Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

Ischemic preconditioning (IPC) is a potential intervention known to protect the heart against ischemia/reperfusion injury, but its role in the no-reflow phenomenon that follows reperfusion is unclear. Dihydrotanshinone I (DT) is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC. Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation (OGD), but the protection was prevented by a ROS scavenger. In addition, DT administration protected the heart against isoprenaline challenge. Mechanistically, PKM2 reacted to transient ROS via oxidization at Cys423/Cys424, leading to glutathionylation and nuclear translocation in dimer form. In the nucleus, PKM2 served as a co-factor to promote HIF-1α-dependent gene induction, contributing to adaptive responses. In mice subjected to permanent coronary ligation, cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection, which was rescued by overexpression of wild-type Pkm2, rather than Cys423/424-mutated Pkm2. In conclusion, PKM2 is sensitive to oxidation, and subsequent glutathionylation promotes its nuclear translocation. Although IPC has been viewed as a protective means against reperfusion injury, our study reveals its potential role in protection of the heart from no-reflow ischemia.

Temporal dependence of neuronal alpha-synuclein oligomerization and nuclear translocation induced by paraquat / 环境与职业医学

Background Paraquat (PQ), one of the environmental poisons associated with sporadic Parkinson's disease (PD), can cause abnormal aggregation of alpha-synuclein (α-syn), but the research on its conformational changes and subcellular localization is limited. Objective To investigate the effect of PQ on α-syn conformation and subcellular localization in dopaminergic neurons. Methods Forty-eight SPF C57BL/6 male mice were selected and randomly divided into a control group and a model group. The model group was intraperitoneally injected with PQ (15 mg·kg−1), and the control group was intraperitoneally injected with 0.9% normal saline, twice a week for eight weeks to construct a PD-like mouse model. The changes of neurobehavior (by open field test and pole climbing test) were observed to evaluate motor ability of mice. Immunohistochemical staining (IHC) was used to detect the expression levels of tyrosine hydroxylase (TH) and α-syn in the midbrain. Western blotting (WB) was used to measure the protein expression levels of TH and α-syn in midbrain. Human neuroblastoma SH-SY5Y cells were used as dopaminergic neuron in vitro models. After the cells were treated with PQ (100 μmol·L−1) for 0, 12, 24, 36 and 48 h, the expressions of α-syn in whole cell, cytoplasm, and nucleus were detected by WB; the expression level of extracellular α-syn was detected by enzyme-linked immunosorbent assay (ELISA); the change of α-syn location was observed by immunofluorescence assay (IFA). Results The neurobehavioral tests' results showed that compared with the control group, the residence time in peripheral area of mice in the PQ model group increased with the increase of exposure time (P<0.05), the residence time and moving distance in the central region decreased (P<0.05), and the pole climbing time increased (P<0.05). The mouse IHC results showed that compared with the control group, the number of TH positive cells in the midbrain decreased in the model group at week 6 and 8 (P<0.05), while the expression level of α-syn increased at week 4, 6, and 8 (P<0.05). The WB results of mouse showed that the relative expression of TH decreased significantly after 6 and 8 weeks of PQ exposure (P<0.05), and the relative expression of oligomer α-syn increased after 4, 6, and 8 weeks of PQ exposure (P<0.05). The WB of in vitro models results showed that the relative expression of α-syn in cells increased with time (R2=0.7440, P<0.05); the relative expression of α-syn in cytoplasm increased firstly and then decreased with time (P<0.05); the relative expression of α-syn in nucleus increased with time (R2=0.7913, P<0.05). The IFA results of in vitro models showed that the expression of oligomerized α-syn increased and translocated to the nucleus (P<0.05). The ELISA results of in vitro models showed that α-syn increased with the increase of PQ exposure time (P<0.05). Conclusion PQ can increase the expression of α-syn in dopaminergic neurons, induce oligomerization and translocation to the nucleus.

Dexmedetomidine attenuates neuroinflammation by inhibiting NF-κB nuclear translocation in rats with traumatic brain injury / 中华急诊医学杂志

Objective:To explore the mechanism of dexmedetomidine (DEX) regulating microglial (MG) polarization and neuroinflammation after traumatic brain injury (TBI) in rats.Methods:Forty-two adult male SD rats were randomly (random number) divided into the sham group, TBI group, TBI+DEX group (further divided into 1 d, 3 d and 7 d subgroups), TBI+NF-κB inhibitor (pyrrolidine dithiocarbamate, PDTC) group and TBI+DEX+PDTC group, with 6 animals in each group. The rat TBI model was established according to the modified Feeney free fall method. PDTC was intraperitoneally injected 1 h after modeling with a dose of 100 mg/kg, and DEX was intraperitoneally injected 2 h after modeling with a dose of 100 μg/kg. Modified neurological severity score (mNSS) was used to evaluate rat neurological function, ELISA was used to detect serum inflammatory factors, and rats’ damaged cortex was collected to detect the phenotype markers of MG and protein expressions of MyD88 and NF-κB p65, and immunofluorescence staining was used to observe the expression and nuclear entry of NF-κB p65 in MG in injured cortex. One-way and two-way ANOVA were used to compare the measurement data among multiple groups.Results:Compared with the sham group, the mNSS score was significantly higher in the TBI group, and DEX treatment significantly decreased the mNSS score of TBI rats ( P<0.05). ELISA and Western blot results showed that in the TBI group, the tumor necrosis factor-α (TNF-α), interleukin (IL)-1β in serum and M1 phenotype marker (TNF-α, IL-1β) in brain were increased, the expression of anti-inflammatory factor IL-10 in serum and M2 phenotype markers (arginase-1 and IL-10) in brain were decreased ( P<0.05), and DEX downregulated the expression of TNF-α, IL-1β in serum and M1 phenotype markers in brain, while upregulated the level of L-10 in serum and the M2 phenotype marker in brain ( P<0.05). In addition, the expression of MyD88 and the nuclear translocation of NF-κB p65 were inhibited in the DEX group, and this effect could be enhanced by PDTC. Conclusions:DEX modulates MG activation in TBI rats by inhibiting NF-κB nuclear translocation and reduces neuroinflammation.

Nuclear cGAS: sequestration and beyond

The cyclic GMP-AMP (cGAMP) synthase (cGAS) has been identified as a cytosolic double stranded DNA sensor that plays a pivotal role in the type I interferon and inflammation responses via the STING-dependent signaling pathway. In the past several years, a growing body of evidence has revealed that cGAS is also localized in the nucleus where it is associated with distinct nuclear substructures such as nucleosomes, DNA replication forks, the double-stranded breaks, and centromeres, suggesting that cGAS may have other functions in addition to its role in DNA sensing. However, while the innate immune function of cGAS is well established, the non-canonical nuclear function of cGAS remains poorly understood. Here, we review our current understanding of the complex nature of nuclear cGAS and point to open questions on the novel roles and the mechanisms of action of this protein as a key regulator of cell nuclear function, beyond its well-established role in dsDNA sensing and innate immune response.

Integrating network pharmacology and pharmacological evaluation for deciphering the mechanism of (-)-epigallocatechin-3-gallate alleviating ethanol-inducedendothelial cells injury / 中国药理学与毒理学杂志

Objective To investigate the potential therapeutic targets and pharmacological mechanism of (-)-epigal?locatechin-3-gallate (EGCG) based on network pharmacology and experimental verification. METHODS The druggability of EGCG was measured by the traditional Chinese medicine systems pharmacology (TCMSP) server, and potential tar?gets of EGCG were identified by Pharm Mapper and Drug Repositioning and Adverse drug Reaction via Chemical-Pro?tein Interactome (DRAR-CPI). The potential targets were imported into GeneMANIA database to obtain the protein-pro?tein direct interaction network, and target physical interaction, co-expression, prediction, genetic interaction, and shared protein domains. The biological process, molecular functions, cellular components and KEGG signaling pathways of potential targets were analyzed using DAVID database. For further study, ethanol was used to establish a model of endothelial injury in vitro. The cell viability was assayed by MTT method, the cellular apoptosis was stained by Annexin V/PI, and the expression levels of Bcl-2, Bax and cleved-caspase-3 were tested by Western blotting. Then, JC-1 and nuclear translocation of NF-κB experiments were used to study the mitochondrial membrane potential and nuclear trans?location. RESULTS The oral availability of EGCG was 55.09％ (≥ 30％) and drug-like index was 0.77 (≥ 0.18), which were considered pharmacokinetically active. 17 potential targetable proteins of EGCG were predicted by Pharm Mapper and DRAR-CPI. Further research showed that 68.13％ displayed similar co-expression characteristics, 26.11％ physical interactions, and 2.74％ shared the same protein domain. The depth network analysis results showed that the biofunc?tions of EGCG were mainly by regulating glutathione derivative biosynthetic process, glutathione metabolic process, nitrogen compound metabolic process etc.. via drug binding, catalytic activity, glutathione transferase activity, anion bind?ing etc.. in sarcoplasmic reticulum, spindle pole, microtubule cytoskeleton and cytoplasm. KEGG enrichment analysis showed that Glutathione metabolism, IL-17 signaling pathway, EGFR tyrosine kinase inhibitor resistance, PI3K-Akt sig?naling pathway and other pathways were involves in the biofunction of EGCG. The above analyses indicated that EGCG exerts its biofunction through antioxidant and anti-inflammatory mechanisms. The experimental results showed that etha?nol 20.0 mmol·L-1 decreased cell viability, Bcl-2 expression, and increased cell apoptosis, the intracellular ROS, as well as the expression of Bax and cleaved-caspase-3 of human endothelial cells. However, treatment of the cells with EGCG can significantly alleviate ethanol induced endothelial cells injury. Further study showed that EGCG significantly allevi?ates ethanol induced mitochondrial depolarization and nuclear translocation of NF-κB. CONCLUSIONS EGCG exerts pharmacological efficacies on ethanol induced endothelial cell injury through multi-target, multi-function and multi-path?way mode. Protective effect of EGCG on ethanol induced cell injury was mainly through alteration of mitochondrial func?tion and NF-κB translocation. Therefore, EGCG have great potential in protecting against endothelial dysfunction of the persons who are chronically abuse of ethanol. This study also provides a new understanding of EGCG in clinical applica?tion on cardiovascular and cerebrovascular diseases.

Activated PKB/GSK-3β synergizes with PKC-δ signaling in attenuating myocardial ischemia/reperfusion injury via potentiation of NRF2 activity: Therapeutic efficacy of dihydrotanshinone-I

@#Disrupted redox status primarily contributes to myocardial ischemia/reperfusion injury (MIRI). NRF2, the endogenous antioxidant regulator, might provide therapeutic benefits. Dihydrotanshinone-I (DT) is an active component in Salvia miltiorrhiza with NRF2 induction potency. This study seeks to validate functional links between NRF2 and cardioprotection of DT and to investigate the molecular mechanism particularly emphasizing on NRF2 cytoplasmic/nuclear translocation. DT potently induced NRF2 nuclear accumulation, ameliorating post-reperfusion injuries via redox alterations. Abrogated cardioprotection in NRF2-deficient mice and cardiomyocytes strongly supports NRF2-dependent cardioprotection of DT. Mechanistically, DT phosphorylated NRF2 at Ser40, rendering its nuclear-import by dissociating from KEAP1 and inhibiting degradation. Importantly, we identified PKC-δ-(Thr505) phosphorylation as primary upstream event triggering NRF2-(Ser40) phosphorylation. Knockdown of PKC-δ dramatically retained NRF2 in cytoplasm, convincing its pivotal role in mediating NRF2 nuclear-import. NRF2 activity was further enhanced by activated PKB/GSK-3β signaling via nuclear-export signal blockage independent of PKC-δ activation. By demonstrating independent modulation of PKC-δ and PKB/GSK-3β/Fyn signaling, we highlight the ability of DT to exploit both nuclear import and export regulation of NRF2 in treating reperfusion injury harboring redox homeostasis alterations. Coactivation of PKC and PKB phenocopied cardioprotection of DT in vitro and in vivo, further supporting the potential applicability of this rationale. Graphical abstract

Intervention Effect of Shenfu Injection on the Nuclear Translocation of HMGB 1 in LPS-induced RAW 264.7 Cells / 中国药房

OBJECTIVE：To investigate the intervention effect of Shenfu i njection（SFI）on the nuclear translocation of high mobility group box 1（HMGB1） in lipopolysaccharide （LPS）-induced RAW 264.7 cells. METHODS ： Using LPS-induced RAW264.7 cells as objects ，the histone deacetylase inhibitor RGFP 966 as positive control ，CCK-8 assay was used to screen drug dosage，and the effects of low ，medium and high doses （3，6，12 μL/mL）of SFI on HMGB 1 nuclear translocation in RAW 264.7 cells were observed by immunofluorescence method ；mRNA expression of HMGB 1 in RAW 264.7 cells were detected by real time fluorescent PCR. Western blotting assay was used to determine protein expression of HMGB 1 and Toll-like receptor 4（TLR4）；the expression of HMGB 1 were compared between nucleus and cytoplasm. The levels of HMGB 1，IL-1β and TNF-α in supernatant of cells were detected by ELISA. RESULTS ：In blank control group ，HMGB1 was mainly located in the nucleus ；after LPS induction， HMGB1 migrated from nucleus to cytoplasm. Compared with blank control group ， mRNA and protein （No.81760738） expression of HMGB 1， protein expression of TLR 4 in RAW264.7 cells as well as the levels of HMGB 1，IL-1β and TNF-α in supernatant of cells were increased significantly in LPS group （P＜0.01）. The protein expression of HMGB 1 was decreased significantly in nucleus while was in creased significantly in cytoplasm （P＜0.01）. After SFI treatment ，the nuclear translocation and secretion of HMGB 1 were inhibited in different degrees ；compared with LPS group ，mRNA and protein expression of HMGB 1 in administration groups ，protein expression of TLR 4 in RAW 264.7 cells of positive control group ，SFI medium- and high-dose groups as well as the levels of HMGB 1，IL-1β and TNF-α in supernatant of cells in administration groups were decreased significantly （P＜0.01）. In positive control group ，SFI medium- and high-dose groups ，the protein expressions of HMGB1 in nucleus were increased significantly ，while protein expressions of HMGB 1 in cytoplasm were decreased significantly （P＜0.01）. CONCLUSIONS ：SFI may inhibit the nuclear translocation and secretion of HMGB 1 in RAW 264.7 cells，thus avoiding the activation of inflammatory pathways and the production of inflammatory factors ，so as to reduce the inflammatory response induced by LPS.

Effect of PARP1 through regulating NFATs on cardiac hypertrophy / 中国药理学通报

Aim To investigate the interaction and mechanism of PARP1 and NFATc3, NFATc4 in ISO-induced pathological cardiac hypertrophy. Methods To establish the model of cardiac hypertrophy in vitro and in vivo, primary neonatal rat cardiomyocytes were treated with ISO (10 jimol • L-1) for 24 h; SD rats were subcutaneously injected with 1. 2 mg • kg-1 • d-1 ISO for 7 d. The nuclear and cytoplasmic proteins were separated by Cellytic Nuclear Extraction Kit. The subcellular localization of NFATc3 and NAFTc4 were detected by Western blot and immunofluorescence. The recombinant adenovirus (Ad-PARPl) infection was used to overexpress PARP1 and knockdown PARP1 by transfecting with siRNA of PARP1 in cardiomyocytes. Results The models of cardiac hypertrophy were successfully built both in vivo and vitro by ISO. It was determined that NFATc3 and NFATc4 were transfered into the nuclear from the cytoplasm in primary neonatal rat cardiomyocytes (NRCMs) after being treated with ISO. And the enzymatic activity of PARP1 was boosted in TSO-trpatpH prmin. OvprpYnrp.ssinn of PARP1 nromo-ted the nuclear translocation of NFATc3 and NFATc4 in cardiomyocytes, while knockdown of PARP1 could reverse the nuclear translocation induced by ISO. PARP1 inhibitor 3AB retarded ISO-induced nuclear transportation of NFATc3 and NFATc4 to some extent. Conclusions ISO leads to the up-regulation of enzymatic activity of PARP1 and promotes nuclear translocation of NFATc3 and NFATc4, thus aggravating car-diac hypertrophy.

Study on the effects of total flavonoids from litchi nucleus on nuclear translocation of nuclear factor-kappa B and related protein expression in rat hepatic stellate cell / 中华肝脏病杂志

Objective@#The effect of total flavonoids of litchi (TFL) on nuclear translocation of nuclear factor-kappa B (NF- kappa B) in rat hepatic stellate cell line (HSC-T6) induced by transforming growth factor - beta 1 (TGF- beta 1) in vitro was studied to explore the mechanism of action of anti-hepatic fibrosis drugs.@*Methods@#HSC-T6 was cultured in vitro, induced by TGFβ1 for 24 h, and then treated with TFL at 125, 250 and 500 μg/ml for 48 h. The effect of TFL on NF-κB nuclear translocation in HSC-T6 was observed by confocal laser microscopy. The effects of TFL on the expression of TLR4, p-IκB ɑ, p-NF-κB p65, NF-κB and Collagen I protein were detected by western blot. The expressions of TLR4 and p-NF-κB p65 were detected by immunofluorescence. Data were presented as mean±SEM. Homogeneity test of variance was performed and then followed by one-way analysis of variance (ANOVA). The multiple comparisons between groups were performed by LSD test. P < 0.05 was considered statistically significant.@*Results@#Confocal laser scanning microscopy showed TFL inhibited the nuclear translocation of NF-κB in activated HSC-T6 in a concentration-dependent manner and TFL down regulated the protein expression levels of TLR4, p-IκB ɑ, p-NF-κB p65, NF-κB and collagen I protein in HSC-T6 in a concentration-dependent manner.@*Conclusion@#The mechanism of TFL against hepatic fibrosis may be related to the inhibition of nuclear translocation of NF-κb in the activated HSC-T6 and the expression of TLR4, P-iκbɑ, P-nf-κb p65, NF-κb and collagen I protein in HSC-T6.

Research progress on the role of epidermal growth factor receptor in human head and neck squamous cell carcinomas / 中国肿瘤临床

Head and neck squamous cell carcinoma(HNSCC)is a common malignant tumor with a relatively high degree of malignan-cy,which often induces recurrence and metastasis.Epidermal growth factor receptor(EGFR)is an important oncogene that is overex-pressed in HNSCC and is negatively correlated with prognosis,making it an important therapeutic target.However,EGFR-targeted ther-apy for HNSCC is not as effective as it is for non-small cell lung cancer.Recent studies have found that EGFR can promote resistance of tumor cells to therapeutic agents via its overexpression,mutation,single nucleotide polymorphism,nuclear translocation,and induc-tion of autophagy.This article will review these aspects and discuss how to utilize EGFR more effectively in treating HNSCC,and also provide a new direction for exploring therapeutic strategies for HNSCC.

Effects of PKC activation on expression of Nrf2 in rabbit RPE cells / 眼科新进展

Objective To investigate whether activation of protein kmase C (PKC) can induce the activation and nuclear translocation of nuclear factor enthroid 2-related factor 2 (Nrf2) in retinal pigment epithelial (RPE) cells in vitro,and explore whether PKC activation may affect the expression of Nrf2 in RPE cells.Methods PKC-specific activator phorbol ester PMA was used to culture rabbit RPE cells and RPE cells pretreated with Nrf2 inhibitor for 24 hours.Immunofluorescence and Western blot were used to detect Nrf2 in the nucleus of the expression of the situation,the data were obtained for statistical analysis.Results The expression of Nrf2 protein in the nucleus of PRE cells was detected by immunofluorescence.Compared with the control group,the expression of Nrf2 protein in the nucleus of RPE cells increased in the experimental group,and the increase of PMA + Nrf2 inhibitor group was lower than that of PMA group.The difference between the two groups was statistically significant (P ＜0.05).Western blot analysis showed that the Nrf2 protein in the nucleus of PRE was quantitatively analyzed by image analysis.The gray value of the control group was significantly different (0.286 ± 0.013 in the control group,1.304 ± 0.033 in the PMA group and 0.671 ± 0.087 in the PMA + Nrf2 inhibitor group,P ＜ 0.05).Conclusion PKC can activate nuclear translocation of Nrf2 in rabbit RPE cytoplasm,and Nrf2 inhibitor can attenuate the effect of PKC.

Summary and prospect of medicinal plant active substances in regulation of p65 nuclear translocation / 中国中药杂志

As the most important nuclear transcription factors in the cells, NF-κB is involved in many intracellular signaling pathways and transcription and regulation of genetic information. The signal transduction pathways mainly include the activation of IκB kinase, degradation of IκB protein and the nuclear translocation of p65. p65 trans-nuclear binding with DNA is the key for NF-κB to play a role. Abnormal activation of NF-κB is a major factor in the induction of oxidative stress, inflammation, cancer and so on. Therefore, maintaining the balance of NF-κB activity and regulating the nuclear translocation of p65 have great significance for further research on related subjects. In this paper, the regulation effects of the main active substances of medicinal plants (such as polyphenols, saponins, and alkaloids) on p65 nuclear translocation and the upstream pathway of NF-κB were discussed, expecting to provide reference for the development of natural active substances for functional food.

The effect of miRNA-7 on chemoresistance in esophageal cancer cell TE-1 / 天津医药

Objective To explore the impacts of over-expression of microRNA-7 (miRNA-7) on the sensitivity of cis-platin in esophageal carcinoma cell line TE-1, and the possible mechanism thereof. Methods Lipofectmin 2000 method was used to transient transfect with miRNA-7 mimic into esophageal cancer cell line TE-1, which was taken as transfection group, mimic negative control was taken as transfection conrtol group. The expressions of miRNA-7 and epidermal growth factor receptor (EGFR) mRNA were detected by RT-PCR in the above two groups and normal control group. The total EGFR and EGFR in cytoplasmic and nucleus were detected with Western blot assay in transfection group and transfection control group. CCK-8 was used to detect IC50 of cisplatin in transfection group and transfection control group. The expression of EGFR was observed with immunofluorescence confocal microscope in two groups. Results The miRNA-7 expression was signifi-cantly increased in transfection group than that of transfection conrtol group and control group. The expression of EGFR mRNA was significantly reduced in transfection group (P<0.001). The total EGFR was significantly decreased in transfec-tion group than that of transfection conrtol group. The level of nuclear EGFR was significantly increased ( P<0.01),and cyto-plasm EGFR expression was significantly decreased in transfection group than that of transfection control group ( P<0.05). CCK-8 results showed that after the over expression of miRNA-7 in TE-1, the IC50 of cisplatin (48 h) increased in transfec-tion group than that of control group (P<0.01). Immunofluorescence results showed that EGFG in nuclear was higher in transfection group than that of transfection control group but its expressions reduced in cell membrane and cytoplasm. Con-clusion The over-expressed miRNA-7 in esophageal cancer cells TE-1 can reduce cisplatin sensitivity by the increased EGFR in nuclear translocation.

Wikstroelide M potently inhibits HIV replication by targeting reverse transcriptase and integrase nuclear translocation / 中国天然药物

AIM@#To evaluate the anti-HIV activity and mechanism of action of wikstroelide M, a daphnane diterpene from Daphne acutiloba Rehder (Thymelaeaceae).@*METHODS@#The anti-HIV activities of wikstroelide M against different HIV strains were evaluated by cytopathic effect assay and p24 quantification assay with ELISA. The inhibitory effect of wikstroelide M on HIV reverse transcription was analyzed by real-time PCR and ELISA. The effect of wikstroelide M on HIV-1 integrase nuclear translocation was observed with a cell-based imaging assay. The effect of wikstroelide M on LEDGF/p75-IN interaction was assayed by molecular docking.@*RESULTS@#Wikstroelide M potently inhibited different HIV-1 strains, including HIV-1IIIB, HIV-1A17, and HIV-19495, induced a cytopathic effect, with EC50 values ranging from 3.81 to 15.65 ng·mL⁻¹. Wikstroelide M also had high inhibitory activities against HIV-2ROD and HIV-2CBL-20-induced cytopathic effects with EC50 values of 18.88 and 31.90 ng·mL⁻¹. The inhibitory activities of wikstroelide M on the three HIV-1 strains were further confirmed by p24 quantification assay, with EC50 values ranging from 15.16 to 35.57 ng·mL⁻¹. Wikstroelide M also potently inhibited HIV-1IIIB induced cytolysis in MT-4 cells, with an EC50 value of 9.60 ng·mL⁻¹. The mechanistic assay showed that wikstroelide M targeted HIV-1 reverse transcriptase and nuclear translocation of integrase through disrupting the interaction between integrase and LEDGF/p75.@*CONCLUSION@#Wikstroelide M may be a potent HIV-1 and HIV-2 inhibitor, the mechanisms of action may include inhibition of reverse trascriptase activity and inhibition of integrase nuclear translocation through disrupting the interaction between integrase and LEDGF/p75.

Verification of Differential Expression Genes after CacyBP/SIP Nuclear Translocation in Colon Carcinoma Cell Line / 天津医药

Objective To verify the genes screened by the polymerase chain reaction (PCR) chip of cell cycle. Methods The colon cancer cells SW480 were randomized into two groups, the test group (with gastrin stimulation) and con-trol group (without gastrin stimulation). The method of Western blot was used to detect the expression of calcylin binding pro-tein/Siah-1 interacting protein (Cacybp/SIP) before and after gastrin stimulation. The differential expression genes, cyclin de-pendent kinase 8 (CDK8) and cyclin dependent kinase subunit (CKS2), were verified by using real-time quantitative PCR (qRT-PCR). Results It was found that before the stimulation, CacyBP/SIP was located and expressed in cytoplasm, and then in both cytoplasm and nucleus after gastrin stimulation. The qRT-PCR results of CDK8 and CKS2 genes were consis-tent with those of microarray detection. The expressions of CDK8 and CKS2 were up-regulated (P < 0.05). Conclusion The stimulation of human gastrin can lead to the nuclear translocation of CacyBP/SIP. The results of microarray are reliable, and the differentially expressed genes screened through gene chip deserve further study.

Promotion of ovarian tumorigenesis by cancer-associated ifbroblasts through Gro-αactivated NF-кB nuclear translocation and high expression of VEGF / 中国癌症杂志

Background and purpose:Ovarian cancer-associated ifbroblasts (CAF) are known to promote epithelial malignancy. The chemoattractant cytokine growth-regulated oncogene alpha (Gro-α) secreted from CAF has been reported to mediate the stroma-epithelia interaction in tumor microenvironment, leading to the development of epithelial ovarian cancer, however, the detailed mechanism is unknown.This study was to determine whether Gro-αcould promote ovarian tumorigenesis through activating NF-кB nuclear translocation and VEGF expression in stromal ifbroblasts. Methods:ELISA was used to measure the levels of Gro-αin two cancer-associated ifbroblasts (CAF) and normal ifbroblasts (NF) isolated from high-grade serous ovarian cancer or normal ovarian tissues. CAF conditioned medium (CM) or Gro-αwas used to treat NF, while PS1145, the inhibitor of NF-кB, was used as control. NF-кB subunit p65 and vascular endothelial growth factor (VEGF) were detected by Western blot in cells after treatment. Xenograft tumors from nude mice were generated by injection of CAF, NF, or OVCA429 alone or OVCA429 mixed with CAF or NF, and by injection of OVCA429 mixed with NF cells that were treated with or without CAF-CM or Gro-α, or with NF cells that were treated with CAF-CM or Gro-αplus PS1145. The tumor growth curve was measured and the blood vessel density in xenograft tumor tissues was examined by histopathological analysis. Results:The levels of Gro-αwere 5-6 folds higher in CAF than in NF. Treatment of NF with CAF-CM or Gro-αstimulated the nuclear translocation of NF-кB subunit p65, and the expression of VEGF, but suppressed the expression of thrombospondin 1, the anti-angiogenesis factor, compared with control cells. However, treatment of NF with the NF-кB inhibitor PS1145 reversed these results. The animal assay revealed that CAF stimulated tumor growth stronger than NF, and NF treated with CAF-CM or Gro-α, but not along with PS1145, enhanced xenograft tumor growth through promoting angiogenesis. Conclusion:Ovarian CAF promotes the nuclear translocation of NF-кB and the expression of VEGF through Gro-αautocrine in tumor microenvironment to facilitate angiogenesis and ovarian cancer development.

A Role for Timely Nuclear Translocation of Clock Repressor Proteins in Setting Circadian Clock Speed

By means of a circadian clock system, all the living organisms on earth including human beings can anticipate the environmental rhythmic changes such as light/dark and warm/cold periods in a daily as well as in a yearly manner. Anticipating such environmental changes provide organisms with survival benefits via manifesting behavior and physiology at an advantageous time of the day and year. Cell-autonomous circadian oscillators, governed by transcriptional feedback loop composed of positive and negative elements, are organized into a hierarchical system throughout the organisms and generate an oscillatory expression of a clock gene by itself as well as clock controlled genes (ccgs) with a 24 hr periodicity. In the feedback loop, hetero-dimeric transcription factor complex induces the expression of negative regulatory proteins, which in turn represses the activity of transcription factors to inhibit their own transcription. Thus, for robust oscillatory rhythms of the expression of clock genes as well as ccgs, the precise control of subcellular localization and/or timely translocation of core clock protein are crucial. Here, we discuss how sub-cellular localization and nuclear translocation are controlled in a time-specific manner focusing on the negative regulatory clock proteins.

Effect of nuclear translocation of E2p45 related factor 2 on the biological activity of melanocytes / 中华皮肤科杂志

Objective To investigate the effect of nuclear translocation of E2p45 related factor 2 (Nrf2)on the biological activity of melanocytes.Methods Plasmid vectors containing wild-type nrf2 gene (pcDNA-nrf2) and nls-deleted nrf2 gene (pcDNA-nrf2△nls) were constructed.B10BR normal murine melanocytes were classified into three groups,i.e.,untransfected group,wild-type nrf2 group transfected with pcDNA-nrf2,and mutated nrf2 group transfected with pcDNA-nrf2△nls.Each of the above groups were further divided into three subgroups:control subgroup receiving no treatment,hydrogen peroxide (H2O2) subgroup treated with H2O2 of 200 μmol/L for 24 hours,and combined subgroup pretreated with tert-butyl hydroquinone (TBHQ) followed by treatment with H2O2 of 200 μmol/L for 24 hours.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of cells,dopa oxidation assay to determine tyrosinase activity,Transwell assay to estimate cell migration ability,Western blot to quantify the expressions of Nrf2 and his tag fusion protein.Results TBHQ significantly enhanced the nuclear expression of Nrf2 in B10BR cells transfected with pcDNA-nrf2 or pcDNA-nrf2△nls (both P ＜ 0.01).No significant difference was observed in tyrosinase activity between untreated wild-type nrf2 group,mutated nrf2 group,and untransfected group (P ＞ 0.05).There was a statistical decrease in tyrosinase activity in the two H2O2-treated transfected groups compared with the untreated transfected groups (both P ＜ 0.05),and the decrease was reversed by TBHQ pretreatment in the wildtype nrf2 group (P ＜ 0.05),but not in the mutated nrf2 group (P ＞ 0.05).Further more,the proliferative activity of B10BR cells experienced no obvious changes in the wild-type nrf2 group (P ＞ 0.05),but was significantly reduced in the untransfected group (P ＜ 0.05) and mutated nrf2 group (P ＜ 0.01) after the H2O2 treatment compared with the corresponding untreated groups.TBHQ could protect the pcDNA-nrf2-transfected B10BR cells,but not pcDNA-nrf2△nls-transfected B10BR cells,from H2O2-induced oxidative damage.Transwell assay showed no significant difference in migration ability among these nine groups (P ＞ 0.05).Conclusions Abnormal nuclear translocation of Nrf2 could affect antioxidant activity of,proliferative activity of and tyrosinase activity in melanocytes.TBHQ may enhance the tyrosinase activity in,proliferative activity and antioxidant activity of melanocytes via activating the nuclear expression of wild type Nrf2.

Effects of propofol on the cell apoptosis and NF-κB p65 in the acute lung injury induced by LPS in rats / 中华急诊医学杂志

Objective To observe the effect of intravenous injection of different doses of propofol on the ceil apoptosis and NF-kB p65 in the acute lung injury(ALl)induced by LPS in rats.Method Sixty SD rats were randomly(random number)divided into five groups,namely,control(NS)group,Au model group and propofol intervention groups(P1,P2,P3 groups).The lung injury was evaluated by using microscopy with hematoxylin and eosin(HE)staining and arterial blood gas,and Western blotting Was applied to evaluating the nuclear translocation of NF-KB P65 in lung tissues.The apoptosis rate of lung tissue Was determined by flow cytometric analysis.Results Lung injury in model group reached the pathologic criteria of acute lung injury,and it was attenuated apparently in propofol intervention groups(P1,P2,P3 groups)in dose-dependent manner.Western blotting results showed that the nuclear translocation of NF-KB P65 and the apoptosis rate increased significantly in ALI model group compared with control group(P<0.05),and decreased in propofol intervention groups compared with ALl model group(P<0.05).Conclusions Propofol Can attenuate acute lung injury induced by LPS in rats,and significantly inhibit the nuclear translocation of NF-KB P65 and the cell apoptosis in lung tissues.The effect of propofol attenuating acute lung injury induced by LPS in rats may be attributed to the inhibition of nuclear translocation of NF-KB P65and ceil apoptosis in lung tissues.

TLR4 mediated the nuclear translocation of transcription factor NF-κB and IRF-3 in HTNV infected vascular endothelial cells / 中华微生物学和免疫学杂志

Objective To observe the nuclear translocation of transcription factor NF-κB and IRF-3 in TLR4 silenced EVC304 cells infected by HTNV and to provide new information for anti-HTNV innate immunity and its signal transduction. Methods TLR4~- cells and TLR4~+ cells were infected by HTNV 76-118, respectively. The cells stimulated by LPS were selected as positive control groups, and the cells without stimulation were selected as negative control groups. After 6 hours, indirect immunofluorescence assay(IFA) was used to detect the nuclear translocation of NF-κB and IRF-3. Results The transcription factor NF-κB and IRF-3 transfered into nuclear 6 hours after stimulated by HTNV 76-118. Conclusion TLR4 may mediate the nuclear translocation of transcription factor NF-κB and IRF-3 in HTNV infected human umbilical vein endothelial cells.