Expression of MMP-2 and NF-κB in myocardium of mice with viral myocarditis

AIM: To observe the effects of TNF-α/nuclear factor-κB(NF-κB)/matrix metalloproteinase-2(MMP-2) pathway on the expression of MMP-2 in the mice with viral myocarditis. METHODS: Six-week-old inbred male mice were randomly assigned to control and myocarditis group. The mice in myocarditis group and control group were intraperitoneally inoculated with 0.1 mL 10^-5.69 TCID50/mL coxsackievirus B3 and vehicle (PBS), respectively. Ten mice were sacrificed at the 4th and 10th days after injection. The blood and heart specimens were harvested. The serum content of TNF-α was measured by ELISA. The myocardial levels of MMP-2, NF-κB p65 and IκBα were determined by Western blot. RESULTS: Compared with control group, the protein expression of MMP-2 and NF-κB p65 in the myocardium and the serum content of TNF-α were significantly increased in myocarditis group (P<0.05). The protein expression of IκBα was lower in myocarditis group than that in control group (P<0.05).CONCLUSION: TNF-α, NF-κB p65 and MMP-2 were higher in the mice with acute viral myocarditis. The increased expression of them might be involved in the pathogenesis of viral myocarditis.     

Effects of burn sera on IκBα degradation and NF-κB activation in monocytes in vitro

AIM: To investigate the effects of burn sera on IκBα degradation, NF-κB activation in peripheral blood monocytes (PBMCs) in order to explore the role of burn sera on activation of monocytes. METHODS: PBMCs isolated from healthy volunteers were stimulated by sera from healthy volunteers and burn patients and by burn sera together with PDTC (pyrrolidine dithioncarbamate). Activation of monocytic NF-κB was tested by electrophoretic mobility shift assay (EMSA) and the degradation of monocytic IκBα was determined by Western blotting. RESULTS: When compared to that in control group, cytosolic IκBα degradation occurred within 30 min after PBMCs stimulated by burn sera, and peaked at 60 min. But IκBα gradually recovered in the cytoplasm after 2 h of stimulation. Meanwhile, activity of monocytic NF-κB was markedly increased, reached the peak at 30 min to 60 min after stimulation, and gradually decreased after 2 h of stimulation. PDTC (an antioxidants) effectively inhibited the monocytic IκBα degradation and activation of NF-κB induced by burn sera. CONCLUSION: Burn sera might induce the degradation of IκBα, then activate NF-κB, which ultimately lead to the secretion of cytokines from the monocytes.     

Advances in IκB protein family research

IκB is an inhibitor of nuclear factor-kappa B (NF-κB) and presents in the majority of cells. Eight structurally related members of the mammalian IκB family have been described:IκBα, IκBβ, IκBε, Bcl-3, IκBγ, IκBδ, p100 and p105. The ankyrin repeat domain of IκB can interact with NF-κB, and sequester NF-κB in the cytoplasm as inactive complexes. Most recently, IκBα has been found to inhibit p53 tumor suppressor protein by binding p53 to form a cytoplasmic p53·IκBα complex and studies have shown that p100 profoundly sensitizes cells to death-receptor-mediated apoptosis. The current review is to describe the members of IκB family, their related signaling pathways, and their application in tumor therapy.     

Expression of nuclear factor-κB in asthmatic guinea pigs and the effect of erigeron breviscapus on it

AIM:To explore the expression of nuclear factor-κB(NF-κB)in asthmatic guinea pigs,and the effect of erigeron breviscapus,a protein kinase C(PKC)inhibitor,on the expression of nuclear factor-κB(NF-κB).METHODS:48 guinea pigs were randomly divided into 6 groups(n=8).Airway resistance and eosinophilic inflammation of airway wall were examined,the expression of NF-κB in the lung tissue was detected by immunohistochemical staining.RESULTS:The expression of NF-κB was mainly found in airway epithelium,all the asthmatic animals showed significantly higher optical densities than that of the normal control group(P＜0.01),and the rats subjected therapeutic treatment for two weeks showed significantly lower NF-κB expression than those of the asthmatic groups(P＜0.01).Positive correlation exist between the airway resistance and the percentage of cells expressing NF-κB in epithelium,and between the amount of eosinophil in airway wall and the percentage of cells expressing NF-κB in epithelium(P＜0101).CONCLUSION:The increased expression of NF-κB in airway epithelium of the asthmatic guinea pigs suggested that NF-κB may be involved in asthma.And result that the increased expression of NF-κB was inhibited significantly by the treatment of the erigeron breviscapus suggested that PKC may play a significant role in the pathogenesis of asthma through NF-κB.     

Activation of Akt signal pathway cascades in kidney tissue in murine chronic graft-versus-host disease lupus nephritis and its regulation by prednisone

AIM:To examine whether Akt signal pathway proteins, including Akt, NF-κB and IκBα, are activated in kidney tissue of murine chronic graft-versus-host disease (GvHD) lupus nephritis in vivo, and whether prednisone suppresses activation of them.METHODS:Akt activity and phosphorylated IκBα were detected by Western-blot. Activation of NF-κB was detected by electropheretic mobility shift assay (EMSA). RESULTS:Activity of Akt, NF-κB and phosphorylated IκBα were significantly increased in kidney tissue of murine chronic graft-versus-host disease (GvHD) in 8th week and 12th week after monocell injection, respectively. However, they were no significant elevation in 16th week, when compared with controls. Prednisone treatment significantly prevented the increase in serum anti-dsDNA antibody level, urinary protein excretion and glomerular cell proliferation in GvHD mice, indicating the beneficial effects of prednisone on this model. Prednisone also significantly suppressed the increase in the activities of glomerular Akt, NF-κB and phosphorylated IκBα. CONCLUSION:This study provides the first evidence of marked increase in glomerular Akt-NF-κB signal pathway activities in murine chronic graft-versus-host disease lupus nephritis. The beneficial effect of prednisone on this lupus nephritis model may be partially mediated by the suppression of abnormal Akt- NF-κB activation.     

Effects of Jiedu-Qingfei mixture on NF-κB and p38 MAPK pathways in lung tissues of rats with Mycoplasma pneumoniae infection

AIM: To observe the therapeutic effect of Jiedu-Qingfei mixture on Mycoplasma pneumoniae (MP)-infected rat lung tissues and to explore its mechanism. METHODS: SD rats (n=40) were randomly divided into 4 groups:blank control group, model group, Jiedu-Qingfei group and positive control group, with 10 rats in each group. The rats in experimental groups were slowly dripped with 1×10⁹ CFU/L MP solution into their nostrils for 4 d. One rat in each group was sacrificed for MP nucleic acid detection at the second day after inoculation, and the other rats were given gavage therapy. The rats in blank control group and model group were intragastrically given the same volume of normal saline, the rats in Jiedu-Qingfei group were given 8 mL/kg Jiedu-Qingfei mixture daily for 4 weeks, and the rats in psoitive control group were given dexmethasone sodium phosphate (0.5 mg·kg^-1·d^-1). After the experiment, the rats were killed. The serum and bronchoalveolar lavage fluid (BALF) were collected for detecting the levels of interleukin-12 (IL-12), IL-13 and TNF-α by ELISA. The right lung tissues were used for pathological observation and HE staining, while the left lung tissues were used to detect the expression of NF-κB p50, I-κBα and p38 mitogen-activated protein kinase (p38 MAPK) at mRNA and protein levels. RESULTS: The results of MP nucleic acid detection showed that all the rats except blank control group were MP nucleic acid positive, indicating that the rat model of MP infection was successfully established. On the 1st day of the treatment, the pathological scores of the lung tissues in model group and Jiedu-Qingfei group were significantly higher than those in blank control group (P<0.05). After treatment, the pathological scores of the lung tissues in mo-del group were significantly higher than those in blank control group and Jiedu-Qingfei group. The levels of IL-12 in the serum and BALF in model group were significantly lower than those in blank control group after MP infection (P<0.05), while those after treatment with Jiedu-Qingfei mixture were significantly higher than those in model group (P<0.05). The levels of IL-13 and TNF-α in the serum and BALF of MP-infected rats were increased significantly, while those after treatment with Jiedu-Qingfei mixture were significantly lower than those in model group (P<0.05). The mRNA expression levels of NF-κB p50 and p38 MAPK in model group were increased significantly (P<0.01). After treatment, the mRNA expression levels of NF-κB p50 and p38 MAPK were decreased significantly compared with model group (P<0.01). The mRNA expression level of I-κBα in model group was significantly lower than that in control group. After treatment, the mRNA expression of I-κBα in Jiedu-Qingfei group was significantly higher than that in model group (P<0.05). The protein levels of NF-κB p50 and p38 MAPK in the lung tissues of model group were significantly higher than those of blank control group. After treatment, the protein expression of NF-κB p50 and p38 MAPK was decreased significantly. The protein level of I-κBα in model group was significantly lower than that in blank control group, and after treatment with Jiedu-Qingfei mixture, the protein expression level of I-κBα was increased significantly (P<0.05). CONCLUSION: Jiedu-Qingfei mixture may attenuate lung tissue inflammation caused by MP through NF-κB and p38 MAPK pathways.     

Protective effect of capsaicin on lipopolysaccharide-induced activation of vascular endothelial cells

AIM:To investigate the effect of capsaicin on lipopolysaccharide (LPS)-induced activation of cultured endothelial cells of mouse aorta in vitro. METHODS:The endothelial cells were isolated from mouse aorta and cultured in vitro, and the specific cell markers of the cells were identified by immunofluorescence staining. The cells were stimulated with LPS (100 μg/L) combined with or without capsaicin, and the cells and supernatant were collected at 12 h, 24 h and 48 h. The levels of soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1) and soluble P-selectin (sP-selectin) in the supernatant were measured by ELISA. The levels of nuclear NF-κB p65 and cytopasmic p-IκBα and IκBα were detected by Western blotting. RESULTS:Compared with control group, the levels of sP-selectin, sICAM-1 and sVCAM-1 in LPS group were significantly increased (P<0.05), and LPS promoted the expression of sICAM-1 and sVCAM-1 in a time-dependent manner. Compared with LPS group at the same time point, capsaicin inhibited the expression of sP-selectin, sICAM-1 and sVCAM-1 in a dose-dependent manner. Compared with control group, the protein levels of NF-κB p65 and p-IκBα in LPS group at 24 h were significantly increased (P<0.05), while the protein level of IκBα in LPS group at 24 h were significantly decreased (P<0.05). Compared with LPS group, capsaicin decreased the protein levels of NF-κB p65 and p-IκBα and increased the protein level of IκBα in a dose-dependent manner. CONCLUSION: Capsaicin has a protective effect on LPS-induced vascular endothelial cell activation, which potentially contributes to the suppression of IκBα degradation and NF-κB p65 nuclear translocation.     

Role of Akt/NF-κB pathway in immune-complexes-induced MCP-1 and CSF-1 expression in murine glomerular mesangial cells

AIM: To explore the role of Akt/NF-κB pathway in immune-complexes-induced monocyte chemoattractant protein-1 (MCP-1) and colony stimulating factor-1 (CSF-1) expression in Mesangial Cells. METHODS: Primary murine glomerular mesangial cells were cultured in vitro and divided into control group, stimulation group and antisense, sense and mismatched oligodeoxynucleotide group. In control group, the cells were stimulated with monomeric IgG after treatment with 0.5% lipofectin for 8 h. In stimulation group, the cells, which had been treated with 0.5% lipofectin for 8 h, were stimulated with aggregated IgG. In antisense, sense and mismatched oligodeoxynucleotide group, being transduced antisense, sense and mismatched oligodeoxynucleotide respectively with 0.5% lipofectin 8 h, the cells were stimulated with AIgG. MCP-1 and CSF-1 in supernatant were deteced with ELISA. In addition, RT-PCR was used to determine MCP-1 and CSF-1 mRNA expression, and EMSA to investigated the activation of NF-κB. RESULTS: Mesangial cells cultured in vitro had a low level NF-κB activation and a low level constitutive expression of MCP-1 and CSF-1. Stimulated with AIgG, activation of NF-κB was markedly increased(0.35±0.06 vs 0.75±0.16, P＜0.01), expression of MCP-1 and CSF-1 mRNA (0.48±0.03 vs 0.72±0.02, P＜0.05; 0.44±0.01 vs 0.59±0.02, P＜0.05), MCP-1 and CSF-1 levels in supernatant(15.52±1.81 vs 43.05±3.18, P＜0.05; 389.06±13.75 vs 764.22±31.78, P＜0.05) were markedly increased. Akt1 antisense oligodeoxynucleotide markedly inhibited immune-complexes-induced NF-κB activation, MCP-1 and CSF-1 mRNA and protein expression. CONCLUSION: Akt/NF-κB pathway mediates immune-complexes-induced MCP-1 and CSF-1 expression in mesangial cells. It suggests that Akt/NF-κB pathway may be a new therapy target for macrophage recruitment and activation in immune complexes nephritis.     

Resveratrol attenuates inflammatory pain induced by complete Freund's adjuvant via NF-κB signaling pathway in a mouse model

AIM: To investigate the anti-inflammatory action of resveratrol (Res) and its correlation with nuclear factor-κB (NF-κB) signaling pathway in a mouse model of inflammatory pain.METHODS: BALB/c mice (n=60) were randomly divided into 6 groups:normal control group, inflammatory pain model group, positive control (dexamethasone, 0.5 mg/kg) group and resveratrol (100, 50 and 25 mg/kg) groups (10 mice in each group). In order to observe the anti-inflammatory pain effects of reseratrol on mice, the paw withdrawal mechanical threshold, paw withdrawal thermal latency and cold withdrawal times were detected. In order to analyze the mechanism of analgesic effect of resveratrol, the expression levels of NF-κB, inhibitor of NF-κB (IκB) α, inhibitor of NF-κB kinase (IKK) β, tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the spinal cord tissues (L4~L6) of the mice were determined by RT-PCR and Western blot.RESULTS: The resveratrol at 100 and 50 mg/kg increased the paw withdrawal mechanical threshold, prolonged the paw withdrawal thermal latency, and decreased the cold withdrawal times in the inflammatory pain mice (P<0.05 or P<0.01). The resveratrol at 100 mg/kg down-regulated the mRNA and protein expression levels of NF-κB, IκBα, IKKβ, TNF-α and IL-1β in the spinal cord tissues (L4~L6) of inflammatory pain mice (P<0.05 or P<0.01).CONCLUSION: Resveratrol ameliorates the inflammatory pain of the mice induced by complete Freund's adjuvant. The mechanism is related to the inhibition of NF-κB signaling pathway.     

Role of NF-κB in inducing HEL cell proliferation and apoptosis during human cytomegalovirus infection

AIM:To investigate whether human cytomegalovirus(HCMV) regulate human embryonic lung fibroblast(HEL) cell proliferation and apoptosis by activating NF-κB.METHODS:Immunohistochemistry and Western blot analysis were used to detect the NF-κB translocation and/or Bcl-2 and the levels of I-κBα during HCMV infection. Apoptotic cell were examined by flow cytometry, and the HEL cell proliferation was determined by MTT.RESULTS:The levels of NF-κB in the nucleus reached highly 48 h postinfection, and the levels of I-κBα were low 24 h postinfection. The activity of NF-κB was inhibited 120 h postinfection. The levels ofbcl-2was accorded with the activity of NF-κB. HCMV promoted HEL cells to proliferate before 72 h postinfection and induced apoptosis 120 h postinfection.CONCLUSION:NF-κB plays a role in HEL cell proliferation and apoptosis during HCMV infection, and it involves in the pathological mechanisms of diseases associated with HCMV infection.     

Inhibition of HMGB1 expression promotes apoptosis of hemangioma endothelial cells

AIM: To investigate the effect of inhibiting high-mobility group box protein 1 (HMGB1) expression on the viability and apoptosis of hemangioma endothelial cells (HemECs). METHODS: Human HemECs were isolated and cultured, and HMGB1 small interfering RNA (HMGB1-siRNA) was transfected into the cells. The cell viability was detected by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) content were analyzed by flow cytometry. The protein levels of HMGB1, NF-κB p65, p-IκBα, cyclin D1 and survivin were determined by Western blot. RESULTS: The protein expression of HMGB1 in the HemECs transfected with HMGB1-siRNA was significantly lower than that in blank control group (P<0.05). Compared with NC group, the cell viability was decreased significantly in the HemECs transfected with HMGB1-siRNA, the apoptotic rate was significantly increased, the content of ROS increased significantly, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were significantly decreased (P<0.05). After exposure to NF-κB signaling pathway inhibitor PDTC, the cell viability was inhibited, the apoptosis was increased, ROS content, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were down-regulated significantly, as compared with si-HMGB1 group (P<0.05). CONCLUSION: Inhibition of HMGB1 reduces the viability of HemECs and induces apoptosis by increasing the content of ROS and down-regulating the activity of NF-κB signaling pathway.     

LPS-induced hyperpermeability in brain vascular endothelial cell is related to NF-κB signaling

AIM: To investigate the role of nuclear factor kappa B (NF-κB) signaling in the changes of permeability in brain-derived microvascular endothelial (bEnd.3) cells induced by lipopolysaccharide (LPS).METHODS: The bEnd.3 cells were randomly divided into 3 groups: bEnd.3 group, bEnd.3/vector group and bEnd.3/muIκBα group. The cells in the latter 2 groups were transfected with pcDNA3.1hygro and DNMu-IκBα (a dominant-negative mutant of IκB) plasmids, respectively. All the cells were exposed to LPS. The activity of NF-κB, monolayer barrier integrity and F-actin distribution were detected by luciferase reporter assay, transendothelial electrical resistance (TEER) assay and rhodamine-phalloidin staining, respectively. The expression of tight junction proteins (ZO-1 and claudin-5) and phosphorylation of myosin light chain (MLC) were determined using Western blotting.RESULTS: In bEnd.3 group and bEnd.3/vector group, the NF-κB activity began to increase obviously as early as 0.5 h after pretreatment with LPS. LPS decreased TEER, and induced F-actin rearrangement and ZO-1 down-regulation in 3 h. Incubation of the cells with LPS for 12 h induced the most significant disruptive effects on the permeability and tight junctions. Moreover, high expression of phosphorylated MLC accompanied with the early damages of tight junctions was observed. However, these destabilizing alterations were suppressed in bEnd.3/muIκBα group by the inhibition of NF-κB activity.CONCLUSION: LPS induces hyperpermeability in brain microvascular endothelial cells. The functions of NF-κB signaling are related to influencing disruptions of tight junctions by regulating the phosphorylation of MLC.     

1,25-dihydroxyvitamin D3 inhibits nuclear factor kappa B signaling pathway in passively sensitized human airway smooth muscle cells

AIM:To investigate the effects of 1,25-dihydroxyvitamin D3 ［1,25-(OH)2D3］ on nuclear factor kappa B (NF-κB) signaling pathway in passively sensitized human airway smooth muscle cells (HASMCs). METHODS:HASMCs were passively sensitized with 10% serum from asthmatic patients. 1,25-(OH)2D3 was used as an interfering factor. Electrophoretic mobility shift assay (EMSA) was used to detect the DNA-binding activity of NF-κB. Immunocytochemical staining was used to observe the nuclear translocation of NF-κB p65. Western blotting was used for IκBα and phosphorylated IκBα protein detection. Real-time fluorescence quantitative PCR was used to determine vitamin D receptor (VDR), vitamin D 24-hydroxylase (CYP24) and IκBα mRNA expression. The mRNA expression of IκBα in HASMCs after actinomycin D treatment was also determined. RESULTS:(1) 1,25-(OH)2D3 significantly attenuated the DNA-binding activity of NF-κB and the nuclear translocation of NF-κB p65 in HASMCs passively sensitized by asthmatic serum. (2) 1,25-(OH)2D3 enhanced IκBα mRNA stability and inhibited IκBα protein phosphorylation in passively sensitized HASMCs, thus increasing IκBα expression in these HASMCs. (3) 1,25-(OH)2D3 up-regulated VDR mRNA level and evoked its functional response in passively sensitized HASMCs. CONCLUSION: 1,25-(OH)2D3 enhanced the expression of IκBα and therefore inhibited NF-κB signaling passway in HASMCs. This effect may be dependent on VDR, and responsible for the inhibitory effect of 1,25-(OH)2D3 on passively sensitized HASMCs.     

Expression and role of CD134 and NF-κB in renal tissue of lupus nephritis

AIM:To investigate the role of CD134 (OX40) and NF-κB in the pathogenesis of lupus nephritis (LN).METHODS:Renal in situ CD134 and NF-κB expression were examined in 40 biopsy specimens from LN patients by immunohistochemistry and microwave-based immunohistochemistry, respectively. The relationship between expression of CD134 and NF-κB was analyzed.RESULTS:The expression of glomerular and tubular CD134 and NF-κB in LN were higher than that in normal control, especially in class Ⅳ LN, where there was intense staining of endothelial cell, distal tubules, and interstitial mononuclear cell. The CD134 expression of glomerular and tubular was closely related to NF-κB expression, respectively (r=0.5542, P＜0.05;r=0.6279, P＜0.05). CONCLUSIONS:The abnormal expression of costimulatory molecule CD134 was well evidenced in LN. Strong expression of renal in situ NF-κB was likely mediated by CD134 signal pathway, which may play an important role in the pathogenesis of LN.     

Combined effect of octreotide and Dachaihu decoction on treatment of severe acute pancreatitis

AIM: To investigate the combined effect of octreotide and Dachaihu decoction on the treatment of severe acute pancreatitis(SAP). METHODS: Wistar rats(n=50) were randomly divided into sham group, SAP group, octreotide group, Dachaihu decoction group and combination group. The quantity of ascites was measured. The levels of amylase, alanine aminotransferase and creatinine in the serum were examined. The morphological changes of the pancreatic tissues were observed by HE staining. The activation of NF-κB and IκBα expression were determined by Western blot. The mRNA expression with ICAM-1 and IL-1 was detected by qPCR.RESULTS: Combined treatment with octreotide and Dachaihu decoction effectively reduced the quantity of ascites and the levels of amylase, alanine aminotransferase and creatinine in the serum in SAP rats. Moreover, combined treatment significantly inhibited SAP-induced activation of NF-κB and decrease in IκBα protein expression, accompanied by a decrease in ICAM-1 and IL-1 mRNA expression. CONCLUSION: Combination of octreotide with Dachaihu decoction effectively attenuates SAP by inhibiting NF-κB signaling pathway and ICAM-1 and IL-1 expression.     

Effects of diterpenoid C from ether extract of Radix Curcumae on nuclear factor-κB activity in LPS-induced human gastric adenocarcinoma SGC7901 cells

AIM: To observe the effects of diterpenoid C from ether extract of Radix Curcumae (RC) on the activity of nuclear factor-κB in human gastric adenocarcinoma SGC7901 cells stimulated with lipopolysaccharide (LPS). METHODS: SGC7901 cells were normally cultured, induced by LPS, or treated with LPS plus RC. The protein expression of IKKα, IKKβ, p65, phosphorylated p65 and phosphorylated IκBα was assayed by Western blotting. NF-κB DNA binding activity was determined by electrophoretic mobility shift assay. RESULTS: RC reduced the protein expression of IKKα, IKKβ, p65, phosphorylated p65 and phosphorylated IκBα induced by LPS. NF-κB DNA binding activity increased much greatly by LPS stimulation, while RC resisted the action of LPS. CONCLUSION: RC may attenuate the secretion of inflammatory cytokines by inhibiting the activation of NF-κB signaling pathway.     

Action of NF-κB p65 in renal interstitium in rats with active Heymann nephritis

AIM: To study the action of NF-κB p65 in tubule-interstitium in rats with active Heymann nephritis(AHN). METHODS: Twenty female Wistar rats in 6-8 weeks of age were divided into two groups. The nephritis was induced with Fx1A/CFA by subcutaneous injection and with CFA as control. After rats were killed, the activation of NF-κB p65 in renal tissue was observed by immune histochemistry. RESULTS: The lesion score of renal interstitium and activation of NF-κB p65 of renal tubule in rats with AHN was higher than those of control group(P<0.01). A positive correlation was found between the positive rate of NF-κB p65 in renal tubule and the lesion score of renal interstitium, the levels of urinary protein (r=0.7138 and 0.6376, P<0.05).CONCLUSION: The activation of NF-κB p65 participates in the damage of pathophysiological functions of tubule-interstitium in AHN.