Semaphorin 7A impairs barrier function in cultured human corneal epithelial cells in a manner dependent on nuclear factor-kappa B

●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0,125,250,or 500 ng/mL for 24,48,or 72h in vitro.Transepithelial electrical resistance(TEER)as well as Dextran-fluorescein isothiocyanate(FITC)permeability assays were conducted to assess barrier function.To quantify tight junctions(TJs)such as occludin and zonula occludens-1(ZO-1)at the mRNA level,reverse transcriptionpolymerase chain reaction(RT-PCR)analysis was performed.Immunoblotting was used to examine the activity of the nuclear factor-kappa B(NF-κB)signaling pathway and the production of TJs proteins.Immunofluorescence analyses were employed to localize the TJs.Enzyme-linked immunosorbent assay(ELISA)and RT-PCR were utilized to observe changes in interleukin(IL)-1βlevels.To investigate the role of NF-κB signaling activation and IL^(-1)βin Sema7A’s anti-barrier mechanism,we employed 0.1μmol/L IκB kinase 2(IKK2)inhibitor IV or 500 ng/mL IL^(-1)receptor(IL-1R)antagonist.●RESULTS:Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time-and dose-dependent manner,as well as altering the localization of TJs.Furthermore,Sema7A stimulated the activation of inhibitor of kappa B alpha(IκBα)and expression of IL-1β.The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists.●CONCLUSION:Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins,as well as the expression of IL-1β.These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.

Delayed hepatocarcinogenesis through antiangiogenic intervention in the nuclear factor-kappa B activation pathway in rats

BACKGROUND: The active form of nuclear factor-kappa B (NF-kappa B) is involved in the initiation, generation, and development of hepatocellular carcinoma (HCC), and is up-regulated in inflammation-associated malignancies. We investigated the dynamic expression of NF-kappa B and its influences on the occurrence of HCC through antiangiogenic (thalidomide) intervention in NF-kappa B activation. METHODS : Hepatoma models were induced with 2-fluorenylacetamide (2-FAA, 0.05%) in male Sprague-Dawley rats, and thalidomide (100 mg/kg body weight) was administered intragastrically to intervene in NF-kappa B activation. The pathological changes in the liver of sacrificed rats were assessed after hematoxylin and eosin staining. NF-kappa B mRNA was amplified by RT-nested PCR. The alterations of NF-kappa B and vascular endothelial growth factor (VEGF) expression were analyzed by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blotting. RESULTS: Rat hepatocytes showed denatured, precancerous, and cancerous stages in hepatocarcinogenesis, with an increasing tendency of hepatic NF-kappa B, NF-kappa B mRNA, and VEGF expression, and their values in the HCC group were higher than those in controls (P<0.001). In the thalidomide-treated group, the morphologic changes generated only punctiform denaturation and necrosis at the early or middle stages, and nodular hyperplasia or a little atypical hyperplasia at the final stages, with the expression of NF-kappa B (chi(2)=9.93, P<0.001) and VEGF (chi(2)=8.024, P<0.001) lower than that in the 2-FAA group. CONCLUSION: NF-kappa B is overexpressed in hepatocarcinogenesis and antiangiogenic treatment down-regulates the expression of NF-kappa B and VEGF, and delays the occurrence of HCC. (Hepatobiliary Pancreat Dis Int 2010; 9: 169-174)

Urinary trypsin inhibitor attenuates hepatic ischemia-reperfusion injury by reducing nuclear factor-kappa B activation

BACKGROUND: Urinary trypsin inhibitor (UTI) inhibits the inflammatory response and protects against ischemia-reperfusion (I/R) injury. The inflammatory response is mediated by nuclear factor-kappa B (NF-kappa B) and its related target genes and products such as vascular endothelial cell adhesion molecule and CXC chemokines. We aimed to assess the roles of those mediators in a UTI-treated mouse model of hepatic I/R injury. METHODS: Treatment group 1 (UTI given 5 minutes prior to liver ischemia), treatment group 2 (UTI given 5 minutes after the anhepatic phase) and a control group were investigated. Blood and liver samples were obtained and compared at 1, 3, 6 and 24 hours after reperfusion. RESULTS: Attenuation of pathological hepatocellular damage was greater in the treatment groups than in the control group (P < 0.05). Compared with the control group, the UTI treatment groups showed significantly lower serum alanine aminotransferase and aspartate aminotransferase levels, decreased myeloperoxidase activity, and reduced NF-kappa B activation. Also downregulated was the expression of tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein-2 at the mRNA level. P-selectin protein and intercellular adhesion molecule-1 protein expression were also downregulated. In addition, the treatment group I showed a better protective effect against I/R injury than the treatment group 2. CONCLUSIONS: UTI reduces NF-kappa B activation and downregulates the expression of its related mediators, followed by the inhibition of neutrophil aggregation and infiltration in hepatic I/R injury. The protective role of UTI is more effective in prevention than in treatment.

Dietary saturated fatty acid and polyunsaturated fatty acid oppositely affect hepatic NOD-like receptor protein 3 inflammasome through regulating nuclear factor-kappa B activation

AIM: To investigate the effect of different dietary fatty acids on hepatic inflammasome activation.METHODS: Wild-type C57BL/6 mice were fed either a high-fat diet or polyunsaturated fatty acid (PUFA)-enriched diet. Primary hepatocytes were treated with either saturated fatty acids (SFAs) or PUFAs as well as combined with lipopolysaccharide (LPS). The expression of NOD-like receptor protein 3 (NLRP3) inflammasome, peroxisome proliferator-activated receptor-&#x003b3; and nuclear factor-kappa B (NF-&#x003ba;B) was determined by real-time PCR and Western blot. The activity of Caspase-1 and interleukine-1&#x003b2; production were measured.RESULTS: High-fat diet-induced hepatic steatosis was sufficient to induce and activate hepatic NLRP3 inflammasome. SFA palmitic acid (PA) directly activated NLRP3 inflammasome and increased sensitization to LPS-induced inflammasome activation in hepatocytes. In contrast, PUFA docosahexaenoic acid (DHA) had the potential to inhibit NLRP3 inflammasome expression in hepatocytes and partly abolished LPS-induced NLRP3 inflammasome activation. Furthermore, a high-fat diet increased but PUFA-enriched diet decreased sensitization to LPS-induced hepatic NLRP3 inflammasome activation in vivo. Moreover, PA increased but DHA decreased phosphorylated NF-&#x003ba;B p65 protein expression in hepatocytes.CONCLUSION: Hepatic NLRP3 inflammasome activation played an important role in the development of non-alcoholic fatty liver disease. Dietary SFAs and PUFAs oppositely regulated the activity of NLRP3 inflammasome through direct activation or inhibition of NF-&#x003ba;B.

Testosterone alleviates tumor necrosis factor-alpha-mediated tissue factor pathway inhibitor downregulation via suppression of nuclear factor-kappa B in endothelial cells

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor(TFPI)gene expression through the androgen receptor in endothelial cells.This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha(TNF-α).Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-α.TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction.To study the cellular mechanism of testosterone’s action,nuclear factor-kappa B(NF-κB)translocation was confirmed by electrophoretic mobility shift assays.We found that after NF-κB was activated by TNF-α,TFPI protein levels declined significantly by 37.3%compared with controls(P<0.001),and the mRNA levels of TFPI also decreased greatly(P<0.001).A concentration of 30 nmol L-1 testosterone increased the secretion of TFPI compared with the TNF-α-treated group.NF-κB DNA-binding activity was significantly suppressed by testosterone(P<0.05).This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-κB activity.

Shuanghuanglian injection downregulates nuclear factor-kappa B expression in mice with viral encephalitis

A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension. Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and protein expression of intelectin-2 and nuclear factor-kappa B in the viral encephalitis and control groups. Nuclear factor-kappa B and intelectin-2 mRNA and protein expression were significantly increased in mice with viral encephalitis. After intraperitoneal injection of Shuanghuanglian at a dose of 1.5 mg/kg for 5 successive days, intelectin-2 and nuclear factor-kappa B protein and mRNA expression were significantly decreased. To elucidate the relationship between intelectin-2 and nuclear factor-kappa B, mice with viral encephalitis were administered an intracerebral injection of 107 pfu recombinant lentivirus expressing intelectin shRNA. Both protein and mRNA levels of intelectin and nuclear factor-kappa B in brain tissue of mice were significantly decreased. Experimental findings suggest that Shuanghuanglian injection may downregulate nuclear factor-kappa B production via suppression of intelectin production, thus inhibiting inflammation associated with viral encephalitis.

Inhibitory effects of Shuanghuanglian injection on nuclear factor-kappa B expression in mice with viral encephalitis in a time-and dose-dependent manner

Previous studies have confirmed that the anti-virus effects of Shuanghuanglian injection may be associated with nuclear factor-kappa B activity. This study observed nuclear factor-kappa B expression in mice with viral encephalitis, and showed significant decreases in nuclear factor-kappa B protein and mRNA levels following Shuanghuanglian injection. The inhibitory effect was more significant with prolonged intervention duration and increased treatment dose. These findings verify that Shuanghuanglian injection plays a therapeutic role in viral encephalitis by reducing expression of nuclear factor-kappa B in a time- and dose-dependent manner.

Effects of nuclear factor-kappaB on rat hepatocyte regeneration and apoptosis after 70% portal branch ligation

AIM： To detect the DNA binding activity of nuclear factor-kappaB （NF-KB） in rat hepatocyte and to investigate the effects of NF-KB on rat hepatocyte regeneration and apoptosis after 70% portal branch Iigation. METHODS： Sixty Wistar rats were randomly divided into control group and portal branch ligation group. The animals were killed 12 h, 1, 2, 3, 7, and 14 d after surgery to determine the contents of plasma ALT. Hepatocytes were isolated and nuclear protein was extracted. DNA binding activity of NF-KB was measured by ENSA. Hepatocyte regeneration and apoptosis were observed under microscope by TUNEL staining. The ultrastructural changes of liver were observed under electron microscope. RESULTS： Seventy percent portal branch ligation produced atrophy of the ligated lobes and the perfused lobes underwent compensatory regeneration, the total liver weight and plasma ALT levels were maintained at the level of sham-operated animals throughout the experiment. After 2 d of portal branch ligation, DNA binding activity of NF-KB in hepatocyte increased and reached its peak, the number of apoptotic hepatocyte in the ligated lobes and the number of mitotic hepatocyte in the perfused lobes also reached their peak. Typical apoptotic changes and evident fibrotic changes in the ligated lobes were observed under electron microscope. CONCLUSION： After 70% portal branch ligation, DNA binding activity of NF-KB in hepatocyte is significantly increased and NF-KB plays an important role in hepatocyte regeneration and apoptosis.

Effects of Five Chitosan Oligosaccharides on Nuclear Factor-kappa B Signaling Pathway

The effects of five chito-oligomers, from dimer to hexamer （chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose） separated from chitosan oligosaccharides, on nuclear factor -kappaB （NF-rd3） signaling pathway were investigated by using luciferase assay and laser scanning microscopy. The expression of NF-rd3 downstream genes （cyclin DI, TNFa and IL-6） were tested by real time PCR. We found that all five chitosan oligosaccharides increased NF-KB-dependent luciferase gene expression and NF-KB downstream genes transcription, and the most significant were chitotetraose and chitohexaose. In addition, laser scanning microscopy experiments showed that chitotetraose and chitohexaose also activated the p65 subunite of NF-kB translocating from cytoplasm to nucleus, which suggested that they were the most potent activators of NF-kB signaling pathway.

Effects of transplantation of microencapsulated rabbit sciatic nerve on nuclear factor-kappa B expression after spinal cord injury in rats

BACKGROUND： It has been reported that nuclear factor-kappa B （NF- κB）, activated after spinal cord injury in rats, plays a key role in inflammatory responses in the central nervous system. OBJECTIVE： To investigate the effects of transplantation of microencapsulated rabbit sciatic nerve on NF- κB expression and motor function after spinal cord injury in rats, and to compare the results with the transplantation of rabbit sciatic nerve alone. DESIGN, TIME AND SETTING： This completely randomized, controlled study was performed at the Department of Neurobiology, Medical College of Nanchang University between December 2007 and July 2008. MATERIALS： A rabbit anti-NF- κB P65 monoclonal antibody was made by the Santa Cruz Company, USA and a streptavidin peroxidase immunohistochemical kit was provided by the Sequoia Company, China. METHODS： Eight rabbits were used to prepare a sciatic nerve cell suspension that was divided into two parts： one stored for transplantation, and the other mixed with a 1.5% sodium alginate solution. One hundred and twenty adult Sprague Dawley rats weighing 220-250 g were randomly divided into four groups： the microencapsulated cell group （n = 36）, the non-encapsulated cell group （n = 36）, the saline group （n = 36） and the sham operation group （n = 12）. The first three groups underwent a right hemisection injury of the spinal cord at the T10 level, into which was transplanted a gelatin sponge soaked with 10 μL of a microencapsulated nerve tissue/cell suspension （microencapsulated cell group）, a tissue/cell suspension （non-encapsulated cell group） or physiological saline （saline group）. In the sham operation group the vertebrae were exposed, but the spinal cord was not injured, and no implantation was given. MAIN OUTCOME MEASURES： Pathological changes were detected using hematoxylin-eosin staining; NF- κB expression was quantified using immunohistochemical staining; motor function was assessed using the Basso, Beattie and Bresnahan （BBB） scale. RESULTS： Spinal cord injuries, such as neuronal death and inflammatory cell infiltration, were found in the microencapsulated cell group, the non-encapsulated cell group and the saline group. However, the damage in the microencapsulated cell group was milder than in the non-encapsulated cell or saline groups. NF- κB expression in the microencapsulated cell group, the non-encapsulated cell group and the saline group was increased after spinal cord injury; it reached a peak after 24 hours, gradually decreased after 3 days, and was close to normal levels after 7 days. NF- κB expression in the microencapsulated cell group was significantly lower than in the saline group and the non-encapsulated cell group （P 〈 0.05）. With time, the motor function of the animals in each group improved to a certain extent, but did not reach normal levels. There were no significant differences in BBB scores between the different groups on post-operative day 3; however, the BBB scores for the microencapsulated cell group and the non-encapsulated cell group were significantly higher than the saline group on post-operative day 7 （P 〈 0.05）. In addition, the motor function recovered better in the microencapsulated cell group than in the non-encapsulated cell group （P 〈 0.05）. CONCLUSION： The transplantation of microencapsulated rabbit sciatic nerve can inhibit NF- κB expression and inflammatory reactions and promote recovery of motor function after spinal cord injury in rats. The effects of microencapsulated cell transplantation are superior to those of transplantation of cells alone.

Influence of edaravone on Notch1 and nuclear factor-kappaB in rats with cerebral ischemia/reperfusion injury

BACKGROUND： It has been demonstrated that edaravone has a a neuroprotective role, inhibits free radical increase, and reduces celt apoptosis. The Notch pathway is a key factor in neurogenesis and cellular apoptosis The proinflammatory transcription factor nuclear factor-kappaB （NF-κB） plays an important role in inflammation and oxidation. OBJECTIVE： To observe the influence of edaravone on Notchl and NF-κB mRNA and protein expression in rats with focal cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING： This randomized controlled neural and molecular biology experiment was performed at the Department of Neurology, the First Affiliated Hospital of Chongqing Medical University, and the Chongqing Key Laboratory of Neurology between July 2007 and May 2008. MATERIALS： Thirty female Wistar rats were used. Edaravone was purchased from Jiangsu Xiansheng Pharmaceutical Limited Company, China. METHODS： Wistar rats were randomly divided into five groups （n = 6）. Thread was inserted into the internal carotid artery of the sham operation group but the middle cerebral artery was not ligated. A focal cerebral ischemia/reperfusion model was established by inserting thread into the right middle cerebral artery. The model rats in the edaravone groups were given tail vein injections of edaravone at 3 mg/kg body weight after ischemia for 2 hours and reperfusion for 12 or 24 hours. Ischemia/reperfusion groups （model group） received intravenous infusion of normal saline at the same volume as the edaravone groups after ischemia for 2 hours and reperfusion for 12 or 24 hours. MAIN OUTCOME MEASURES： The volume of the ischemic region was measured by 2,3,5-triphenyltetrazolium chloride staining. Notchl and NF-κB protein and mRNA expression were measured by immunohistochemistry and RT-PCR. Protein expression was represented by the absorbance value. RESULTS： Edaravone greatly reduced the focal infarct volume. Notchl and NF-κB protein and mRNA expression were rapidly upregulated following cerebral ischemia/reperfusion injury in model and edaravone groups compared with the sham operation group （P 〈 0.01 ）. In addition, edaravone treatment significantly upregulated Notchl expression but down-regulated NF-κB expression compared with model groups （P 〈 0.01）. CONCLUSION： Edaravone possibly protects brain tissue from ischemia/reperfusion injury by upregulating Notchl expression and regulating NF-κB expression.

Activation of nuclear factor-kappa B and effects of pyrrolidine dithiocarbamate on TNBS-induced rat colitis

AIM: To explore the changes of nuclear factor-kappa B (NF-κB) DNA-binding activity, the expression of intercellular adhesion molecule-1(ICAM-1) regulated by IMF-κB at various times and to evaluate the effects of pyrrolidine dithiocarbamate (PDTC) on trinitrobenzene sulfonic acid (TNBS)-induced rat colitis. METHODS: TNBS of 0.6 mL was mixed with ethanol of 0.3 mL solution and instilled into the lumen of the rat colon. The rat models were divided into 6 groups, which were killed at 24 h, 3, 7,14, and 21 d after enema. Colonic inflammation and damage were assessed by macroscopical and histological criteria. Activity of NF-κB DNA-binding was analyzed by electrophoresis mobility shift assays (EMSA). Expression of ICAM-1 was detected by in situ hybridization (ISH) and immunohistochemistry (IH). Then various doses of PDTC were injected into rat abdomen 30 min before enema with TNBS/ethanol as pretreatment. The rats were killed 4 h after enema and the colonic inflammation, myeloperoxidase (MPO) activity, malondialdehyde (MDA) level, and DNA-binding activity of NF-κB were assessed. Finally, PDTC was injected intraperitoneally after colitis was induced. Changes of morphology were assayed. RESULTS: During the first week, hyperemia, hemorrhage, edema and ulceration of the colonic mucosa appeared with predominant infiltration of leukocytes. Neutrophils, macrophages, lymphocytes infiltrated in mucosa and submucosa 14 d later. Fibroblasts and granuloma-like structures were also obviously seen. The binding activity of NF-κB began to increase at 24 h time point and reached a peak at 14 d, then decreased but still was higher than control group at 21 d (P<0.01). Levels of ICAM-1 mRNA and protein significantly elevated at 24 h and the peak was at 21 d. Pretreatment with PDTC could attenuate the development of inflammation but not by reducing NF-KB activity. This attenuation of inflammation had a positive relationship with the dose of PDTC. PDTC at the dose of 100 mg/kg had no therapeutic effect after colitis was induced. CONCLUSION: NF-κB activation is an important event that may be involved in acute and chronic inflammation development and may contribute to self-protection against early inflammation damage. NF-κB also regulates ICAM-1 expression during colonic inflammation. Pretreatment of PDTC may attenuate the inflammation development. But PDTC has no therapeutic effect after the colitis is induced.

Effect of Sirpα1 on the expression of nuclear factor-kappa B in hepatocellular carcinoma

BACKGROUND:Signal regulatory protein alpha1(Sirpα1) is a member of Sirps families containing four immunoreceptor tyrosine-based inhibitory motifs(ITIMs) domains in the cytoplasm of and an activated substrate of receptor tyrosine kinase(RTK),that negatively regulates the RTK-dependent cell proliferating signal transduction pathway.Previously we found that Sirpα1 was closely associated with the occurrence and development of hepatocellular carcinoma(HCC)as well as liver regeneration.Since it is unclear about the regulatory mechanisms,we established the cell line transfected Sirpα1 gene and preliminarily clarified the mechanisms by which Sirpα1 negatively regulates the carcinogenesis and development of HCC. METHODS:Liver cancer Sk-Hep1 cell was respectively transfected with plasmids of pLXSN,pLXSN-Sirpα1 and pLXSN-Sirpα1Δ4Y 2 ,screened with the drug of G418(1200 μg/ml),and various transfected Sk-Hep1 cell lines were obtained.The protein expressions of P65,P50,IκBα,cyclin D1 and Fas in various Sk-Hep1 cell lines were determined by Western blotting,and P65 and P50 were localized by the immunofluorescence technique. RESULTS:Sirpα1 could significantly upregulate the protein expression of IκBα(vs.other cell lines,P<0.05) in the Sk-Hep1 cell,and downregulate the protein expressions of P65,P50 and cyclin D1(vs.other cell lines, P<0.05)in the Sk-Hep1 cell.P65 protein expression was mainly localized in the cytoplasm in the pLXSN Sk-Hep1 cell,and in the nucleus of the Sk-Hep1 cell with mutantSirpα1Δ4Y 2 ,but in nucleus of the Sk-Hep1 cell with wild Sirpα1.P50 protein expression was localized in the cytoplasm and nucleus of the pLXSN Sk-Hep1 cell,but in the nucleus of the Sk-Hep1 cell with wild Sirpα1 and mutant Sirpα1Δ4Y 2 plasmid. CONCLUSIONS:Sirpα1 might negatively regulate and control the abnormal proliferation of liver cancer cells by influencing the protein content and localization of nuclear factor-kappa B,then influence the expression of cyclins such as cyclin D1 in the signal transduction pathway.It may be one of the important mechanisms by which Sirpα1 negatively regulates the carcinogenesis and development of HCC.

Nuclear factor-kappaB activation on the reactive oxygen species in acute necrotizing pancreatitic rats

AIM： To investigate the potential role of nuclear factor kappa-B （NF-κB） activation on the reactive oxygen species in rat acute necrotizing pancreatitis （ANP） and to assess the effect of pyrrolidine dithiocarbamate （PDTC, an inhibitor of NF-κB）.METHODS： Rat ANP model was established by retrograde injection of 5% sodium taurocholate into biliopancreatic duct. Rats were randomly assigned to three groups （10 rats each）： Control group, ANP group and PDTC group. At the 6^th of the model, the changes of the serum amylase,nitric oxide （NO）, malondialdehyde （MDA）, superoxide dismutase （SOD） and pancreatic morphological damage were observed. The expressions of inducible nitric oxide （iNOS） were observed by SP immunohistochemistry. And bhe expressions of NF-κB p65 subunit mRNA were observed by hybridization in situ.RESULTS： Serum amylase and NO level decreased significantly in ANP group as compared with PDTC administrated group [（7 170.40＋1 308.63） U/L vs（4 074.10＋1 719.78） U/L,P〈0.05], [（76.95±9.04） μmol/L vs （65.18±9.02） μmol/L,P〈0.05] respectively. MDA in both ANP and PDTC group rose significantly over that in control group [（9.88＋1.52）nmol/L, （8.60±1.41） nmol/L, vs （6.04：hl.78） nmol/L,P〈0.05], while there was no significant difference between them. SOD levels in both ANP and PDTC group underwent a significant decrease as compared with that in control[（3 214.59±297.74） NU/mL, （3 260.62±229.44） NU/mL,vs（3 977.80＋309.09） NU/mL, P〈0.05], but there was no significant difference between them. Though they were still higher bhan those in Control group, pancreas destruction was slighter in PDTC group, iNOS expression and NF-κB p65 subunit mRNA expression were lower in PDTC group as compared with ANP group.CONCLUSION： We conclude that correlation among NF-κB activation, serum amylase, reactive oxygen species level and tissue damage suggests a key role of NF-κB in the pathogenesis of ANP. Inhibition of NF-κB activation may reverse the pancreatic damage of rat ANP and the production of reactive oxygen species.

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.

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.

Effect of Nuclear Factor-kappa B on Vascular Endothelial Growth Factor mRNA Expression of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia

In order to investigate the effect of nuclear factor kappa B (NF κB) on vascular endothelial growth factor (VEGF) mRNA expression of human pulmonary artery smooth muscle cells (HPASMCs) in hypoxia, the cultured HPASMCs in vitro were stimulated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF κB. The NF κB p65 nuclei positive expression was detected by immunocytochemical technique. The IκBα protein expression was measured by Western blot. RT PCR was used to detect the VEGF mRNA expression of HPASMCs. The results showed that no significant change was observed in the NF κB p65 nuclei positive expression of cultured HPASMCs during 6 h-24 h in normoxia, but the levels of NF κB p65 nuclei positive expression of cultured HPASMCs were significantly increased in hypoxia groups as compared with those in all normoxia groups ( P <0.05). The IκBα protein expression of cultured HPASMCs showed no significant change during 6 h-24 h in normoxia, but significantly decreased in hypoxia as comapred with that in normoxia groups ( P <0.05). PDTC (1 to 100 μmol/L) could inhibit the VEGF mRNA expression of HPASMCs in a concentration dependent manner in hypoxia. In conclusion, NF κB can be partly translocation activated from cytoplasm into nuclei in the cultured HPASMCs under hypoxia. The inhibition of NF κB activation can decrease the VEGF mRNA expression. It is suggested that the activation of NF κB is involved in the VEGF mRNA expression of HPASMCs under hypoxia.

Treatment of COVID-19 by Controlling the Activity of the Nuclear Factor-Kappa B

Heavy infection of the virus leads to overproduction of cytokines. The overproduction of cytokine (cytokines storms) is responsible for the critical cases and deaths of COVID-19. The nuclear factor kappa-B stimulates the expression of the genes, which is responsible for cytokines storm and RNA transcription. The COVID-19 virus can be controlled by inhibition of nuclear factor kappa-B. Nuclear factor kappa-B is controlled by inhibition of hydrogen peroxide and inhibitor kappa-B kinase enzyme.

Interon-gamma Enhances the Antitumor Effect of All-trans Retinoic Acid on Hepatocellular Carcinoma Cells by Inhibiting the Expression of Nuclear Factor-kappaB

Objective： To explore the combination effects of all-trans retinoic acid （ATRA） with interferon-gamma （IFN-γ） on human hepatocarcinoma cell line SMMC-7721 and the mechanism of action. Methods： SMMC-7721 cells were divided into treated group and control group. The cells were treated with ATRA or ATRA＋ IFN-γ in the former and added with PBS in the latter. The inhibition rate of SMMC-7721 cell proliferation was detected by MTT, the cell change in morphology was observed by electron microscope. The apoptosis was detected by flow cytometry and the expression changes of nuclear factor-kappaB （NF-κB） was analyzed by Western blotting when the SMMC-7721 cells were treated with ATRA and IFN-γ. Results： The SMMC-7721 cell proliferation was suppressed and apoptosis was induced after the cells were treated with ATRA treatment, and these effects were enhanced when ATRA was combined with IFN-γ. The expression of NF-κB was reduced after SMMC-7721 cell was treated with ATRA, and reduced significantly when the cells were treated with the combination of ATRA and IFN-γ. Conclusion： IFN-γ can enhance the inhibiting effects of ATRA on cell proliferation and inducing apoptosis on SMMC-7721 cell and these effects might be mediated by inhibiting the expression of NF-κB.

Benchmark experiment on slab^(238)U with D-T neutrons for validation of evaluated nuclear data

A benchmark experiment on^(238)U slab samples was conducted using a deuterium-tritium neutron source at the China Institute of Atomic Energy.The leakage neutron spectra within energy levels of 0.8-16 MeV at 60°and 120°were measured using the time-of-flight method.The samples were prepared as rectangular slabs with a 30 cm square base and thicknesses of 3,6,and 9 cm.The leakage neutron spectra were also calculated using the MCNP-4C program based on the latest evaluated files of^(238)U evaluated neutron data from CENDL-3.2,ENDF/B-Ⅷ.0,JENDL-5.0,and JEFF-3.3.Based on the comparison,the deficiencies and improvements in^(238)U evaluated nuclear data were analyzed.The results showed the following.(1)The calculated results for CENDL-3.2 significantly overestimated the measurements in the energy interval of elastic scattering at 60°and 120°.(2)The calculated results of CENDL-3.2 overestimated the measurements in the energy interval of inelastic scattering at 120°.(3)The calculated results for CENDL-3.2 significantly overestimated the measurements in the 3-8.5 MeV energy interval at 60°and 120°.(4)The calculated results with JENDL-5.0 were generally consistent with the measurement results.