Objective To investigate the effects of simvastatin on lung tissue in septic rats by observing the protein expression of nuclear factor kappa B ( NF-κB) and pathologic changes in lung tissue at different time points. Methods 90 healthy male Sprague-Dawley rats were randomly divided into three groups ( n =30 in each group) . All the rats received administration by caudal vein and capacity volume is 2 mL. The rats in the control group were treated with saline ( 2 mL) . The rats in the LPS group were treated with LPS ( 5 mg/kg ) . The rats in the simvastatin group were treated with LPS ( 5 mg/kg) and simvastatin ( 20 mg/kg) . Six rats in each group were killed randomly at 2, 4, 6, and 12 hours after the injection, and the right middle lobe of lung was taken out. Pathological changes of lung tissue wee investigated under light microscope. The expression of NF-κB in lung tissue was determined by immunohistochemistry ( IHC) method. Results Microscopic studies showed that there were not pathological changes in the lung tissue of rats in the control group. While in the LPS group, the alveolar spaces were narrowed and the alveolar wall were thickened. Furthermore, severe interstitial edema of lung and proliferation of epithelial cells were observed. In the simvastatin group, the degree of the infiltration of leukocytes and the lung interstitial edema were less severe than those in the simvastatin group. In the control group, the expression of NF-κB protein in most of lung tissue was negative. In the LPS group, the expression of NF-κB protein was detected at 2h, andreached the peak at 6h, then decreased at 12h. In the Simvastatin group, the NF-κB expression was significantly lower than that in the LPS group at all time points ( P lt; 0. 01) . Conclusion Simvastatin can ameliorate pathological lesions and decrease expression of NF-κB in lung tissue of septic rats.
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.
【Abstract】 Objective To investigate the protective role of recombinant human growth hormone (rhGH )in ischemic reperfusion injury of rat liver and its mechanism. Methods One hundred Male rats were randomly divided into two groups: the rhGH group and the control group. In the rhGH group, rhGH were injected （0.2U/100g weight） to rats seven days before the ischemic reperfusion injury, and in the control group, normal saline was injected instead. Serum levels of ALT， TNF-α and IL-1α were tested. Hepatic tissue was sectioned for to detect the level of EC and MDA, the expression of NF-κB and ICAM-1 mRNA on SEC. Ultrastructural characteristics histopathological characteristics were determined also. Results Serum levels of ALT, TNF-α, IL-1α and the contents of MDA in the control group were significantly higher than those in the rhGH group （P＜0.05）. Comparied with control group, rhGH also decreased NF-κB activation, and reduced the expression of ICAM-1 mRNA of SEC in the liver cells （P＜0.05）. Electronic microscopic revealed that the hepatic sinusoidal endothelial cells and the hepatocellular mitochondria were injured in the control group. Pretreatment with the rhGH was able to significantly improved the pathological changes. Conclusion rhGH might confer the protection to ischemic reperfusion injury of rat liver through reducing the expression of NF-κB to down-regulate cytokine (IL-1α,TNF-α), MDA and inhibition the expression of ICAM-1 mRNA.
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 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.
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.
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 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 changes of TLR2 mRNA expression in lung of a mouse model of Chlamydia Pneumoniae pneumonitis, and to explore the possible mechanism of signal transduction. Methods Ninety-six male C3H/HeJ mice were randomly divided into four groups as follows: a control group, a model group, a SB203580 intervened group, and a pyrrolidine dithiocarbamate( PDTC) intervened group. Chlamydia Pneumoniae pneumonitis was induced by intranasally inoculated with 4. 0 ×106 IFU/mL of C. Pneumoniae per mouse in the model group and two intervened groups. Then the intervened groups were intraperitoneally injected with the p38MAPK inhibitor SB203580 and nuclear factor kappa B ( NF-κB)inhibitor PDTC, respectively. Six mice in each group were randomly killed in 1st, 4th, 7th and 14th day. The expressional changes of TLR2 mRNA in the mice lung tissue were measured by semi-quantitative RT-PCR. The concentrations of TNF-α in the lung homogenate were measured by ELISA. Results TLR2 mRNA expression in the lung tissue significantly increased after C. Pneumoniae infection, peaking at 4th and 7th days, then decreased after 14th day. Tumor necrosis factor-α( TNF-α) was also elevated in the lung tissue after C. Pneumoniae challenging. Both SB203580 and PDTC treatment effectively inhibited TNF-αand TLR2 mRNA expressions in lung. The inhibitory effect was more obvious by SB203580 treatment. Conclusion C. Pneumoniae can upregulate the expressions of TLR2 and TNF-α in lung, and TLR2/MAPK and TLR2 /NF-κB signal pathways may be involved in Chlamydia Pneumoniae pneumonitis.
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.