A Nonradioactive Method for Detecting DNA-binding Activity of Nuclear Transcription Factors

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-κB were labled with DIG by terminal transferase After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8 % nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film The results showed that nuclear proteins binded specifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-κB in PMA group was more than that in PMA+PDTC group It is suggested that detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories

Effect of Sirpα1 on the expression of nuclear factor-kappa B in hepatocellular carcinoma

BACKGROUND:Signal regulatory protein alpha1(Sirpα1) is a member of Sirps families containing four immunoreceptor tyrosine-based inhibitory motifs(ITIMs) domains in the cytoplasm of and an activated substrate of receptor tyrosine kinase(RTK),that negatively regulates the RTK-dependent cell proliferating signal transduction pathway.Previously we found that Sirpα1 was closely associated with the occurrence and development of hepatocellular carcinoma(HCC)as well as liver regeneration.Since it is unclear about the regulatory mechanisms,we established the cell line transfected Sirpα1 gene and preliminarily clarified the mechanisms by which Sirpα1 negatively regulates the carcinogenesis and development of HCC. METHODS:Liver cancer Sk-Hep1 cell was respectively transfected with plasmids of pLXSN,pLXSN-Sirpα1 and pLXSN-Sirpα1Δ4Y 2 ,screened with the drug of G418(1200 μg/ml),and various transfected Sk-Hep1 cell lines were obtained.The protein expressions of P65,P50,IκBα,cyclin D1 and Fas in various Sk-Hep1 cell lines were determined by Western blotting,and P65 and P50 were localized by the immunofluorescence technique. RESULTS:Sirpα1 could significantly upregulate the protein expression of IκBα(vs.other cell lines,P<0.05) in the Sk-Hep1 cell,and downregulate the protein expressions of P65,P50 and cyclin D1(vs.other cell lines, P<0.05)in the Sk-Hep1 cell.P65 protein expression was mainly localized in the cytoplasm in the pLXSN Sk-Hep1 cell,and in the nucleus of the Sk-Hep1 cell with mutantSirpα1Δ4Y 2 ,but in nucleus of the Sk-Hep1 cell with wild Sirpα1.P50 protein expression was localized in the cytoplasm and nucleus of the pLXSN Sk-Hep1 cell,but in the nucleus of the Sk-Hep1 cell with wild Sirpα1 and mutant Sirpα1Δ4Y 2 plasmid. CONCLUSIONS:Sirpα1 might negatively regulate and control the abnormal proliferation of liver cancer cells by influencing the protein content and localization of nuclear factor-kappa B,then influence the expression of cyclins such as cyclin D1 in the signal transduction pathway.It may be one of the important mechanisms by which Sirpα1 negatively regulates the carcinogenesis and development of HCC.

Effect of oridonin-mediated hallmark changes on inflammatory pathways in human pancreatic cancer(BxPC-3) cells

AIM: To investigate the effect of oridonin on nuclear transcription factors and to study the relationship between biological behavior and inflammatory factors in human pancreatic cancer (BxPC-3) cells.

Nuclear transcription factors and lipid homeostasis in liver

The liver plays a major role in the regulation , glucose, lipid and energy metabolism. Increasd hepatic fat deposit is a very common feature in obese, insulin-resistant patients, in metabolic syndrome, alcoholic steatohepatitis （ASH） and nonalchoholic fatty liver disaseas （NAFLD）. As a central organ for whole body lipid metabolism, disruption of the normal mechanisms for synthesis, transport and removal/ metabolism of long-chain fatty acids and triglycerides are the basis for the development of fatty liver. The exact mechanisms that link hepatic lipid accumulation, impaired glucose metabolism, and insulin resistance are unknown, but increasing evidence suggest that nuclear transcription factors play important roles. Members of the nuclear receptor superfamily, especially the peroxisome proliferator-activated receptors （PPARs） and the liver X receptor （LXR）, other factors such as sterol regulatory element binding proteins （SREBPs）, carbohydrate- response element-binding protein （ChREBP）, and nuclear transcription fator-κB （NF-κB） have emerged as dominant regulators of these processes, but the relative role of each of these factors in fatty liver disease is still undefined.

Huaiqihuang Granules(槐杞黄颗粒)Reduce Proteinuria by Enhancing Nephrin Expression and Regulating Necrosis Factor κB Signaling Pathway in Adriamycin-Induced Nephropathy

Objective To investigate the effects of Huaiqihuang Granules (槐杞黄颗粒, HQH), a mixture of Chinese herbs including Trametes robiniophila Murr, Fructus Lycii and Polygonatum sibiricum, on adriamycininduced nephropathy (ADRN) in rats and its underlying mechanisms. Methods Rats with ADRN were divided into four groups: the sham group, the model group (distilled water), the low-dose HQH-treated (2 g/kg) group, and the high-dose HQH-treated (4 g/kg) group. Body weight and 24-h urinary protein (Upro) were checked every week. After 5-week intervention, at the end of the study, the rats were sacrificed and blood samples were collected for examination of biochemical parameters, including glomerular morphological makers, podocyte shape, cellular apoptosis, expressions of nephrin, inflammatory and apoptosis markers. Results HQH ameliorated the rat’s general status, proteinuria, renal morphological appearance and glomerulosclerosis. The decreased expression of nephrin in ADRN rats was increased by HQH, as well as the impaired podocyte foot process fusion. Cytosolic levels of p65 and inhibitor of nuclear factor κBα (IκBα) were decreased in ADRN rats, and recovered by the treatment of HQH. Consistently, the induced expression of tumor necrosis factor α (TNF-α), phosphorylated nuclear factor κB p65 (p-NFκB p65) and IκBα in ADRN were markedly suppressed by HQH. In addition, induction of Bax, cleaved caspase-3 and cytochrome C in ADRN rats were suppressed by HQH, indicating the amelioration of apoptosis. Conclusion HQH could ameliorate renal impairments in ADRN rats by increasing nephrin expression, inhibiting NF-κB signaling pathway via the down-regulation of p-NF-κB p65 and p-IκBα, and suppression of glomerular and tubular apoptosis.

Andrographolide as An Anti-H1N1 Drug and the Mechanism Related to Retinoic Acid-Inducible Gene-I-Like Receptors Signaling Pathway

Objective： To observe the anti-virus effects of andrographolide （AD） on the retinoic acid-inducible gene-I （RIG-I）-Iike receptors （RLRs） signaling pathway when immunological cells were infected with HIN1. Methods： Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line （16HBE）. The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium （MTT） assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma （IFN- ~, ） and interleukin-4 （IL-4） enzyme-linked immunosorbent assay （ELISA） detection while immunological cells for real-time polymerase chain reaction （RT-PCR）. Results： The optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-13 promoter stimulator-1 （IPS-1）, interferon regulatory factor （IRF）-7, IRF-3 and nuclear transcription factor K B （NF- K B） mRNA levels increased significantly （P〈0.05）. When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF- K B decreased significantly in each group with significant statistic differences （P〈0.05）. Conclusions： The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Effects of Bushen Huoxue Yin(补肾活血饮) on brain NF-κB and NO content in the parkinson's disease model mouse

OBJECTIVE:To observe the effects of Bushen Huoxue Yin(补肾活血饮,BSHXY) on nuclear transcription factor kappa B(NF-κB) and nitric oxide(NO) in the brain of the Parkinson's disease(PD) model mouse.METHODS:Forty-five C57BL/6 mice were randomly divided into three groups;normal,model and BSHXY treatment groups.Concentrations of NF-κB and NO in mouse brain tissue were determined by ELISA and spectrophotometry,respectively.RESULTS:NF-κB concentration in brain tissue in the model group was 14.04±4.38 μg· L-1,which was higher than that in normal(P<0.01) and BSHXY(P< 0.05) groups.NO content in brain tissue in the model group was 5.93±0.79 μmol · gprot-1,which was also higher than that in model(P<0.01) and BSHXY(P<0.01) groups.However,there were no significant differences in the content of NF-κB and NO between BSHXY and normal groups(P>0.05).CONCLUSION:The mechanism of BSHXY for treatment of PD is possibly related to inhibition ofNF-κB activation and decreased NO content in the brain.

Human Recombinant PLD2 Can Repress p65 Activity of Guinea Pigs of Chronic Asthma in vivo

This article is to investigate the effect of human recombinant phospholipase D2 （rhPLD2） in vivo on the expression of nuclear transcription factor p65 in chronic asthma of guinea pigs. After treating the guinea pigs with chronic asthma by rhPLD2, the crude nuclear extraction was assayed with TransAM Transcription Factor Assay Kit for the activity of pulmo tissue nuclear transcription factor p65. Compared with the healthy guinea pigs, the activity of nuclear transcription factor p65 in guinea pigs of chronic asthma is much higher than that of control groups. Our results showed that rhPLD2 markedly depressed the activity of p65 when the guinea pigs were attacked by chronic asthma.