NF-kappaB P65 Protein Research Articles

Abstract A16: Regulation of TRAIL and NF-kappaB pathways in ovarian cancer.

Abstract Objective: The equilibrium between apoptotic and survival pathways determines the fate of cells. Dysregulation in any one mechanism often leads to cancer. The present study was designed to determine the crosstalk between TRAIL and NF-kappaB pathways through study of the much controversial role of DcR2 in the induction of NF-kappaB. Methodology: A total of 50 epithelial ovarian tissue samples were used for immunohistochemical staining of DcR2 and NF-kappaB (p65) proteins along with the other players of TRAIL pathway including DR4, DR5, DcR1, FLIP and TRAIL. Correlations were drawn and survival analysis was performed using Kaplan Meier's method. Results: A total of 72% ovarian carcinomas showed positive DR4 expression, 68% showed positive DR5 expression, 42% showed positive DcR1 expression, 48% showed positive DcR2 expression, 44% had positive FLIP expression, 46% had positive TRAIL expression in epithelium while 28% had positive TRAIL expression in stroma. NF-kappaB was positive in 46% of the tissue samples. A strong but non-significant correlation was seen between DcR2 and NF-kappaB expression. Interestingly a similar correlation was also found between DR4 and NF-kappaB expression. Survival analysis showed better survival for patients with positive NF-kappaB expression as compared to negative expression. Conclusion: Coexpression of DcR2 and NF-kappaB suggests a potential therapeutic approach of targeting NF-kappaB through TRAIL pathway by using anti-DcR2 antibodies. However, the study may be elaborated with increased sample size to get significant results. Note: This abstract was not presented at the conference. Citation Format: Mariam Anees, Umaima Batool, Sidra Bukhari, Sohail Yousaf. Regulation of TRAIL and NF-kappaB pathways in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A16.

Sodium butyrate protects against toxin-induced acute liver failure in rats

Acute liver failure (ALF) is a serious clinical syndrome with high mortality. Sodium butyrate has been shown to alleviate organ injury in a wide variety of preclinical models of critical diseases. The aim of this study was to investigate the protective effect of sodium butyrate on ALF in rats. All rats were randomly divided into control, model and sodium butyrate treatment groups. Except the control group, the rats were induced ALF animal model by subcutaneous injection of human serum albumin+ D-galactosamine+lipopolysaccharide. After induction of ALF, the rats in the treatment group received sodium butyrate (500 mg/kg) at 12-hour or 24-hour time point. Fourty-eight hours after ALF induction, the animals were sacrificed and samples were harvested. Serum endotoxin, high mobility group box-1 (HMGB1), liver function parameters, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were measured. The expression of HMGB1 and nuclear factor-kappa B (NF-kappaB) p65 protein in liver tissue was detected by Western blotting. The histological changes of liver and intestine were examined. The survival duration was also observed. Serum endotoxin, alanine aminotransferase, HMGB1, TNF-alpha and IFN-gamma were significantly increased and the liver histology showed more severe histopathological injury in the model group compared with the control group (P<0.05). Compared to the model group, sodium butyrate treatment significantly improved the histopathological changes in the liver and intestine, reduced serum endotoxin and inflammatory cytokines, suppressed HMGB1 and NF-kappaB p65 proteins in liver tissue, and prolonged the survival duration regardless of treatment at 12 hours or 24 hours after induction of ALF (P<0.05). Sodium butyrate protected the liver from toxin-induced ALF in rats. The mechanisms may be due to direct hepatoprotection and decreased intestinal permeability.

Study on protective mechanism of kushenin injection on colonic mucosa of experimental colitis rats

To study the effect of NOD2 on colitis pathogenesis in experimental rats, and discuss therapeutical effect and mechanism of kushenin injection (OMT) on colitis in experimental rats. Fourty Sprague-Dawley (SD) rats were randomly divided into four groups: the normal control group, the model group, the SASP group, and the OMT group, with 10 rats in each group. Except the normal control group, models were established in the remaining three groups with TNBS. The OMT group was injected with kushenin injection, the SASP group was orally administered with mesalazine suspension, the model group and the normal group were orally administered with distilled water for 15 days. Colon lesion score and histological score of experimental rats were observed. Expression of NOD2, NF-kappaB p65 protein in rats colonic mucous was detected by immunohistochemistry. Expression of IL-6 in rat colon mucous was detected by ELISA. Compared with normal control group, the expression of NOD2, NF-kappaB p65 and IL-6 in colonic mucosa of the model group were significantly increased (P < 0.01). The SASP group and the OMT group showed lower expressions of NOD2, NF-kappaB p65 and IL-6 in colonic mucosa than the model group (P < 0.01, P < 0.05). The over expression of colonic mucosa proteins NOD2 and NF-kappaB p65 and increasing secretion of IL-6 take part in the appearance and development of ulcerative colitis. OMT can attenuate ulcerative colitis and protect colonic mucosa by inhibiting expression of NOD2, NF-kappaB p65 and decreasing IL-6.

Research of therapeutical effect and immunologic mechanism of Jiawei Foshou San on model rats of endometriosis

To explore the effect and mechanism of Jiawei Foshou San on the accretion ectopic endometrium of rats. Endometriosis model was established by surgical implant of endometrial tissue which belongs to its body in rats. Jiawei Foshou San was administrated to the model rats. Twenty-eight days later, the length of ectopic endometrium was measured by vernier caliper, the spleen was weighed by electronic balance, the content of tumor necrosis factor (TNF)-alpha in blood serum and peritoneal fluid was detected by ELISA test, the expression of interleukin (IL)-8 in ectopic endometrium was detected by immunohistochemical method, the expression of NF-kappaB p65 protein and inhibitory KBalpha (IkappaBalpha) protein in ectopic endometrium were analyzed by western blot. Jiawei Foshou San 0.045, 0.09, 0.18 g x kg(-1) group reduced the volume of ectopic endometrium. Jiawei Foshou San 0.18 g x kg(-1) group raised the spleen exponent of model rats. Jiawei Foshou San 0.09, 0.18 g x kg(-1) group decreased the content of TNF-alpha in blood serum and peritoneal fluid, and the content of IL-8 in ectopic endometrium was also decreased. Jiawei Foshou San can decrease the expression of NF-KB p65 and increase the expression of IkappaBalpha in ectopic endometrium. Jiawei Foshou San can inhibit the accretion of endometriosis implants of rats, and its mechanism might be associated with improving the environment of body.

Ursolic acid, a naturally occurring triterpenoid, suppresses migration and invasion of human breast cancer cells by modulating c‐Jun N‐terminal kinase, Akt and mammalian target of rapamycin signaling

Metastasis is one of the most important factors related to breast cancer therapeutic efficacy. Ursolic acid, a naturally occurring triterpenoid, has various anticancer activities. In this study, we first observed that ursolic acid exerted a dose- and time-dependent inhibitory effect on the migration and invasion of highly metastatic breast MDAMB231 cells at non-cytotoxic concentrations. This effect was associated with reduced activities of metalloproteinase-2 (MMP-2) and u-PA, which correlated with enhanced expression of tissue inhibitor of MMP-2 and plasminogen activator inhibitor-1, respectively. Ursolic acid suppressed the phosphorylation of Jun N-terminal kinase, Akt and mammalian target of rapamycin, but had no effect on the phosphorylation of ERK and p38. Ursolic acid also strongly reduced the levels of NFkappaB p65, c-Jun and c-Fos proteins in the nucleus of MDAMB231 cells. A time-dependent inhibition of the protein levels of Rho-like GTPases, growth factor receptor-bound protein 2, Ras and vascular endothelial growth factor in cytosol by ursolic acid treatment was also observed. In conclusion, we demonstrated that the anti-invasive effects of ursolic acid on MDAMB231 cells might be through the inhibition of Jun N-terminal kinase, Akt and mammalian target of rapamycin phosphorylation and a reduction of the level of NFkappaB protein in the nucleus, ultimately leading to downregulation of MMP-2 and u-PA expression. These results suggest that ursolic acid has potential as a chemopreventive agent for metastatic breast cancer.

Mutation analysis of tumor suppressor gene PTEN in patients with gastric carcinomas and its impact on PI3K/AKT pathway

The aim of this study was to clarify the participation of PTEN mutation in gastric carcinogenesis and its impact on PI3K/AKT pathway. All nine exons of PTEN were screened for mutations by direct sequencing in 144 patients with pathologically proven gastric carcinoma and their corresponding normal mucosae, followed by Western blotting to detect the changes in PI3K/AKT pathway. Direct sequencing indicated there were 27 cases with mutations among 144 patients consisting of 15 cases (55.6%) of missense mutation, nine nonsense mutations (33.3%), two 1-bp deletion (7.4%), and a mutation within intron 6 (3.7%). The mutation hot spots at codons 36, 75, 232 and 393 had not been observed previously, and the mutation sites in exons 3, 5, 6 and 8 were not found, suggesting that there might be some unique characteristic of PTEN inactivation mechanism in the Shanghai population. The PTEN mutation rate was significantly higher at pTMN stages III and IV than that at stages I and II (P<0.005), and it was higher in poorly differentiated gastric cancer than in well or moderately differentiated types (P<0.05). PTEN and E-cadherin protein expression in gastric cancer was significantly down-regulated comparing with that in paracancerous tissues, while the PI3K, AKT, MMP-2, MMP-9 and NF-kappaBp65 protein were overexpressed in cancer tissues. Our results implicated that the mutations of PTEN did not occur at a significant rate in gastric carcinoma in Shanghai, but might play a role in tumorigenesis. The mutation status of PTEN was significantly relevant to pTNM staging and degree of cell differentiation, hinting that PTEN might be a prognostic biomarker of gastric cancer. The decreased expression of PTEN and E-cadherin, together with the overexpression of PI3K, AKT, MMP-2, MMP-9 and NF-kappaBp65, contributed cooperatively to the accelerated progress of gastric cancer.

Effect of retroviral vector mediating RNA interference on the release of tumor necrosis factor-alpha in lipopolysaccharide-stimulating macrophage

To explore the effect of small interfering RNA (siRNA) targeting NF-kappaB signal pathway on the expression level of tumor necrosis factor alpha (TNF-alpha) released by lipopolysaccharides (LPS)-stimulating-macrophages. Human monocytic THP-1 cell was induced by phorbol myristate acetate (PMA) and transformed into macrophage. Two groups of macrophage were infected by siRNA retroviral expression vector specific to NF-kappaB functional subunit P65 (siRNA group) and Scramble control vector (Scramble control group) constructed by molecular cloning technology. Lipopolysaccharide (50 microg/ml) was used to treat the macrophages continuously. RT-PCR was performed to detect the expression level of NF-kappaB P65 mRNA and TNF-alpha mRNA at different time-points of LPS stimulation. Western blotting was used to analyze the protein level of NF-kappaB P65. Enzyme-linked immunosorbent assay was applied to analyze the expression level of TNF-alpha released by LPS-stimulated macrophages. At Hours 12 and 24 after LPS stimulation, the expression level of NF-kappaB P65 mRNA in siRNA group (0.97 +/- 0.02, 0.89 +/- 0.01) was significantly less than that in Scramble control group (1.01 +/- 0.03, 0.97 +/- 0.01, both P < 0.05). At Hours 24 and 72 after LPS stimulation, the expression level of NF-kappaB P65 protein in siRNA group (0.95 +/- 0.04, 0.94 +/- 0.01) was obviously less than that in Scramble control group (1.07 +/- 0.06, 1.03 +/- 0.05, both P < 0.05). At Hours 4, 8, 12 and 24 after LPS stimulation, TNF-alpha mRNA released by siRNA group macrophages was far less than that by Scramble control group macrophages (0.92 +/- 0.02 vs 0.98 +/- 0.01, 0.86 +/- 0.02 vs 1.00 +/- 0.01, 0.79 +/- 0.03 vs 1.01 +/- 0.01, 0.78 +/- 0.03 vs 1.02 +/- 0.01, all P < 0.05). At Hours 2, 4, 8, 24, 36, 48, 54 and 72 after LPS stimulation, the TNF-alpha content in culture medium supernatant in siRNA group macrophage was less than that in scramble control group (P < 0.05). NF-kappaB P65 siRNA inhibits the functional activity of NF-kappaB signal pathway in PMA-induced macrophage. Then it blocks the activation of macrophage and the excessive release of TNF-alpha due to endotoxin stimulation. The RNA interference technology may be applied to prevent and treat excessive inflammatory reaction in acute lung injury.

Hepatocyte nuclear factor-kappa β (NF-κB) activation is protective but is decreased in the cholestatic liver with endotoxemia

Obstructive jaundice (OJ) is an important clinical consideration associated with a high risk of bacteremia. Hepatocyte nuclear factor-kappa B (NF-kappaB) activation confers an antiapoptotic function. Although the occurrence of hepatocyte apoptosis has been shown in OJ, the activation and role of NF-kappaB over the time course of OJ in conjunction with endotoxemia have not yet been well defined. We hypothesized that NF-kappaB activation may be decreased over the time course of OJ and endotoxemia, which leads to severe liver injury. The aim of the current study was to examine whether NF-kappaB activation can decrease hepatocyte apoptosis and liver injury over the time course of OJ in response to lipopolysaccharide (LPS) administration. Male C57BL/6 mice were subjected to bile duct ligation and were administered LPS intravenously at 3 days (OJ3) or 14 days (OJ14) after bile duct ligation. NF-kappaB activation; protein expressions of NF-kappaB p65, IkappaB-alpha, Ikappabeta-b, and Pin1; immunohistochemistry of poly adenosine diphosphate (ADP)-ribose polymerase p85 fragment (PARP); and serum alanine transaminase (ALT) levels were examined. Hepatocyte NF-kappaB activation was observed during OJ. After LPS administration, the hepatic NF-kappaB activation defined by electrophoretic mobility shift assay was decreased in the OJ14 group compared with the OJ3 group, which is consistent with a decrease in NF-kappaB p65 protein expression. Changes in phosphorylated Ikappa-B-beta but not phosphorylated IkappaB-alpha mirrored these results. Significant hepatocyte apoptosis defined by PARP immunohistochemistry was observed in the LPS-treated OJ14 relative to the LPS-treated OJ3. Hepatic expressions of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the LPS OJ14 mice were upregulated relative to those in the LPS OJ3. Serum ALT levels increased significantly in the LPS OJ14 relative to other mice. The survival rate was significantly less in the LPS OJ14 relative to other mice. After prolonged OJ, exposure to endotoxemia was associated with a decrease in hepatocyte NF-kappaB activation and an increase in hepatocyte apoptosis and secondary necrosis, thus resulting in liver dysfunction.

Effect of probiotics on pro-inflammatory cytokines and NF-κB activation in ulcerative colitis

To demonstrate the therapeutic effect of probiotics in patients with ulcerative colitis (UC), and their effect on inflammatory mediators and nuclear factor (NF)-kappaB activation in these patients. Thirty patients with mild to moderate UC were randomly classified into two groups: sulfasalazine group, who received sulfasalazine 2400 mg/d; and probiotic group, who received sulfasalazine 2400 mg/d with probiotic. The patients were investigated before and after 8 wk of treatment with probiotic (Lactobacillus delbruekii and Lactobacillus fermentum). Colonic activity of myeloperoxidase (MPO) was assayed with UV spectrophotometry, the colonic content of interleukin (IL)-6 was determined by enzyme-linked immunosorbent assay (ELISA), fecal calprotectin was determined by ELISA, and expression of NF-kappaB p65 and tumor necrosis factor (TNF)-alpha proteins in colonic tissue was identified by immunohistochemistry and reverse transcription polymerase chain reaction, respectively. At the start of the study, colonic mucosal injury and inflammation were demonstrated in UC patients by hematoxylin and eosin staining, and an increase in colonic MPO activity, fecal calprotectin, and expression of colonic TNF-alpha and NF-kappaB p65 proteins. The use of probiotic for 8 wk significantly ameliorated the inflammation by decreasing the colonic concentration of IL-6, expression of TNF-alpha and NF-kappaB p65, leukocyte recruitment, as demonstrated by a decrease in colonic MPO activity, and the level of fecal calprotectin compared to sulfasalazine group and the control group (P < 0.05). Oral supplementation with probiotics could be helpful in maintaining remission and preventing relapse of UC.

Expression and significance of TLR4 and HIF-1α in pancreatic ductal adenocarcinoma

To investigate the expression of toll-like receptor (TLR) 4, nuclear factor-kappaB (NF-kappaB) p65 and hypoxia-inducible transcription factor 1alpha (HIF-1alpha) in pancreatic ductal adenocarcinoma and their clinical significance. The mRNA of TLR4 and HIF-1alpha were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues, and expression of TLR4, NF-kappaB p65 and HIF-1alpha protein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent tissues. The relationship between TLR4 or HIF-1alpha and pathologic features, as well as the association between TLR4 and HIF-1alpha, were also analyzed. Kaplan-Meier method was used to assess the impact of expression of TLR4 and HIF-1alpha on survival of patients with pancreatic cancer. The relative quantification of TLR4 and HIF-1alpha mRNA in tumor tissues was 0.81 +/- 0.10 and 0.87 +/- 0.11, respectively, significantly higher than that in adjacent tissues (0.81 +/- 0.10 vs 0.70 +/- 0.16, P = 0.002; 0.87 +/- 0.11 vs 0.68 +/- 0.13, P = 0.000). The protein expression of TLR4, NF-kappaB p65 and HIF-1alpha in tumor tissues was 69.20%, 66.15% and 70.80%, respectively, being significantly higher than that in adjacent normal tissues (69.20% vs 39.50%, P = 0.003; 66.15% vs 31.58%, P = 0.001; 70.80% vs 36.80%, P = 0.001). There was no significant correlation between TLR4 or HIF-1alpha expression and the age, gender, tumor location, the degree of tumor differentiation in the patients (P > 0.05). However, there was significant correlation between the expression of TLR4 or HIF-1alpha and tumor size, lymph node metastasis, venous invasion and clinical staging (P < 0.05). The expression of TLR4 and HIF-1alpha had a significant impact on survival of patients with pancreatic adenocarcinoma. TLR4, NF-kappaB p65 and HIF-1alpha are overexpressed in pancreatic adenocarcinoma, TLR4 may be partly involved in up-regulating HIF-1alpha, and both synergestically promote development of pancreatic adenocarcinoma.

Uncovering an IL‐10‐dependent NF‐KB recruitment to the IL‐1ra promoter that is impaired in STAT3 functionally defective patients

The interleukin 1 receptor antagonist (IL-1ra) is an important negative regulator of the inflammatory response, whose genetic deficiency has been recently shown to cause a severe autoinflammatory syndrome in humans. In this study we characterized the molecular mechanisms whereby interleukin 10 (IL-10) potentiates IL-1ra transcription in LPS-stimulated monocytes and neutrophils. Using chromatin immunoprecipitation, we show that although NF-kappaBp65 and NF-kappaBp50 proteins accumulate into the nuclei and bind to the IkappaB alpha promoter during LPS stimulation, they are not recruited to the kappaB sites of the IL-1ra promoter. However, in response to LPS plus IL-10, which were found to induce chromatin acetylation, recruitment of both NF-kappaBp65 and NF-kappaBp50 to the IL-1ra promoter efficiently occurs in a STAT3-dependent manner. Accordingly, in neutrophils from hyper-IgE syndrome patients, who carry a nonfunctional STAT3, IL-10 failed to promote NF-kappaBp65 recruitment to the IL-1ra promoter and consequently to potentiate LPS-induced IL-1ra transcription. Altogether our findings uncover a novel mechanism whereby IL-10-activated STAT3 modulates IL-1ra transcription in LPS-treated phagocytes by making IL-1ra promoter accessible to readily available nuclear NF-kappaB.

Choline supplementation reduces oxidative stress in mouse model of allergic airway disease

Asthma is a multi-factorial inflammatory disease associated with increased oxidative stress and altered antioxidant defences. We have evaluated the effect of choline on oxidative stress in a mouse model of airway disease. Balb/c mice were sensitised with 100 microg of ovalbumin on days 0 and 14, and challenged with aerosolized ovalbumin on days 25-27. Mice were administered 1 mg kg(-1) of choline via oral gavage or intranasal route on days 14-27. Mice were also administered 100 mg kg(-1) of alpha-lipoic acid as standard antioxidant. Total cell counts, eosinophils and eosinophil peroxidase (EPO) activity were determined in bronchoalveolar lavage (BAL) fluid. Reactive oxygen species (ROS), lipid peroxidation and isoprostanes levels were measured in BAL fluid. IL-13 and tumour necrosis factor-alpha (TNF-alpha) levels were also measured in BAL fluid and spleen cell culture supernatant. Nuclear factor kappaB (NFkappaB) p65 protein expression was measured after last ovalbumin challenge in nuclear and cytosolic extracts of lungs. Compared with ovalbumin-challenged mice, choline and alpha-lipoic acid treated mice had significantly reduced eosinophilic infiltration and EPO activity in BAL fluid. Choline and alpha-lipoic acid treatment reduced ROS production and isoprostanes level significantly in BAL fluid and thus suppressed oxidative stress. Choline and alpha-lipoic acid administration by either route decreased lipid peroxidation levels and down regulated NFkappaB activity. Further, choline and/or alpha-lipoic acid treatment suppressed TNF-alpha level significantly as compared with that of ovalbumin-challenged mice. Choline administration reduces oxidative stress possibly by modulating the redox status of the cell and inhibits inflammatory response in a mouse model.

Effects of total glucosides of paeony on oxidative stress in the kidney from diabetic rats

TGP, extracted from the traditional Chinese herb root of Paeonia lactiflora pall, has been shown to have therapeutic effect in experimental diabetic nephropathy. However, its mechanism is not fully understood. In this study, the effects of TGP on oxidative stress were investigated in the kidney of diabetic rats induced by streptozotocin. TGP (50, 100, 200mg/kg) was orally administered once a day for 8 weeks. TGP treatment in all three doses significantly lowered 24 h urinary albumin excretion rate in diabetic rats and attenuated glomerular volume. TGP treatment with 100 and 200mg/kg significantly reduced indices for tubulointerstitial injury in diabetic rats. The level of MDA was significantly increased in the kidney of diabetic rats and attenuated by TGP treatment at the dose of 200mg/kg. TGP treatment in a dose-dependent manner decreased the level of 3-NT protein of the kidney which increased under diabetes. T-AOC was significantly reduced in diabetic rat kidney and remarkably increased by TGP treatment at the dose of 100 and 200mg/kg. Activity of antioxidant enzyme such as SOD, CAT was markedly elevated by TGP treatment with 200mg/kg. Western blot analysis showed that p-p38 MAPK and NF-kappaB p65 protein expression increased in diabetic rat kidney, which were significantly decreased by TGP treatment. It seems likely that oxidative stress is increased in the diabetic rat kidneys, while TGP can prevent diabetes-associated renal damage against oxidative stress.

Effect of artesunate on inhibiting proliferation and inducing apoptosis of SP2/0 myeloma cells through affecting NFκB p65

The initial treatment of multiple myeloma (MM) experienced a paradigm shift, in the past decade, with the introduction of novel agents such as thalidomide, lenalidomide and bortezomib, leading to improved outcomes. High dose therapy and autologous stem cell transplantation remain an important therapeutic option for patients with MM eligible for the procedure. However, most of these treatment regimens are too expensive for Chinese patients. Therefore, we investigated the effects of artesunate, which is commonly used in the treatment of severe malaria, on inhibition of proliferation and induction of apoptosis of a mouse myeloma cell line SP2/0. The growth inhibition of SP2/0 cell proliferation induced by artesunate (ART) treatment was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and the rate of apoptosis and cell cycle changes induced by ART were analyzed by flow cytometry. ART-induced morphology changes of apoptosis in SP2/0 cells, as observed by light and transmission electron microscopy. Additionally, DNA laddering, which is a hallmark of apoptosis, was observed by agarose gel electrophoresis of DNA harvested from SP2/0 cells treated with ART. The levels of nuclear factor kappa B p65 (NFkappaB p65) protein in nucleus and the inhibitor of NFkappaB (IkappaBalpha) in the cytoplasm were measured by western blot analysis and ELISA to evaluate NFkappaB p65 transcription activity indirectly. The results show that artesunate inhibited the proliferation and induced apoptosis of SP2/0 cells in a dose- and time-dependent manner. Artesunate also increased the proportion of SP2/0 cells in G0/G1 phase, while decreased the proportion of cells in G2/M or S phase. Additionally, artesunate treatment decreased the level of NFkappaB p65 protein in the nucleus, while increased the level of IkappaBalpha protein in the cytoplasm. The present result is the first report to show that artesunate may be useful in the treatment of MM.

Effect of salvia miltiorrhizae on apoptosis and NF‐κB p65 expression in the liver of rats with severe acute pancreatitis or obstructive jaundice

To investigate the therapeutic effects and mechanism of salvia miltiorrhizae in the treatment of severe acute pancreatitis (SAP) or obstructive jaundice (OJ). SAP rat models were prepared and randomly divided into the model control group and treated group. The sham-operated group was also set. At 3 h, 6 h and 12 h after operation, the mortality rate, the pathological changes in the liver, the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, the expression levels of Bax and NF-kappaB p65 proteins in the liver, and the apoptosis index of hepatic cells in SAP rats in each group were observed. On days 7, 14, 21 and 28 after operation, the above parameters and the contents of TBILI (total billirubin), DBILI (direct bilirubin) and r-GT (r-glutamyl transpeptidase) in serum in OJ rats were observed. The contents of serum ALT (at 6 h and 12 h after operation) and AST (at 3 h and 12 h after operation) as well as the staining intensity of NF-kappaB p65 protein (at 12 h after operation) in the liver of SAP rats in the treated group were significantly lower than those in model control group (all P < 0.01). The pathological severity scores (on 21 d and 28 d after operation) in the liver, the contents of serum ALT (on 14 d and 21 d after operation), AST (on 21 d after operation), TBILI (on 21 d and 28 d after operation), DBILI (on 28 d after operation) and r-GT (on 21 d after operation), and the apoptosis index of hepatic cells in OJ rats in treated group were significantly lower than those in model control group (all P < 0.05). The positive rates of Bax protein (on 28 d after operation) in treated group was significantly lower than model control group (P < 0.05). Salvia miltiorrhizae is able to improve the liver function of SAP or OJ rats, suppress the expression of NF-kappaB p65 protein in the liver of SAP rats, and inhibit apoptosis in OJ rats, thereby showing some protective effects on the liver of SAP or OJ rats.

Suppression of NF-κB Activity by Parthenolide Induces X-Ray Sensitivity through Inhibition of Split-Dose Repair in TP53 Null Prostate Cancer Cells

We have shown that parthenolide, a sesquiterpene lactone, is a radiation sensitizer for human CGL1 hybrid cells that have constitutively activated NF-kappaB and wild-type p53. Since many malignant cells have nonfunctional p53, we investigated whether parthenolide could alter the X-ray sensitivity of PC-3 prostate cancer cells, a p53 null cell line with constitutively activated NF-kappaB. The addition of 5 microM parthenolide induced non-apoptotic cell death, inhibited PC-3 proliferation, and increased the population doubling time from 23+/-1 h to 49+/-4 h. Parthenolide also inhibited constitutive and radiation-induced NF-kappaB binding activity and enhanced the X-ray sensitivity of these p53 null PC-3 cells by a dose modification factor of 1.7. Cell cycle analysis of PC-3 cells treated with parthenolide showed only small alterations in G1 and G2/M cells, and these appeared to be insufficient to explain the observed radiosensitization. Split-dose studies using clinically relevant 2- and 4-Gy fractions demonstrated that parthenolide completely inhibited split-dose repair in PC-3 cells. We hypothesized that inhibition of NF-kappaB activity by parthenolide was responsible for the observed X-ray sensitization and inhibition of split-dose repair. To test this hypothesis, we knocked down the expression of NF-kappaB p65 protein, an active component of NF-kappaB in both PC-3 and CGL1 cells, by siRNA. Inhibition of NF-kappaB activity by knockdown of p65 increased radiation sensitivity and completely inhibited split-dose repair in both cell lines in a nearly identical manner as parthenolide treatment alone. Treating p65-depleted PC-3 cells with 5 microM parthenolide did not further increase their radiation sensitivity or the inhibition of split-dose repair. We propose that the suppression of radiation-induced NF-kappaB activity by parthenolide leads to X-ray sensitization through inhibition of split-dose repair in p53 null PC-3 prostate cancer cells.

Effects of gut barrier dysfunction and NF‐κB activation on aggravating mechanism of severe acute pancreatitis

To study the effects of gut-derived endotoxin translocation and NF-kappaB activation on the aggravating mechanism of severe acute pancreatitis (SAP) and of treatment with pyrrolidine dithiocarbamate (PDTC) on rats with SAP. SD rats were randomly divided into sham operation group (SO), SAP group, SAP + lipopolysaccharide(LPS) group, pyrrolidine dithiocarbamate (PDTC) treatment group and LPS group. Biochemical parameters and cytokines were examined in the serum. Multiple organs pathological slices were examined. Expression of NF-kappaB mRNA in the liver tissue was detected by RT-PCR. Activation of NF-kappaB by the method of streptomycin avidin-peroxidase (SP) and expression of NF-kappaB p65 protein and its binding activity were analyzed by Western blot and electrophoretic mobidity shift assay (EMSA). Compared with sham operation group, the concentration of TNF-alpha, alanine aminotransferase (ALT), and diamine oxidase (DAO) in serum significantly increased in SAP + LPS group (P < 0.05). Pathological changes were markedly observed in tissues and the expression of NF-kappaB mRNA in the liver significantly increased (P < 0.05) also, the activation of NF-kappaB and binding activity of NF-kappaB p65 protein in the liver markedly increased (P < 0.01) in SAP + LPS group. Treatment with PDTC markedly reduced concentration of ALT, DAO and TNF-alpha, and the expression of NF-kappaB, and the pathologic scores, as well as significantly decreased the expression of NF-kappaB p65 protein. The activation and overexpression of NF-kappaB may participate in the aggravating mechanism of SAP. Treatment with PDTC has a protective effect on multiple organs damage in SAP.

Human Corneal Epithelium-Derived Thymic Stromal Lymphopoietin Links the Innate and Adaptive Immune Responses via TLRs and Th2 Cytokines

To explore the crucial role of the human corneal epithelium-derived proallergic cytokine thymic stromal lymphopoietin (TSLP) in initiation and regulation of immune responses. Primary corneal epithelial cells, established from donor limbal explants, were treated with 11 microbial ligands and proinflammatory and Th2 cytokines, alone or in combination. TSLP mRNA and protein were determined by real-time PCR and ELISA, respectively. NF-kappaB activation was detected by immunostaining and Western blot. TSLP was found to be expressed by human corneal epithelium and its cultures. TSLP in corneal epithelial cells were largely induced in a concentration-dependent fashion by polyI:C, flagellin, and FSL-1, the ligands for toll-like receptor (TLR)-3, -5, and -6, respectively. Compared with the control, TSLP mRNA was increased by 60-, 12- and 8-fold and TSLP protein increased by 67-, 19- and 7-fold by these three ligands, respectively. The proinflammatory (TNF-alpha and IL-1beta) and Th2 (IL-4 and IL-13) cytokines moderately induced TSLP expression and production. IL-4 and -13 strongly synergized with PolyI:C, flagellin, and TNF-alpha to promote TSLP production in ex vivo tissues and in vitro cultures of corneal epithelium. PolyI:C, flagellin, or TNF-alpha also induced NF-kappaB p65 protein nuclear translocation. The NF-kappaB inhibitor quinazoline blocked p65 nuclear translocation and further suppressed TSLP expression and production induced by these stimuli. These findings provide the first evidence of TSLP induction in the human eye and suggest the novel phenomenon that human corneal epithelium-derived TSLP may serve as a link between the innate and adaptive immune responses. TSLP may become a novel therapeutic target for allergic and inflammatory ocular surface diseases.

Influence of Baicalin and Octreotide on NF-κB and P-Selectin Expression in Liver and Kidney of Rats with Severe Acute Pancreatitis

To observe the influence of Baicalin and Octreotide on liver and kidney of rats with severe acute pancreatitis (SAP) and discuss the related mechanism. SAP rats were randomly divided into model control, Baicalin treated and Octreotide treated group (n = 45). The same number of normal rats were included in sham-operated group (n = 45). In all groups, the mortality rate, pathological changes as well as expression levels of NF-kappaB p65 and P-selectin protein in liver and kidney were observed at 3, 6 and 12 h after operation. The survival rate of treated group was 100% at 12 h significantly higher than that of model control group (P < 0.05). The pathological changes of liver and kidney in treated groups were alleviated to different degrees, the NF-kappaB protein expression levels and pathological severity scores in liver and kidney of treated groups were significantly lower than those of model control group (P < 0.05 or P < 0.001). The hepatic P-selectin protein expression level in Baicalin treated group was significantly lower than that of model control group at 3 h (P < 0.01), and renal P-selectin expression level in Baicalin treated group at 3 and 6 h were significantly lower than those of model control group and Octreotide treated group (P < 0.01). (1) Early treatment with Baicalin or Octreotide have obvious protecting effects on liver and kidney injuries in SAP with their mechanisms associated to inhibiting NF-kappaB and P-selectin expression of liver and kidney. (2) Comparing the pharmacologic effects of Octreotide and Baicalin, we believe Baicalin as a new drug with its protecting effects on liver and kidney of SAP rats similar to Octreotide is worth further studying. (3) The advantages of tissue microarrays in pathological examination include time and energy saving and highly efficient. But the restriction of small diameter weakens the representation of tissues to various extents, which may lead to the deviation of analysis.

Nonalcoholic steatohepatitis induced by a high‐fat diet promotes diethylnitrosamine‐initiated early hepatocarcinogenesis in rats

It has been suggested that patients with nonalcoholic steatohepatitis (NASH) may have high risk for liver cancer. However, it is unknown whether high-fat diet (HFD) induced NASH promotes hepatocarcinogenesis. In this study, Sprague-Dawley rats were injected with a low dose of hepatic carcinogen diethylnitrosamine (DEN) and then fed either Lieber-DeCarli control diet (CD) or HFD for 6 weeks. Liver histology and the hepatic placental form of glutathione S-transferase (P-GST) positive foci were examined. Expression levels of proliferating cell nuclear antigen (PCNA), cyclinD1, phosphorylated mitogen-activated protein kinase (MAPK) including extracellular signal-regulated kinase (ERK) and p38, as well as tumor necrosis factor-alpha (TNF-alpha), and nuclear factor-kappaB (NF-kappaB) were measured in the liver. Induction of lipid peroxidation end products (malondialdehyde plus 4-hydroxynonenal) in liver and apoptotic hepatocytes were also assessed. Results showed that HFD-fed rats developed significantly higher incidence and multiplicity of P-GST positive foci along with more fat accumulation, infiltration of inflammatory cells and higher lipid peroxidation in the liver, when compared with rats fed the CD. This high prevalence of hepatic lesions in the liver was accompanied by greater PCNA expression and cyclinD1 protein concentration but little change in hepatocyte apoptosis. HFD feeding elevated hepatic phosphorylated ERK but reduced phosphorylated p38 when compared with the CD feeding. In addition, a significantly higher expression of TNF-alpha mRNA and nuclear NF-kappaB p65 protein were observed in HFD group than in CD group. These data clearly demonstrate that NASH induced by HFD promoted DEN-initiated early hepatocarcinogenesis, which was associated with elevated TNF-alpha/NF-kappaB signaling and MAPK related hepatocyte proliferation.