Mechanistic insights into the amelioration effects of lipopolysaccharide-induced acute lung injury by baicalein: An integrated systems pharmacology study and experimental validation.

BACKGROUND: Acute lung injury is an acute progressive respiratory failure caused by several of non-cardiogenic factors which involves in excessive amplification or uncontrolled inflammatory response. OBJECTIVES: In this study, we investigated the protective effect of baicalein against acute lung injury induced by LPS and explored the underlying mechanisms. METHODS: Forty-eight SPF male C57BL/6 mice were randomly divided into normal group, model group, dexamethasone group and baicalein low-dose, medium-dose and high-dose groups. After 5 days of adaptive feeding, the mice were intraperitoneally injected with LPS and dissected after 12 h. Hematoxylin-eosin staining, ELISA assay, immunofluorescence assay and Western-Blot were applied to appraise microstructural changes and protein expressions of lung tissues. Systems pharmacology study was used to evaluate the protection of baicalein on acute lung injury. FINDINGS: The results showed that baicalein administration could significantly inhibit LPS-induced lung morphological changes, inhibit inflammatory response and pyroptosis. A total of forty-three potential targets of baicalein and acute lung injury were obtained. And PI3K-Akt, TNF and NF-κB were mainly signaling pathways. It is worth mentioning that this experiment also confirmed that NLRP3, caspase-1 and other inflammasome are involved in pyroptosis. CONCLUSION: Baicalein has protected against LPS-induced lung tissues injury via inhibiting inflammatory response and pyroptosis.

Emerging Benefits: Pathophysiological Functions and Target Drugs of the Sigma-1 Receptor in Neurodegenerative Diseases.

The sigma-1 receptor (Sig-1R) is encoded by the SIGMAR1 gene and is a nonopioid transmembrane receptor located in the mitochondrial-associated endoplasmic reticulum membrane (MAM). It helps to locate endoplasmic reticulum calcium channels, regulates calcium homeostasis, and acts as a molecular chaperone to control cell fate and participate in signal transduction. It plays an important role in protecting neurons through a variety of signaling pathways and participates in the regulation of cognition and motor behavior closely related to neurodegenerative diseases. Based on its neuroprotective effects, Sig-1R has now become a breakthrough target for alleviating Alzheimer's disease and other neurodegenerative diseases. This article reviews the most cutting-edge research on the function of Sig-1R under normal or pathologic conditions and target drugs of the sigma-1 receptor in neurodegenerative diseases.

The Effects of Lysimachia christinae Hance Extract Fractions on Endothelium-Dependent Vasodilatation.

BACKGROUND: Endothelium-dependent dilatation is a predictor for vascular function. NADPH oxidase-derived O2- can inactivate nitric oxide and induce vascular injury. METHOD: The crude ethanolic extract of Lysimachia christinae Hance were separated out 4 fractions of different olarities by petroleum ether, ethyl acetate, n-butanol (NB), and aqueous. The endothelial integrity was appraised by vascular tension measurement. Dihydroethidium was utilized to observe the vascular reactive oxygen species (ROS) production. Western-blot was adopted to detect protein expression. RESULTS: Among the 4 fractions of L. christinae Hance, the NB fraction showed the most potent capacity of promoting endothelium-dependent vascular relaxation and inhibiting ROS formation in aortic rings, which were likely attributed by suppressing the expression of NAD(P)H oxidase subunit (gp91phox, p47phox, and p67phox) and enhancing the phosphorylation of endothelial NOS in vascular tone. CONCLUSIONS: These results suggest that the NB fraction possess the strongest vascular pharmacological activities among the crude ethanolic extract of L. christinae Hance, which may help us for purifying bioactive constituents and discovering new drugs from this herb in future.

Evaluation of the antioxidant and endothelial protective effects of Lysimachia christinae Hance (Jin Qian Cao) extract fractions.

BACKGROUND: Lysimachia christinae Hance is a traditional Chinese medicine with diuretic, detumescent, and detoxifying effects. Our aimed to optimize the extraction protocol to maximize the yield of flavonoids from Lysimachia christinae Hance, and evaluate the pharmacological activities of four fractions, namely, petroleum ether (PE), ethyl acetate (EA), n-butanol (NB), and aqueous (AQ) fractions, of the ethanolic extract of Lysimachia christinae Hance. METHODS: The flavonoid monomers in the crude extract were characterized via high performance liquid chromatography (HPLC), were used as markers for extract quality control and standardization. The total flavonoid, total phenolic, and total polysaccharide contents of each fraction were determined by spectrophotometry. Further, the in vitro free radical (diphenylpicrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), superoxide, and hydroxyl radicals) scavenging activities, and antioxidant capacity in endothelial cells were evaluated for each fraction. RESULTS: After optimizing the extraction protocol to maximize the total flavonoid yield from L. christinae Hance, the NB fractions had the highest total flavonoid (39.4 ± 4.55 mg RE/g), total phenolic (41.1 ± 3.07 mg GAE/g) and total polysaccharide (168.1 ± 7.07 mg GE/g); In addition, the NB fraction of the ethanolic extract of L. christinae Hance reveal the strongest radical-scavenging activity, antioxidant activity and protective effects against H2O2-induced injury in HUVECs. CONCLUSIONS: Among the four fractions of L. christinae Hance, the NB fraction showed the most potent antioxidant and endothelial protective effects, which may be attributed to its high flavonoid, phenolic contents and optimal portfolio of different active ingredients of NB fractions of the ethanolic extract of L. christinae Hance. This study might improve our understanding of the pharmacological activities of L. christinae Hance, thereby facilitating its use in disease prevention and treatment.

A switch from hBrm to Brg1 at IFNγ-activated sequences mediates the activation of human genes.

The SWI/SNF chromatin-remodeling complexes utilize energy from ATP hydrolysis to reposition nucleosomes and regulate the expression of human genes. Here, we studied the roles of human Brahma (hBrm) and Brahma-related gene 1 (Brg1), the ATPase subunits of the SWI/SNF complexes, in regulating human genes. Our results indicate that both hBrm and Brg1 interact with Signal transducer and activator of transcription (Stat) 1 in vitro. However, Stat1 in its native form only recruits hBrm to IFNγ-activated sequences (GAS) of individual genes; by contrast, in a stress-induced phosphorylated form, Stat1 mainly binds to Brg1. Under basal conditions, hBrm is recruited by native Stat1 to the GAS and exists in a mSin3/HDAC co-repressor complex on the hsp90α gene, which shows a compact chromatin structure. Upon heat-shock, hBrm is acetylated by p300 and dissociates from the co-repressor complex, which the phosphorylated Stat1 is increased, and binds and recruits Brg1 to the GAS, leading to elevated induction of the gene. This hBrm/Brg1 switch also occurs at the GAS of all of the three examined immune genes in heat-shocked cells; however, this switch only occurs in specific cell types upon exposure to IFNγ. Regardless of the stimulus, the hBrm/Brg1 switch at the GAS elicits an increase in gene activity. Our data are consistent with the hypothesis that the hBrm/Brg1 switch is an indicator of the responsiveness of a gene to heat-shock or IFNγ stimulation and may represent an "on-off switch" of gene expression in vivo.

Role of acetylated p53 in regulating the expression of map2 in retinoic acid-induced P19 cells.

OBJECTIVE: To investigate the regulatory mechanisms of acetylated p53 in the expression of microtubule-associated protein-2 (MAP2) in neuronal differentiation of P19 cells induced by all-trans retinoic acid (RA). METHODS: Neuronal differentiation of P19 cells was initiated with 4-day RA treatment. Immunofluorescence, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, and map2 promoter driven luciferase assay were performed to detect the expression and relative promoter activity of MAP2 in those RA-treated cells. Real-time PCR-based chromatin immunoprecipitation assay (ChIP) was carried out to reveal the specific recruitment of acetylated p53 onto its binding sites on map2 promoter. RESULTS: The expression of MAP2 was markedly increased in RA-induced P19 cells. The map2 mRNA increased 34-fold after 4 days of RA treatment and 730-fold 2 days after the treatment, compared with the cells without RA treatment (control). p53 was recruited to the promoter of map2 gene in acetylated form and thereby enhanced its promoter activity. p300/CBP associated factor (PCAF) was found induced in RA-treated cells and enriched in the nucleus, which might contribute to the acetylation of p53 in the regulation of map2 gene. CONCLUSIONS: Acetylated p53 may participate in regulating the expression of map2 in RA-induced differentiation of P19 cells. PCAF is possibly involved in this process by mediating the acetylation of p53.

CARM1 activates myogenin gene via PCAF in the early differentiation of TPA-induced rhabdomyosarcoma-derived cells.

CARM1/PRMT4 is a member of the protein arginine methyltransferase (PRMT) family. CARM1 as a transcriptional coactivator plays an active role on mammalian genes. Here, we show that CARM1 can be recruited to the promoter of myogenin gene to enhance its transcriptional activation via PCAF at the early stage of TPA-induced RD cell differentiation. By adding adenosine dialdehyde, AdOx, to inhibit the PRMT in RD cells, the TPA-induced recruiting of p300, PCAF and the Brg1 at the myogenin promoter is abolished and myogenic differentiation is blocked. More specifically, the expression of PCAF and its nucleation are prohibited when CARM1 is knockdown by its specific siRNA. We suggest that the physical interaction of CARM1 and PCAF is likely pivotal for the activation of PCAF in the downstream of CARM1 pathway for inducing myogenin under TPA-induced differentiation. The findings shed lights on novel therapeutic targets in the treatment of rhabdomyosarcoma patients.

HDAC3 augments the autoregulation of neuroD gene in P19 cells.

NeuroD, a basic helix-loop-helix transcription factor, is capable of converting embryonic epidermal cells into neuronal cells. However, whether histone deacetylases (HDACs) are involved in the autoregulation of neuroD or not is unclear. In this study, neuroD expression was found to be significantly increased in the all-trans retinoid acid-treated P19 cells. Meanwhile, neuroD could itself enhance its promoter activity and mRNA expression. By using specific inhibitors to histone modification enzymes, HDAC3 was identified to specifically augment the autoactivation of neuroD in P19 cells. The data suggest that the elevation of HDAC3 and neuroD in all-trans retinoid acid-treated cells exponentially increases the neuroD expression and mediates an early commitment of P19 cells for neuronal differentiation.

An inhibitory role of p53 via NF-kappaB element on the cyclin D1 gene under heat shock.

Little is known about the mechanisms underlying heat shock-mediated inhibition of cyclin D1 transcription. Here, we report that NF-kappaB site-mediated cyclin D1 transcription is inhibited by heat shock. The mRNA level of cyclin D1 decreased under heat shock (40-60%). This inhibition of transcription is promoter activity dependent and is mediated by the proximal NF-kappaB site. However, P65 overexpression did not influence the heat-inducible inhibitory pattern and heat shock did not significantly change the binding activity of p65. P53 can inhibit cyclin D1 promoter activity via an NF-kappaB site-dependent manner and its binding activity increased after heat shock. Importantly, p53 overexpression can prevent cyclin D1 promoter activation by p65. Therefore, we can deduce that p53 inhibits promoter activity under heat shock. These results reveal that the mechanism of heat shock-mediated inhibition of cyclin D1 transcription involves an NF-kappaB site. The data presented provide a new insight into the underlying heat shock inhibition of gene transcription.

Histone marks and chromatin remodelers on the regulation of neurogenin1 gene in RA induced neuronal differentiation of P19 cells.

Neurogenin1 is an important bHLH protein that plays crucial role in neurogenesis. We first show that the expression of ngn1 increases drastically in RA induced neuronal differentiation. During which, a three successive stages of the epigenetic changes surrounding the ngn1 gene are found correlated with a repression to activation of the gene in P19 cells. Recruiting of a repressive histone code H3K27me3 on the ngn1 gene is the dominant change in first repression stage, which is followed by the binding of the active codes of H3K9ac, H3K14ac, and the H3K4me3 in the second and third stages of RA treatment. Additionally, BRM but not BRG1 is specifically recruited to ngn1 gene at the third stage and is positively involved in the RA induced ngn1 expression. We propose that histone modifiers and chromatin remodelers are pivotal in the activation of the ngn1 gene in RA induced differentiation of P19 cells.

Sequential recruitment of PCAF and BRG1 contributes to myogenin activation in 12-O-tetradecanoylphorbol-13-acetate-induced early differentiation of rhabdomyosarcoma-derived cells.

Myogenin and its upstream regulator MyoD are known to be required for myogenic cell differentiation. Although both of them can be expressed in rhabdomyosarcoma-derived RD cells, the cells are unable to undergo full-scale terminal myogenic differentiation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) has been found to be functional in the induction of RD cell differentiation, whereas its mechanism is not fully understood. By using quantitative real-time-based chromatin immunoprecipitation and real-time reverse transcription-PCR-based promoter activity assays, we examined the activation mechanism of the myogenin gene during TPA-induced differentiation of the RD cells. We have shown that a histone acetyltransferase PCAF and ATPase subunit BRG1 of the SWI/SNF chromatin remodeling complex are sequentially recruited to the promoter of the myogenin gene. Both PCAF and BRG1 are also involved in the activation of the myogenin gene. In addition, we have found that the p38 mitogen-activated protein kinase is required for BRG1 recruitment in TPA-mediated myogenin induction. We propose that there are two distinct activation steps for the induction of myogenin in TPA-induced early differentiation of RD cells: 1) an early step that requires PCAF activity to acetylate core histones and MyoD to initiate myogenin gene expression, and 2) a later step that requires p38-dependent activity of the SWI/SNF remodeling complex to provide an open conformation for the induction of myogenin. Our studies reveal an essential role for epigenetic regulation in TPA-induced differentiation of RD cells and provide potential drug targets for future treatment of the rhabdomyosarcoma.

Diverse effects of Stat1 on the regulation of hsp90alpha gene under heat shock.

Stat1 has been known as a regulator of gene expression and a mediator of IFNgamma signaling in mammalian cells, while its effect in a heat shock response remains unclear. We used RNAi knockdown, point mutations, ChIP and promoter activity assays to study the effect of Stat1 on the heat-shock induction of the hsp90alpha gene under heat shock conditions. We found that Stat1 regulates the heat shock induction of its target genes, the hsp90alpha gene in a heat shock response while the constitutive activity of the gene remains unaffected. The result of Stat1 in complex with Stat3 and HSF1 that bound at the GAS to lead a moderate heat shock induction was designated as an "intrinsic" induction of the hsp90alpha gene. Additionally a reduced or an elevated level of heat shock induction was also controlled by the Stat1 on hsp90alpha. These diverse effects on the hsp90alpha gene were a "reduced" induction with over-expressed Stat1 elicited by transfection of wild-type Stat1 or IFNgamma treatment, bound at the GAS as homodimer; and an "enhanced" heat shock induction with a mutation-mediated prohibition of Stat1/GAS binding. In conclusion, the status and efficacy of Stat1 bound at the GAS of its target gene are pivotal in determining the impact of Stat1 under heat shock. The results provided the first evidence on the tumor suppressor Stat1 that it could play diverse roles on its target genes under heat shock that also shed lights on patients with fever or under thermotherapy.

Resistance to geldanamycin-induced apoptosis in differentiated neuroblastoma SH-SY5Y cells.

Geldanamycin (GA) is a specific inhibitor of the 90 kDs heat shock protein (Hsp90) in the cytoplasm of mammalian cells, which binds directly to Hsp90 and promotes proteolytic degradation of its client proteins. As an antitumor drug, GA antagonizes the protecting effects of Hsp90 on cell survival, while its mechanisms remain unclear. Here, we show that GA induces apoptosis in a human neuroblastoma cell line, SH-SY5Y. Treatment of the cells with all trans retinoic acid (RA) generates a neuron-like, morphological change of differentiation, and results in the activation of ERK and Akt pathways, an inhibition of the nuclear translocation of p53 induced by GA, and induces higher resistance to the GA-induced apoptosis. These results provide the first evidence for the requirement of p53 nucleation in SH-SY5Y cells to counteract GA in neuron survival.

A negative regulatory element-dependent inhibitory role of ITF2B on IL-2 receptor alpha gene.

Despite the fact that the negative regulatory element (NRE) within the upstream regulatory region of human IL-2 receptor alpha (IL-2Ralpha) gene has been identified two decades ago, mechanisms of the NRE function on the gene are hitherto unknown. In this paper, we report for the first time that the immunoglobulin transcription factor 2B (ITF2B) encoded by transcription factor 4 (TCF4) gene is a NRE binding protein. The full-length TCF4 cDNA clone was obtained from a HTLV-1 transformed human peripheral T cell MACHERMAKER cDNA library with NRE as the bait in yeast one-hybrid system. The NRE binding ability of ITF2B was further confirmed in chromatin-immunoprecipitation assay. Competitive RT-PCR-based promoter activity assay showed that over-expression of ITF2B protein inhibited the expression of IL-2Ralpha gene in Jurkat cells in an NRE-dependent manner. The function of ITF2B on the inhibition of both the IL-2Ralpha and the 5'LTR activity of HIV-1 shed light on the essence of NRE binding protein as a potential target for immune therapy and treatment in AIDS patients.

[Screening, cloning, and analyzing for hSNF5 binding proteins in human fetal brain].

OBJECTIVE: To identify novel binding proteins of hSNF5, a subunit of chromatin remodeling complex in human fetal brain. METHODS: The yeast two-hybrid system was used for this study. Positive cDNA clones were sequenced. Sequence homology and putative functional domains were analyzed and compared with databank. RESULTS: Nine positive clones obtained were analyzed, among which the sequence of one clone was 97% homologous to the 3' mRNA of a hypothetical protein FLJ20643, while other four clones were related to protein coding sequences existed in the GenBank. The rest four clones were not in frame with any known protein coding sequence. CONCLUSIONS: Clones encoding for hSNF5 binding protein exists in cDNA library of human brain. These proteins may recruit chromatin remodeling complex via hSNF5 to modulate the transcription of their target gene and the related cell functions.

[Heat shock induced transcription of hsp90alpha gene on chromatin template].

OBJECTIVE: A CAT reporter plasmid (pBLCAT3alpha1) driven by the promoter of hsp90alpha was in vitro assembled into chromatin to investigate the transcription activity of the reporter gene upon heat shock. METHODS: A competitive RT-PCR-based technique was used to quantify the promoter activity of hsp90alpha gene on chromatin or naked DNA templates in vitro. RESULTS: The in vitro transcription efficiency was first optimized by using different amounts of whole cell extracts from heat shock-treated HeLa cells. In vitro chromatin assembly was carried out with purified components of chromatin assembly associated factors, core histones and CAT reporter gene driven by the promoter of hsp90alpha gene. Results showed that chromatin formation repressed the in vitro transcription of the gene. CONCLUSION: The heat shock induced transcription of hsp90alpha gene on chromatin template is more efficient than that on naked DNA in vitro.

[Enhanced action of a BTB/POZ domain protein on the expression of hsp90alpha gene in heat shock].

OBJECTIVE: To study the role of a BTB/POZ domain protein in the expression of hsp90alpha gene. METHODS: The eukaryotic expression plasmids of sense- and antisense-GAGA related protein (GRP) or empty vector were transfected into Jurkat cells with pREP4 episomal vector plasmids carrying the hsp90alpha promoter sequence from -1756 to +37 and control plasmids pMCAT. Total RNA was extracted. The relative promoter activity of hsp90alpha-CAT reporter gene was determined by competitive RT-PCR assay. RESULTS: GRP markly increased the relative promoter activity of hsp90alpha-CAT reporter gene during heat shock. CONCLUSION: GRP may promote the expression of hsp90alpha gene by participating in chromatin remolding.

Repression of hsp90beta gene by p53 in UV irradiation-induced apoptosis of Jurkat cells.

Tumor suppressor p53 has been implicated in cell stress response and determines cell fate of either growth arrest or apoptosis. Heat shock proteins (Hsps) expressed under stress usually confer survival protection to the cell or interruption in the apoptotic pathways. Although Hsp90 can physically interact with p53, whether or not the hsp90 gene is influenced downstream of p53 in UV irradiation-induced apoptosis remains unclear. We have found that the level of p53 is elevated with the decline of Hsp90 in UV-irradiated cells and that malfunction of Hsp90, as inhibited by geldanamycin, enhances the p53-involved UV irradiation-induced apoptosis. In addition, the expression of the hsp90beta gene was reduced in both UV-irradiated and wild type p53-transfected cells. These results suggest a negative correlation between the trans factor p53 and a chaperone gene hsp90beta in apoptotic cells. Mutation analysis demonstrated that the p53 binding site in the first exon was indispensable for p53 regulation on the hsp90beta gene. In addition, with p53 bound at the promoter of the hsp90beta gene, mSin3a and p300 were differentially recruited in UV irradiation-treated or untreated Jurkat cells in vivo. The evidence of p53-repressed hsp90beta gene expression in UV-irradiated cells shed light on a novel pathway of Hsp90 in the survival control of the stressed cells.

[Chromatin immunoprecipitation and its preliminary application for gene regulation].

OBJECTIVE: To verify the binding of p53 to p21WAF1/CIP1 gene promoter and detect its binding to hsp90 beta gene promoter in vivo. METHODS: Chromatin immunoprecipitation and PCR analysis were used to measure specific gene regulation sequence and Western blot analysis to investigate p53 protein. RESULTS: The p53 binding sequences on the promoters of p21WAF1/CIP1 and hsp90 beta gene were found in the p53 antibody immunoprecipitated DNA fragments and p53 was detected in the immunoprecipitated samples. CONCLUSIONS: p53 binds to promoters of p21WAF1/CIP1 and hsp90 beta gene in vivo, and regulates the expression of the two genes.

PKC epsilon is a unique regulator for hsp90 beta gene in heat shock response.

An early event in cellular heat shock response is the transmittance of stress signals from the cell surface into the nuclei, resulting in the induction of heat shock proteins (Hsps). Protein kinase C (PKC) has been implicated as a key player in transducing stress signals. However, mechanism(s) by which PKC regulates heat shock-induced events remains largely unknown. Here we present data that pan-PKC inhibitor GF109203X, but not classic PKC inhibitor Gö6976, specifically repressed heat shock-induced accumulation of mRNA as well as promoter activity of hsp90 beta, but not hsp90 alpha, in Jurkat cells. Subcellular fractionation studies revealed that heat shock exclusively induced PKC-epsilon membrane translocation. Consistently, expression of a constitutively active PKC-epsilon(A159E) resulted in an enhanced promoter activity of hsp90 beta upon heat shock, whereas a dominant-negative PKC-epsilon(K437R) abolished this effect. In contrast, constitutively active-PKC-alpha or dominant-negative-PKC-alpha had no effects on heat shock induction of the gene. The effect of PKC-epsilon on hsp90 beta expression seems to be stimuli-specific, as phorbol myristate acetate-mediated hsp90 beta expression was PKC-epsilon-independent. We conclude that PKC-epsilon is specifically required in the signaling pathway leading to the induction of hsp90 beta gene in response to heat shock.