Objective To investigate the expression changes of nuclear factor kappa B (NF-κB) and matrix metalloproteinase-9 (MMP-9) in the cultured hepatocellular carcinoma cells 9204 (HCC9204) transfected with inhibitory kappa B alpha（IκB-α)vector. Methods After pcDNA3-IκB-α vector and pcDNA3 were transfected into HCC9204 by lipofectamine method, Western-blot and RT-PCR analysis were used to detect the expressions of NF-κB and MMP-9. Migration and invasion of tumor cells were assayed by fundus membrane invaded by them. Results When pcDNA3-IκB-α was transfected into HCC9204, the expression of NF-κB was decreased at the protein level, and the expression of MMP-9 mRNA and the invision and metastasis ability of transfected cells were obviously decreased. Conclusion When the activity of NF-κB is inhibited, the ability of invasion and metastasis in HCC9204 cells decrease, which could be related to the decreased the expression of MMP-9.
Objective To explore the effects of asiaticoside on the activation of nuclear factor kappa B ( NF-κB) and cytokines expression in RAW264. 7 cells induced by lipopolysaccharide ( LPS) . Methods RAW264. 7 cells were allocated to 5 groups, ie. a blank group, a model group stimulated by LPS at dose of 10 μg/mL, and three asiaticoside treatment groups stimulated by LPS and different doses of asiaticoside simultaneously. The effects of asiaticoside ( 10 - 7 , 10 - 6 , 10 - 5 mol /mL) on the proliferation of cells were examined by MTT assay. The activation of NF-κB was detected and analyzed by the laser scanning confocal microscope( LSCM) ,meanwhile the concentrations of TNF-α, IL-1, and IL-10 in supernatants were quantified by ELISA. Results MTT assay showed that asiaticoside ( 10 - 7 , 10 - 6 ,10 - 5 mol /mL) had no effects on the proliferation of RAW264. 7 cells. Asiaticoside significantly decreased the activation of NF-κB, downregulated the secretion of TNF-αand IL-1, and upregulated IL-10 secretion in a dose dependent manner. According to LSCM, the ratio of NF-κB activation was ( 3. 5 ±1. 5) % , ( 75. 7 ±9. 1) % , ( 66. 8 ±7. 1) % , ( 58. 9 ±9. 0) % , and ( 40. 1 ±8. 8) % in the blank, model, and asiaticoside( 10 - 7 , 10 - 6 , 10 - 5 mol /mL) treatment groups respectively. The contents of TNF-α in supernatants were ( 171. 12 ±35. 42, 1775. 45 ±193. 97,1284. 63 ±162. 13,1035. 22 ±187. 97, 598. 90 ±107. 73) pg/mL respectively and IL-1 were ( 5. 66 ±0. 98,26. 93 ±3. 48,22. 41 ±2. 84, 17. 05 ±1. 70, 10. 64 ±1. 29) ng/mL respectively, while IL-10 were ( 25. 23 ±2. 17,71. 75 ±8. 31, 82. 82 ±6. 00, 98. 70 ±8. 84, 119. 97 ±9. 13) pg/mL respectively. Conclusion The antiinflammation mechanism of asiaticoside may be mediated by downregulating inflammatory factors throughNF-κB signal pathway and keeping the balance between proinflammatory and antiinflammatory system.
Objective To investigate the effects of nuclear factor kappa B decoy oligodeoxynucleotides ( NF-κB decoy ODN) transfection on biological characteristics of mature dendritic cells ( mDCs) in mice. Methods Immature DCs were harvested from Balb / c mice bone marrow, followed by the incubation with antigen OVA and LPS, and mature DCs were evaluated by the expressions of CD11c and MHC-Ⅱ detected by FACS. Mature DCs were transfected with NF-κB decoy ODN and the changes of NF-κB activity after the transfection were detected by EMSA. The expressions of the costimulatory molecules( CD40,CD80 and CD86) on DCs were detected by FACS and the proliferation of T cells was tested by mixed lymphocyte reaction( MLR) . Results The mature DCs were cultured successfully. The NF-κB activity of NF-κB decoy ODN transfected DCs was decreased significantly( P lt; 0. 05) . There was no difference in the expressions of CD40 and CD80, but the expression of CD86 was decreased significantly in NF-κB decoy ODN transfection group( P lt; 0. 05) . MLR test showed that the proliferation of T lymphocyte cells was inhibited by NF-κB decoy ODN transfected DCs, but was stimulated bly by the DCs of other groups. Conclusions Mature DCs transfected with NF-κB decoy ODN could inhibit the proliferation and activation of antigenspecical T cells, which was probably related to the down-regulation of CD86 on DCs. This modified DCs might be a promising vaccine for the treatment of asthma in the future.
Objective To observe the expression levels of nuclear factor kappa B （NF-κB）， vascular endothelial growth factor （VEGF）， and CD31 in portal vein and surrounding tissues of rats during the formation process of cavernoustransformation of portal vein （CTPV）， and try to search the relationship between NF-κB， VEGF， and the angiogenesisof portal areas， as well as the significance and the role of NF-κB and VEGF in the formation process of CTPV. Methods One hundred and ten Sprague-Dawley （SD） rats were randomly （random number method） divided into sham operation group and model group. The partial constriction operations on portal vein were performed in model rats with a blunt 21Gcaliber to establish CTPV animal models （model group）， while the exploratory operations on portal vein， not constriction，were performed in rats of sham operation group. All specimens （portal vein and surrounding tissues） were fixed in formalinand made into paraffin blocks. Each specimen was tested by immunohistochemistry for the expressions of NF-κB， VEGF， and CD31， then optical density （OD） of NF-κB expression and the mean integral optical density （IOD） of VEGF expressionwere measured by using Image Pro Plus 6.0 software， and microvessel density （MVD） was calculated under microscope. Results Nucleoplasm ratio of OD value of NF-κB， mean IOD value of VEGF， and MVD value in 1， 2， 3， 4， and 6 weeks after operation didn’t significantly differed from that of before operation in sham operation group （P＞0.05）， but higher at all time points after operation in model group （P＜0.01）. Compared with sham operation group， nucleoplasm ratio of OD value of NF-κB， mean IOD value of VEGF， and MVD value were significantly higher in 1， 2， 3， 4， and 6 weeks after operation in model group （P＜0.01）. NF-κB and VEGF， NF-κB and MVD， VEGF and MVD were positively correlated with each other （r=0.654 6，P＜0.01；r=0.620 7， P＜0.01；r=0.636 9， P＜0.01） in model group. Conclusion NF-κB and VEGF may relate to the formation of CTPV， and may involve in the angiogenesis.
Objective Series of compl icated molecule signal pathway are involved in the bone regeneration. To explore the possibil ity of nuclear factore kappa B (NF-κB) which is taken as the “key activation” during the fracture healing and provide the laboratory evidence for the gene therapy of nonunion or delayed union of fractures. Methods Thirtythree adult male Wistar rats (weighing 180-220 g) were selected and divided randomly into 4 groups: group A (the control group, n=3), the rigth lower segments of radius were injected with normal sal ine 0.3 mL for 7 days, once per day; group B (Bay 11-7082 injection group, n=6), the middle and distal radius were injected with normal sal ine containing 50 μmol/L NF- κB inhibitor Bay 11-7082 0.3 mL for 7 days, once per day; group C (fracture group, n=12), the right middle and distal radius were cut by a sharp scissors to form per fracture model; and group D (Bay 11-7082 treatment group, n=12), based on group C, 0.3 mL of 50 μmol/L Bay 11-7082 were injected into the fracture site for 7 days, once per day. The callus tissues were harvested at 3, 7, 14, and 28 days after fracture for Western blot analysis, alkal ine phosphatase (ALP) activity assessment, prostaglandins E2 (PGE2) production assay, and histological observation. Results The rats of all groups were survivaltill the experiment completion. At 3 and 7 days after injection, there was no significant difference in the ALP activity and PGE2 production between group B and group A (P gt; 0.05); but group C was significantly higher than group A (P lt; 0.01) and group D was significantly lower than group A (P lt; 0.01). The expressions of NF-κB p65, bone morphogenetic protein 7 (BMP-7), and inhibitor of DNA binding 2 (Id2) were observed at fracture sites of 4 groups. There was no significant difference in the expressions of NF-κB p65, BMP-7, and Id2 between group B and group A (P gt; 0.05); the expressions of NF-κB p65 and BMP-7 were significantly higher and the expression of Id2 was significantly lower in group C than group A (P lt; 0.01); and the expressions of NF-κB p65 and BMP-7 were significantly lower and the expression of Id2 was significantly higher in group D than group A (P lt; 0.01). The histological observation showed that a lot of osseous callus formed in group C at 14 and 28 days, but osseous callus just began to form in group D at 28 days. Conclusion NF-κB p65 can facil itate early fracture heal ing of rat radius by elevating the PGE2 production and regulating BMP-7 and Id2 expression.
ObjectiveTo investigate the effects of naringenin on the production of chemokines and its mechanism in human bronchial epithelial (HBE) cells. MethodsHBE cells were divided into a control group, a TNF-αgroup, a low-dose naringenin group, a moderate-dose naringenin group and a high-dose naringenin group. The Naringenin groups were incubated with different doses of naringenin (10, 5 and 2.5μmol/L, respectively) for 2 h. Then the naringenin groups and the TNF-αgroup were incubated with TNF-α. After 24 h of incubation, the levels of eotaxin and RANTES were determined by ELISA method, and IκBαdegradtion was detected by Western blot method. After incubated with TNF-αfor 30 min, NF-κB DNA-binding activity was detected by EMSA method. ResultsCompared with the control group, the levels of eotaxin and RANTES were significantly increased in the HBE cells stimulated with TNF-α. Naringenin had inhibitory effects on the expression of these chemokines. Naringenin abolished IκBαdegradation and reduced the DNA-binding activity of NF-κB. ConclusionNaringenin may inhibit the production of chemokines through inhibiting NF-κB pathway.
Objective To investigate the roles of NF-κB and EGFR in hepatolithiasis associated with intrahepatic cholangiocarcinoma. Methods Ninety cases of liver tissue specimens from hepatectomies performed in the 2nd Affiliated Hospital of Sun Yat-sen University between August 1989 and June 2009 were enrolled in the study. Among them, 33 cases of hepatolithiasis associated with intrahepatic cholangiocarcinoma were considered as observing group, 32 cases of hepatolithiasis as control group, and 25 cases of normal bile duct tissues as normal control group. The SP method of immunohistochemical staining was applied to detect the expressions of NF-κB and EGFR in intrahepatic biliary ducts epithelial cells, and their relations with clinicopathologic factors and the accumulated survival rate of hepatolithiasis associated with intrahepatic cholangiocarcinoma were analyzed. Results Expression rates of NF-κB and EGFR were gradually raised from normal control group, control group to observing group (Plt;0.01). Expression of EGFR in tumor patients was related to histopathologic differentiation grading and the depth of tumor invasion (Plt;0.05), but not to gender, age, or lymph node metastasis (Pgt;0.05)； there were no significant relationships between the expression of NF-κB and factors described above (Pgt;0.05). The survival rate of patients with tumor expressed EGFR was significantly lower than that of patients with tumor non-expressed EGFR (Plt;0.01). Conclusions NF-κB expression is in the early stage during intrahepatic cholangiocarcinoma genesis. NF-κB and EGFR play cooperating roles during hepatolithiasis carcinogenesis process. Over expression of EGFR is related with poor differentiation and prognosis of tumor.
Objective To investigate the molecular mechanism of multiple cellular factors expressed shortly after ischemia reperfusion (IR) injury from the pathway of nuclear factor kappa B (NF κB). Methods The isolated heart models were established and sixty six rats were randomly divided into experimental group and control group. The deoxyribonucleic acid (DNA) binding activities of NF κB, the inhibitory kappa B (IκBα) levels in cytoplasm and tumor necrosis factor α (TNF α) messenger ribonucleic acid (mRNA) expressions were determined after 5, 15 min ischemia in experimental group, both after 0, 5, 15, 30 min ischemia and concomitantly 5, 15, 30, 45, 60 min reperfusion in control group. Results Augment of DNA binding activities of NF κB and reduction of IκBα in cytoplasm shortly after ischemia results were observed in control group. The level of IκBα was restored after reperfusion, the DNA binding activities of NF κB was further augmented. DNA binding activities of NF κB and TNF α mRNA expressions were lower in experimental group than those in control group. Conclusions NF κB in IR myocardium is activated by two different pathways: p65 p50 heterodimers and p50 p50 homodimers. In addition, the results suggest that early activation of NF κB induced by ischemia in the myocardium could be a signal mechanism for controlling and regulating immediate gene expressions during ischemia reperfusion.
ObjectiveTo determine the nuclear factor kappa B (NFkB) activity in peripheral blood mononuclear cells (PBMC) in patients with acute cholangitis of severe type (ACST) and correlate the degree of NFkB activation with severity of biliary tract infection and clinical outcome.MethodsTwenty patients with ACST were divided into survivor group (14 cases) and nonsurvivor group (6 cases). Other 10 patients undergoing elective gastrectomy or inguinal hernia repair were selected as control group. Peripheral blood samples were taken 24 hours after operation, PBMC was separated and nuclear proteins were isolated from PBMC, and NFkB was determined with electrophoretic mobility shift assay (EMSA). The levels of TNFα, IL6 and IL10 in plasma were determined by using an enzymelinked immunoassay (ELISA). ResultsThe NFkB activity was 5.02±1.03, 2.98±0.51 and 1.02±0.34 respectively in three groups. It was increased in all patients with ACST, versus the control group (P＜0.05), and the patients of nonsurvivor group had higher levels of NFkB activation than those of survivor group (P＜0.05). The levels of TNFα and IL6 were (496.28±52.35) ng/L and (578.13±67.72) ng/L in nonsurvivor group; (284.47±39.41) ng/L and (318.67±34.92) ng/L in survivor group; (89.43±10.39) ng/L and (101.27±13.47) ng/L in control group. All patients with ACST had increased levels of TNFα and IL6, which were many fold greater than that of control group, and there was an evidence of significantly higher levels in nonsurvivor group than in survivor group (P＜0.05). All patients had also increased levels of IL10 as compared to control group (P＜0.05), but the IL10 concentrations in plasma were not significantly higher in nonsurvivors than that of in those survivors (Pgt;0.05). ConclusionNFkB activation in PBMCs in patients with ACST
Objective To investigate the effects of 1, 25-( OH) 2D3 on the expression of matrix metalloprotease-9 ( MMP-9) and nuclear factor κB ( NF-κB) activity in a murine model of chronic asthma. Methods BALB/ c mice were sensitized and challenged with ovalbumin to establish chronic asthmatic model. The animals were randomly divided into a control group, an asthma group and a VD group. Lung sections from the mice were stained by HE and Masson’s trichrome, respectively. Morphometric analysis of the stained sections was performed using computerized image analysis system. Nuclear translocation of NF-κB p65 was examined using Western blot. The level of IκBαwas detected with real-time quantitative PCR ( RTPCR) and Western blot. In addition, the expression of MMP-9 in both activity and mRNA level was detected by gelatin zymograph and RT-PCR, respectively. Results Prominent airway remodeling developed in the asthma group, including the inflammatory cell infiltration, subepithelial collagen deposition and increased airway smooth muscle mass. In contrast, 1, 25-( OH) 2D3 attenuated these established structural changes of the airways. Stimulation with OVA induced a 7. 87-fold increase in the MMP-9 activity compared with that in the control group, and 1, 25-( OH) 2D3 treatment only induced a 3. 46-fold increase in the MMP-9 activity compared with that in the control group ( P lt;0. 05) . The mRNA level of MMP-9 in the VD group ( 3.16 ± 0.09) was decreased compared with the asthma group ( 5.74 ±0.13) ( P lt;0.05) , but itwas still higher than that in the control group ( 0.57 ±0.08) ( P lt;0.05) . 1, 25-( OH) 2D3 reduced the nuclear translocation of NF-κB p65 while up-regulated the IκBα level in lung tissue of chronic asthma. Conclusions 1, 25- ( OH) 2D3 can inhibit the NF-κB activity and down-regulate the expression of MMP-9 in lung tissue of chronic asthma, thus alleviating the established chronic asthma-induced airway remodeling.