视网膜缺血再灌注损伤后HSP70、NF-κB的表达和细胞凋亡的关系

目的探讨大鼠视网膜缺血再灌注损伤后热休克蛋白70(HSP70)、核蛋白因子-κB(NF-κB)表达和损伤神经细胞凋亡的关系。方法前房加压法制作大鼠视网膜缺血再灌注损伤模型,28只大鼠随机分为正常组和缺血再灌注组,后者又按照再灌注后不同时间段分为1、6、12、24、48、72h组。应用末端脱氧核酸转移酶介导的脱氧三磷酸尿苷缺口末端标记(TUNEL)法检测视网膜神经细胞凋亡指数(AI),过氧化物酶标记的链酶卵白素(SP)免疫组织化学法检测视网膜组织中HSP70、NF-κB的表达。结果视网膜神经细胞的凋亡出现于再灌注后6h,并逐渐递增,24h达到高峰,48h开始下降,72h不明显。HSP70在再灌注后1h即有表达,随时间段延长表达逐渐增加,至再灌注后24h达到高峰,之后渐下降。NF-κB的表达变化与凋亡细胞变化的规律基本一致。结论视网膜缺血再灌注损伤导致神经节细胞的凋亡;HSP70和NF-κB在视网膜缺血再灌注损伤中表达升高,对神经细胞的凋亡起着重要的调节作用。

The nuclear signaling of NF-KB： current knowledge, new insights, and future perspectives

The nuclear factor-kappa B （NF-κB） transcription factor plays a critical role in diverse cellular processes associated with proliferation, cell death, development, as well as innate and adaptive immune responses. NF-κB is normally sequestered in the cytoplasm by a family of inhibitory proteins known as inhibitors of NF-κB （IκBs）. The signal pathways leading to the liberation and nuclear accumulation of NF-κB, which can be activated by a wide variety of stimuli, have been extensively studied in the past two decades. After gaining access to the nucleus, NF-κB must be actively regulated to execute its fundamental function as a transcription factor. Recent studies have highlighted the importance of nuclear signaling in the regulation of NF-κB transcriptional activity. A non-Rel subunit of NF-κB, ribosomal protein S3 （RPS3）, and numerous other nuclear regulators of NF-κB, including Akirin, Nurrl, SIRT6, and others, have recently been identified, unveiling novel and exciting layers of regulatory specificity for NF-κB in the nucleus. Further insights into the nuclear events that govern NF-κB function will deepen our understanding of the elegant control of its transcriptional activity and better inform the potential rational design of therapeutics for NF-κB-associated diseases.

Heme oxygenase-1 prevents liver fibrosis in rats by regulating the expression of PPAR_γ and NF-_κB

AIM:To investigate the effects of heme oxygenase(HO)-1 on liver fibrosis and the expression of peroxisome proliferator-activated receptor gamma(PPARγ) and nuclear factor-kappa B(NF-κB) in rats.METHODS:Sixty Wistar rats were used to construct liver fibrosis models and were randomly divided into 5 groups:group A(normal,untreated),group B(model for 4 wk,untreated),group C(model for 6 wk,untreated),group D [model for 6 wk,treated with zinc protoporphyrin Ⅸ(ZnPP-Ⅸ) from week 4 to week 6],group E(model for 6 wk,treated with hemin from week 4 to week 6).Next,liver injury was assessed by measuring serum alanine aminotransferase(ALT),aspartate aminotransferase(AST) and albumin levels.The degree of hepatic fibrosis was evaluated by measuring serum hyaluronate acid(HA),type Ⅳ collagen(Ⅳ-C) and by histological examination.Hydroxyproline(Hyp) content in the liver homogenate was determined.The expres-sion levels of alpha-smooth muscle actin(α-SMA) in liver tissue were measured by real-time quantitative polymerase chain reaction(RT-PCR).The expression levels of PPARγ and NF-κB were determined by RT-PCR and Western blotting.RESULTS:The expression of HO-1 increased with the development of fibrosis.Induction of HO-1 by hemin significantly attenuated the severity of liver injury and the levels of liver fibrosis as compared with inhibition of HO-1 by ZnPP-Ⅸ.The concentrations of serum ALT,AST,HA and Ⅳ-C in group E decreased compared with group C and group D(P < 0.01).Amount of Hyp and α-SMA in the liver tissues in group E decreased compared with group C(0.62 ± 0.14 vs 0.84 ± 0.07,1.42 ± 0.17 vs 1.84 ± 0.17,respectively,P < 0.01) and group D(0.62 ± 0.14 vs 1.11 ± 0.16,1.42 ± 0.17 vs 2.56 ± 0.37,respectively,P < 0.01).The expression of PPARγ at levels of transcription and translation decreased with the development of fibrosis especially in group D;and it increased in group E compared with groups C and D(0.88 ± 0.15 vs 0.56 ± 0.19,0.88 ± 0.15 vs 0.41 ± 0.11,respectively,P < 0.01).The expression of NF-κB increased with the development of fibrosis especially in group D;and it decreased in group E compared with groups C and D(1.43 ± 0.31 vs 1.89 ± 0.29,1.43 ± 0.31 vs 2.53 ± 0.54,respectively,P < 0.01).CONCLUSION:Our data demonstrate a potential mechanism that HO-1 can prevent liver fibrosis by enhancing the expression of PPARγ and decreasing the expression of NF-κB in liver tissues.

PDTC干预对左向右分流大鼠模型肺动脉压力的影响

目的探讨核蛋白因子κB(NF-κB)在左向右分流大鼠模型肺动脉内皮细胞的活性及应用阻断剂(PDTC)干预后肺动脉压力的变化。方法 50只4周龄Wistar雄性大鼠随机分为4组:手术分流组(Tn组)15只,手术+PDTC阻断组(Ti组)15只,假手术对照组(Co组)10只,阴性对照组(Cn组)10只。将Tn组和Ti组30只大鼠通过颈总动脉-颈外静脉吻合术建立左向右分流型肺动脉高压模型,其中Ti组15只大鼠于术前1 h开始腹腔注射PDTC,剂量为120 mg.kg-1.d-1,持续2周,Co组大鼠除了不进行颈总动脉-颈外静脉吻合外,其余手术程序与实验组完全相同。连续饲养12周后,采用心导管术测量右心室收缩压(RVSP);分离大鼠肺动脉内皮细胞,通过凝胶迁移率实验(EMSA)测定各组内皮细胞NF-κB与特定基因识别序列相结合的活性。结果 Tn组NF-κB活性明显高于Cn组(P<0.01),Ti组NF-κB活性低于Cn组(P<0.01),而Co组与Cn组相比无统计学差异。Tn组大鼠肺血管MT%与Cn组相比明显升高(P<0.01);而Ti组与Cn组相比无统计学差异(P>0.05);Co组与Cn组相比较无统计学差异(P>0.05)。结论高肺血流所致的大鼠肺血管收缩和结构重建与NF-κB的活性增强有关,阻断剂PDTC可以通过阻断高血流所致的NF-κB信号通路从而干预肺血管重建。

枸杞多糖对颅内动脉瘤大鼠NF-κB信号通路及血管内皮组织炎性损伤的影响

目的探讨枸杞多糖(LBP)对颅内动脉瘤(IA)大鼠核蛋白因子-κB(NF-κB)蛋白通路及血管内皮组织炎性损伤的影响。方法取SD雌性大鼠50只,采用切除双侧卵巢并结扎左侧颈总动脉及双侧肾动脉后支以构建IA模型,随机分为IA模型组、LBP低(5 mg/kg)、中(10 mg/kg)、高(20 mg/kg)剂量组,另取10只大鼠,只暴露卵巢、颈总动脉、双侧肾动脉后支,不进行摘除和结扎,作为对照组(Control组);各组均于手术1周后,开始给药,Control组、IA模型组经灌胃给予生理盐水,LBP各剂量组灌胃给予相应剂量药物,1次/d,共给药30 d;末次给药12 h后处死,取大鼠血液用酶联免疫吸附法(ELISA)检测血清炎性因子白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)及血管内皮损伤标志物血管内皮生长因子(VEGF)、血管内皮素(ET)水平;分离取出大鼠脑动脉(Wills)环,在40倍显微镜下检查大脑动脉(Wills)环病理改变,并检测动脉瘤血管壁厚度及动脉瘤体积;取脑动脉瘤血管组织,用苏木精-伊红染色(HE)检测动脉瘤血管组织病理变化;蛋白免疫印迹法(Western blot)检测动脉瘤血管组织NF-κB,Toll样受体-4(TLR4)蛋白表达水平。结果与Control组比较,IA模型组大鼠Willis环上凸起、管壁厚度和肿动脉瘤体积、动脉瘤内皮细胞空泡变性及炎性细胞浸润等病理损伤程度、血清IL-6,TNF-α,VEGF,ET水平、动脉瘤血管组织NF-κB和TLR4蛋白表达水平明显升高(P<0.05);与IA模型组比较,LBP低、中、高剂量组大鼠Willis环上凸起、管壁厚度和动脉瘤体积、动脉瘤内皮细胞空泡变性及炎性细胞浸润等病理损伤程度、血清IL-6,TNF-α,VEGF,ET水平、动脉瘤血管组织NF-κB和TLR4蛋白表达水平明显降低(P<0.05),且LBP各剂量组上述指标水平呈剂量依赖性降低。结论 LBP可能通过抑制TLR4/NF-κB通路激活、降低炎症反应来减轻IA血管内皮组织损伤。

Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λ production by blocking the nuclear factor-κB nuclear translocation

Porcine epidemic diarrhea virus（PEDV） is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon（IFN） signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid（N） protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid（poly（I：C）） in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly（I：C）-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB（NF-κB） nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.

Effect of Lingguizhugan decoction on myocardial Nuclear factor kappa B protein expression in rats with chronic heart failure

OBJECTIVE: To investigate the effect of Lingguizhugan decoction (LGZGD) on changes of cardiac structure and function, and its putative mechanism of action, by investigating mRNA and protein expression of myocardial nuclear factor kappa B(NF-κB), and the plasma content of NF-κB in rats with chronic heart failure.METHODS: The chronic heart failure (CHF) model in rats was induced by coronary artery ligation.Sham operation was performed in control rats. Six weeks after the procedure, rats were randomly classified into the various treatment groups: model CHF, Captopril (4.4 mg/kg), low LGZGD dose (2.1 g/kg), medium LGZGD dose (4.2 g/kg), and high LG-ZGD dose (8.4 g/kg). Treatments continued for 4 consecutive weeks. Changes of hemodynamic indices were observed by the PowerLab data acquisition and analysis system. Morphological changes of myocardium were observed by hematoxylin and eosin staining, and Masson staining. The mRNA and protein expression of myocardial NF-κB were detected by reverse transcription-polymerase chain reaction and western blotting, respectively. The plasma content of NF-κB was detected by enzyme-linked immuno-sorbent assay.RESULTS: CHF rats showed significant dysfunction in hemodynamic indices and in cardiac structure.Compared with the sham operation group, mRNA expression of myocardial NF-κB and plasma content of NF-κB of the model group was significantly increased. All three doses of LGZGD, and Captopril,improved the hemodynamic dysfunction, and inhibited the change of cardiac structure while significantly improving the survival rate. Furthermore,compared with the model group, mRNA expression of myocardial NF-κB and plasma content of NF-κB were significantly reduced by all dosage groups of LGZGD as well as the\Captopril group.CONCLUSION: In CHF rats, LGZGD improves changes of cardiac structure and function via its inhibition of NF-κB.

Nuclear factor-κB in signal conduction of protein kinase C in T lymphocytes from an asthmatic guinea pig model

Objective To explore the role of nuclear factor κB (NF κB) in the signal conduction of protein kinase C (PKC) regulated proliferation, apoptosis and expression of Th2 cytokines - interleukin 4 (IL 4) and interleukin 5 (IL 5) of T lymphocytes in the bronchial alveolus lavage fluid (BALF) Methods T lymphocytes were isolated and purified from BALF of asthmatic guinea pigs in normal and asthmatic groups, and were stimulated with PKC agitator phorbol 12 myristate 13 acetate (PMA) and NF κB inhibitor pyrrolidine dithiocarbamate (PDTC), respectively The expressions of NF κB, IL 4 and IL 5 mRNA and protein, the proliferation and apoptosis of T lymphocytes were observed by immunohistochemistry, in situ hybridization, ELISA, MTT and TUNEL, respectively Results The activation of NF κB, proliferation response, and expression of IL 4 and IL 5 mRNA and protein in T lymphocytes stimulated by PMA were significantly higher than those of their blank control ( P 【0 01), while those indexes of T lymphocytes stimulated by PMA and PDTC simultaneously were significantly lower than those stimulated by PMA alone ( P 【0 01) The apoptotic index of T lymphocytes stimulated with PMA were significantly lower than that of their blank control ( P 【0 01), and the apoptotic index of asthmatic guinea pig T lymphocytes stimulated with PMA and PDTC simultaneously were significantly higher than that stimulated by PMA alone ( P 【0 01) The significant positive correlations were found between the activation of NF κB and the proliferation ( r =0 64, P 【0 001), and the expression of IL 4 and IL 5 mRNA and protein of T lymphocytes, respectively ( r =0 55-0 68, P 【0 001) There was also significant negative correlation between the activation of NF κB and apoptosis of T lymphocytes ( r =0 62, P 【0 001) Conclusions NF κB may participate in the signal conduction of PKC regulated proliferation, apoptosis and expression of IL 4 and IL 5 of T lymphocytes in asthma The activation of NF κB in PKC signal conduction pathway of T lymphocytes may play an important role in the pathogenesis of asthma

Effects of moxibustion on the P2X7R/STAT3/VEGF pathway in rats with colitis-associated colon cancer

Objective:To observe the effects of herb-partitioned moxibustion and ginger-partitioned moxibustion on the growth of colon tumors in rats with colitis-associated colon cancer(CACC),and explore the mechanism of moxibustion intervening CACC through the purinergic receptor P2X ligand-gated ion channel 7(P2X7R)/signal transducer and activator of transcription 3(STAT3)/vascular endothelial growth factor(VEGF)pathway.Methods:A total of 26 male Sprague-Dawley rats were selected.According to the random number table method,6 rats were selected as the normal group.The remaining 20 rats were injected intraperitoneally with azoxymethane(AOM)combined with oral dextran sodium sulfate(DSS)to prepare the CACC model.After the model was successfully established,2 rats were randomly selected for model identification.The remaining 18 rats which were successfully modeled were randomly divided into a model group,a herb-partitioned moxibustion group and a ginger-partitioned moxibustion group,with 6 rats in each group.Moxibustion intervention was performed in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group at Qihai(CV 6)and bilateral Tianshu(ST 25).Moxibustion was performed twice at each point each time,once a day,at a 1-day interval after 6 consecutive interventions,for a total of 30 interventions.After intervention,the colon tumor load,pathological change and histopathological score were observed.Immunohistochemistry was used to detect the expressions of VEGF,P2X7R,phospho-STAT3(p-STAT3),and nuclear factor-kappa B p65(NF-κB p65)proteins in rat colon tissue.Western blot was used to detect the levels of p-STAT3 and NF-κB p65 proteins in rat colon tissue.Results:Compared with the normal group,the colon tumor load and histopathological score in the model group were significantly increased(both P<0.001),and different grades of dysplasia were observed in colon tissue from the model group,reaching the degree of adenocarcinoma;the expression level of P2X7R protein in colon tissue was significantly decreased(P<0.001),and the expression levels of p-STAT3,NF-κB p65 and VEGF proteins were significantly increased(all P<0.001)in the model group.Compared with the model group,the colon tumor load,colon histopathological score and the levels of p-STAT3,NF-κB p65 and VEGF proteins in colon tissue were significantly decreased(all P<0.05)in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group while the expression levels of P2X7R protein in colon tissue were significantly increased(both P<0.05).Conclusion:Both herb-partitioned moxibustion and ginger-partitioned moxibustion can reduce the colon tumor load in CACC rats and delay the progression of colon adenomas.The mechanism may be mediated by the P2X7R/STAT3 pathway to inhibit STAT3 phosphorylation,thereby reducing VEGF protein expression.

Effect of herb-partitioned moxibustion in improving tight junctions of intestinal epithelium in Crohn disease mediated by TNF-α-NF-κB-MLCK pathway

Objective:To explore the effect of herb-partitioned moxibustion(HPM)on tight junctions(TJs)of intestinal epithelial cells in Crohn disease(CD)mediated by tumor necrosis factor-α(TNF-α)-nuclear factor kappa B(NF-κB)-myosin-light-chain kinase(MLCK)pathway.Methods:Forty-eight male Sprague-Dawley rats were randomly divided into a normal control(NC)group,a model control(MC)group,an HPM group and a mesalazine(MESA)group,with 12 rats in each group.Trinitrobenzene sulfonic acid(TNBS)was administered to establish CD models.When the model was confirmed a success,the HPM group rats were treated with HPM at Tianshu(ST 25)and Qihai(CV 6),while the MESA group rats were given MESA solution by lavage.When the intervention finished,the colonic epithelial tissues were separated,purified and cultured in each group to establish the intestinal epithelial barrier model in vitro,and TNF-αwas added(100 ng/mL)in the culture medium and maintained for 24 h to establish an increased epithelial permeability model.Transepithelial electrical resistance(TEER)was used to examine the permeability of the barrier;Western blot was used to observe the expressions of the proteins related to TJs of intestinal epithelial cells mediated by TNF-α-NF-κB-MLCK pathway;immunofluorescence staining was used to observe the expressions and distributions of tight junction proteins in the intestinal epithelium.Results:After TNF-αinduction,compared with the MC+TNF-αgroup,the TEER value increased significantly in the HPM+TNF-αand MESA+TNF-αgroups(both P<0.001);the expressions of nuclear factor kappa B(NF-κB)p65,MLCK,myosin light chain(MLC),tumor necrosis factor receptor-associated factor 6(TRAF6)and receptor interaction protein-1(RIP1)decreased significantly(P<0.01 or P<0.05),and the expression of zinc finger protein A20(A20)increased significantly(P<0.01);the expressions of occludin,claudin-1,zonula occludens protein 1(ZO-1)and F-actin also increased significantly(all P<0.01).Compared with the MESA+TNF-αgroup,the expressions of MLC,occludin,claudin-1,ZO-1 and F-actin increased significantly in the HPM+TNF-αgroup(P<0.01 or P<0.05).Conclusion:HPM can protect or repair the damage of intestinal epithelial barrier in CD rats,which may be achieved through modulating the abnormal TJs in intestinal epithelium mediated by TNF-α-NF-κB-MLCK pathway.

人参皂苷Rg1调节N2a/APP695细胞β分泌酶活性的可能机制

目的 观察人参皂苷Rg1能否通过核蛋白因子κB（NF-κB）影响N2a/APP695细胞β淀粉样肽（Aβ）的生成和代谢,从源头上缓解和（或）减轻阿尔茨海默病（AD）病人的神经病理改变.方法 利用稳定表达突变型APP695的N2a细胞（N2a/APP695）,通过RT-PCR、蛋白免疫印迹、激光共聚焦显微镜等方法检测人参皂苷Rg1对β分泌酶活性及上游转录因子NF-κB活性的影响.结果 2.5 mol/L人参皂苷Rg1作用6～12 h后,N2a/APP695细胞外液Aβ1-40和Aβ1-42水平较Rg1作用前均有不同程度的下降[最低值分别为（13.3±4.3 ）ng/ml及（12.0±5.4） ng/ml]；BACE1的蛋白表达水平下降（BACE1/β-肌动蛋白比值为0.26±0.05）；NF-κB p65蛋白表达水平明显增加（磷酸化p65/总p65比值为0.93±0.02）并诱导促进NF-κB的核转位；NFκB的抑制剂quinazoline预处理后可见人参皂苷Rg1对BACE1 mRNA和蛋白表达的抑制作用明显减弱.结论 人参皂苷Rg1能激活NF-κB,促进NF-κB的核转位,增加与BACE1启动子相应位点的结合,导致β分泌酶的活性减少.