Effect of Tetrandrine on LPS-induced NF-κB activation in isolated pancreatic acinar cells of rat

AIM: To investigate the effect of Tetrandrine (Tet) on LPS-induced NF-κB activation and cell injury in pancreatic acinar cells and to explore the mechanism of Tetrandrine preventing LPS-induced acinar cell injury.METHODS: Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to LPS (10mg/L), Tet (50 μmol/L, 100 μmol/L) or normal media. At different time point (30 min, 1 h, 4 h, 10 h) after treatment with the agents, cell viability was determined by MTT, the product and nuclear translocation of subunit p65 of NF-κB was visualized by immunofluorescence staining and nuclear protein was extracted to perform EMSA which was used to assay the NF-κB binding activity.RESULTS: LPS induced cell damage directly in a time dependent manner and Tet attenuated LPS-induced cell damage (50 μmol/L, P ＜ 0.05; 100 μmol/L, P ＜ 0.01).NF-κB p65 immunofluorescence staining in cytoplasm increased and began showing its nuclear translocation within 30 min and the peak was shown at 1 h of LPS 10 mg/L treatment. NF-κB DNA binding activity showed the same alteration pattern as p65 immunofluorescence staining. In Tet group, the immunofluorescence staining in cytoplasm and nuclear translocation of NF-κB were inhibited significantly.CONCLUSION: NF-κB activation is an important early event that may contribute to inflammatory responses and cell injury in pancreatic acinar cells. Tet possesses the protective effect on LPS-induced acinar cell injury by inhibiting NF-κB activation.     

Effect of BN52021 on NFκ-Bp65 expression in pancreatic tissues of rats with severe acute pancreatitis

AIM: To investigate dynamic changes and significance of expression of NF-κBp65 in pancreatic tissues of rats with severe acute pancreatitis (SAP), as well as BN52021 effects.METHODS: Wistar male rats were randomly divided into negative control group (NC group, n = 60), SAP-model group (SAP group, n = 60), and BN52021-treated group (BN group, n = 60), and each of the above groups was respectively divided into 6 subgroups at different time points after operation (1 h, 2 h, 3 h, 6 h, 12 h, and 24 h) (n = 10). By RT-PCR and Western blot, NF-KBp65 mRNA and its protein expression in pancreatic tissues of rats were detected respectively.RESULTS: The expression of NF-KBp65 mRNA dynamically changed in both SAP groups and BN groups. The mRNA level was higher in SAP groups than NC groups at 2 h, 3 h, 12 h, and 24 h after operation (P < 0.05), higher in BN groups than NC groups at all time points (P < 0.05), and higher in BN groups than SAP group at 1 h (P < 0.05). The NF-jcBp65 protein level was higher in SAP groups than NC groups at 1 h, 3 h, and 6 h (P < 0.01), and 2 h, 12 h, and 24 h (P < 0.05), higher in BN groups than NC groups at all time points (P < 0.05), and lower in BN groups than SAP groups at 1 h, 3 h, and 6 h (P < 0.05).CONCLUSION: The expression of NF-icBp65 in pancreatic tissues is dynamically changed and the changes play an important role in pathogenesis of SAP. BN52021 exerts therapeutic effects through reducing the expression level of NF-κBp65 protein in the early stage of SAP.     

Effect of nuclear factor kappa B on intercellular adhesionrnolecule-1 expression and neutrophil infiltration in lung injury induced by intestinal ischemia/reperfusion in rats

AIM: To investigate the role of nuclear factor kappa B(NF-κB) in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion (I/R), and its effect on intercellular adhesion molecule-1 (ICAM-1) expression and neutrophil infiltration.METHODS: Twenty-four Wistar rats were divided randomly into control, I/R and pyrrolidine dithiocarbamate (PDTC) treatment groups, n = 8 in each. I/R group and PDTC treatment group received superior mysenteric artery (SMA) occluding for 1 h and reperfusion for 2 h. PDTC group was administrated with intraperitoneal injection of 2% 100 mg/kg PDTC 1 h before surgery. Lung histology and bronchia alveolus lung fluid (BALF) protein were assayed. Serum IL-6, lung malondialdehyde (MDA) and myeloperoxidase (MPO) as well as the expression level of NF-κB and ICAM-1 were measured.RESULTS: Lung injury induced by intestinal I/R, was characterized by edema, hemorrhage and neutrophil infiltration as well as by the significant rising of BALF protein. Compared to control group, the levels of serum IL-6 and lung MDA and MPO increased significantly in I/R group (P=0.001). Strong positive expression of NF-κB p65 and ICAM-1 was observed. After the administration of PDTC, the level of serum IL-6, lung MDA and MPO as well as NF-κB and ICAM-1 decreased significantly(P＜ 0.05) when compared to I/R group.CONCLUSION: The activation of NF-κB plays an important role in the pathogenesis of lung injury induced by intestinal I/R through upregulating the neutrophil infiltration and lung ICAM-1 expression. PDTC as an inhibitor of NF-κB can prevent lung injury induced by intestinal I/R through inhibiting the activity of NF-κB.     

Anti-inflammatory effects of bifidobacteria by inhibition of LPS-induced NF-κB activation

AIM: Different strains of bifidobacteria were analysed for their effects on HT-29 intestinal epithelial cells (IECs)in in vitro models both of the non-inflamed and inflamed intestinal epithelium.METHODS: A reporter gene system in HT-29 cells was used to measure levels of NF-κB activation after challenge with bifidobacteria or after bacterial pre-treatment following LPS challenge. IL-8 protein and pro-inflammatory gene expression was investigated using normal HT-29 cells.RESULTS: None of the bifidobacteria tested induced activation of nuclear factor KB (NF-κB) indicating that bifidobacteria themselves do not induce inflammatory events in IECs. However, six out of eight bifidobacteria tested inhibited lipopolysaccharide- (LPS-) induced NFκB activation in a dose- and strain-dependent manner. In contrast, NF-κB activation in response to challenge with tumor necrosis factor-α (TNF-α) was affected by none of the tested bifidobacteria, indicating that the inhibitory effect of bifidobacteria is specific for LPS-induced infiammation in IECs. As shown with two of the six inhibitionpositive bifidobacteria, LPS-induced inhibition of NFκB activation was accompanied by a dose-dependent decrease of interleukin 8 (IL-8) secretion and by lower mRNA levels for IL-8, TNF-α, cyclooxygenase 2 (Cox-2),and intercellular adhesion molecule 1 (ICAM-1).CONCLUSION: Some strains of bifidobacteria are effective in inhibiting LPS-induced inflammation and thus might be appropriate candidates for probiotic intervention in chronic intestinal inflammation.     

Evaluation of the effect of pyrrolidine dithiocarbamate in suppressing inflammation in mice with dextran sodium sulfate-induced colitis

AIM: To evaluate the effect of pyrrolidine dithio- carbamate (PDTC; an NF-κB inhibitor) administered at low (50 mg/kg) and high (100 mg/kg) doses in suppressing colitis in mice with dextran sodium sulfate (DSS)-induced colitis. METHODS: Mice were divided into a DSS-untreated group (normal group), DSS-treated control group, DSS+PDTC-treated groupⅠ(low-dose group), and DSS+PDTC-treated groupⅡ (high-dose group). In each group, the disease activity index score (DAI score), intestinal length, histological score, and the levels of activated NF-κB and inflammatory cytokines (IL-1β and TNF-α) in tissue were measured. RESULTS: The DSS+PDTC-treated groupⅡ exhibited suppression of shortening of intestinal length and reduction of DAI score. Activated NF-κB level and IL-1β and TNF-α levels were significantly lower in DSS+PDTC- treated groupⅡ. CONCLUSION: These findings suggest that PDTC is useful for the treatment of ulcerative colitis.     

Anti-inflammatory properties of the short-chain fatty acids acetate and propionate： A study with relevance to inflammatory bowel disease

AIM: To compare the anti-inflammatory properties of butyrate with two other SCFAs,namely acetate and propionate,which have less well-documented effects on inflammation. METHODS: The effect of SCFAs on cytokine release from human neutrophils was studied with ELISA. SCFA-dependent modulation of NF-κB reporter activity was assessed in the human colon adenocarcinoma cell line,Colo320DM. Finally,the effect of SCFAs on gene expression and cytokine release,measured with RT-PCR and ELISA,respectively,was studied in mouse colon organ cultures established from colitic mice. RESULTS: Acetate,propionate and butyrate at 30 mmol/L decreased LPS-stimulated TNFα release from neutrophils,without affecting IL-8 protein release. All SCFAs dose dependently inhibited NF-κB reporter activity in Colo320DM cells. Propionate dose-dependently suppressed IL-6 mRNA and protein release from colon organ cultures and comparative studies revealed that propionate and butyrate at 30 mmol/L caused a strong inhibition of immune-related gene expression,whereas acetate was less effective. A similar inhibition was achieved with the proteasome inhibitor MG-132,but not the p38 MAPK inhibitor SB203580. All SCFAs decreased IL-6 protein release from organ cultures. CONCLUSION: In the present study propionate and butyrate were equipotent,whereas acetate was less effective,at suppressing NF-κB reporter activity,immune-related gene expression and cytokine release in vitro. Our findings suggest that propionate and acetate,in addition to butyrate,could be useful in the treatment of inflammatory disorders,including IBD.     

Cortex cinnamomi extract prevents streptozotocin- and cytokine-induced β-cell damage by inhibiting NF-κB

AIM: To clarify the mechanism underlying the antidiabetic activities of cortex cinnamomi extract (CCE).METHODS: To induce in vivo diabetes, mice were injected with streptozotocin (STZ) via a tail vein (100 mg STZ/kg body weight). To determine the effects of CCE,mice were administered CCE twice daily for 7 d by oral gavage starting 1 wk before the STZ injection. Blood glucose and plasma insulin concentration were measured as an index of diabetes. Also, to induce cytotoxicity of RINm5F cells, we treated with cytokines (IL-1β (2.0 ng/mL) and IFN-γ (100 U/mL)). Cell viability and nitric oxide production were measured colorimetrically.Inducible nitric oxide synthase (iNOS) mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. The activation of NF-KB was assayed by using gel mobility shift assays of nuclear extracts.RESULTS: Treatment of mice with STZ resulted in hyperglycemia and hypoinsulinemia, which was further evidenced by immunohistochemical staining of islets. However, the diabetogenic effects of STZ were completely prevented when mice were pretreated with CCE. The inhibitory effect of CCE on STZ-induced hyperglycemia was mediated through the suppression of iNOS expression. In rat insulinoma RINm5F cells,CCE completely protected against interleukin-1β and interferon-y-mediated cytotoxicity. Moreover, RINm5F cells incubated with CCE showed significant reductions in interleukin-1β and interferon-y-induced nitric oxide production and in iNOS mRNA and protein expression,and these findings correlated well with in vivo observations.CONCLUSION: The molecular mechanism by which CCE inhibits iNOS gene expression appears to involve the inhibition of NF-κB activation. These results reveal the possible therapeutic value of CCE for the prevention of diabetes mellitus progression.     

Role of Kupffer cells in acute hemorrhagic necrotizing pancreatitis-associated lung injury of rats

AIM:To investigate the role of Kupffer cells(KCs)inacute hemorrhagic necrotizing pancreatitis-associatedlung injury(AHNP-LI).METHODS:Forty-two rats were allocated to fourgroups[sham operation,AHNP model,gadoliniumchloride(GdCl_3)pretreatment,GdCl_3 control].In GdCl_3pretreatment group,GdCl_3 was administered by caudalvein injection 24 h before the AHNP model induction.Blood from the iliac artery,alveolar macrophages andtissues from the pancreas and lung,were collected insix animals per group 3 and 6 h after acute pancreatitisinduction.TNF-α,IL-1 of serum,myeloperoxidase(MPO)of lung tissue,NF-kB activation of alveolar macrophageswere detected.Serum AST and ALT in sham operationgroup and GdCl_3 control group were tested.In addition,histopathological changes of the pancreas and lung wereobserved under light microscope.RESULTS:MPO of lung tissue and TNF-α,IL-1 levelsof serum were all reduced significantly in GdCl_3pretreatment group compared to those in AHNP group(P<0.01).NF-kB activation of alveolar macrophageswas also attenuated significantly in GdCl_3 pretreatmentgroup compared to that in AHNP group(P<0.01).Thepathological injury of the lung was ameliorated obviouslyin GdCl_3 pretreatment group compared to that in AHNPgroup.Nevertheless,the serum amylase level did notreduce and injury of the pancreas was not prevented inGdCl_3 pretreatment group.CONCLUSION:Pulmonary injury induced by AHNPis mediated by KC activation and AHNP-LI can be significantly ameliorated by pretreatment with GdCl_3 andKCs play a vital role in AHNP-LI.     

Gene therapy that inhibits NF-κB results in apoptosis of human hepatocarcinoma by recombinant adenovirus

AIM: To investigate whether the recombinant adenovirus induces the TNF-α-mediated apoptosis in vivo.METHODS: Human hepatocarcinoma cell line (HepG2)cells were transfected into BALB/c nude mice, and the tumor growth curve was drawn. We analyzed apoptosis in HepG2 cells by TUNEL, HE staining and electron microscopy.RESULTS: AdIκBαM was expressed stably and efficiently in HepG2 and could not be degraded by induction of TNF-α. Tumor growth in mice could be reduced remarkably if treated by AdIκBαM plus TNF-α. There was apoptosis of ＞ 70% of cells treated with AdIκBαM plus TNF-α and about 50% of cells treated with AdIκBαM. In contrast, there was few cell apoptosis in HepG2 cells treated with phosphate buffered saline and AdIκBα. HepG2 cells in mice also exhibited a high level of apoptosis after in vivo injection with AdIκBαM. The tumor growth curve indicated the tumor transfected with AdIκBαM could be restrained.CONCLUSION: AdIκBαM gene therapy greatly enhances apoptosis due to inhibition of an NF-κB-mediated antiapoptosis signaling pathway.     

NF-κB及其抑制蛋白I-κBα与大肠癌侵袭和转移的相关性

目的 探讨NF-κB和I-κBα在大肠癌中的表达特点、相互关系及其与大肠癌进展的相关性.方法 免疫组化和Western印迹方法检测48例大肠癌患者手术切除的癌组织以及配对的癌旁正常组织标本中NF-κB,I-κBα和p-IκBα的表达情况,分析这3种蛋白的表达与大肠癌侵袭和转移的相关性.结果 大肠癌组织中NF-κB的表达水平明显高于正常组织(P＜0.01),且随着肿瘤Dukes分期的进展,NF-κB的表达水平呈逐渐升高趋势.大肠癌组织中I-κBα的表达水平明显低于正常组织(P＜0.01),且随着肿瘤Dukes分期的进展,I-κBα的表达水平呈逐渐降低趋势,同时,I-κBα的磷酸化(p-IκBα)水平逐渐升高.结论 在大肠癌组织中I-κBα的磷酸化降解与NF-κB表达升高有助于肿瘤的进展和不良预后.     

Hepatic preconditioning of doxorubicin in stop-flow chemotherapy:NF-κB/IκB-α pathway and expression of HSP72

AIM: To provide hepatic protection through administration of doxorubicin before stop-flow chemotherapy (SFC) and to investigate the expression of heat shock protein 72 (HSP72) and role of nuclear factor kappa B (NF-κB) in this effect. METHODS: The hepatic preconditioning of doxorubicin was established in a porcine model by injection of doxorubicin (1 mg/kg) before SFC. The experimental animals were randomized into two groups: groups receiving doxorubicin (DOX) and normal saline (NS). Serial serum and tissue samples were taken from both groups to evaluate the protection of doxorubicin. Western blot and immunoprecipitation were applied to detect the expression of HSP72, NF-κB p65 protein, inhibitor κB-α (IκB-α) and phosphorylated IκB-α as well. The expression of tumor necrosis factor α (TNF-α) was estimated by semiquantitative RT-PCR. And the extent of the hepatic injury was estimated with the level of serum aminotransferases. RESULTS: An abundance production of HSP72 in porcine liver was observed after 24 h of intravenous administration of doxorubicin, but without any change in the expression of NF-κB p65 subunit in cytoplasm. NF-κB p65 subunit accumulated in nuclei at the end of SFC and reached its highest level at 30 min after the restoration of the abdominal circulation and decreased gradually during the 6 h after SFC in NS group, while there was little change in DOX group. There was also a slight decrease of IκB-α at 30 min after the restoration of the abdominal circulation in NS group accompanying with the appearance of phosphorylated IκB-α. The expression of TNF-α was significantly higher in NS group than that in DOX group (average 1.40±0.17 vs 0.62±0.22, P<0.01) at serial time points after SFC. Serum ALT and AST levels of NS group were higher after 24 h than those of DOX group (93.2±7.8 IU/L vs 53.3±13.9 IU/L, 217.0±29.4 IU/L vs 155.0±15.6 IU/L for ALT and AST respectively, P<0.05) and after 48 h than those of DOX group (66.6±18.1 IU/L vs 43.3±16.7 IU/L, 174.4±21.3 IU/L vs 125.7±10.5 IU/L for ALT and AST respectively, P<0.05). CONCLUSION: Doxorubicin renders the liver to be tolerant to the hepatic influence in SFC in a porcine model through the NF-κB/IκB-αpathway with the expression of HSP72.     

核因子-κB抑制剂与肝纤维化关系

核因子-κB(NF-κB)是Sen和Baltimore等(1986)首先发现的一种能够与B细胞免疫球蛋白κ轻链基因的增强子-κB序列特异结合的核蛋白因子,与许多靶基因的转录启动有关,在细胞和生物体生长、分泌、细胞系定向等过程中起非常重要的作用.     

Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were trans-fected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-κB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.     

三氧化二砷联合Ad-IκBαM诱导胃癌细胞凋亡的研究

目的研究三氧化二砷（As2O3）对胃癌SGC-7901细胞作用过程中细胞内核转录因子KB（NF-κB）的激活及重组腺病毒Ad—IκBαM通过抑制NF-κB的活化增强As2O3的诱导凋亡作用。方法培养胃癌SGC-7901细胞,以非感染组及感染Ad—IκBα组为对照,采用凝胶电泳迁移率实验（EMSA）及免疫组化法检测As2O3处理后细胞核内NF-κB的激活情况,以及感染Ad—IκBαM对NF-κB活性的影响;四甲基偶氮唑盐（MTT）法、Hoechest染色、原位末端标记（TUNEL）法分别检测感染Ad—IκBαM对As2O3诱导细胞凋亡的影响。结果EMSA及免疫组化法显示As2O3作用于胃癌细胞可使细胞内NF-κB激活,感染Ad—IκBαM使NF-κB活性受到明显抑制;MTT法证明,As2O3作用后,感染Ad—IκBαM细胞的凋亡率（59．2±2．5）％较感染Ad-IκBα组（47．5±2．3）％及未感染组（40．0±1．2）％明显升高,各组间比较P〈0．01;Hoechest法显示,感染Ad—IκBαM组的凋亡率为（27.7±2．6）％,明显高于感染Ad—IκBα组（18．3±1．5）％及未感染组（11.0±1．7）％（P〈0．05）。TUNEL法结果与Hoechest法一致,感染Ad—IκBαM组的凋亡率为（31．1±2．5）％,高于感染Ad-IκBα组（20.7±2．1）％及未感染组（13.0±1、7）％（P〈0．05）。可见,感染Ad—IκBαM可明显提高As2O3诱导的细胞凋亡。结论As2O3作用于胃癌细胞可使细胞内NF-κB激活,从而表明NF-κB激活可能为胃癌细胞抗诱导凋亡作用的重要机制;感染Ad．IKBctM可有效抑制NF-κB的活性,并增强As2O3的诱导凋亡作用。     

肾上腺皮质肿瘤中NF-κB的表达及意义

目的探讨NF-κB在肾上腺皮质肿瘤中的表达及其临床意义。方法用免疫组化法检测肾上腺疾病标本(肾上腺皮质癌15例,肾上腺皮质瘤30例,肾上腺皮质增生11例,正常肾上腺皮质组织7例)中NF-κB的表达。结果 NF-κB在肾上腺皮质癌组中阳性表达为9/15例,其中2例(+++),4例(++),3例(+),6例(-);在肾上腺皮质瘤组中,NF-κB阳性表达8/30例,其中0例(+++),2例(++),6例(+),22例(-);增生组有2例阳性,均为(+);在正常组中未检测出阳性。在肾上腺皮质癌组中的表达明显高于在其他组中的表达,肾上腺皮质癌组与肾上腺皮质瘤组相比较,差异有统计学意义(P0.01)。结论 NF-κB对肾上腺皮质癌的诊断具有重要意义。     

NF-κB在肺间质纤维化中的作用

核因子 κB(NF κB)是一个结合DNA的蛋白因子家族 ,参与许多前炎症介质分子转录水平的调控 ,包括细胞因子 (如IL 1β、TNF a、IL 6、IL 8、TGF等 )、粘附分子 (ICVA 1/CD54、ECVA 1/E 选择素、CCVA 1/CD10 6)、酶 (诱导型一氧化氮合成酶、环氧合酶 2 )。这些分子大多是促进肺间质性疾病发展的重要因子。     

NF-κB及其调控基因在胃肠道肿瘤发生发展中的作用

核转录因子NF-κB通过调控众多相关基因的转录表达,可介导广泛的生物学作用,参与炎症、应激、免疫反应等.近来研究发现,NF-κB通过调节其下游基因转录,参与多种实体肿瘤的进展.NF-κB活性与肿瘤的关系已成为目前研究的热点,通过IκB的基因治疗或应用抗氧化剂、蛋白酶体抑制剂来抑制NF-κB活性,有望成为肿瘤治疗的新靶点.