人脑胶质瘤与人脑转移瘤中NF-κBP65的表达及意义

目的研究人脑胶质瘤(HBG)与人脑转移瘤(HBMC)中核转录因子κBP65(NF-κBP65)的表达及其在肿瘤生物学行为中的作用。方法应用SABC方法进行免疫组织化学染色,观察6例正常人脑组织、18例HBG组织与12例HBMC组织中NF-κBP65的表达及与肿瘤生物学行为之间的关系。结果正常脑细胞中NF-κBP65仅微量表达,在HBG细胞与HBMC细胞中呈不同程度的表达;在高级别HBG、HBMC和复发型HBG中,表达水平较低级别HBG细胞的高(P<0.01)。结论NF-κBP65表达的增强与HBG和HBMC恶性程度的增高、肿瘤浸润关系密切,并在HBG的复发中起重要作用。

Clinical significance of SQSTM1/P62 and nuclear factor-κB expression in pancreatic carcinoma

BACKGROUND Overexpression of SQSTM1(sequestosome 1,P62)and nuclear factor-κB(NF-κB)plays an important role in the invasion and metastasis of a variety of malignant tumors.AIM To explore the expression of P62 and NF-κB in pancreatic cancer and their relationship with clinicopathological features.METHODS The expression levels of P62 and NF-κB were analyzed by immunohistochemistry with a tissue chip containing 40 cases of human pancreatic carcinoma.Then we analyzed the correlation among P62 expression,phospho-P65 expression,and clinicopathological features of pancreatic carcinoma samples.RESULTS P62 expression was mainly observed in the cytoplasm of pancreatic carcinoma cells.Phosphorylated P65(phospho-P65)was mainly expressed in the nucleus and cytoplasm of pancreatic carcinoma cells.There was a significant difference in P62 expression among T stages.And a significant difference in phosphor-P65 expression among pathology types was noted.In the cases with strongly positive P62 expression,significant differences were found in age.And there were significant differences in T stage and tumor-node-metastasis stage in the cases with strongly positive phosphor-P65 expression.CONCLUSION In pancreatic carcinoma,P62 expression is significantly correlated with T stage.It may be a valuable malignant indicator for human pancreatic carcinoma.

Effect of NF-κB p65 antisense oligodeoxynucleotide on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2

AIM：To study the inhibition of nuclear factor kappa-B p65（NF-κB p65）antisense oligodeoxynucleotide（ASODN）on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2（TGF-β2）.·M ETHODS：NF-κBp65ASODNand NF-κBp65missense oligodeoxynucleotide（MSODN）were designed and synthesized.Human lens epithelial cell line（HLE B-3）cells were prepared for study and divided into 7 groups.Control group was HLE B-3 cells cultured in dulbecco’s modified eagle medium（DMEM）.T1,T2,and T3 group were HLE B-3 cells cultured in DMEM with 10 ng/m L TGF-β2 for 6h,12h,24h respectively.A＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2for 24h after transfected by NF-κB p65 ASODN for 24h.M＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h.The negative control group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after cultured with transfer agent（Hi Per Fect）for 24h.Cell morphology was observed at different time points using an inverted microscope.The expression of NF-κB p65 m RNA was detected with reverse transcription-polymerase chain reaction（RT-PCR）,and the expression ofα-smooth muscle actin（α-SMA）protein was assayed with ELISA.·RESULTS：With the TGF-β2 stimulation prolongation,the expression of NF-κB p65 m RNA and a-SMA protein increased in T1,T2,T3 groups compared with the control group,and the difference was statistically significant（〈0.05）.NF-κB p65 ASODN lowered the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.NF-κB p65 MSODN and Hi Per Fect did not lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.The difference between control group and A＋T group was not statistically significant（〉0.05）,but the difference among A＋T group and other groups was statistically significant（〈0.05）.·CONCLUSION：NF-κB p65 ASODN could lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2,and antagonized TGF-β2-induced transdifferentiation of HLE B-3.NF-κB p65ASODN could be used as a new biological therapeutic target of posterior capsular opacification.

Oxymatrine Improves TNBS-induced Colitis in Rats by Inhibiting the Expression of NF-κB p65

Inflammatory bowel disease is thought to be regulated by the balance between Th1 and Th2 cytokines secreted by T cells, and NF-κB p65 also plays a predominant role in the intestinal inflammation. We evaluated the potency of oxymatrine, one of active components of Sophora Root, in inhibiting the immune responses and inflammation in 2,4,6-trinitrobenzene sulfonic acid （TNBS）-induced colitis. The inflammation was markedly ameliorated in the oxymatrine-treated rats. The level of IL-2 was increased and that of IL-10 was decreased in colon tissue in the rat model, which was reversed by the treatment of oxymatrine. Moreover, the elevated expression of NF-κB p65 in colon tissue in the model was also improved by oxymatrine treatment. Our results suggest that oxymatrine might be beneficial for the abnormal immune responses and inflammation by regulating the unbalance of Th1 and Th2 cytokines secretion and inhibiting the expression of NF-κB p65 in colon tissue.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

青藤碱对缺血再灌注大鼠脑保护的作用机制

目的探讨青藤碱(Sin)对缺血再灌注(IR)大鼠脑保护的作用机制。方法80只Wistar大鼠随机分为假手术组、IR组、Sin高剂量(60mg/kg)组和Sin低剂量(30mg/kg)组;Sin高剂量组和Sin低剂量组分别腹腔注射相应剂量Sin;30min后线栓法制备局灶性IR模型;IR后24h,应用2,3,5-三苯基氯化四氮唑(TTC)和HE染色观察脑梗死体积及脑组织病理学变化;干湿重法检测脑含水量;免疫组化法检测各组大鼠额顶部皮质核转录因子(NF)-κBp65、细胞间黏附分子(ICAM)-1表达及髓过氧化物酶(MPO)活性。结果与IR组比较,Sin预处理后脑组织病理学改变明显减轻,Sin高剂量组缺血性改变更轻;Sin高、低剂量组脑梗死体积明显缩小,脑含水量明显降低,且Sin高剂量组更明显(均P<0.05)。假手术组皮质可见少量NF-κBp65表达于胞质,IR组、Sin高、低剂量组NF-κBp65表达增加(均P<0.05),且表达于胞核;与IR组比较,Sin高、低剂量组NF-κBp65核表达明显减少,Sin高剂量组减少更显著(均P<0.05)。IR组及Sin高、低剂量组MPO活性较假手术组明显增加(均P<0.05);与IR组比较,Sin高、低剂量组ICAM-1表达和MPO活性明显降低,Sin高剂量组降低更显著(均P<0.05)。结论Sin通过抑制脑组织NF-κBp65及ICAM-1表达和MPO活性,减轻IR后脑组织的炎症反应和脑水肿;其脑保护作用呈剂量依赖性。

梓醇对脑缺血急性期及亚急性期的神经保护作用及作用机制

目的：探讨梓醇对永久性脑缺血模型急性期脑组织梗死体积、含水量及亚急性期炎性反应的影响。方法：采用化学刺激法建立局灶性脑缺血（MCAT）模型，检测急性期（24 h）神经行为学症状、脑梗死面积和脑含水量；线栓法制作永久性大鼠脑缺血（pMCAO）模型，酶联免疫吸附法（ELISA）测定缺血侧脑组织白细胞介素-6（IL-6）、白细胞介素-10（IL-10）和核转录因子Bp65（NF-κBp65）的含量。结果：大鼠MCAT后24 h，梓醇15-60 mg＆#183;kg-1剂量组可以显著改善模型动物神经症状损伤（P〈0.01或P〈0.001），梓醇15 mg＆#183;kg-1组可显著降低模型动物梗塞区面积（P〈0.05），30 mg＆#183;kg-1组和60 mg＆#183;kg-1组可显著降低脑水肿（P〈0.05）；大鼠pMCAO术后7天开始，梓醇30 mg＆#183;kg-1组或60 mg＆#183;kg-1组开始改善模型动物神经症状损伤；术后14天，与假手术组比较，缺血侧脑组织IL-10和核转录因子NF-κBp65的含量变化与模型组已经不明显，IL-6水平显著降低（P〈0.05），梓醇15 mg＆#183;kg-1灌胃14天可以降低模型动物缺血侧脑组织NF-κBp65含量（P〈0.05）。结论：梓醇能改善局灶性脑缺血模型动物急性期及亚急性期神经症状损伤，缩小梗死灶，减轻脑部水肿，其作用可能与抑制脑缺血引起的炎症损伤无关。

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

AIM:To investigate the expression of toll-like receptor(TLR) 4,nuclear factor-κB(NF-κB) p65 and hypoxiainducible transcription factor 1α(HIF-1α) in pancreatic ductal adenocarcinoma and their clinical significance.METHODS:The mRNA of TLR4 and HIF-1α were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues,and expression of TLR4,NF-κB p65 and HIF-1α 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-1α and pathologic features,as well as the association between TLR4 and HIF-1α,were also analyzed.Kaplan-Meier method was used to assess the impact of expression of TLR4 and HIF-1α on survival of patients with pancreatic cancer.RESULTS:The relative quantif ication of TLR4 and HIF-1α mRNA in tumor tissues was 0.81±0.10 and 0.87±0.11,respectively,signif icantly 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-κB p65 and HIF-1α in tumor tissues was 69.20%,66.15% and 70.80%,respectively,being signif icantly 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 signif icant correlation between TLR4 or HIF-1α expression and the age,gender,tumor location,the degree of tumor differentiation in the patients(P>0.05).However,there was signif icant correlation between the expression of TLR4 or HIF-1α and tumor size,lymph node metastasis,venous invasion and clinical staging(P<0.05).The expression of TLR4 and HIF-1α had a signif icant impact on survival of patients with pancreatic adenocarcinoma.CONCLUSION:TLR4,NF-κB p65 and HIF-1α are overexpressed in pancreatic adenocarcinoma,TLR4 may be partly involved in up-regulating HIF-1α,and both synergestically promote development of pancreatic adenocarcinoma.

(5R)-5-hydroxytriptolide inhibits the inflammatory cascade reaction in astrocytes

Many studies have shown that(5R)-5-hydroxytriptolide is the optimal modified analogue of triptolide, possessing comparable immunosuppressive activity but much lower cytotoxicity than triptolide. Whether(5R)-5-hydroxytriptolide has preventive effects on neuroinflammation is unclear. This study was designed to pretreat primary astrocytes from the brains of neonatal Sprague-Dawley rats with 20, 100 and 500 nM(5R)-5-hydroxytriptolide for 1 hour before establishing an in vitro neuroinflammation model with 1.0 μg/mL lipopolysaccharide for 24 hours. The generation of nitric oxide was detected by Griess reagents. Astrocyte marker glial fibrillary acidic protein was measured by immunohistochemical staining. The levels of tumor necrosis factor-α and interleukin-1β in the culture supernatant were assayed by enzyme linked immunosorbent assay. Nuclear factor-κB/p65 expression was examined by immunofluorescence staining. The phosphorylation of inhibitor of nuclear factor IκB-α and the location of nuclear factor-κB/P65 were determined using western blot assay. Our data revealed that(5R)-5-hydroxytriptolide inhibited the generation of nitric oxide, tumor necrosis factor-α and interleukin-1β from primary astrocytes activated by lipopolysaccharide, decreased the positive reaction intensity of glial fibrillary acidic protein, reduced the expression of tumor necrosis factor alpha and interleukin-1β in culture supernatant, inhibited the phosphorylation of IκB-α and the translocation of nuclear factor-κB/P65 to the nucleus. These results have confirmed that(5R)-5-hydroxytriptolide inhibits lipopolysaccharide-induced glial inflammatory response and provides cytological experimental data for(5R)-5-hydroxytriptolide in the treatment of neurodegenerative diseases.

Expression of STAT6 and NF-κB p65 in the colon mucosa ofpatients with ulcerative colitis

The expression of signal transducer and activator of transcription 6(STAT6)and nuclear factor-κB(NF-κB)in the colonic mucosa of patients with ulcerative colitis(UC)was examined.Real-time polymer-ase chain reaction and immunohistochemistry were used to detect the expression of STAT6 and NF-κB p65 at both mRNA and protein levels in the colonic mucosa of patients with UC and healthy volunteers.The results showed that the expression levels of STAT6 and NFκB p65 in the colonic mucosa of patients with UC were significantly higher than in normal controls at both mRNA and protein levels.These data suggest that STAT6 and NFκB p65 perhaps play an important role in the pathogenesis of UC and underscore the potential value of anti-UC strategies in the clinical management of this disease.

Effect of compound Sophorae Flavescentis Jiechangrongcapsule on expression of NF-κB p65 and STAT6 in theintestinal mucosa of patients with ulcerative colitis

The effects of compound Sophorae Flavescen-tis Jiechangrong capsule(CSFJC)on the expression of nuclear factor-κB p65(NF-κB p65)and signal transducer and activator of transcription 6(STAT6)in the intestinal mucosa of patients with ulcerative colitis and the possible mechanism were investigated.Eighteen patients with ulcerative colitis were randomly divided into a traditional Chinese medicine(TCM)group(n=11)treated by CSFJC and a western medicine(WM)group(n=7)treated by Sulfasalazine tablets.The treatment duration lasted eight weeks.Before and after the treatment,the symptoms and the physical signs were observed,and the routine stool test,the colonoscopy,and pathological examination were performed in the two groups.The expression levels of NF-κB p65 and STAT6 were detected by using immuno-histochemistry.The results showed that the total effective rate of the curative effectiveness in TCM and WM groups was 100%and 71.4%,respectively,and the total effective rate of colonic mucosa lesion in TCM and WM groups was 90.9%and 71.4%,respectively,with the differences being significant(all P<0.05).The total effective rate of syndromes of damp-heat blocking according to the TCM in TCM and WM groups was 90.9%and 71.4%,respectively.After the treatment,the expression of NF-κB p65 and STAT6 in the two groups was decreased,and the decrease of NF-κB p65 and STAT6 expression in TCM group was more significant than in WM group(P<0.05).It was concluded that CSFJC can inhibit the activation and expression of NF-κB p65 and STAT6 in the intestinal mucosa of patients with ulcerative colitis,which is a possible mechanism for CSFJC treating ulcerative colitis.

Protective Effect of Hydroxysafflor Yellow A on Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Rats

Objective： To observe the effect of hydroxysafflor yellow A （HSYA）, an active ingredient of a traditional Chinese herbal medicine Carthamus tinctorius L., on lung inflammation and pulmonary fibrosis induced by bleomycin （BLM） in rats. Methods： Animals were divided into 6 groups including normal group, model group, three HSYA groups and dexamethasone （DXM） group. Three doses of HSYA （35.6, 53.3, and 80.0 mg？kg–1？day–1） were intraperitoneally （i.p.） injected in rats for 3 weeks after BLM administration and DXM was used as the positive control （n=8 or 12）. Arterial blood gas was assayed and morphological changes were observed. Lung mRNA expressions of tumor necrosis factor （TNF）-α, interleukin （IL）-1β, IL-6 and some cytokines in lung tissue were detected by real-time polymerase chain reaction. Nuclear factor-κB p65 or α-smooth muscle actin （α-SMA） protein distribution in rat lung tissue was observed by immunohistochemistry. Results： On the 7th day after BLM administration, lung tissue showed serious inflammation. Treatment with HSYA or DXM ameliorated lung inflammation. After treatment with HSYA or DXM, oxygen partial pressure （PaO2） increased （HSYA 80.0 mg？kg–1, P〈0.01） and CO2 partial pressure （PaCO2） decreased （HSYA 53.3, 80.0 mg？kg–1, P〈0.05）. Moreover, the mRNA expression of TNF-α, IL-1β, and IL-6; and the number of NF-κB p65 positive cells was lower in HSYA 53.3 and 80.0 mg？kg–1 groups than those in the model group （all P〈0.05）. Twenty-one days after BLM administration, HSYA or DXM treatment ameliorated fibrosis, increased PaO2 （HSYA 53.3, 80.0 mg？kg–1, P〈0.01）, and decreased PaCO2 （53.3 and 80.0 mg？kg–1, P〈0.05）. Further, the mRNA expression of TGF-β1, α-SMA, and collagen Ⅰ as well as the number of α-SMA positive cells increased in the model group and HSYA can attenuate these changes （53.3, 80.0 mg？kg–1, P〈0.05）. Hematoxylin and eosin and Masson＇s trichrome staining indicated that the fibrosis and collagen deposition were ameliorated in HSYA groups （53.3, 80.0 mg？kg–1, P〈0.05）. Conclusion： HSYA could alleviate acute lung inflammation and chronic pulmonary fibrosis induced by BLM in rats.

Interleukin-1β-induced inflammatory signaling in C20 human microglial cells

Aim: Increased inflammatory signaling in microglia is implicated in the pathogenesis of neurodegenerative diseases, trauma, psychiatric disorders, and anxiety/depression. Understanding inflammatory signaling in microglia is critical for advancing treatment options. Studying rodent-derived microglia has yielded substantial information, yet, much remains to better understand inflammatory signaling in human microglia. Hence, there is great interest in developing immortalized human microglial cell lines. The C20 human microglial cell line was recently developed and our primary objective was to advance our knowledge of inflammatory signaling in these cells. Methods: Expression of the microglia specific marker transmembrane protein 119 (TMEM119) was assessed by western blot analysis. Lipopolysaccharide (LPS)- and interleukin-1β (IL-1β)-induced cytokine/chemokine expression was determined by ELISA. Phosphorylation of inhibitory kappa B alpha (IκBα), nuclear factor (NF)-κB p65, and p38 mitogen-activated protein kinase (p38 MAPK) was measured by western blot analysis. Results: TMEM119 was expressed in unstimulated C20 cells, and to a greater extent in IL-1β-stimulated cells. IL-1βsignificantly induced IL-6, monocyte chemoattractant protein-1/CCL2, and interferon-γ inducible protein 10/CXCL10 expression. LPS induced CCL2 expression, but not IL-6 or CXCL10 expression. IL-1β induced inflammatory signaling as indicated by increased phosphorylation of IκBα, NF-κB p65 and p38 MAPK. Conclusion: We provide the first evidence that C20 microglia express TMEM119. This is the initial report of IL-1β-induced activation of IκBα, NF-κB p65, and p38 MAPK and subsequent CXCL10, CCL2 and IL-6 secretion in C20cells. These findings advance our understanding of inflammatory signaling in C20 cells and support the value of this cell line as a research tool.