Helicobacter pylori tumor necrosis factor-α inducing protein promotes cytokine expression via nuclear factor-κB

AIM:To study the effects of Helicobacter pylori(H. pylori)tumor necrosis factor-α(TNF)inducing protein (Tip-α)on cytokine expression and its mechanism. METHODS:We cloned Tip-αfrom the H.pylori strain 26695,transformed Escherichia coli with an expression plasmid,and then confirmed the expression product by Western blotting.Using different concentrations of Tip-αthat affected SGC7901 and GES-1 cells at different times,we assessed cytokine levels using enzyme-linked immunosorbent assay.We blocked SGC7901 cells with pyrrolidine dithiocarbamate(PDTC),a specific inhibitor of nuclear factorκB(NF-κB).We then detected interleukin(IL)-1βand TNF-αlevels in SGC7901 cells. RESULTS:Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein,which indicated that recombinant Tip-αprotein was recombined successfully.The levels of IL-1β,IL-8 and TNF-αwere sig-nificantly higher following Tip-αinterference,whether GES-1 cells or SGC-7901 cells were used(P<0.05).However,the levels of cytokines(including IL-1β,IL-8 and TNF-α)secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-αat the same concentration and for the same duration(P<0.05).After blocking NF-κB with PDTC, the cells(GES-1 cells and SGC-7901 cells)underwent interference with Tip-α.We found that IL-1βand TNF-αlevels were significantly decreased compared to cells that only underwent Tip-αinterference(P<0.05). CONCLUSION:Tip-αplays an important role in cyto-kine expression through NF-κB.

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.

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.

Hepatitis B virus X protein up-regulates tumor necrosis factor-α expression in cultured mesangial cells via ERKs and NF-κB pathways

Objective:To investigate the effects of hepatitis B virus(HBV)X protein(HBx)on the expression of tumor necrosis factor-α(TNF-α)in glomerular mesangial cells(GMCs)and the underlying intracellular signal pathways.Methods:The plasmid pCI-neo-X that carries the X gene of hepatitis B virus was transfected into cultured GMCs.HBx expression in the transfected GMCs was assessed by Western-blot.TNF-αprotein and mRNA were assessed by ELISA and semi-quantitative RT-PCR,respectively.Three kinase inhibitors-U0126,an inhibitor of extracellular signal-regulated kinases(ERKs);lactacvstin,an inhibitor of nuclear factor-κB(NF-κB);and SB203580,a selective inhibitor of p38 MAP kinase(p38 MAPK)were used to determine which intracellular signal pathways may underlie the action of HBx on TNF-αexpression in transfected GMCs.Results:A significant increase in HBx expression in pCI-neo-X transfected GMCs was detected at 36 h and 48 h,which was not affected by any of those kinase inhibitors mentioned above.A similar increase in the expression of both TNF-αprotein and mRNA was also observed at 36 h and 48 h,which was significantly decreased in the presence of U0126 or lactacytin,but not SB203580.Conclusions:HBx upregulates TNF-αexpression in cultured GMCs,possibly through ERKs and NF-κB pathway,but not p38 MAPK pathway.

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis

AIM To determine the effects of ω-3 fatty acids(ω-3FA) on the toll-like receptor 4(TLR4)/nuclear factor κB p56(NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis(SAP).METHODS A total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay. RESULTS The expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group(P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h(P < 0.05).CONCLUSION During the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

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.

Nuclear Factor kappa B p65 Expression in Mouse Cochlea

Nuclear factor kappa B(NF-κB) is one of the best-characterized transcription factors playing important roles in many cellular responses to a large variety of stimuli,including inflammatory cytokines, phorbol esters, growth factors, and bacterial and viral products. The aim of this study is to demonstrate NF-κB expression in the mouse cochlea and its enhancement in response to lipopolysaccharides(LPS) and kanamycin(KA) treatment. Methods KA treatment consisted of subcutaneous KA injections at 700 mg/kg twice a day with an eight-hour interval between the two injections for 3 or 7 days. For animals in the LPS treatment group, a single dose of 0.3 mg LPS dissolved in 0.2 ml sterile saline were injected into both bullae through the tympanic membrane and kept there for 3 hours. Animals in the control group received subcutaneous saline injection for 7 days. Following immmunohistochemichal processing with rabbit polyclonal anti-NF-κB p65 antibodies, cryosections of the cochlea were examined for expression of NF-κB p65 in various structures in the cochlea. Results NF-κB p65 expression, identified by presence of brown reaction products characteristic of DAB immunohistochemistry, was visible in the spiral ligament, spiral prominence, tectorial membrane(TM), spiral ganglion and nerve fibers. Relatively weak NF-κB p65 expression was also visualized in the organ of Corti. Within the organ of Corti, the inner hair cells(IHC), outer hair cells(OHC), inner pillar cells(IP), outer pillar cells (OP), Deiter’s cells(DC), and Boettcher’s cells exhibited stronger staining than the inner sulcus cells, Hensen’s cells(HC) and Claudius’cells. No NF-κB p65 expression was seen in the nucleus of the IHC and OHC. NF-κB p65 expression was increased in animals exposed to LPS or KA, demonstrating significant differences in the staining between control animals and LPS/KA-treated animals. NF-κB p65 expression was not significantly different between LPS treated and KA treated animals or between 3 and 7 days in KA-treated animals. Conclusion LPS and KA exposure increases expression of NF-κB p65 in the mouse cochlea.

血管活性肠肽抑制帕金森病MPTP模型小鼠纹状体中核因子-κB p65、肿瘤坏死因子α和单核细胞趋化蛋白-1的上调

目的:研究血管活性肠肽(VIP)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的帕金森病(PD)模型小鼠纹状体神经炎症及核因子-κB p65(NF-κB p65)水平的影响。方法:C57BL/6J雄性小鼠30只随机分为生理盐水(NS)组、MPTP组、MPTP+VIP组。免疫组织化学法观察纹状体酪氨酸羟化酶(TH)、胶质纤维酸性蛋白(GFAP)和离子钙结合蛋白(Iba-1)的水平变化;ELISA法检测肿瘤坏死因子α(TNF-α)及单核细胞趋化蛋白-1(MCP-1)的含量;免疫印迹检测NF-κB p65的水平变化。结果:与NS组相比,MPTP组小鼠纹状体TH水平显著减少,GFAP和Iba-1水平显著上升,TNFα和MCP-1的含量及NF-κB p65的水平显著升高。给予VIP后,纹状体TH水平明显增加,GFAP和Iba-1水平显著下降,TNF-和MCP-1的含量及NF-κB p65的水平明显降低。结论:VIP可能通过降低NF-κB p65的水平从而抑制MPTP诱导的PD小鼠纹状体的神经炎症反应。

贲门腺癌组织中RKIP表达变化及其与血清NF-κB p65蛋白、T淋巴细胞亚群的关系

目的观察Raf激酶抑制蛋白(RKIP)在贲门腺癌组织中的表达变化,进一步探讨贲门腺癌的发生、发展机制。方法收集160例贲门腺癌患者(腺癌组)的腺癌组织及其癌旁组织,应用免疫组织化学SP法检测RKIP表达;采用ELISA法和流术细胞术分别检测腺癌组和50例查体健康者(对照组)的血清NF-κB p65蛋白、T淋巴细胞亚群水平。采用Spearman秩和相关分析法分析指标间的关系。结果贲门腺癌及癌旁组织中RKIP表达阳性率分别为38.7%、52.5%(P<0.05),在有、无淋巴结转移者中的表达阳性率分别为24.4%、53.1%(P<0.05,t=4.34);腺癌组血清总NF-κB p65蛋白水平高于对照组(P<0.05),但RKIP表达阳性与阴性者水平无显著差异(P>0.05),有淋巴结转移者高于无淋巴结转移者(P<0.05);腺癌组细胞免疫功能低于对照组,尤以RKIP表达阴性者为著(P<0.05)。结论 RKIP在贲门腺癌组织中表达降低并与肿瘤发生、发展、浸润转移有关,可能机制为抑制NF-κB活性及细胞免疫功能,使肿瘤细胞逃避机体的免疫监视。

甲状腺癌组织中NF-κBp65、NF-κBp50及HMGB1表达及意义

目的探讨甲状腺癌组织中核因子-κB(NF-κB)p65、NF-κB p50及高迁移率族蛋白B1(HMGB1)的表达水平及临床意义。方法选择解放军171医院2013年1月—2015年12月切取的甲状腺组织标本212例进行研究,其中甲状腺癌组织122例,甲状腺腺瘤组织30例,甲状腺肿组织30例,正常甲状腺组织30例,比较各组NF-κB p65、NF-κB p50及HMGB1表达,并分析其与甲状腺癌临床特征的关系,以及三者之间的相关性。结果甲状腺癌组、甲状腺腺瘤组和甲状腺肿组的HMGB1、NF-κB p50和NF-κB p65阳性率均高于正常甲状腺组(P<0.01),甲状腺癌组和甲状腺腺瘤组均高于甲状腺肿组(P<0.01),甲状腺癌组高于甲状腺腺瘤组(P<0.01)。肿瘤直径≥1.0 cm和有淋巴结转移的甲状腺癌组织中NF-κB p65、NF-κB p50及HMGB1表达阳性率高(P<0.01,P<0.05)。NF-κB p65、NF-κB p50及HMGB1三者间呈显著正相关(r=0.356、0.645和0.275,P<0.05)。结论甲状腺癌组织中NF-κB p65、NF-κB p50及HMGB1均呈高表达,并且与肿瘤直径和淋巴结转移有关。

葫芦巴碱调节PI3K/Akt/NF-κB信号通路对变应性接触性皮炎大鼠免疫反应的影响

目的探讨葫芦巴碱对变应性接触性皮炎(ACD)大鼠免疫、炎性反应及PI3K/Akt/NF-κB信号通路的影响。方法60只SD大鼠随机分为正常组、ACD组、葫芦巴碱(低、中、高)剂量组(20、40、80 mg/kg)和PI3K抑制剂(LY294002)组(40 mg/kg),每组10只。除正常组外,其余组大鼠采用2,4二硝基氟苯(DNFB)诱导ACD模型。给药结束后,通过录像观察大鼠挠痒行为;HE染色检测大鼠耳皮肤组织病理学变化;ELISA检测大鼠血清IgE及Th1、Th2、Th17型细胞因子(IFN-γ、IL-4、IL-17)水平;Western blot检测大鼠耳皮肤组织中炎性因子(IL-1β、IL-6)蛋白及PI3K/Akt/NF-κB信号通路相关蛋白表达。结果与正常组比较,ACD组大鼠挠痒次数增加,血清IgE、IFN-γ、IL-4、IL-17水平及耳皮肤组织中IL-1β、IL-6蛋白表达和p-PI3K/PI3K、p-Akt/Akt、p-NF-κB/NF-κB比值升高(P<0.05),耳皮肤组织角化过度且可见大量炎性细胞浸润;与ACD组比较,葫芦巴碱(低、中、高)剂量组和LY294002组大鼠挠痒次数减少,血清IgE、IFN-γ、IL-4、IL-17水平及耳皮肤组织中IL-1β、IL-6蛋白表达和p-PI3K/PI3K、p-Akt/Akt、p-NF-κB/NF-κB比值降低(P<0.05),耳皮肤组织病理损伤均有所改善,且葫芦巴碱各给药组呈剂量依赖效应;葫芦巴碱高剂量组和LY294002组大鼠上述指标比较,差异无统计学意义(P>0.05)。结论葫芦巴碱可抑制ACD大鼠皮肤组织中PI3K/Akt/NF-κB信号通路激活,调控Th1、Th2、Th17型免疫应答并减轻皮肤局部和全身炎性反应。

Maraviroc promotes recovery from traumatic brain injury in mice by suppression of neuroinflammation and activation of neurotoxic reactive astrocytes

Neuroinflammation and the NACHT,LRR,and PYD domains-containing protein 3 inflammasome play crucial roles in secondary tissue damage following an initial insult in patients with traumatic brain injury(TBI).Maraviroc,a C-C chemokine receptor type 5 antagonist,has been viewed as a new therapeutic strategy for many neuroinflammatory diseases.We studied the effect of maraviroc on TBI-induced neuroinflammation.A moderate-TBI mouse model was subjected to a controlled cortical impact device.Maraviroc or vehicle was injected intraperitoneally 1 hour after TBI and then once per day for 3 consecutive days.Western blot,immunohistochemistry,and TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)analyses were performed to evaluate the molecular mechanisms of maraviroc at 3 days post-TBI.Our results suggest that maraviroc administration reduced NACHT,LRR,and PYD domains-containing protein 3 inflammasome activation,modulated microglial polarization from M1 to M2,decreased neutrophil and macrophage infiltration,and inhibited the release of inflammatory factors after TBI.Moreover,maraviroc treatment decreased the activation of neurotoxic reactive astrocytes,which,in turn,exacerbated neuronal cell death.Additionally,we confirmed the neuroprotective effect of maraviroc using the modified neurological severity score,rotarod test,Morris water maze test,and lesion volume measurements.In summary,our findings indicate that maraviroc might be a desirable pharmacotherapeutic strategy for TBI,and C-C chemokine receptor type 5 might be a promising pharmacotherapeutic target to improve recovery after TBI.

NIBP和NF-κB p65在胃癌中的表达及其临床意义

目的探讨NIBP和NF-κB p65在胃癌中的表达及其临床意义。方法采用实时荧光定量PCR法、Western blot法和免疫组化SP法,检测55例胃癌、癌旁及正常胃组织中NIBP和NF-κB p65蛋白及mRNA的表达。结果胃癌组织中NIBP和NF-κB p65mRNA和蛋白阳性表达率均高于癌旁及正常组织(P<0.05);胃癌组织中NIBP和NF-κB p65的mRNA及蛋白相对表达水平均高于癌旁及正常组织(P<0.05)。胃癌组织中NIBP与NF-κB p65呈正相关(P<0.05)。胃癌组织中NIBP和NF-κB p65阳性表达率与肿瘤浸润程度、分化程度、有无远处转移及临床分期有关(P<0.05)。结论 NIBP和NF-κB p65在胃癌组织中高表达,可能参与了胃癌的发生和发展,且NIBP激活NF-κB p65信号通路途径可能在胃癌的浸润和转移过程中起重要作用。

青藤碱对PKC-α/NF-κB-p65通路的抑制作用及机制

目的:探讨过表达蛋白激酶C-α(protein kinase C-α,PKC-α)对HEK-293T细胞中核因子-κB-p65(nuclear factor-κB-p65,NF-κB-p65)磷酸化水平的影响,并检查青藤碱(sinomenine,SIN)对PKC-α/NF-κB-p65信号通路的抑制作用。方法:采用PCR技术扩增大鼠PKC-α基因蛋白质编码区(complete sequence coding,CDS)序列,将其插入到pIRES2-EGFP空载质粒中,构建野生型(wide type,WT)PKC-α过表达质粒(pIRES2-PKC-α-WT,PKC-α^(WT));在PKC-αWT质粒的基础上,将PKC-α第25位丙氨酸(A)与368位赖氨酸(K)分别突变为谷氨酸(E)和精氨酸(R),即构建持续活化(constitutively active,CA)突变型PKC-α过表达质粒(pIRES2-PKC-α-A25E,PKC-α^(CA))和显性负性(dominant negative,DN)突变型PKC-α过表达质粒(pIRES2-PKC-α-K368R,PKC-α^(DN))。本课题组前期构建的大鼠野生型NF-κB-p65过表达质粒(pIRES2-NF-κB-p65,p65^(WT))分别与上述各PKC-α过表达质粒共同转染HEK-293T细胞,免疫印迹法检测PKC-α、NF-κB-p65的表达及磷酸化水平。再将上述不同质粒转染HEK-293T细胞,46 h后予50 ng/mL SIN处理细胞2 h,再行免疫印迹实验分析SIN对PKC-α、NF-κB-p65磷酸化的影响。结果:菌液PCR及测序证实,上述PKC-α过表达质粒构建成功。在HEK-293T中过表达PKC-αWT或PKC-αCA可促进NF-κB-p65的磷酸化,且过表达PKC-αCA时尤为显著,而过表达PKC-αDN后NF-κB-p65的磷酸化无明显变化。SIN处理可抑制PKC-α^(WT)、PKC-α^(CA)和PKC-α^(DN)转染组HEK-293T细胞中PKC-α的磷酸化,同时也能抑制过表达PKC-α^(WT)上调的NF-κB-p65磷酸化修饰,但不影响过表达PKC-αCA和PKC-αDN对NF-κB-p65的磷酸化作用。结论:过表达PKC-α可促进HEK-293T细胞中NF-κB-p65的磷酸化,SIN处理HEK-293T细胞后可通过抑制PKC-α的磷酸化下调NF-κB-p65的磷酸化修饰。

Role of nuclear factor kappa B in central nervous system regeneration

Activation of nuclear factor kappa B （NF-κB） is a hallmark of various central nervous system （CNS） pathologies. Neuron-specific inhibition of its transcriptional activator subunit RelA, also referred to as p65, promotes neuronal survival under a range of conditions, i.e., for ischemic or excitotoxic insults. In macro- and microglial cells, post-lesional activation of NF-κB triggers a growth-permissive program which contributes to neural tissue inflammation, scar formation, and the expression of axonal growth inhibitors. Intriguingly, inhibition of such inducible NF-~B in the neuro-glial compartment, i.e., by genetic ablation of RelA or overexpression of a trans- dominant negative mutant of its upstream regulator IκBa, significantly enhances functional recovery and promotes axonal regeneration in the mature CNS. By contrast, depletion of the NF-κB subunit p50, which lacks transcriptional activator function and acts as a transcriptional repressor on its own, causes precocious neuronal loss and exacerbates axonal degeneration in the lesioned brain. Collectively, the data imply that NF-κB orchestrates a multicellular pro- gram in which κB-dependent gene expression establishes a growth-repulsive terrain within the post-lesioned brain that limits structural regeneration of neuronal circuits. Considering these subunit-specific functions, interference with the NF-κB pathway might hold clinical potentials to improve functional restoration following traumatic CNS injury.

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.

L-and T-type Ca^(2+)channels dichotomously contribute to retinal ganglion cell injury in experimental glaucoma

Retinal ganglion cell apoptotic death is the main pathological characteristic of glaucoma,which is the leading cause of irreversible blindness.Disruption of Ca^(2+)homeostasis plays an important role in glaucoma.Voltage-gated Ca^(2+)channel blockers have been shown to improve vision in patients with glaucoma.However,whether and how voltage-gated Ca^(2+)channels are involved in retinal ganglion cell apoptotic death are largely unknown.In this study,we found that total Ca^(2+)current densities in retinal ganglion cells were reduced in a rat model of chronic ocular hypertension experimental glaucoma,as determined by whole-cell patch-clamp electrophysiological recordings.Further analysis showed that L-type Ca^(2+)currents were downregulated while T-type Ca^(2+)currents were upregulated at the later stage of glaucoma.Western blot assay and immunofluorescence experiments confirmed that expression of the Ca_(V)1.2 subunit of L-type Ca^(2+)channels was reduced and expression of the Ca_(V)3.3 subunit of T-type Ca^(2+)channels was increased in retinas of the chronic ocular hypertension model.Soluble tumor necrosis factor-α,an important inflammatory factor,inhibited the L-type Ca^(2+)current of isolated retinal ganglion cells from control rats and enhanced the T-type Ca^(2+)current.These changes were blocked by the tumor necrosis factor-αinhibitor XPro1595,indicating that both types of Ca^(2+)currents may be mediated by soluble tumor necrosis factor-α.The intracellular mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and nuclear factor kappa-B signaling pathway mediate the effects of tumor necrosis factor-α.TUNEL assays revealed that mibefradil,a T-type calcium channel blocker,reduced the number of apoptotic retinal ganglion cells in the rat model of chronic ocular hypertension.These results suggest that T-type Ca^(2+)channels are involved in disrupted Ca^(2+)homeostasis and apoptosis of retinal ganglion cells in glaucoma,and application of T-type Ca^(2+)channel blockers,especially a specific CaV3.3 blocker,may be a potential strategy for the treatment of glaucoma.

CARMA3: A novel scaffold protein in regulation of NF-κB activation and diseases

CARD recruited membrane associated protein 3 (CARMA3) is a novel scaffold protein. It belongs to the CARMA protein family, and is known to activate nuclear factor (NF)- κB. However, it is still unknown which receptor functions upstream of CARMA3 to trigger NF-κB activation. Recently, several studies have demonstrated that CARMA3 serves as an indispensable adaptor protein in NF-κB signaling under some G protein-coupled receptors (GP- CRs), such as lysophosphatidic acid (LPA) receptor and angiotensin (Ang) Ⅱ receptor. Mechanistically, CARMA3 recruits its essential downstream molecules Bcl10 and MALT1 to form the CBM (CARMA3-Bcl10-MALT1) signalosome whereby it triggers NF-κB activation. GPCRs and NF-κB play pivotal roles in the regulation of various cellular functions, therefore, aberrant regulation of the GPCR/NF-κB signaling axis leads to the development of many types of diseases, such as cancer and atherogenesis. Recently, the GPCR/CARMA3/NF-κB signaling axis has been confirmed in these specific diseases and it plays crucial roles in the pathogenesis of disease progression. In ovarian cancer cell lines, knockdown of CARMA3 abolishes LPA receptor-induced NF-κB activation, and reduces LPA-induced ovarian cancer invasion. In vascular smooth cells, downregulation of CARMA3 substantially impairs Ang-Ⅱ-receptor-induced NF-κB activation, and in vivo studies have confirmed that Bcl10- deficient mice are protected from developing Ang-Ⅱ-receptor-induced atherosclerosis and aortic aneurysms. In this review, we summarize the biology of CARMA3, describe the role of the GPCR/CARMA3/NF-κB signaling axis in ovarian cancer and atherogenesis, and speculate about the potential roles of this signaling axis in other types of cancer and diseases. With a significant increase in the identification of LPA- and Ang-Ⅱ-like ligands, such as endothelin-1, which also activates NF-κB via CARMA3 and contributes to the development of many diseases, CARMA3 is emerging as a novel therapeutic target for various types of cancer and other diseases.

Isoflavone Attenuates the Nuclear Transcription Factor Kappa B (NF-<i>κ</i>B) Activation on MPP⁺-Induced Apoptosis of PC12 Cells

Objective: To explore the underlying molecular mechanisms of cellular response to the challenge by 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells, an in vitro cell model for Parkinson’s disease, and the effect of NF-κB activation on the protection of Parkinson’s disease by Isoflavone (I). Methods: PC12 cells were used to establish the cell model of Parkinson’s disease, and are divided into five groups: control group;MPP+ group;I (Isoflavone) + MPP+ group;I group;SN-50 + MPP+ group. The content of NF-κB in PC12 cells was determined by immunocytochemistry;The viability of PC12 cells after treated with cell-permeable NF-κB inhibitor SN-50 and cell viability were measured by MTT assay;the expression levels of NF-κB p65 in cytoplasm and nuclear fractions were evaluated by western blot analysis;the mRNA expression of NF-κB p65 was analyzed by in situ hybridization (ISH). Results: Compared with the control group, the protein of NF-κB p65 both in cytoplasm and in nuclei was significantly higher than in I + MPP+ and MPP+ groups;similarly, the mRNA expression level of NF-κB p65 gene was also significantly higher;moreover, the protein expression of NF-κB p65 was much lower in I group (P + group, the protein of NF-κB p65 was significantly lower in I + MPP+ group, the mRNA expression level of NF-κB p65 gene was also significantly lower, and the protein expression level of NF-κB p65 was much lower in I + MPP+ group (P + group (P > 0.05). Conclusion: NF-κB activation is essential to MPP+-induced apoptosis in PC12 cells;but Isoflavone can inhibit the cell damage to some extent to execute its protective function, which may be involved in nigral neurodegeneration in patients with Parkinson’s disease.