A bacteriophage against Citrobacter braakii and its synergistic effect with antibiotics.

A bacteriophage BD49 specific for Citrobacter braakii was screened out and purified by double-layer plate method. It consists of a polyhedral head of 93.1 ± 1.2 nm long and 72.9 ± 4.2 nm wide, tail fibers, collar, sheath and baseplate. The bacteriophage was identified by morphology observed with transmission electron microscope (TEM), whole genome sequencing carried out by Illumina next generation sequencing (NGS) technique, and gene annotation based on Clusters of Orthologous Groups of proteins (COG) database. It was identified primarily as a member of Caudovirales by morphology and further determined as Caudovirales, Myoviridae, and Citrobacter bacteriophage by alignment of its whole genome sequence with the NCBI database and establishment of phylogenetic tree. The bacteriophage showed good environmental suitability with optimal multiplicity of infection (MOI) of 0.01, proliferation time of 80 min, optimum living temperature of 30-40 °C, and living pH of 5-10. In addition, it exhibited synergistic effect with ciprofloxacin against C. braakii in antibacterial tests.

[Identification of chemical components and network pharmacology of Huanglian Decoction based on UPLC-Q-TOF-MS/MS technology].

The chemical components of Huanglian Decoction were identified by ultra-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS) technology. The gradient elution was conducted in Agilent ZORBAX Extend-C_(18) column(2.1 mm×100 mm, 1.8 µm) with the mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B) at a flow rate of 0.3 mL·min~(-1) and the column temperature of 35 ℃. The MS adopted the positive and negative ion mode of electrospray ionization(ESI), and the MS data were collected under the scanning range of m/z 100-1 500. Through high-resolution MS data analysis, combined with literature comparison and confirmation of reference substances, this paper identified 134 chemical components in Huanglian Decoction, including 12 alkaloids, 23 flavonoids, 22 terpenes and saponins, 12 phenols, 7 coumarins, 12 amino acids, 23 organic acids, and 23 other compounds, and the medicinal sources of the compounds were ascribed. Based on the previous studies, 7 components were selected as the index components. Combined with the network pharmacology research and analysis me-thods, the protein and protein interaction(PPI) network information of the intersection targets was obtained through the STRING 11.0 database, and 20 core targets of efficacy were screened out. In this study, UPLC-Q-TOF-MS/MS technology was successfully used to comprehensively analyze and identify the chemical components of Huanglian Decoction, and the core targets of its efficacy were discussed in combination with network pharmacology, which laid the foundation for clarifying the material basis and quality control of Huanglian Decoction.

Bacteria-based nanodrug for anticancer therapy.

Bacteria-based immunotherapy has become a promising strategy to induce innate and adaptive responses for fighting cancer. The advantages of bacteriolytic tumor therapy mainly lie in stimulation of innate immunity and colonization of some bacteria targeting the tumor microenvironment (TME). These bacteria have cytotoxic proteins and immune modulating factors that can effectively restrain tumor growth. However, cancer is a multifactorial disease and single therapy is typically unable to eradicate tumors. Rapid progress has been made in combining bacteria with nanotechnology. Using the nanomolecular properties of bacterial products for tumor treatment preserves many features from the original bacteria while providing some unique advantages. Nano-bacterial therapy can enhance permeability and retention of drugs, increase the tolerability of the targeted drugs, promote the release of immune cell mediators, and induce immunogenic cell death pathways. In addition, combining nano-bacterial mediated antitumor therapeutic systems with modern therapy is an effective strategy for overcoming existing barriers in antitumor treatment and can achieve satisfactory therapeutic efficacy. Overall, exploring the immune antitumor characteristics of adjuvant clinical treatment with bacteria can provide potential efficacious treatment strategies for combatting cancer.

[Identification of chemical components based on UPLC-Q-TOF-MS/MS and network pharmacology of Zhuru Decoction].

This study analyzed the main chemical components of Zhuru Decoction via ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS), and then predicted the mechanism of Zhuru Decoction in clearing heat, resolving phlegm, detoxifying, and treating vomiting and alcohol-related vomiting caused by heat in stomach based on network pharmacology. The gradient elution was conducted in Agilent ZORBAX extend-C_(18) column(2.1 mm×100 mm, 1.8 µm) with the mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B) at a flow rate of 0.3 mL·min~(-1) and the column temperature of 35 ℃. The MS adopted the positive and negative ion mode of electrospray ionization(ESI), and the data were collected in the scanning range of m/z 100-1 500. A total of 98 compounds in Zhuru Decoction were identified via BATMAN, SYMMAP, TCMSP, and relevant literature, including 36 flavonoids, 7 triterpenoids, 8 gingerols, 20 organic acids, 5 amino acids, and 22 other compounds. On the basis of the available studies, 9 components were selected as index components, and the protein-protein interaction(PPI) network of the common targets was established with STRING 11.0. Finally, 10 core targets associated with the pharmacodynamic effect were screened out. This study established the UPLC-Q-TOF-MS/MS method for identifying the chemical components in the classic prescription Zhuru Decoction, and employed network pharmacology to explore the core targets of its efficacy, which provided a refe-rence for the quality control and the research of the pharmacodynamic substances of Zhuru Decoction.

[Study on characteristic chromatogram and content determination of Wuzhuyu Decoction reference sample].

In this study, the critical quality attributes of Wuzhuyu Decoction reference sample were explored by using characteristic chromatogram, index component content and dry extract rate as indexes.The dissemination relationship of quantity value between medicinal materials-decoction pieces-reference sample was investigated to preliminarily formulate the quality standard of the reference sample.The characteristic chromatogram of 15 batches of Wuzhuyu Decoction was established by high performance liquid chromatography(HPLC) and the similarity analysis was conducted.Common peaks were demarcated and assigned to medicinal materials.Moreover, quantitative determination of limonin, evodiamine, rutaecarpine and ginsenoside Rb_1 of Wuzhuyu Decoction were performed.The dissemination of quantity value was explored combined with dry extract rate, similarity of characteristic chromatogram and transfer rate of index component content.A total of 18 common peaks were identified in the corresponding materials of Wuzhuyu Decoction reference sample, with the similarity of characteristic chromatogram greater than 0.9, and Fructus Evodiae, Radix Ginseng, Rhizoma Zingiberis Recens and Fructus Jujubae contributed 9, 5, 8 and 2 chromatographic peaks, respectively.The index component content of corresponding materials and the transfer rates of medicinal materials-decoction pieces and decoction pieces-reference sample of different batches of Wuzhuyu Decoction reference sample were as follows: the content of limonin was 0.16%-0.51%, and the transfer rates were 83.66%-115.60% and 38.54%-54.58%, respectively; the content of evodiamine was 0.01%-0.11%, the transfer rated were 80.80%-116.15% and 3.23%-12.93%, respectively; the content of rutaecarpine was 0.01%-0.05%, the transfer rates were 84.33%-134.53% and 5.72%-21.24%, respectively; the content of ginsenoside Rb_1 was 0.06%-0.11%, and the transfer rates were 90.00%-96.92% and 32.45%-67.24%, respectively.The dry extract rate of the whole prescription was 22.58%-29.89%.In this experiment, the dissemination of quantity value of Wuzhuyu Decoction reference sample was analyzed by the combination of characteristic chromatogram, index component content and dry extract rate.A scientific and stable quality evaluation method of the reference sample was preliminarily established, which provided basis for the subsequent development of Wuzhuyu Decoction and the quality control of related preparations.

[Quality value transmitting of substance benchmarks of Zhuru Decoction].

A total of 18 batches of Zhuru Decoction samples were prepared. Chromatographic fingerprints were established for Zhuru Decoction and single decoction pieces, the content of which was then determined. The extraction rate ranges, content, and transfer rate ranges of puerarin, liquiritin, and glycyrrhizic acid, together with the common peaks and the similarity range of the fingerprints, were determined to clarify key quality attributes of Zhuru Decoction. The 18 batches of Zhuru Decoction samples had 25 common peaks and the fingerprint similarity higher than 0.95. Puerariae Lobatae Radix, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens had 21, 3, and 1 characteristic peaks, respectively. The 18 batches of samples showed the extraction rates within the range of 18.45%-25.29%. Puerarin had the content of 2.20%-3.07% and the transfer rate of 38.5%-45.9%; liquiritin had the content of 0.24%-0.85% and the transfer rate of 15.9%-37.5%; glycyrrhizic acid had the content of 0.39%-1.87% and the transfer rate of 16.2%-32.8%. In this paper, the quality value transmitting of substance benchmarks of Zhuru Decoction was analyzed based on chromatographic fingerprints, extraction rate, and the content of index components. A scientific and stable method was preliminarily established, which provided a scientific basis for the quality control and formulation development of Zhuru Decoction.

[Quality value transfer of substance benchmarks in Huanglian Decoction].

Following the preparation of substance benchmarks in Huanglian Decoction from 18 batches, the method for detecting their characteristic spectra was established to identify the similarity range and peak attribution. The content and transfer rate ranges of the index components coptisine, palmatine, berberine, liquiritin, glycyrrhizic acid, 6-gingerol, and cinnamaldehyde and the extraction amount were combined for analyzing the quality value transfer from the Chinese medicinal pieces to substance benchmarks and clarifying the key quality attributes of substance benchmarks in Huanglian Decoction. The results showed that the substance benchmarks in Huang-lian Decoction of 18 batches exhibited good similarity in characteristic spectra(all greater than 0.98). There were 17 characteristic peaks identified in the substance benchmarks of Huanglian Decoction, including 10 from Coptidis Rhizoma, 3 from Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle(processed with water), 1 from Zingiberis Rhizoma, and 3 from Cinnamomi Ramulus. The contents and average transfer rates of the index components were listed as follows: coptisine 2.20-6.46 mg·g~(-1) and 18.50%±2.93%; palmatine 3.03-8.13 mg·g~(-1) and 26.56%±4.69%; berberine 7.71-22.29 mg·g~(-1) and 17.34%±3.00%; liquiritin 0.88-2.18 mg·g~(-1) and 9.88%±4.88%; glycyrrhizic acid 1.83-4.44 mg·g~(-1) and 8.50%±3.72%; 6-gingerol 0.56-1.43 mg·g~(-1) and 11.36%±2.37%; cinnamaldehyde 1.55-3.48 mg·g~(-1) and 19.02%±4.36%. The extraction amount of the substance benchmarks from the 18 batches was controlled at 10.65%-13.88%. In this paper, the quality value transfer of substance benchmarks in Huanglian Decoction was analyzed based on the characteristic spectra, the index component contents and the extraction amount, which has provided a basis for the subsequent development of Huanglian Decoction and the quality control of its related preparations.

[Mechanism of "herb soaking with exact amount of water" during moistening process of ginseng based on needle pressure sensor].

In this study, the texture analyzer acupuncture pressure sensor was used to objectively characterize the "herb soaking with exact amount of water" for moistening process of ginseng. The single factor rotation experiment was used to investigate the effects of puncture speed, puncture depth and puncture site on puncture force and work. According to ginseng processing method in Chinese Pharmacopoeia, ginseng medicinal materials with diameters of about 1 cm and 2 cm were selected, and puncture experiments were carried out at the set measurement time to determine the hardness, work and water absorption of the ginseng moistening process. The endpoint threshold for the ginseng softening process was determined and verified. To reflect the actual internal conditions of the ginseng softening process, the puncture depth was preferably 70%, and the puncture speed was 30 mm·min~(-1). In the ginseng moistening process, the softening hardness and the puncture work were in accordance with the first-order kinetic equation y=a×exp(-k×x). The 0 h initial hardness a of 1 cm and 2 cm ginseng herbs were 289.8 N and 1 227 N, and the rate constants K were 0.149 4 N·h~(-1) and 0.100 7 N·h~(-1), respectively. After the ginseng was completely softened, the force required for puncture was 10 N, which can be used as the standard for "drug penetration". At this time, the water absorption rate of ginseng was 70%-100%. The softening time of ginseng with a diameter of 1 cm was about 20-22 h, and the softening time of ginseng with a diameter of 2 cm was about 40-46 h. A needle-type pressure sensor was used to accurately determine the end point of the softening process of ginseng and reduce the loss of active ingredients. The study results provide reference for the softening process kinetics and the process intelligent monitoring of other dried roots and rhizomes.

YiQi Tongluo Granule against Cerebral Ischemia/Reperfusion Injury in Rats by Freezing GluN2B and CaMK II through NMDAR/ERK1/2 Signaling.

Yiqi Tongluo Granule (YQTL) is a kind of proprietary Chinese medicine, manufactured by China Shineway Pharmaceutical Group Ltd., under the authority of China Food and Drug Administration (CFDA) treating cardiovascular and cerebrovascular diseases such as ischemic stroke in China, however the underlying mechanism of YQTL on treating ischemic stroke has not been revealed. This study is aimed to evaluate the protective effect of YQTL on cerebral ischemia/reperfusion (I/R) injury and inquire into its underlying mechanisms. Cerebral I/R injury was induced by occluding the middle cerebral artery for 2 h followed by 24 h reperfusion. And regional cerebral flow was monitored by Laser Doppler flow during ischemia phase. The infarct volume was evaluated by Triphenyte-trazolium chloride staining. The protective effects of YQTL were assessed by a number of parameters, including neurological scores, regional cerebral blood flow, pathological changes of neuron in hippocampuses and hippocampus calcium level. The proteins of extracellular signal-regulated kinase (ERK), N-methyl D-aspartate receptor subtype 2B (GluN2B) and p-calcium-dependent protein kinaseII (CaMKII) response were assayed by Western blotting. I/R caused significant change in neurological deficit scores, regional cerebral flow and infarct volume. However results in YQTL groups and Nimodipine Tablets (NMDP) group were reversed. Subsequently YQTL reduced I/R-induced calcium influx. Results of hematoxylin-eosin staining manifested that YQTL significantly improved neuronal injury after I/R in rats. Meanwhile, microdialysis data demonstrated that extracellular glutamate was increased in the striatum during ischemia reperfusion, which was reduced by YQTL. YQTL and mitogen-activated protein extracellular kinase (MEK) inhibitor suppressed the I/R-mediated over-expression of GluN2B, p-ERK, ERK and p-CaMKII proteins expression. Putting these together, our results suggest that YQTL played a neuroprotective role in cerebral I/R injury, which might be exerted by inhibiting the excitotoxicity and expression of GluN2B, p-CaMKII and MEK/ERK signal pathway.

[A network pharmacology approach to explore mechanisms of Buyang Huanwu decoction for treatment of cerebral infarction].

The point of this study is to explore and investigate mechanisms of Buyang Huanwu decoction for treatment of cerebral infarction (CI) using a network pharmacology approach. First, TCMSP database, DrugBank database and PharmMapper server were used and combined with oral bioavailability and drug analysis to screen the components of Buyang Hanwu decoction and predict the potential targets. Then, Cytoscape 3.5.1 software was used to construct compounds-targets network and the protein-protein interaction (PPI) networks for targets of compounds and CI-related targets and merge the two PPI networks to acquire active targets. Finally, gene ontology (GO) and KEGG pathway analysis of active targets were carried out by DAVID online analysis tool and KOBAS 3.0 software. In total of 150 screened compounds and 232 potential targets were obtained. And in total of 208 active targets were finally determined by merging networks. Results indicated that Buyang Huanwu decoction might have a role in treating CI by regulating some biological processes including response to drug, aging, response to hypoxia, and blood coagulation, and some molecular function, such as protein binding, enzyme binding and serine-type endopeptidase activity. The mechanisms might be concerned with PI3K-Akt signaling pathway, TNF signaling pathway, HIF-1 signaling pathway and cAMP signaling pathway. Among them, the regulation of PI3K-Akt signaling pathway might be one of the most crucial mechanisms.

[Calcium mobilizing effect of hawthorn leaf procyanidins in vascular endothelial cells].

The hawthorn leaves have the effect of activating blood, removing blood stasis, regulating qi through the veins, dissolving turbidity and lowering lipid. Procyanidinis is one of its main active components and plays an important role in regulating vasoactivity. Previous studies showed that the regulating effect of procyanidins was related to its regulation on nitric oxide secretion from vascular endothelial cells, and this effect was dependent on the extracellular calcium concentration, suggesting that the changes in intracellular calcium ion concentration in endothelial cells may play a key role in this process. However, the research on this issue is still insufficient so far. This study is aimed to observe the effect of hawthorn leaf oligomeric procyanidins (HLP) on calcium mobilization of vascular endothelial cells, and investigate the underlying mechanism. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro and labeled with Fura-2. HUVEC were treated with HLP at concentrations of 6.25, 12.5, 25 and 50 mg·L⁻¹, and the intracellular calcium concentrations were measured with a living cell microscope for 30 min. HLP increased the intracellular calcium concentration of HUVEC in a concentration dependent manner; and the intracellular calcium concentrations in 25 and 50 mg·L⁻¹ HLP groups were significantly higher than that in the normal group. With the use of calcium-free incubation buffer, addition of calcium chelating agent EGTA in incubation buffer, or use of inhibitors for sodium calcium exchanger, the effect of HLP was significantly inhibited. On the other hand, the effect of HLP could also be weakened by inhibiting the calcium release from the intracellular storage. In conclusion, these results suggest that HLP can elicit calcium mobilization in vascular endothelial cells, which may be one of the mechanisms for its vascular modulatory activity; and this calcium mobilizing effect may be achieved through promoting both extracellular calcium influx and intracellular calcium release, additionally the former may be related to activating the reverse transport of Na⁺-Ca²âº exchangers on the cell membrane.

Salvianolic acid B induced upregulation of miR-30a protects cardiac myocytes from ischemia/reperfusion injury.

BACKGROUND: MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. This study was designed to ascertain if miR-30a is involved in the cardioprotective actions of salvianolic acid B (Sal B) against myocardial ischemia-reperfusion (I-R) injury through suppression of autophagy. METHODS: Murine myocardial cells that had undergone primary culture were induced by I-R and incubated with Sal B (25, 50, 100 µM) in the presence of a miR-30a mimic or miR-30a inhibitor. Expression of miR-30a, beclin-1, LC3-II and p-Akt protein, cell viability, and lactic acid dehydrogenase (LDH) release were assessed. RESULTS: miR-30a expression was down-regulated remarkably in I-R cells, and this suppression could be reversed by Sal B in a dose-dependent manner. Sal B repressed autophagy in I-R myocardial cells. Sal B improved cell viability and reduced the rate of LDH leakage, which suggested that autophagy suppression was beneficial for cell survival. Knockdown of miR-30a with a miR-30a inhibitor could reverse the anti-autophagy effect of Sal B against I-R injury. Furthermore, we confirmed that Sal B has a protective role in miR-30a-mediated autophagy through the PI3K/Akt signaling pathway, which was abrogated by the PI3K inhibitor LY294002. CONCLUSIONS: These data suggest that miR-30a is involved in Sal B-mediated cardioprotection against I-R injury through the PI3K/Akt signaling pathway.

Ginsenoside Rg1 protects starving H9c2 cells by dissociation of Bcl-2-Beclin1 complex.

BACKGROUND: Autophagy can result in cellular adaptation, as well as cell survival or cell death. We investigated how ginsenoside Rg1(G-Rg1) regulates the relationship between autophagy and apoptosis induced by continuous starvation. METHODS: H9c2 cells under continuous starvation were treated with or without ginsenoside Rg1, and autophagy and apoptosis related proteins were assessed over a continuous time course by Western blot. Dynamic fluorescence intensity of green fluorescent protein (GFP)-LC3 was used to assess autophagosome formation by live cell imaging. Cyan fluorescent protein (CFP) -Beclin1(BECN1) and yellow fluorescent protein (YFP) -Bcl-2 were co-transfected into cells to observe ginsenoside Rg1 regulation of BECN1/Bcl-2 interaction using Fluorescence Resonance Energy Transfer (FRET). Immunoprecipitation was also used to assess BECN1/Bcl-2 interaction over a continuous time course. RESULTS: In H9c2 cells, starvation induced both apoptosis and autophagy. Cell apoptosis was significantly attenuated in ginsenoside Rg1-treated conditions, while autophagy was promoted. Ginsenoside Rg1 weakened the interaction between Beclin1 and Bcl-2, inhibiting apoptosis while promoting autophagy. Our results suggest that autophagy is beneficial to starved cardiac cells over a period of time. Furthermore, we describe the effect of ginsenoside Rg1 on the relationship between autophagy and apoptosis during starvation. CONCLUSIONS: Our findings provide valuable evidence for employing ginsenoside Rg1 as a specific promoter of autophagy and inhibitor of apoptosis.

Cardioprotection of recombinant human MG53 protein in a porcine model of ischemia and reperfusion injury.

Ischemic heart disease is a leading cause of death in human population and protection of myocardial infarction (MI) associated with ischemia-reperfusion (I/R) remains a challenge. MG53 is an essential component of the cell membrane repair machinery that protects injury to the myocardium. We investigated the therapeutic value of using the recombinant human MG53 (rhMG53) protein for treatment of MI. Using Langendorff perfusion of isolated mouse heart, we found that I/R caused injury to cardiomyocytes and release of endogenous MG53 into the extracellular solution. rhMG53 protein was applied to the perfusion solution concentrated at injury sites on cardiomyocytes to facilitate cardioprotection. With rodent models of I/R-induced MI, we established the in vivo dosing range for rhMG53 in cardioprotection. Using a porcine model of angioplasty-induced MI, the cardioprotective effect of rhMG53 was evaluated. Intravenous administration of rhMG53, either prior to or post-ischemia, reduced infarct size and troponin I release in the porcine model when examined at 24h post-reperfusion. Echocardiogram and histological analyses revealed that the protective effects of rhMG53 observed following acute MI led to long-term improvement in cardiac structure and function in the porcine model when examined at 4weeks post-operation. Our study supports the concept that rhMG53 could have potential therapeutic value for treatment of MI in human patients with ischemic heart diseases.

Berberine alleviates ox-LDL induced inflammatory factors by up-regulation of autophagy via AMPK/mTOR signaling pathway.

BACKGROUND: Inflammation induced by oxidized low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis. Recently, roles of autophagy against inflammation in the process of atherosclerosis have drawn increasing attention. Here, we tested the possible molecular mechanisms by which berberine confers an anti-inflammatory effect in macrophages by upregulation of autophagy. METHODS: J774A.1 macrophages were incubated with various doses of ox-LDL for various times. We evaluated the inflammatory factors and autophagy proteins (LC3II/LC3I, and SQSTM1/p62) to ascertain the optimal dose and time. Ox-LDL-induced inflammatory factors and autophagy in J774A.1 cells were tested by the AimPlex multiplex assay, Western blotting, confocal microscopy, and transmission electron microscopy in the presence of berberine or chloroquine (CQ). Adenosine 5'-monophosphate-activated protein kinase (AMPK) inhibitor compound C was used to evaluate the AMPK/mTOR signaling pathway. RESULTS: Berberine dose- and time-dependently reduced ox-LDL-induced inflammation and increased the ratio of LC3II/LC3I, and SQSTM1/p62 in J774A.1 cells. CQ significantly attenuated the berberine-induced autophagy and anti-inflammation. In addition, berberine increased the ratio of p-AMPK/AMPK and decreased the ratio of p-mTOR/mTOR. AMPK inhibitor compound C abolished berberine-induced autophagy and promoted p-mTOR/mTOR expression in J774A.1 cells. CONCLUSION: Berberine treatment inhibits inflammation in J774A.1 cells by inducing autophagy, which is mediated through activation of the AMPK/mTOR signaling pathway. Importantly, this study provides new insight into berberine's molecular mechanism and its therapeutic potential in the treatment of atherosclerosis.

Dasatinib enhances cisplatin sensitivity in human esophageal squamous cell carcinoma (ESCC) cells via suppression of PI3K/AKT and Stat3 pathways.

The clinical efficacy of cisplatin in esophageal squamous cell carcinoma (ESCC) treatment remains undesirable. Src, a non-receptor tyrosine kinase involved in multiple fields of tumorigenesis, recently has been indicated as a promising therapeutic target in the treatment of solid tumors including ESCC. However, whether inhibition of Src activity can increase cisplatin efficacy in ESCC cells remains unknown. The present study found that inhibition of Src by its inhibitor-dasatinib sensitized ESCC cells to cisplatin in vitro. Our data also suggest a likely mechanism for this synergy that dasatinib reduces expression of critical oncogenic members of the signaling pathways, such as AKT or Stat3, and cisplatin-resistant molecules, such as ERCC1 and BRCA1, under the control of Src. Furthermore, dasatinib could sensitize ESCC cells to another platin-based agent, carboplatin. Therefore, this study provides a potential target for improving cisplatin efficacy in ESCC therapy.

Amelioration of ischemia-reperfusion-induced muscle injury by the recombinant human MG53 protein.

INTRODUCTION: Ischemia-reperfusion injury (I-R) in skeletal muscle requires timely treatment. METHODS: Rodent models of I-R injury were used to test the efficacy of recombinant human MG53 (rhMG53) protein for protecting skeletal muscle. RESULTS: In a mouse I-R injury model, we found that mg53,-/- mice are more susceptible to I-R injury. rhMG53 applied intravenously to the wild-type mice protected I-R injured muscle, as demonstrated by reduced CK release and Evans blue staining. Histochemical studies confirmed beneficial effects of rhMG53. Of interest, rhMG53 did not protect against I-R injury in rat skeletal muscle. This was likely due to the fact that the plasma level of endogenous MG53 protein is high in rats. CONCLUSIONS: Our data suggest that rhMG53 may be a potential therapy for protection against muscle trauma. A mouse model appears to be a better choice than a rat model for evaluating potential treatments for protecting skeletal muscle.

Ginsenoside Rd promotes neurogenesis in rat brain after transient focal cerebral ischemia via activation of PI3K/Akt pathway.

AIM: To investigate the effects of ginsenoside Rd (Rd) on neurogenesis in rat brain after ischemia/reperfusion injury (IRI). METHODS: Male SD rats were subjected to transient middle cerebral artery occlusion (MCAO) followed by reperfusion. The rats were injected with Rd (1, 2.5, and 5 mg·kg(-1)·d(-1), ip) from d 1 to d 3 after MCAO, and with BrdU (50 mg·kg(-1)·d(-1), ip) from d 3 to d 6, then sacrificed on 7 d. The infarct size and neurological scores were assessed. Neurogenesis in the brains was detected by BrdU, DCX, Nestin, and GFAP immunohistochemistry staining. PC12 cells subjected to OGD/reperfusion were used as an in vitro model of brain ischemia. VEGF and BDNF levels were assessed with ELISA, and Akt and ERK phosphorylation was measured using Western blotting. RESULTS: Rd administration dose-dependently decreased the infarct size and neurological scores in the rats with IRI. The high dose of Rd 5 (mg·kg(-1)·d(-1)) significantly increased Akt phosphorylation in ipsilateral hemisphere, and markedly increased the number of BrdU/DCX and Nestin/GFAP double-positive cells in ischemic area, which was partially blocked by co-administration of the PI3 kinase inhibitor LY294002. Treatment with Rd (25, 50, and 100 µmol/L) during reperfusion significantly increased the expression of VEGF and BDNF in PC12 cells with IRI. Furthermore, treatment with Rd dose-dependently increased the phosphorylation of Akt and ERK, and significantly decreased PC12 cell apoptosis, which were blocked by co-application of LY294002. CONCLUSION: Rd not only attenuates ischemia/reperfusion injury in rat brain, but also promotes neurogenesis via increasing VEGF and BDNF expression and activating the PI3K/Akt and ERK1/2 pathways.

Involvement of oxidative stress in SAMP10 mice with age-related neurodegeneration.

Age-related changes in the brain tissue are reflected in many aspects. We sought to determine the morphology, Nissl bodies, behavioral appearance and oxidative stress in the brain using SAMP10 mice, a substrain of the senescence-accelerated mouse. SAMP10 mice groups divided by different ages (3, 5, 8 and 14 months) were compared with those of control groups with the above corresponding ages. Cortical thickness, Nissl bodies, behavioral appearance and oxidative stress were evaluated through image software, thionine staining, step-down test and colorimetry, respectively. The weight and cortical thickness of the brain in SAMP10 mice significantly reduced from 8 months of age. The results showed that the number of Nissl bodies decreased or Nissl bodies shrank with dark staining in histology. The same result appeared in a step-down test. As the SAMP10 mice grew older, the oxidative stress-related markers superoxide dismutase decreased and malondialdehyde increased after 8 months. Glutathione peroxidase activities showed no age-related changes. The changes of brain morphology and productions of oxidative stress in the brain tissue might contribute to the behavioral abnormality. Deceleration of age-related production of oxidative stress might be expected to be a potent strategy for anti-aging interventions.

[Effect of Guanmaitong Tablet on ERK and p38 Protein of TLR2 Pathway Expression in Cerebral Ischemia/Reperfusion Rats: an Experimental Study].

OBJECTIVE: To explore the inflammatory cascade mechanism through Toll like receptor 2 (TLR2) pathway after cerebral ischemia/reperfusion, and to study molecular mechanisms of Guanmaitong (GMT) Tablet for protecting brain damage. METHODS: We used bolt-line method to block/release the middle cerebral artery, causing cerebral ischemia/reperfusion (I/R) injury model. GMT Tablet was given by gastrogavage. Rats were then divided into the high dose GMT group (1200 mg/kg), the middle dose GMT group (600 mg/kg), the low dose GMT group (300 mg/kg), the positive control group (Tanakan, 20 mg/kg). Their right brain tissues were fixed in 10% neutral formalin. TLR2 expressions were detected by immunofluorescence staining. The total protein was extracted from right brain tissues by ultrasonica- tion. Expression levels of extracellular regulated protein kinases (ERK), phospho-extracellular regulated protein kinases (p-ERK), p38-mitogen activated protein kinases (p-ERK), phospho-p38-mitogen activated protein kinases [p-p38-MAPKs(p-p38)] were assessed by Western blot. Abdominal aortic blood was withdrawn. IL-6 and IL-1ß levels were detected by ELISA in brain tissues and serum. RESULTS: Compared with the sham-oepration group, expression levels of TLR2, ERK, p-ERK, p38, p-p38 protein were up-regulated (P < 0.05, P < 0.01), and contents of IL-6 and IL-1ß in brain tissues and serum were increased in the model group (P < 0.01). Expression levels of TLR2, ERK, p-ERK, p38, p-p38 were down-regulated (P < 0.05, P < 0.01), and contents of IL-6 and IL-1ß were reduced in brain tissues and serum in middle and high dose GMT groups (P < 0.05, P < 0.01). CONCLUSIONS: TLR2 pathway was involved in cerebral I/R injury. GMT protected neurons by down-regulating protein expressions of TLR2, ERK, p-ERK, p38, p-p38 and contents of IL-1ß and IL-6.