Inhibition of TRPA1 Attenuates Oxidative Stress-induced Damage After Traumatic Brain Injury via the ERK/AKT Signaling Pathway.

Neuron apoptosis is a feature of secondary injury after traumatic brain injury (TBI). Evidence implies that excess calcium (Ca2+) ions and reactive oxidative species (ROS) play critical roles in apoptosis. In reaction to increased ROS, the anti-oxidative master transcription factor, Transient receptor potential Ankyrin 1 (TRPA1) allows Ca2+ ions to enter cells. However, the effect of TBI on the expression of TRPA1 and the role of TRPA1 in TBI are unclear. In the present study, TBI in the mouse brain was simulated using the weight-drop model. The process of neuronal oxidative stress was simulated in HT22 neuronal cells by treatment with hydrogen peroxide. We found that TRPA1 was significantly upregulated in neurons at 24 h after TBI. Neuronal apoptosis was increased in the in vivo and in vitro models; however, this increase was reduced by the functional inhibition of TRPA1 in both models. After TBI, TRPA1 was upregulated via nuclear factor, erythroid 2 like 2 (Nrf2) in neurons. TRPA1-mediated neuronal apoptosis after TBI might be achieved in part through the CaMKII/AKT/ERK signaling pathway. To sum up, TBI-triggered TRPA1 upregulation in neurons is mediated by Nrf2 and the functional blockade of TRPA1 attenuates neuronal apoptosis and improves neuronal dysfunction, partially mediated through the activation of the calcium/calmodulin dependent protein kinase II (CaMKII) extracellular regulated kinase (ERK)/protein kinase B (AKT) signaling pathway. Our results suggest that functional blockade of TRPA1 might be a promising therapeutic intervention related to ROS and Nrf2 in TBI.

S100A8 Promotes Inflammation via Toll-Like Receptor 4 After Experimental Traumatic Brain Injury.

INTRODUCTION: S100 calcium-binding protein A8 (S100A8) is also known as macrophage-related protein 8, which is involved in various pathological processes in the central nervous system post-traumatic brain injury (TBI), and plays a critical role in inducing inflammatory cytokines. Accumulating evidences have indicated that toll-like receptor 4 (TLR4) is considered to be involved in inflammatory responses post TBI. The present study was designed to analyze the hypothesis that S100A8 is the key molecule that induces inflammation via TLR4 in TBI. METHODS: The weight-drop TBI model was used and randomly implemented on mice that were categorized into six groups: Sham, NS, S100A8, S100A8+TAK-242, TBI, and TBI+TAK-242 groups. In the S100A8+TAK-242 and TBI+TAK-242 groups, at half an hour prior to the intracerebroventricular administration of S100A8 or TBI, mice were intraperitoneally treated with TAK-242 that acts as a selective antagonist and inhibitor of TLR4. Furthermore, the protein recombinant of S100A8 was injected into the lateral ventricle of the brain of mice in the S100A8 and S100A8+TAK-242 groups. Sterile normal saline was injected into the lateral ventricle in the NS group. To evaluate the association between S100A8 and TLR4, Western blot, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and Nissl staining were employed. Simultaneously, the neurological score and brain water content were assessed. In the in vitro analysis, BV-2 microglial cells were stimulated with lipopolysaccharide LPS or S100A8 recombinant protein, with or without TAK-242. The expression of the related proteins was subsequently detected by Western blot or enzyme-linked immunosorbent assay. RESULTS: The levels of S100A8 protein and pro-inflammatory cytokines were significantly elevated after TBI. There was a reduction in the neurological scores of non-TBI animals with remarkable severe brain edema after the intracerebroventricular administration of S100A8. Furthermore, the TLR4, p-p65, and myeloid differentiation factor 88 (MyD88) levels were elevated after the administration of S100A8 or TBI, which could be restored by TAK-242. Meanwhile, in the in vitro analysis, due to the stimulation of S100A8 or LPS, there was an upregulation of p-p65 and MyD88, which could also be suppressed by TAK-242. CONCLUSION: The present study demonstrated that the TLR4-MyD88 pathway was activated by S100A8, which is essential for the development of inflammation in the brain after TBI.

CKIP-1 silencing promotes new bone formation in rat mandibular distraction osteogenesis.

OBJECTIVE: This study investigated the effects and possible molecular mechanism of casein kinase-2 interacting protein-1 (CKIP-1) silencing on bone regeneration during rat mandibular distraction osteogenesis (DO). STUDY DESIGN: CKIP-1 silencing by chitosan/si-CKIP-1 was employed and analyzed both in rat mandibular DO models in vivo and in cultured rat mandible bone marrow stromal cells (BMSCs) in vitro. RESULTS: Gross observation, micro-computed tomography analysis, and hematoxylin and eosin (H&E) staining revealed that new bone formation in the distraction gap of the chitosan/si-CKIP-treated group was better compared with the chitosan/si-NC and phosphate buffered saline-treated groups in both quantity and quality. Proliferation assay, flow cytometry, and alizarin red staining indicated that CKIP-1 silencing significantly inhibited apoptosis, but promoted osteogenic differentiation of cultured BMSCs. Additionally, CKIP-1 silencing significantly promoted the expression of Wnt3 a, ß-catenin, and osteocalcin both in new bone formation of DO models in vivo and in the osteogenic differentiation process of BMSCs in vitro. CONCLUSIONS: Promotion of bone formation after CKIP-1 silencing in rat mandibular distraction osteogenesis appears to be mediated through the Wnt3 a/ß-catenin signaling pathway.

Targeting P38 Pathway Regulates Bony Formation via MSC Recruitment during Mandibular Distraction Osteogenesis in Rats.

Distraction osteogenesis (DO) is a widely used self-tissue engineering. However, complications and discomfort due to the long treatment period are still the bottleneck of DO. Novel strategies to accelerate bone formation in DO are still needed. P38 is capable of regulating the osteogenic differentiation of both mesenchymal stem cells (MSCs) and osteoblasts, which are crucial to bone regeneration. However, it is not clear whether targeting p38 could regulate bony formation in DO. The purpose of the current work was to investigate the effects of local application of either p38 agonist anisomycin or p38 inhibitor SB203580 in a rat model of DO. 30 adult rats were randomly divided into 3 groups: (A) rats injected with DMSO served as the control group; (B) rats injected with p38 agonist anisomycin; (C) rats injected with p38 inhibitor SB203580. All the rats were subjected to mandibular distraction and the injection was performed daily during this period. The distracted mandibles were harvested on days 15 and 30 after surgery and subjected to the following analysis. Micro-computed tomography and histological evaluation results showed that local application of p38 agonist anisomycin increased new bone formation in DO, whereas p38 inhibitor SB203580 decreased it. Immunohistochemical analysis suggested that anisomycin promoted MSC recruitment in the distraction gap. In conclusion, this study demonstrated that local application of p38 agonist anisomycin can increase new bone formation during DO. This study may lead to a novel cell-based strategy for the improvement of bone regeneration.

Role of α7-nicotinic acetylcholine receptor in nicotine-induced invasion and epithelial-to-mesenchymal transition in human non-small cell lung cancer cells.

Nicotine via nicotinic acetylcholine receptors (nAChRs) stimulates non-small cell lung cancer (NSCLC) cell invasion and epithelial to mesenchymal transition (EMT) which underpin the cancer metastasis. However, the receptor subtype-dependent effects of nAChRs on NSCLC cell invasion and EMT, and the signaling pathway underlying the effects remain not fully defined. We identified that nicotine induced NSCLC cell invasion, migration, and EMT; the effects were suppressed by pharmacological intervention using α7-nAChR selective antagonists or by genetic intervention using α7-nAChR knockdown via RNA inference. Meanwhile, nicotine induced activation of MEK/ERK signaling in NSCLC cells; α7-nAChR antagonism or MEK/ERK signaling pathway inhibition suppressed NSCLC cell invasion and EMT marker expression. These results indicate that nicotine induces NSCLC cell invasion, migration, and EMT; the effects are mediated by α7-nAChRs and involve MEK/ERK signaling pathway. Delineating the effect of nicotine on the NSCLC cell invasion and EMT at receptor subtype level would improve the understanding of cancer biology and offer potentials for the exploitation of selective ligands for the control of the cancer metastasis.

[Molecular cloning and characterization of a novel DoSWEET1 gene from Dendrobium officinale].

SWEET (sugars will be eventually exported transporters) constitute a large and conserved gene family of sugar transporters in eukaryotes, which are important in the cellular metabolisms, growth and development, and plant-microbe interaction in plants. In the present study, a full length cDNA of SWEET encoding gene, designed as DoSWEET1 (GenBank accession No. KT957550), was identified in Dendrobium officinale using RT-PCR and RACE approaches. DoSWEET1 was 1 150 bp in length and encoded a 262-aa protein with a molecular weight of 29.18 kD and an isoelectric point of 9.49. The deduced DoSWEET1 protein contained seven transmembrane regions and two conserved MtN3-slv domains (11-94, 130-212）. Multiple sequence alignment revealed that DoSWEET1 had high identities 45%-54.6%） with SWEET proteins from various plants. A neighbor joining phylogenetic analysis suggests that DoSWEET1 belonged to the class Ⅱ subgroup of the SWEET evolutionary tree, and was closely related to rice OsSWEET13, OsSWEET14, and OsSWEET15. qPCR analysis demonstrated that DoSWEET1 gene was differentially expressed in the three included organs of D. officinale, and the expression was most abundant in the roots at 9.88 fold over that of the stems, followed by that of the leaves with 2.85 fold higher. In the 3rd symbiotic germinating seeds infected by Tulasnella sp., the transcipts were dramatically induced by 1 359.06 fold over that in the ungerniamted control seeds, suggesting a vital role of the gene in the D. officinale symbiotic germination process. Molecular cloning and characterization of the novel DoSWEET1 gene provides a foundation for the functional study of the gene in sugar translocation during the D. officinale symbiotic germination process.

[Correlation analysis between effectiveness of element in rhizonsphere soil and quality of Danshen (Salvia miltiorrhiza)].

In order to investigate the content and distribution of available element in the rhizonsphere soil of the growing areas of Salvia miltiorrhiza Bunge, the contents of available element (N,P,K,B,Cu,Zn,Fe,Mn) in 26 soil samples were tested and evaluated. The results showed that the contents of available P and Fe were very plentiful, available K, Cu and Zn were rich, available N and Mn were deficient, available B was extremely deficient in all growing areas of S. miltiorrhiza of eight provinces in China. The correlation analysis showed that the contents of eight kinds of available elements were varying degree correlation. The stepwise regression analysis between the contents of available elements of rhizonsphere soil and ten kinds of active ingredients of Danshen (Salviae Miltiorrhizae Radix et Rhizoma) were researched. The results showed that the rates of contribution of available N,B,Mn and Fe to quality of Danshen were relatively large and they were the significant factors, and the other factors did not show statistical significance. The recommended fertilizing strategies is that the usage of N,B and Mn fertilizers should be controlled according to different stages of growth of S. miltiorrhiza, and P fertilizer should be reduced in all growing areas of S. miltiorrhiza.

Adrenergic DNA damage of embryonic pluripotent cells via ß2 receptor signalling.

Embryonic pluripotent cells are sensitive to genotoxicity though they need more stringent genome integrity to avoid compromising multiple cell lineages and subsequent generations. However it remains unknown whether the cells are susceptible to adrenergic stress which can induce somatic cell genome lesion. We have revealed that adrenergic stress mediators cause DNA damage of the cells through the ß2 adrenergic receptor/adenylate cyclase/cAMP/PKA signalling pathway involving an induction of intracellular reactive oxygen species (ROS) accumulation. The adrenergic stress agonists adrenaline, noradrenaline, and isoprenaline caused DNA damage and apoptosis of embryonic stem (ES) cells and embryonal carcinoma stem cells. The effects were mimicked by ß2 receptor-coupled signalling molecules and abrogated by selective blockade of ß2 receptors and inhibition of the receptor signalling pathway. RNA interference targeting ß2 receptors of ES cells conferred the cells the ability to resist the DNA damage and apoptosis. In addition, adrenergic stimulation caused a consistent accumulation of ROS in the cells and the effect was abrogated by ß2 receptor blockade; quenching of ROS reversed the induced DNA damage. This finding will improve the understanding of the stem cell regulatory physiology/pathophysiology in an adrenergic receptor subtype signalling mechanism.

[Effect of licorice flavonoids on kainic acid-induced seizure in mice].

OBJECTIVE: To investigate the effect of licorice flavonoid (LF) on kainic acid (KA)-induced seizure in mice and its mechanism. METHODS: Male adult ICR mice were injected with 25 mg/kg KA to induce temporal lobe seizure. LF was administrated 7 d before seizure induction (pre-treatment) or 24 h after seizure induction (post-treatment) for 7 d. Acute seizure latency, seizure stage and duration were observed and compared between LF- and vehicle-treated mice. From d2 on, mice with status epilepticus were video-monitored for spontaneous seizures, 10 h/d for 6 w. Immunohistochemical analysis of BrdU and Timm staining was conducted to detect the neurogenesis and mossy fiber sprouting, respectively. RESULTS: No significant difference was observed in acute seizure latency, seizure stage and duration between LF-and vehicle-treated mice. KA-induced acute seizure resulted in spontaneous seizure in mice, and the seizure frequency was increased with time. Pre- and post-treatment with LF decreased seizure frequency from w3 after modeling [(0.58±0.15)/d, (0.38±0.38)/d vs (1.23±0.23)/d, P <0.05]. Furthermore, KA-induced seizure resulted in robust neurogenesis and mossy fiber sprouting, while treatment with LF both pre- and post- KA injection significantly inhibited neurogenesis (15.6±2.6, 17.1±3.1 vs 28.9±3.5, P <0.05) and mossy fiber sprouting (1.33±0.31, 1.56±0.42 vs 3.0±0.37, P <0.05). CONCLUSION: LF has no significant anti-seizure effect. However, it can decrease epileptogenesis through inhibition of neurogenesis and mossy fiber sprouting.

ß2-Adrenoreceptor-Mediated Proliferation Inhibition of Embryonic Pluripotent Stem Cells.

Adrenoreceptors (ARs) are widely expressed and play essential roles throughout the body. Different subtype adrenoceptors elicit distinct effects on cell proliferation, but knowledge remains scarce about the subtype-specific effects of ß2-ARs on the proliferation of embryonic pluripotent stem (PS) cells that represent different characteristics of proliferation and cell cycle regulation with the somatic cells. Herein, we identified a ß2-AR/AC/cAMP/PKA signaling pathway in embryonic PS cells and found that the pathway stimulation inhibited proliferation and cell cycle progression involving modulating the stem cell growth and cycle regulatory machinery. Embryonic stem (ES) cells and embryonal carcinoma stem (ECS) cells expressed functional ß-ARs coupled to AC/cAMP/PKA signaling. Agonistic activation of ß-ARs led to embryonic PS cell cycle arrest and proliferation inhibition. Pharmacological and genetic analyzes using receptor subtype blocking and RNA interference approaches revealed that this effect selectively depended on ß2-AR signaling involving the regulation of AKT, ERK, Rb, and Cyclin E molecules. Better understanding of the effects of ß2-ARs on embryonic PS cell proliferation and cycle progression may provide new insights into stem cell biology and afford the opportunity for exploiting more selective ligands targeting the receptor subtype for the modulation of stem cells.

Simultaneous determination of the repertoire of classical neurotransmitters released from embryonal carcinoma stem cells using online microdialysis coupled with hydrophilic interaction chromatography-tandem mass spectrometry.

Dynamic, continuous, and simultaneous multi-analysis of transmitters is important for the delineation of the complex interactions between the neuronal and intercellular communications. But the analysis of the whole repertoire of classical transmitters of diverse structure is challenging due to their different physico-chemical properties and to their high polarity feature which leads to poor retention in traditional reversed-phase columns during LC-MS analysis. Here, an online microdialysis coupled with hydrophilic interaction chromatography-tandem mass spectrometry (online MD-HILIC-MS/MS) detection method was developed for the simultaneous measurement of the repertoire of classical transmitters (acetylcholine, serotonin, dopamine, norepinephrine, glutamate, GABA, and glycine). Stable isotope labeled internal standards and authentic matrix have been applied to guarantee reliable results. The method was successfully employed to reveal the characteristics of transmitter release from embryonal carcinoma stem cells. The method features simple procedure (no sample preparation), high recovery (≥ 73%), high accuracy (89.36%≤RE≤116.89%), good reproducibility (2.18%≤ RSD ≤14.56%), and sensitive limits of detection (2 pg for acetylcholine, serotonin, and glutamate, 10 pg for dopamine, norepinephrine, GABA, and glycine). It can be flexibly applied to determine the contents of the classical transmitters in other biological matrix samples with minor changes.

[Characteristics of inorganic elements in Panacis Majoris Rhizoma and relationship with its Daodi habitat].

OBJECTIVE: To investigate the Daodi habitat of Panacis Majoris Rhizoma by analyzing the characteristics of inorganic elements in Panacis Majoris Rhizoma from different habitats. METHODS: The contents of inorganic elements in Panacis Majoris Rhizoma from different habitats were determined by ICP-AES. The characteristics of inorganic elements in Panacis Majoris Rhizoma were analyzed by correlation analysis, principal component analysis and cluster analysis. RESULTS: It was showed that there was a correlation between the contents of inorganic elements and the medicine quality of Panacis Majoris Rhizoma; Fe, Cr, Al, Mg, Cd, Ca and Zn were principal components of Panacis Majoris Rhizoma; and the contents of inorganic elements in Panacis Majoris Rhizoma existed regional differences. CONCLUSION: The contents of inorganic elements Ca, Fe and Zn,especially the content of the essential trace elements Fe and Zn, can be used as one of the key reference for medicinal quality evaluation of Panacis Majoris Rhizoma; as well, Shaanxi Province is probably the Daodi habitat of Panacis Majoris Rhizoma.

[Influence of soil factors on quality of panacis majoris rhizoma].

OBJECTIVE: To research and screen the soil factors which influenced the quality of Panacis Majoris Rhizoma. METHODS: By determining the effective constituent of saponin of Panacis Majoris Rhizoma from different habitats, and the soil nutrient and heavy metal content, using correlation analysis, stepwise regression analysis and other statistical methods to analyze the soil factors on the quality of Panacis Majoris Rhizoma. RESULTS: The contents of ginsenoside Ro and chikusetsusaponin NVa of samples from different habitats were different, the corresponding soil nutrient and heavy metal content were also different. The content of ginsenoside-Ro was positively related with the soil pH value (P <0. 01) and negatively related with the soil available N(P < 0.05). The content of chikusetsusaponin IVa was positively related with the soil pH (P < 0.01) and copper content (P < 0.05), and negatively with the soil available N(P < 0.05) CONCLUSION: Soil factors are the leading factors that influence the quality of Panacis Majoris Rhizoma, soil pH and soil available N were the dominating factors.

Non-neuronal release of gamma-aminobutyric Acid by embryonic pluripotent stem cells.

γ-Aminobutyric acid (GABA), the principle inhibitory transmitter in the mature central nervous system, is also involved in activities outside the nervous system. Recent studies have shown that functional GABA receptors are expressed in embryonic stem (ES) cells and these receptors control ES cell proliferation. However, it is not clear whether ES cells have their own GABAergic transmission output machinery that can fulfill GABA release or whether the cells merely process the GABA receptors by receiving and responding to the diffused GABA released elsewhere. To get further insight into this unresolved problem, we detected the repertoire of components for GABA synthesis, storage, reaction, and termination in ES and embryonal carcinoma stem cells by biological assays, and then directly quantified released GABA in the intercellular milieu from these pluripotent stem (PS) cells by an analytical chemical assay based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). We found that embryonic PS cells processed a GABAergic circuit machinery and spontaneously released GABA, which suggests the potential that embryonic PS cells could autonomously establish a GABA niche via release of the transmitter.

[Clinical observation of icotinib hydrochloride for patients with advanced non-small cell lung cancer].

OBJECTIVE: To explore the efficacy and side effects of icotinib hydrochloride in the treatment of patients with advanced non-small cell lung cancer (NSCLC). METHODS: The efficacy and side effects of icotinib hydrochloride in treatment of 59 cases with stage IV NSCIC and followed-up from March 2009 to January 2012 were retrospectively analyzed. RESULTS: Twenty seven patients (45.8%) showed partial response (PR), 17 patients (28.8%) achieved SD, and 15 (25.4%) had progressive disease. The objective response rate (ORR) was 45.8% (27/59), and disease control rate (DCR) was 74.6% (44/59). Among the 23 patients with EGFR mutation, ORR was 73.9% (17/23), and DCR was 95.7% (22/23). Thirty six patients (61.0%) achieved remission of symptoms to varying degrees. The main symptoms relieved were cough, asthmatic suffocating, pain and hoarseness. The major adverse events were mild skin rash (35.6%) and diarrhea (15.3%). Others were dry skin, nausea and stomach problems. The efficacy of icotinib hydrochloride were related to the ECOG performance status, smoking history, EGFR mutation and rash significantly (P < 0.05). CONCLUSIONS: Monotherapy with icotinib hydrochloride is effective and tolerable for patients with advanced NSCLC, especially with EGFR mutation.

[Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells].

OBJECTIVE: To investigate the effects of DNA hypomethylation on mRNA and protein expression of perforin promotor in T cells. METHODS: T cells were isolated from the peripheral venous blood of healthy donors by density gradient centrifugation. CD4(+) and CD8(+) subsets were isolated using Miltenyi beads and protocols provided by the manufacturer. Where indicated the T cells were stimulated with PHA for 24 h, then treated with 5-azaC for an additional 72 h. Genomic DNA, mRNA, and protein were isolated from untreated and 5-azaC-treated T cells. Purified DNA was treated with sodium bisulfite, the desired sequences were amplified in sequential fragments using nested PCR. The amplified fragments were cloned into bacteria DH5 alpha and 5 independent clones for each of the amplified fragments were sequenced. The expression of perforin was determined using real time RT-PCR and Western blot. RESULTS: The perforin mRNA and protein in the CD4(+) and CD8(+) subsets treated with 5-azaC were significantly higher than those in the untreated subsets (P<0.05). The results of bisulfite genomic sequencing showed that the methylation of perforin promotor was significantly reduced in the treated cells compared with the untreated cells (P<0.05). CONCLUSION: The mRNA and protein expression of perforin significantly increases in the CD4(+) and CD8(+) T cells treated with 5-azaC,which is associated with DNA hypomethylation of perforin promoter in T cells.