co., Ltd., China. METHODS： Ganoderma lucidum spore powder was intragastrically administered at a dose of 10.0 mL/kg, once a day for 28 days. In the epilepsy and control groups, an equivalent volume of normal saline was intragastrically administered. MAIN OUTCOME MEASURES： Immunoreactivity for IGF-1 and NF-κB/P65 were detected by immunohistochemical staining. Neuronal apoptosis was detected using TUNEL methods. RESULTS： The hippocampus and cerebral cortex of rats with PTZ-induced epilepsy exhibited a higher number of apoptotic cells at high magnification （×400）, compared with the control group. Expression of IGF-1 and NF-κB were higher in the epilepsy group, compared with the control group （P 〈 0.01）. In Ganoderma lucidum spore-treated rats, fewer apoptotic cells were observed in the hippocampus and cerebral cortex, expression of NF-κB/P65 was lower, and immunoreactivity to IGF-1 increased more distinctly, compared with the epilepsy group. In addition, seizure latency was longer on 17, 21, and 25 days post-PTZ treatment in the Ganoderma lucidum spore powder group, compared with the epilepsy group （P 〈 0.05-0.01）. CONCLUSION： Ganoderma lucidum spore powder down-regulated expression of NF-κB in brain tissues of rats with PTZ-induced epilepsy, increased immunoreactivity to IGF-1, and inhibited neuronal apoptosis. These results indicated that Ganoderma lucidum spore powder has a neuroprotective effect.

Antidepressants can treat inflammatory bowel disease through regulation of the nuclear factor-κB/nitric oxide pathway and inhibition of cytokine production:A hypothesis

Inflammatory bowel disease (IBD) is a group of inflammatory disorders mainly affecting the colon and small intestine. The main types of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). UC is restricted to the large intestine whereas CD can affect any part of the gastrointestinal tract. Treating this disorder depends on the form and level of severity. Common treatment involves an anti-inflammatory drug, such as mesalazine, and an immunosuppressant, such as prednisone. Several signaling pathways, including nuclear factor (NF)-κB and nitric oxide (NO), and genetic and environmental factors are believed to play an important role in IBD. Amitriptyline is a commonly used antidepressant with known anti-inflammatory activities. Amitriptyline also acts on the NF-κB/NO pathway or cytokine production. Therefore, we hypothesize that antidepressants like amitriptyline can be pioneered and considered effective as an innovative and effective therapeutic in the treatment and attenuation of development of IBD in adjusted doses.

Iguratimod promotes transformation of mononuclear macrophages in elderly patients with rheumatoid arthritis by nuclear factor-κB pathway

BACKGROUND The role of macrophages in rheumatoid arthritis(RA)and its mechanism have attracted much attention in RA pathogenesis.Macrophages accumulate in the synoviums of RA,and the proportion of M1 type pro-inflammatory macrophages is higher than that of M2 type anti-inflammatory macrophages,leading to the secretion of inflammatory molecules and the aggravation of inflammatory reaction,which has made macrophages a potential target of RA drugs.Iguratimod is a kind of cyclo-oxygenase-2 inhibitor that affects macrophage polarity.It is speculated that its anti-inflammatory and anti-rheumatic effects may be related to the regulation of macrophage M1/M2 ratio.AIM To investigate the effects of Iguratimod on the polarity of mononuclear macrophages in elderly patients with RA.METHODS Elderly patients with RA and joint effusion were selected,including 10 men and 25 women,with an average age of 66.37±4.42 years.Patients were treated with oral administration of 25 mg Iguratimod(Iremod,State Food and Drug Administration Approval No.H20110084)twice daily for 12 wk.Disease Activity Score 28 and Health Assessment Questionnaire score were collected according to the disease severity before and after treatment.Venous blood and joint effusion fluid were collected,mononuclear macrophages were extracted and expression of cell surface markers CD86,CD64,CD163,and CD206 was analyzed by flow cytometry.The concentration of inflammatory factors interleukin(IL)-6,IL-1β,transforming growth factor-β,and IL-4 in the joint effusion fluid was analyzed by enzyme-linked immunosorbent assay.Expression of mononuclear cells inhibitor of nuclear factor-κB(IκB)and phosphorylated IκB in peripheral blood was analyzed by western blotting.RESULTS Disease Activity Score 28 score and Health Assessment Questionnaire score of patients treated with Iguratimod decreased significantly.The percentage of cell surface markers CD86 and CD64 decreased significantly,and the percentage of CD163 and CD206 increased significantly(P<0.05).The inflammatory factors IL-6 and IL-1βdecreased significantly,and transforming growth factor-βand IL-4 increased significantly.Western blot analysis showed that mononuclear cell inhibitor of nuclear factor-κB in peripheral blood was significantly increased after treatment,and its phosphorylation level was significantly decreased(P<0.05).CONCLUSION Iguratimod can promote the transformation of mononuclear macrophages from M1 to M2 in elderly patients with RA by inhibiting the nuclear factor-κB pathway,thus improving symptoms of RA.

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.

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.