Dual regulatory effects of resveratrol on activation of NF-κB and cell proliferation in human embryonal kidney 293 cells

Resveratrol (3,4‘,5-trihydroxystilbene, Res), a naturally occurring polyphenol, exhibits antioxidant, antiinflammatory, potential chemopreventive and chemotherapeutic properties in preclinical studies. To further understand its potential clinical efficacy and safety, effect of Res at 10^-9-10^-4 mol/L on human embryonal kidney (HEK293) cell proliferation and its potential mechanism were investigated in present study. Cell viability was detected by using trypan blue dye exclusion method. Cell cycle and apoptosis were analyzed by flow cytometry with propidium iodide stain.Activation of nuclear factor-κB (NF-κB) was determined by luciferase reporter gene assay using stably transfected HEK293/κB-luc cells. Secretion of human interleukin-8(hlL-8) was measured by ELISA. Our results show that HEK293 cell proliferation was significantly stimulated by 10^-7 mol/L Res after treatment for 48 hours, or by 10^-8-10^-7mol/L Res combinated with 10 ng/mL TNFα for 24 h, but was suppressed by 10^-4 mol/L Res with or without TNFα. Both endogenous and TNFα-induced NF-κB activation were downregulated by Res at 10^-7 mol/L, but were upregulated at 10^-4 mol/L. With 10^-4 mol/L Res, the content of secreted IL-8 was increased, and apoptosis rate was increased from lessthan 5 % to 10%, together with significant cell-cycle arrest in S phase. TNFα has coordinative effects with Res on HEK293 cell apoptosis, cell-cycle arrest and IL-8 secretion. These results indicate that Res promotes cell proliferation at low concentration through down-regulation of NF-κB activation in HEK293, but suppresses its growth at high concentration through up-regulation of NF-κB activation, increasing IL-8 and cell-cycle arrest. As resveratrol has dual regulatory effect on cell proliferation in vitro, comprehensive evaluation of its potential clinical utility is needed.     

Activation of proteinkinase ERK mediates induction of macrophage MMP-12 by OxLDL

The present study was undertaken to investigate the effect of oxidized low density lipoprotein (oxLDL) on the expression of maerophage matrix metalloproteinase-12 (MMP-12), and the possible mechanisms. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was detected by Western blot analysis.Enzymatic activity of MMP-12 was determined by β-casein zymography. RT-PCR analysis was used to measure the mRNA expression level of MMP-12. OxLDL-stimulated macrophages produced increased casein-degrading activities and oxLDL also significantly increased the mRNA level of MMP-12 in a dose-dependent manner.OxLDL stimulated the phosphorylation of ERK1/2 in macrophages. The use of the specific inhibitor indicated that the ERK1/2 signaling pathway was required for the induction of MMP-12.These data demonstrated that oxLDL induced MMP-12 expression in macrophages through an ERK1/2-dependent pathway.     

Neuronal growth inhibitory factor inhibits pheochromocytoma PC12 in vitro

Neuronal growth inhibitory factor (GIF), named Metallothioneins-Ⅲ (MT-Ⅲ), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombinant GIF on the growth of neuroblastoma SY5Y and pheochromocytoma PC12 by the MTT (Thiazolyl blue) reduction assay. Recombinant GIF inhibited PC12 in vitro; the inhibitory rate was about 25% when GIF was at 100 mg/L; and the inhibitory rate was about 50% when GIF was at 300 mg/L. It is shown that PC12 could serve as a proper model for detecting neuronal growth inhibitory activity of GIF. Recombinant GIF did not inhibit neuroblastoma SY5Y in vitro, a common model of neuroma; it is also shown that GIF could not inhibit neuromata extensively. The reason for GIF inhibiting PC12 may be that PC12 have some properties of cholinergic neuron. It must play an important role in discovering the mechanism of GIF’s neuronal growth inhibitory activity.     

Isolation and purification of BVⅠ-2H from bee venom and analysis of its biological action

The medical use of bee venom for rheumatoid arthritis ( RA ) has a very long tradition. In this study, isolation and purification of polypeptides from bee venom were carried out on sephadex chromatography, heparin sepharose CL-6B chromatography and HPLC. Several fractions were extracted, and their effects on activation of splenocyte and THP-1 cell were studied. The inhibitory fraction was selected for further studies. Finally, BVⅠ-2H that the HPLC elution profiles was a single peak was isolated by C8 column. ESI- MS detection results showed that BVⅠ-2H was a fraction of bee venom, and the molecular weight of the major component was 644.8. BVⅠ-2H could inhibit ConA-induced splenocyte proliferation, IL-1 production and interfere with splenocyte cycle in mice. Moreover, BVⅠ-2H could inhibit PMA-induced TNFα production in THP-1 cells, which was due to its inhibitory effects on TNFα mRNA expression and protein phosphorylation of IκBα. Our studies indicated that BVⅠ-2H was one of the anti-inflammatory components of bee venom.     

Synergic Inhibition of Lung Carcinoma 95-D Cell Proliferation and Invasion by Combination with(—)-Epigallocatechin-3-Gallate and Ascorbic Acid

In this study, the anti-invasion effects of(-)-epigallocatechin-3-gallate(EGCG) mixed with ascorbic acid(Vc) on human lung carcinoma 95-D cells in vitro were examined and the synergism of the combination of EGCG and Vc was evaluated. Soft agar colony formation assay, cell migration assay, invasion assay, western blot analysis of NF-κB, in situ detection of cellular oxidative stress, and statistical analysis were assessed. The results showed that combining EGCG with Vc could inhibit clone forming rate of 95-D cell by 73.2%, reduce the migration ability of 95-D cell by 65.9%, and decrease the intracellular reactive oxygen species(ROS) level by 76.8%. The results of western blot proved that Vc enhanced the activity of EGCG in inhibiting NF-κB localization. It is speculated that the combination of EGCG and Vc can strongly suppress the proliferation and metastasis of lung carcinoma cells in a synergic manner, possibly with a mechanism associated with the scavenging of reactive oxygen species.     

Cardiac protective role of a novel erythrocyte-derived depressing factor on rats and its Ca^2＋ mechanism

The cardiac protective role of a novel erythro-cyte-derived depressing factor (EDDF) on spontaneous hy-pertensive rats (SHR), calcium overload (CaO) rats and Wistar rats and its mechanism was evaluated. Mean artery pressure (MAP), heart rate (HR) and LVdp/dtmax were measured by physiological recorder. The effect of EDDF on the Ca2+-ATPase activity in myocardial sarcoplasmic reticu-lum (SR) of CaO rats was determined by inorganic phos-phate assay. Calcium transport in myocytes was measured by 45Ca2+ radioactive isotope measurement. The phosphoryla-tion levels of extracellular signal-regulated protein kinases (ERK1/2) in myocardial tissue of SHR and CaO rats were measured by Western blot method. And the ultrastructures of cardiac muscle cells were observed with the transmission electron microscope. The results indicated that EDDF could significantly decrease MAP, HR and LVdp/dtmax in a dose dependent manner (P < 0.05). It seems that the mechanism might relate with activating the Ca2+-APTase, enhancing the uptake and release of Ca2+ from SR (P < 0.05), decreasing the phosphorylation levels of ERK1/2 of myocytes (P < 0.01) and lightening the ultrastructural lesion of cardiac muscle cells. In CaO rats, the Ca2+-ATPase activity decreased clearly com-pared to control (64.99 7.16 vs 94.48 7.68 nmol·min-1 ·mg-1 protein, P < 0.01), while EDDF (100 mg/mL) could significantly increase the activity (87.93 ?9.54 vs 64.99 ?7.16, P < 0.05, n = 7). Both uptake and release rate of Ca2+ (祄ol 45Ca2+/g protein/min) from myocardial SR of CaO rats re-markably decreased compared to control (32.40 ?2.70 and 15.46 ?1.49 vs 61.09 ?10.89 and 25.47 ?4.29, P < 0.05); EDDF (100 mg/mL) could significantly stimulate their activi-ties (50.48 6.76 and 21.76 2.75 vs 32.40 2.70 and 15.46 1.49, P < 0.05). EDDF could evidently down-regulate the phosphorylation of ERK1/2 in myocardial tissue from SHR and CaO rats (P < 0.01), lighten the ultrastructural lesion of cardiac muscle cells of SHR as well. It is concluded that EDDF seems to play protective roles on both structure and function of heart, which closely related with amelioration of Ca2+ transport and inhibition of Ca2+-MAP kinase pathway.     

Platelet-derived growth factor receptor-β in myocyte was upregulated by angiotensin Ⅱ

To observe the regulation of platelet-derived growth factor (PDGF) receptor-βin myocyte stimulated by angiotensin II (AngII) at both integrated and cellular levels and reveal the signal transduction mechanism in cell, two kidneys, one clip (2K1C) renal hypertension were performed by placing a sliver clip around the left renal artery. Blood pressure and the ratio of left ventricular weight to body weight were measured at 4 and 8 weeks after operation. The content of AngII in heart was detected by radioimmunology assay; the protein level of PDGF receptor-βin heart was measured by Western blot analysis. The alteration of PDGF receptor-βstimulated by AngII and several inhibitors was observed on cultured neonatal rat ventricular myocyte (NRVM). The content of AngII in heart of 2K1C renal hypertensive rat at 4 and 8 weeks after operation was increased. Compared with sham group, 4 and 8 weeks after operation, PDGF receptor-βin heart of 2K1C group was upregulated by 100.3% and 127.1% (P < 0.05), respectively. This upregulation could be inhibited by captopril. For cultured myocyte, PDGF receptor-βwas increased by 47.1% after being stimulated by AngII and this upregulation could be inhibited by losartan which was an inhibitor of AT1 receptor. PLC inhibitor (U73122) and MEK inhibitor (PD98059) could partly inhibit PDGF receptor-βupregulation induced by AngII. These results suggested that AngII could upregulate PDGF receptor-βin myocyte by its AT1 receptor and this effect was at least partly dependent on PLC and extracellular signal-regulated kinase (ERK).     

Lymphotactin enhances the in-vitro immune efficacy of dendritoma formed by dendritic cells and mouse hepatocellular carcinoma cells

Objective: To investigate the in-vitro antitumor immune responses of dendritoma formed by mouse hepatocellular carcinoma (HCC) cells and lymphotactin (Lptn) gene modified dendritic cells (DCs). Method: DCs prepared from mouse bone marrow were genetically modified by lymphotactin adenovirus, and fused with H22 cells by polyethylene glycol (PEG). RT-PCR and ELISA were employed to identify lymphotactin expression at mRNA and protein level. Cell phenotypes and fusion efficiency was detected by FACS. The stimulatory effect of DC on T cells was detected by mixed lymphocyte reaction. The cytotoxicity activity against H22 cells was assayed by LDH method. Results: Lymphotactin could be efficiently expressed by DCLptn/H22 hybridoma. DCLptn/H22 cells could induce potent T cell proliferation effect and generate strong cytotoxic T lymphocyte (CTL) reaction against allogenic H22 cells. Conclusion: Lymphotactin genetic modification could enhance the in vitro immune activity of the dendritoma.     

Losartan reduced connexin43 expression in left ventricular myocardium of spontaneously hypertensive rats

Objective： To assess the effect of angiotensin II type 1 （AT1） receptor antagonist losartan on myocardium connexin43 （Cx43） gap junction （GJ） expression in spontaneously hypertensive rats （SHRs） and investigate possible mechanisms. Methods： Sixteen 9-week-old male SHRs and 8 age-matched male Wistar-Kyoto （WKY） rats were included in this study. SHRs were randomly divided into two groups to receive losartan at 30 mg/（kg·d） by oral gavage once daily for 8 weeks （SHR-L） or vehicle （0.9% saline） to act as controls （SHR-V）; WKY rats receiving vehicle for 8 weeks served as normotensive controls. At the end of the experiment, rats were sacrificed and the hearts were removed. Expressions of Cx43 and nuclear factor-kappaB p65 （NF-κB p65） proteins in all three groups were observed and further investigations on the effect of angiotensin II type 1 receptor antagonist losartan （30 mg/（kg·d）, 8 weeks） on Cx43 expression were conducted with Western blot and immunohistochemistry. NF-κB p65 protein in nuclear extracts was determined by Western blot. Results： Left ventricular （LV） hypertrophy was prominent in SHRs, Cx43 and NF-κB p65 protein expressions were obviously upregulated and Cx43 distribution was dispersed over the cell surface. Treatment with losarton reduced the over-expressions of Cx43 and NF-κB p65 in LV myocardium. The distribution of Cx43 gap junction also became much regular and confined to intercalated disk after losartan treatment. Conclusion： Cx43 level was upregulated in LV myocardium of SHR during early stage of hypertrophy. Angiotensin II type 1 receptor antagonist losartan prevented Cx43 gap junction remodeling in hypertrophied left ventricles, possibly through the NF-κB pathway.     

Triphenyltin inhibition of the proteasome activity and its influence on substrate protein levels in nerve cells

Triphenyltin (TPT) widely exists as environmental pollutant, but its toxicity towards nerve cells and the related mechanism remain unclear. In this research, SHSY-5Y cells were exposed to TPT, and the cellular proteasome activity, Iκb proteins, Bax and α-synuclein levels were investigated. The results show that TPT can inhibit the cellular proteasome activity, and result in the accumulation of ubiquitinized proteins. TPT exposure can change the protein levels of IκB, Bax, and α-synuclein, affect NFκB pathwa...     

Construction and<Emphasis Type="Italic">in vitro</Emphasis> study of an E1B-defective adenovirus

An E1B-defective adenovirus named rl/Ad was constructed by homologous recombination.The construction,selection and propagation of recombinant virus was done in the human embryonic kidney 293 cells (HEK293).The in vitro study demonstrated that the recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as the human glioblastoma tumor cells (U251) and human bladder tumer cells (EJ) but not in the normal cells with functional p53 such as the human fibroblast cells (MRC-5).Also,based on the cytopathic effect (CPE),it was demonstrated that the U251 cells were more sensitive to the infection of rl/Ad than that of EJ cells under identical conditions.In this paper,it was found that rl/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus.This may help to determine the safety of using any E1B-defective adenoviruses in cancer gene therapy.     

Development of a functional cell-based HTS assay for identification of NKCC1-negative modulators

Na–K–Cl cotransporter 1(NKCC1) cotransports Na+, K+, and Cl-ions across the plasma membrane into cells. Accumulation of Cl-ions in dorsal root ganglion neurons induces depolarizing GABAA receptors, which mediate presynaptic inhibition and filtration of sensory noise. The activity of the Na–K–Cl cotransporter is modulated by high-dose loop diuretics, such as furosemide and bumetanide. To identify NKCC1 modulators, we developed a functional cell-based assay feasible for highthroughput screening(HTS), in which the activity of NKCC1 was detected by a BTC-AM dye-based thallium transportation assay. We demonstrated that the influx of Tl?was mediated by NKCC1, which required the existence of Cl-ions and could be inhibited by bumetanide and furosemide. Our results demonstrated that the assay was stable, reproducible, and suitable for HTS of negative modulators for NKCC1.     

Regulations of Thyroid Hormone on Cardiac Protein Kinase C Signal Pathway in vitro

The experiments were conducted to assess the influences of thyroid hormone on cardiac protein kinase C(PKC) signal pathway with cultured cardiac myocytes and fibroblasts as the models. Cells were pretreated with 1% newborn calf serum (NCS) or angiotensin Ⅱ (Ang Ⅱ), and then following by a triiodothyronine (T3) treatment. The PKC activity, PKCa and PKCε expressions were analyzed and compared. In 1% NCS pretreatment, T3 could inhibit PKC activity and PKCε expression in cardiac myocytes. The AngⅡ pretreatment led to an increase of PKC acUvity and PKCε expression in cardiac myocytes, and an increase of PKC activity in cardiac fibroblasts. Following by T3 treatment, the increased PKC activity and PKCε expression in cardiac myocytes were markedly decreased. In conclusion, whether in 1% NCS or in Ang lI pretreatment, T3 could inhibit PKC activity and PKCε expression in cardiac myocytes.     

Glycyrrhetinic acid-modified nanoparticles for drug delivery: Preparation and characterization

In this work, glycyrrhetinic acid-modified chitosan （mGA-suc-CTS） used as liver targeted carrier for drug delivery, was prepared via hemisuccinate as a bridged group. The structure of the product was confirmed by IR and NMR methods and the degree of substitution （DS） of glycyrrhetinic acid groups was estimated via elemental analysis. Nanoparticles were formed by ionic gelation methold. The drug-loading and release behavior of the nanoparticles were investigated using BSA as the model drug. The results indicated that the carrier with a highest DS of 5.19% could be got and the DS was controlled by changing reaction temperature or feed ratio. BSA could be entrapped into the nanoparticles with the drug-loading ratio of 26.3% and the encapsulation efficiency of 81.5%. A sustained release over an 11-day period was observed in pH 7.4 in vitro.     

Isolation and purification of BVⅠ-2H from bee venom and analysis of its biological action

The medical use of bee venom for rheumatoid arthritis (RA) has a very long tradition. In this study, isolation and purification of polypeptides from bee venom were carried out on sephadex chromatography, heparin sepharose CL-6B chromatography and HPLC. Several fractions were extracted, and their effects on activation of splenocyte and THP-1 cell were studied. The inhibitory fraction was selected for further studies. Finally, BVⅠ-2H that the HPLC elution profiles was a single peak was isolated by C8 column. ESI- MS detection results showed that BVⅠ-2H was a fraction of bee venom, and the molecular weight of the major component was 644.8. BVⅠ-2H could inhibit ConA-induced splenocyte proliferation, IL-1 production and interfere with splenocyte cycle in mice. Moreover, BVⅠ-2H could inhibit PMA-induced TNFa production in THP-1 cells, which was due to its inhibitory effects on TNFa mRNA expression and protein phosphorylation of IkBa. Our studies indicated that BVⅠ-2H was one of the anti-inflammatory components of bee venom.     

Expression of human clotting factor Ⅸ mediated by recom- binant lentiviral vector in cultured cells and hemophilia B mice

To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMV腞8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.     

Expression of recombinant human lysozyme in the milk of transgenic mice

Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal of improving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic mice was 0.2mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.