马立克氏病病毒pp38基因和1.8kb转录子之间双向启动子的特性研究

从马立克氏病病毒(MDV)基因组DNA复制原点区某一点,将介于MDVpp38基因和1 8kb转录子之间的双向启动子分割成两个单方向的启动子。以pp38为报告基因,pUC18质粒为载体,构建了含不同方向完整启动子序列的pProfpp38和pProrpp38质粒,以及含分割后单方向启动子序列的pdProfpp38和pdProrpp38质粒。4种质粒分别转染鸡胚成纤维细胞(Chickenembryofibroblast,CEF)后,均能检测到pp38基因的表达。进一步以氯霉素乙酰转移酶(Chloramphenicolacetyltransferase,CAT)为报告基因,构建了含不同方向完整双向启动子的pProfCAT和pProrCAT质粒,以及含分割后单方向启动子序列的pdProfCAT和pdProrCAT质粒。通过转染试验,定量分析了完整启动子和分割后启动子在两个方向上的启动活性。实验结果表明,分割后的启动子在两个方向上的启动活性均比相应方向上完整启动子的活性低,其中1 8kb转录子方向上的活性下降了4

旋覆花内酯抑制AD模型大鼠海马环加氧酶2和核转录因子κB的表达

目的观察旋覆花内酯（l-O-acetylbfitannilactone，ABL）对Aβ25-35海马内注射所致阿尔采末病（Alzheimers disease，AD）模型大鼠海马环加氧酶2（COX-2），核转录因子KB（NF-kB）表达的影响，探讨其对AD大鼠的治疗作用及机制。方法30只SD大鼠随机分为对照组、模型组及药物处理组，Aβ25-35海马内注射制备AD大鼠模型，药物处理组给予ABL26mg·kg^-1·d^-1，模型组给予等量溶剂，分两次灌胃，共21d。用Morris水迷宫测定大鼠的学习记忆能力，免疫组化及Western blot测定大鼠海马COX-2，NF-kB蛋白表达。结果AD大鼠在定位航行试验中，逃避潜伏期延长，单位时间内跨越原平台次数减少，空间记忆力受损，与对照组及药物处理组大鼠比较差异有统计学意义（P〈0．05）；于试验d2、3、4药物处理组与对照组大鼠比较差异无统计学意义（P〉0．05）。模型组海马COX-2、NF-kB表达升高（P〈0．05），分别是对照组的2．8倍和1．6倍，药物处理组COX-2、NF—kB表达下降，与模型组比较差异有统计学意义（P〈0．05）。结论ABL的抗炎作用可能是其改善AD大鼠学习记忆能力的作用机制之一。

Nuclear factor-KB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM： To evaluate the protective effect of NF-kB decoy oligodeoxynucleotides （ODNs） on ischemia/reperfusion （I/R） injury in rat liver graft. METHODS： Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kB decoy ODNs or scrambled ODNs. NF-kB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kB activity in the liver graft was analyzed by electrophoretic mobility shift assay （EMSA）. Hepatic mRNA expression of TNF-α, IFN-γ and intercellular adhesion molecule-1 （ICAM-1） were determined by semiquantitative RT-PCR. Serum levels of TNF-α and IFN-γ were measured by enzyme-linked immunosorbent assays （ELISA）. Serum level of alanine transaminase （ALT） was measured using a diagnostic kit. Liver graft myeloperoxidase （MPO） content was assessed. RESULTS： NF-kB activation in liver graft was induced in a time-dependent manner, and NF-kB remained activated for 16 h after graft reperfusion. NF-kB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kB decoy ODNs significantly suppressed NF-kB activation as well as mRNA expression of TNF-α, IFN-γ, and ICAM-1 in the liver graft. The hepatic NF-kB DNA binding activity [presented as integral optical density （IOD） value] in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat （2.16±0.78 vs 36.78 ±6.35 and 3.06±0.84 vs 47.62± 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P〈0.001）. The hepatic mRNA expression level of TNF-α, IFN-γ and ICAM-1 rpresented as percent of β-actin mRNA （%）] in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat （8.31 ±3.48 vs 46.37±10.65 and 7.46± 3.72 vs 74.82±12.25 for hepatic TNF-α mRNA, 5.58±2.16 vs 50.46±9.35 and 6.47±2.53 vs 69.72±13.41 for hepatic IFN-γ mRNA, 6.79 ±2.83 vs 46.23±8.74 and 5.28±2.46 vs 67.44±10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P〈0.001）. Administration of NF-kB decoy ODNs almost completely abolished the increase of serum level of TNF-α and IFN-γ induced by hepatic ischemia/reperfusion, the serum level （pg/mL） of TNF-α and in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat （42.7±13.6 vs 176.7±15.8 and 48.4±15.1 vs 216.8±17.6 for TNF-α level, 31.5±12.1 vs 102.1±14.5 and 40.2±13.5 vs 118.6±16.7 for IFN-γ level after 4 and 8 h of reperfusion, respectively, P〈0.001）. Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kB decoy ODNs, the hepatic MPO content （A655） and serum ALT level （IU/L） in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat （0.17±0.07 vs 1.12±0.25 and 0.46±0.17 vs 1.46±0.32 for hepatic MPO content, 71.7±33.2 vs 286.1±49.6 and 84.3±39.7 vs 467.8±62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P〈0.001）. CONCLUSION： The data suggest that NF-kB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kB activation and subsequent expression of proinflammatory mediators.

Andrographolide inhibits NF-KB activation and attenuates neointimal hyperplasia in arterial restenosis

The NF-kB transcription factors modulate the expression of tissue factor （TF）, E-selectin （CD62E） and vascular cell adhesion molecule-1 （VCAM-1）, which are essential for thrombosis and inflammation. We have previously shown that andrographolide （Andro） covalently modifies the reduced cysteine^62 of p50-a major subunit of NF-kB transcription factors, thus blocking the binding of NF-kB transcription factors to the promoters of their target genes, preventing NF-kB activation and inhibiting inflammation in vitro and in vivo. Here we report that Andro, but not its inactive structural analog 4H-Andro, significantly suppressed the proliferation of arterial neointima （-60% reduction） in a murine model of arterial restenosis. Consistently, p50^-/- mice manifested attenuated neointimal hyperplasia upon arterial ligation. Notably, the same dosage of Andro did not further reduce neointimal formation in p50^-/- mice, which implicates the specificity of Andro on p50 for treating experimental arterial restenosis. The upregulation of NF-kB target genes, including TF, E-selectin and VCAM-1, and the increased deposition of leukocytes （mainly CD68^＋ macrophages） were clearly detected within the injured arterial walls, all of which were significantly abolished by treatment with Andro or genetic deletion of p50. The expression ofTF, E-selectin and VCAM-I was also markedly upregulated in the patient sample of thrombotic vasculitis, indicating the clinical relevance of NF-kB activation in the pathogeneses of occlusive arterial diseases. Our data thus indicate that, by the downregulation of the NF-kB target genes that are critical in thrombosis and inflammation, specific inhibitors of p50, such as Andro, may be therapeutically valuable for preventing and treating thrombotic arterial diseases, including neointimal hyperplasia in arterial restenosis.

自锁托槽矫正器对牙周炎患者正畸治疗过程中龈沟液内RANKL、OPG表达的影响

分析自锁托槽矫正器对牙周炎患者正畸治疗过程中龈沟液(GCF)内核因子кB受体活化子配体(RANKL)及骨保护素(OPG)表达的影响。方法:选取2018年1月—2018年9月期间,在院进行正畸治疗的轻中度牙周炎患者48例作为研究对象。根据随机数字表原则,将患者分为对照组和观察组,每组各24例。对照组患者接受传统直丝弓托槽矫正器治疗,观察组患者接受自锁托槽矫正器治疗。检测GCF中RANKL、OPG表达情况,分析RANKL、OPG及RANKL/OPG变化趋势。结果:T1~T6时刻,观察组患者RANKL水平和RANKL/OPG低于对照组,OPG水平高于对照组,并且,相比于对照组患者,观察组患者GCF中RANKL、OPG及RANKL/OPG变化更早。结论:自锁托槽矫正器可有效降低牙周病患者正畸过程中GCF的RANKL水平,提高OPG水平,从而更有利于牙周炎患者正畸过程中的牙周环境。

Tumor metastasis and the reciprocal regulation of heparanase gene expression by nuclear factor kappa B in human gastric carcinoma tissue

AIM: To investigate whether NF-kB is activated in human gastric carcinoma tissues and, if so, to study whether there is any correlation between NF-kB activity and heparanase expression in gastric carcinoma. METHODS: NF-kB activation was assayed by immunohistochemical staining in formalin-fixed, paraffin-embedded specimens from 45 gastric carcinoma patients. Electrophoretic mobility shift assay (EMSA) method was used for nuclear protein from these fresh tissue specimens. Heparanase gene expression was quantified using quantitative RT-PCR. RESULTS: The nuclear translocation of RelA (marker of NF-kB activation) was significantly higher in tumor cells compared to adjacent and normal epithelial cells [(41.3±3.52)% vs (0.38±0.22) %, t=10.993, P= 0.000<0.05; (41.3±3.52)% vs(0±0.31)%, t=11.484, P= 0.000<0.05]. NF-kB activation was correlated with tumor invasion-related clinicopathological features such as lymphatic invasion, pathological stage, and depth of invasion (Z= 2.148, P= 0.032<0.05; t = 8.758, P= 0.033<0.05; t = 18.531, P = 0.006<0.05). NF-KB activation was significantly correlated with expression of heparanase gene (r= 0.194, P=0.046<0.05). CONCLUSION: NF-KB RelA (p65) activation was related with increased heparanase gene expression and correlated with poor clinicopathological characteristics in gastric cancers. This suggests NF-kB as a major controller of the metastatic phenotype through its reciprocal regulation of some metastasis-related genes.

TRIM22 Inhibits the TRAF6-stimulated NF-κB Pathway by Targeting TAB2 for Degradation

Tripartite motif containing 22 （TRIM22）, a member of the TRIM/RBCC family, has been reported to activate the nuclear factor-kappa B （NF-kB） pathway in unstimulated macrophage cell lines, but the detailed mechanisms governing this activation remains unclear. We investigated this mechanism in HEK293T cells. We found that overexpression of TRIM22 could activate the NF-kB pathway and conversely, could inhibit the tumor necrosis factor receptor-associated factor 6 （TRAF6）-stimulated NF-kB pathway in HEK293T cells. Further experiments showed that TRIM22 could decrease the self-ubiquitination of TRAF6, and interact with and degrade transforming growth factor-13 activated kinase 1 binding protein 2 （TAB2）, and that these effects could be partially rescued by a TRIM22 RING domain deletion mutant. Collectively, our data indicate that overexpression of TRIM22 may negatively regulate the TRAF6-stimulated NF-rd3 pathway by interacting with and degrading TAB2.

Hepatitis C virus non-structural 5A protein can enhance full-length core protein-induced nuclear factor-κB activation

AIM： To study the effects of hepatitis C virus （HCV） core and non-structural 5A （NS5A） proteins on nuclear factor- k B （NF- k B） activity for understanding their biological function on chronic hepatitis caused by HCV infection. METHODS： Luciferase assay was used to measure the activity of NF-kB in three different cell lines cotransfected with a series of deletion mutants of core protein alone or together with NS5A protein using pNF- k B-Luc as a reporter plasmid. Western blot and indirect immunofluorescence assays were used to confirm the expression of proteins and to detect their subcellular localization, respectively. Furthermore, Western blot was also used to detect the expression levels of NF- k B/p65, NF- k B/p50, and inhibitor k B-a（k B-a）. RESULTS： The wild-type core protein （C191） and its mutant segments （C173 and C158） could activate NF- k B in Huh7 cells only and activation caused by （C191） could be enhanced by NS5A protein. Moreover, the full-length core protein and its different deletion mutants alone or together with NS5A protein did not enhance the expression level of NF- k B. The NF- k B activity was augmented due to the dissociation of NF-k： B-I k： B complex and the degradation of Ik B-a. CONCLUSION：NF- k B is the key transcription factor that can activate many genes that are involved in the cellular immune response and inflammation. Coexpression of the full-length core protein along with NS5A can enhance the NF- k B activation, and this activation may play a significant role in chronic liver diseases including hepatocellular carcinoma associated with HCV infection.

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.

2型糖尿病伴牙周炎患者龈沟液及血清中RANKL/OPG的表达与相互关系

目的:分析2型糖尿病伴牙周炎患者龈沟液及血清中骨保护素(osteoprotegerin,OPG)、核因子-κB受体活化因子配体(Receptor activator of nuclear factor-κB ligand,RANKL)的表达情况。方法:设置益阳医专附属医院内分泌门诊的40例单纯糖尿病患者为DM组,糖尿病伴牙周炎患者40例为DM+PD组,另设置益阳医专附属口腔医院抽取的40例单纯牙周病为PD组,对这120例患者空腹血糖、随机血糖、糖化血红蛋白、龈沟液及血清中RANKL、OPG水平,计算RANKL/OPG。结果:PD+DM组患者空腹血糖为(11.21±1.32)mmol/L,随机血糖为(15.21±1.42)mmol/L,糖化血红蛋为(10.45±1.36)%,高于DM组和PD组(P<0.05);DM+PD组患者龈沟液RANKL水平为(1.78±0.21)pg/m L,RANKL/OPG水平为(0.92±0.12),高于DM组,OPG水平为(2.04±0.56)pg/m L;血清RANKL水平为(122.45±14.16)pg/m L,OPG水平为(10.16±2.32)pg/m L,RANKL/OPG水平为(11.68±1.21),高于PD组(P<0.05)。结论:2型糖尿病合并牙周炎可导致患者血糖水平增加;与单纯糖尿病患者相比,2型糖尿病合并牙周炎患者龈沟液RANKL、OPG和RANKL/OPG表达增强,与单纯牙周病患者相比,2型糖尿病合并牙周炎患者龈血清RANKL、OPG和RANKL/OPG表达增强,提示RANKL/OPG比例失衡是导致2型糖尿病合并慢性牙周炎患者病情加重的原因之一。

甲氨蝶呤对类风湿关节炎成纤维样滑膜细胞表达RANKL的影响

目的探讨类风湿关节炎(rheumatoid arthritis,RA)滑膜细胞中表达核因子κB受体活化子配体(RANKL)的细胞以及甲氨蝶呤(MTX)对其表达的影响。方法收集原代RA及正常滑膜细胞,用免疫磁珠法筛选滑膜CD68+和CD68-细胞,CD44免疫组织化学染色观察以及RANKL免疫荧光检测;细胞体外培养并在培养液中加入最终浓度为1mg/L的MTX,用双夹心法酶联免疫吸附试验(ELISA)检测培养液中RANKL含量以及评估每一细胞RANKL表达量。结果在滑膜细胞中,CD68+滑膜细胞为巨噬样细胞,CD44、RANKL染色阴性;CD68-滑膜细胞为纤维样细胞,CD44、RANKL染色阳性。RA滑膜CD44+/CD68-纤维样细胞RANKL蛋白表达量显著增高,MTX对其RANKL表达有抑制作用(P<0.01)。正常滑膜纤维样细胞低表达RANKL,MTX对其RANKL表达抑制作用来自对细胞增殖的抑制。结论滑膜组织中有RANKL表达,这些细胞为CD44+/CD68-纤维样细胞,RA滑膜纤维样细胞RANKL分泌增高并受到MTX抑制。MTX可通过抑制RANKL减少关节周围骨破坏作用。

筋骨胶囊对骨质疏松大鼠骨髓细胞OPG和RANKL mRNA表达量的影响

目的:观察筋骨胶囊对骨质疏松大鼠骨髓细胞护骨素(OPG)和核因子kB受体活化子配体(RANKL)mRNA表达量的影响,从分子水平上进一步探讨筋骨胶囊治疗骨质疏松症的有效机制。方法:取健康6月龄雌性SD大鼠48只,采用摘除双侧卵巢去势方法造成大鼠骨质疏松模型,随机分为假手术组、模型组、阿仑膦酸钠组(0.5mg/kg)、筋骨胶囊组(0.27g/kg)4组。用药12周后,处死所有大鼠,并取双侧股骨骨髓为标本,运用现代分子生物学RT-PCR技术,测量各大鼠骨髓基质细胞的OPG和RANKL mRNA表达量。结果:模型组OPG和RANKL mRNA表达量均明显高于假手术组(P<0.01);阿仑膦酸钠组OPG mRNA表达量与模型组对比差异无统计学意义(P>0.05),阿仑膦酸钠组RANKL mRNA表达量明显低于模型组(P<0.01);筋骨胶囊组OPG mRNA表达量明显高于模型组(P<0.01),筋骨胶囊组RANKL mRNA表达量明显低于模型组(P<0.01);筋骨胶囊组OPG mRNA表达量明显高于阿仑膦酸钠组(P<0.01),筋骨胶囊RANKL mRNA表达量与阿仑膦酸钠组相比差异无统计学意义(P>0.05)。结论:筋骨胶囊能有效地上调OPG mRNA表达量,并抑制RANKL mRNA的表达,通过影响OPG/RANKL系统,抑制骨吸收,并促进骨形成,能有效地防治骨质疏松症。

Hydrogen sulfide from a NaHS source attenuates dextran sulfate sodium(DSS)-induced inflammation via inhibiting nuclear factor-κB

This study investigated the alleviating effects of hydrogen sulfide （H2S）, derived from sodium hydrosulfide （NariS）, on inflammation induced by dextran sulfate sodium （DSS） in both in vivo and in vitro models. We found that NariS injection markedly decreased rectal bleeding, diarrhea, and histological injury in DSS-challenged mice. NariS （20 pmol/L） reversed DSS-induced inhibition in cell viability in Caco-2 cells and alleviated pro-inflammation cytokine expression in vivo and in vitro, indicating an anti-inflammatory function for H2S. It was also found that H2S may regulate cytokine expression by inhibiting the nuclear factor-KB （NF-KB） signaling pathway. In conclusion, our results demon- strated that H2S alleviated DSS-induced inflammation in vivo and in vitro and that the signal mechanism might be associated with the NF-KB signaling pathway.