[Mechanism of n-butanol fraction of Wenxia Formula combining with gefitinib in treating non-small cell lung cancer based on network pharmacology and in vitro experiment].

This study combined network pharmacology, molecular docking, and in vitro experiments to explore the potential mechanism of the active components of the n-butanol fraction of Wenxia Formula(NWXF) combined with gefitinib(GEF) in treating non-small cell lung cancer(NSCLC). Ultra-performance liquid chromatography-quadrupole Orbitrap mass spectrometry(UPLC-Q-Orbitrap MS) was employed to detect the main chemical components of NWXF. The active components of NWXF were retrieved from SwissADME, and the candidate targets of these active components were retrieved from SwissTargetPrediction. Online Mendelian Inheritance in Man(OMIM) and GeneCards were searched for the targets of NSCLC. Cytoscape 3.9.0 and STRING were employed to build the protein-protein interaction(PPI) network with the common targets shared by NWXF and NSCLC. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment were performed in DAVID to predict the potential mechanisms. Finally, molecular docking between the main active ingredients and key targets was conducted in SYBYL-X 2.0. The methyl thiazolyl tetrazolium(MTT) assay was employed to evaluate the inhibitory effects of NWXF and/or GEF on the proliferation of human non-small cell lung cancer cells(A549 and PC-9). Additionally, the impact of NWXF on human embryonic lung fibroblast cells(MRC-5) was assessed. The effectiveness of the drug combination was evaluated based on the Q value. The terminal-deoxynucleoitidyl transferase mediated nick-end labeling(TUNEL) assay was employed to examine the apoptosis of A549 and PC-9 cells treated with NWXF and/or GEF. Quantitative real-time PCR(qRT-PCR) was employed to measure the mRNA levels of epidermal growth factor receptor(EGFR), c-Jun N-terminal kinase(JNK), and Bcl2-associated X protein(Bax) in the A549 and PC-9 cells treated with NWXF and/or GEF. Western blot was employed to determine the protein levels of EGFR, p-EGFR, JNK, p-JNK, and Bax in the A549 and PC-9 cells treated with NWXF and/or GEF. A total of 77 active components, 488 potential targets, and 49 key targets involved in the treatment of NSCLC with NWXF were predicted. The results of GO annotation showed that NWXF may treat NSCLC by regulating the biological processes such as cell proliferation, apoptosis, and protein phosphorylation. KEGG enrichment revealed that the key targets of NWXF in treating NSCLC were enriched in the mitogen-activated protein kinase(MAPK), phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT), hypoxia-inducible factor-1(HIF-1), and microRNA-related signaling pathways. Molecular docking results showed that 91.9% of the docking scores were greater than 5, indicating the strong binding capability between main active components and key targets. The cell experiments demonstrated that NWXF combined with GEF synergistically inhibited the proliferation, promoted the apoptosis, decreased p-EGFR/EGFR and p-JNK/JNK values, down-regulated the mRNA levels of EGFR and JNK, and up-regulated the mRNA and protein levels of Bax in A549 and PC-9 cells. In conclusion, NWXF combined with GEF can regulate the EGFR/JNK pathway to promote the apoptosis of NSCLC cells, thus treating NSCLC.

Pontibacterium sinense sp. nov., a nitrate-reducing and thiosulphate-oxidizing bacterium, isolated from coastal sediment.

A novel bacterial strain, N1Y112T, was isolated from coastal sediment collected in Weihai, PR China. This Gram-stain-negative, facultatively anaerobic, motile rod-shaped bacterium exhibited the ability to oxidize thiosulphate to sulphate and reduce nitrate to ammonia through its Sox system and nitrate reduction pathway, respectively. The strain grew at 20-35 °C (optimum, 28 °C), pH 6.0-10.0 (optimum, pH 7.5) and in the presence of 1.0-5.0 % (w/v) NaCl (optimum, 3.0 %). Major fatty acids present in the strain included summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c) and C16 : 0. Its polar lipid profile consisted of one phosphatidylethanolamine, two unknown aminolipids, one aminophosphoglycolipid, one diphosphatidylglycerol, one phosphatidylglycerol, two unknown phospholipids and two unknown lipids. Strain N1Y112T contained ubiquinone-7 and ubiquinone-8 as isoprenoid quinones, with a genomic G+C content of 50.6 mol%. Based on phylogenetic analysis, strain N1Y112T clustered with Pontibacterium granulatum JCM 30316T being its closest relative at 97.1 % 16S rRNA gene sequence similarity. The average nucleotide identity and digital DNA-DNA hybridization values were 77.1 and 20.7 %, respectively, which suggest significant differences between genomes of N1Y112T and P. granulatum JCM 30316T. Based on the findings from its phenotypic, genotypic and phylogenetic analyses, N1Y112T is considered to represent a novel species of the genus Pontibacterium, for which the name Pontibacterium sinense sp. nov. is proposed. The type strain is N1Y112T (=KCTC 72927T=MCCC 1H00429T).

Costunolide alleviates atherosclerosis in high-fat diet-fed ApoE-/- mice through covalently binding to IKKß and inhibiting NF-κB-mediated inflammation.

Costunolide (CTD) is a sesquiterpene lactone isolated from costus root and exhibits various biological activities including anti-inflammation. Since atherosclerosis is a chronic inflammatory disease, we herein investigated the anti-atherosclerotic effects of CTD and the underlying mechanism. Atherosclerosis was induced in ApoE-/- mice by feeding them with a high-fat diet (HFD) for 8 weeks, followed by administration of CTD (10, 20 mg ·kg-1·d-1, i.g.) for 8 weeks. We showed that CTD administration dose-dependently alleviated atherosclerosis in HFD-fed ApoE-/- mice. Furthermore, we found that CTD dose-dependently reduced inflammatory responses in aortas of the mice, as CTD prevented infiltration of inflammatory cells in aortas and attenuated oxLDL uptake in macrophages, leading to reduced expression of pro-inflammatory and pro-fibrotic molecules in aortas. Similar results were observed in oxLDL-stimulated mouse primary peritoneal macrophages (MPMs) in vitro. We showed that pretreatment with CTD (2.5, 5. 10 µM) restrained oxLDL-induced inflammatory responses in MPMs by blocking pro-inflammatory NF-κB/p65 signaling pathway. We further demonstrated that CTD inactivated NF-κB via covalent binding to cysteine 179 on IKKß, a canonical upstream regulator of NF-κB, reducing its phosphorylation and leading to conformational change in the active loop of IKKß. Our results discover IKKß as the target of CTD for its anti-inflammatory activity and elucidate a molecular mechanism underlying the anti-atherosclerosis effect of CTD. CTD is a potentially therapeutic candidate for retarding inflammatory atherosclerotic diseases.

Effects of ketoconazole, voriconazole, and itraconazole on the pharmacokinetics of apatinib in rats.

We investigated the effect of azole antifungal drugs (ketoconazole, voriconazole, and itraconazole) on the pharmacokinetics of apatinib in rats. The rats in ketoconazole, voriconazole, and itraconazole groups received single-dose apatinib 30 mg/kg after the oral administration of ketoconazole, voriconazole, and itraconazole, respectively. Co-administration of ketoconazole or voriconazole significantly increased the apatinib Cmax and AUC(0-t) and decreased the clearance. Co-administration of itraconazole did not significantly affect the pharmacokinetics parameters of apatinib. It could be concluded that both ketoconazole and voriconazole significantly increase the exposure of apatinib, and affect the pharmacokinetics of apatinib in rat. Apatinib can be co-administered with itraconazole, but ketoconazole and voriconazole should be avoided if possible or be underwent therapeutic drug monitoring of apatinib. A further clinical study should be conducted to investigate the inhibitory effect of azole antifungal drugs on the apatinib plasma concentration.

Cerebrospinal fluid neuron specific enolase, interleukin-1ß and erythropoietin concentrations in children after seizures.

PURPOSE: In the present study, the levels of neuron-specific enolase (NSE), interleukin-1ß (IL-1ß), and erythropoietin (EPO) in cerebrospinal fluid (CSF) in children with idiopathic epilepsy were measured to illuminate the relationships between these markers with idiopathic epilepsy. METHODS: Eighty-five children from 6 months to 12.5 years of age with single, previously undiagnosed, and untreated idiopathic epilepsy were participated in this study. The concentrations of CSF NSE, 1L-1ß, and EPO were measured by specific ELISA methods. RESULTS: The mean concentrations of CSF NSE, IL-1ß, and EPO in the epileptic groups showed a significant increase (P < 0.01) compared with those in the control groups. Besides, the mutual correlations of NSE, 1L-1ß, and EPO were also analyzed. Results showed that there were positive correlations between the levels of IL-1ß, NSE, and EPO. CONCLUSIONS: The changes of NSE, 1L-1ß, and EPO level in CSF may be beneficial for the pathophysiology study of epileptic seizures and the identification and diagnosis of a seizure clinically.

Effect of Evodiamine on CYP Enzymes in Rats by a Cocktail Method.

The aim of this study was to assess the influence of evodiamine on the activities of the drug-metabolizing enzymes cytochrome P450 (CYP) 1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in rats. The activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were measured using specific probe drugs. After pretreatment for 1 week with evodiamine or physiological saline (control group) by oral administration, probe drugs phenacetin (5.0 mg/kg; CYP1A2 activity), tolbutamide (1.0 mg/kg; CYP2C9 activity), omeprazole (10 mg/kg; CYP2C19 activity), metoprolol (20 mg/kg; CYP2D6 activity) and midazolam (10 mg/kg; CYP3A4 activity) were administered to rats by oral administration. The blood was then collected at different times for ultra-performance liquid chromatography-tandem mass spectrometry analysis. The data showed that evodiamine exhibits an inhibitory effect on CYP1A2, CYP2C9 and CYP2D6 by increasing t(1/2), Cmax and AUC(0-∞), and decreasing CL/F compared with those of the control group. However, no significant changes in CYP2C19 and CYP3A4 activities were observed. In conclusion, the results indicated that evodiamine could inhibit CYP1A2, CYP2C9 and CYP2D6, which may affect the disposition of medicines primarily dependent on these pathways. Our work may be the basis of related herb-drug interactions in the clinic.

Inhibitory Effect of Apigenin on Losartan Metabolism and CYP2C9 Activity in vitro.

BACKGROUND: CYP2C9 is one of the most important phase I drug-metabolizing enzymes in liver. The objective of this work was to investigate the effects of apigenin on the metabolism of losartan and human CYP2C9 and rat CYP2C11 activity in vitro. METHODS: Different concentrations of apigenin were added to a 100 mmol/l Tris-HCl reaction mixture containing 2 pmol/ml recombinant human CYP2C9.1, 0.25 mg/ml human liver microsomes or 0.5 mg/ml rat liver microsomes to determine the half maximal inhibition or a half-maximal inhibitory concentration (IC50) on the metabolism of losartan. In addition, diclofenac used as CYP2C9 substrate was performed to determine the effects of apigenin on CYP2C9. RESULTS: The results showed that apigenin has the inhibitory effect on the metabolism of losartan in vitro, the IC50 was 7.61, 4.10 and 11.07 µmol/l on recombinant CYP2C9 microsomes, human liver microsomes and rat liver microsomes, respectively. Meanwhile, apigenin's mode of action on human CYP2C9 activity was competitive for the substrate diclofenac. In contrast to its potent inhibition of CYP2C9 in humans (9.51 µmol/l), apigenin had lesser effects on CYP2C11 in rat (IC50 = 15.51 µmol/l). CONCLUSION: The observations imply that apigenin has the inhibitory effect on the metabolism of losartan and CYP2C9 activity in vitro. More attention should be paid as to when losartan should be administrated combined with apigenin.

The CYP3A inducer dexamethasone affects the pharmacokinetics of sunitinib by accelerating its metabolism in rats.

Sunitinib, a novel anti-tumor small molecule targeting VEGFR, is prescribed for advanced RCC and GISTs. Sunitinib is primarily metabolized by the CYP3A enzyme. It is well-known that dexamethasone serves as a potent inducer of this enzyme system. Nonetheless, the effect of dexamethasone on sunitinib metabolism remains unclear. This study examined the effect of dexamethasone on the pharmacokinetics of sunitinib and its metabolite N-desethyl sunitinib in rats. The plasma levels of both compounds were measured using UHPLC-MS/MS. Pharmacokinetic parameters and metabolite ratio values were calculated. Compare to control group, the low-dose dexamethasone group and high-dose dexamethasone group decreased the AUC(0-t) values of sunitinib by 47 % and 45 %, respectively. Meanwhile, the AUC(0-t) values of N-desethyl sunitinib were increased by 2.2-fold and 2.4-fold in low-dose dexamethasone group and high-dose dexamethasone group, respectively. The CL values for sunitinib were both approximately 45 % higher in the two dexamethasone groups. Remarkably, metabolite ratio values increased over 5-fold in both low-dose dexamethasone group and high-dose dexamethasone group, indicating a significant enhancement of sunitinib metabolism by dexamethasone. Moreover, the total levels of sunitinib and its metabolite are also significantly increased. The impact of interactions on sunitinib metabolism, as observed with CYP3A inducers such as dexamethasone, is a crucial consideration for clinical practice. To optimize the dosage and prevent adverse drug events, therapeutic drug monitoring can be employed to avoid the toxicity from such interactions.

Effects of CYP3A inhibitors ketoconazole, voriconazole, and itraconazole on the pharmacokinetics of sunitinib and its main metabolite in rats.

Sunitinib is a small molecule inhibitor of multiple receptor tyrosine kinases such as platelet derived growth factor receptor, vascular endothelial growth factor receptor, kit receptor and other receptors. The US Food and Drug Administration (FDA) has approved sunitinib for the treatment of advanced renal cell carcinoma and gastrointestinal stromal tumors. It has been reported that sunitinib was mainly metabolized by CYP3A but its pharmacokinetic interactions have not been revealed. In this study, we investigated whether CYP3A inhibitors (ketoconazole, voriconazole, and itraconazole) could influence the pharmacokinetics of sunitinib and its equipotent metabolite N-desethyl sunitinib in a drug-drug interaction study in Sprague Dawley (SD) rats. The results showed that ketoconazole and voriconazole significantly increased the exposure of sunitinib, decreased the exposure of N-desethyl sunitinib, and inhibited the metabolism of sunitinib in rats. However, itraconazole showed only a weak effect on pharmacokinetics and metabolism. Coadministration of sunitinib with ketoconazole and voriconazole should be avoided if possible or if not, there should be therapeutic drug monitoring of the levels of sunitinib and N-desethyl sunitinib. Therefore, drug-drug interaction should be considered when sunitinib is administered in conjunction with CYP3A inhibitors, which might lead to toxicity.

[Comparison of preoperative T staging by oral contrast enhanced ultrasonography and double contrast enhanced ultrasonography in advanced gastric carcinoma].

OBJECTIVE: To compare the accuracy of preoperative T staging of gastric cancer by oral and intravenous contrast-enhanced gastric ultrasonography. METHODS: One hundred and forty three patients who had been diagnosed as gastric cancer by endoscopic biopsy and confirmed by pathology after operation were examined by oral and intravenous contrast-enhanced gastric ultrasonography, and they were divided into satisfied group and non-satisfied group according to the 2-D image quality of lesion. The results were compared with postoperative pathologic findings. RESULTS: All the patients with gastric cancer presented regional gastric wall thickening. Among them, 117 cases were clearly presented with good image quality. The remaining 26 cases were presented with vague profile, the ulcerative surface of lesion was filled with hyperechogenicity combined with rear shadow. The accuracy of oral contrast-enhanced ultrasonography in determining the T stage of gastric cancer was 74.1%. The accuracy in satisfied group and non-satisfied group was 78.6% and 53.8%, respectively. The enhancement pattern of 143 cases was showed as hyperenhancement during the arterial phase and hypoenhancement during the portal phase in DCUS. The accuracy of double contrast-enhanced ultrasongraphy in determining the T stage of gastric cancer was 86.7%, but the accuracy in satisfied group and non-satisfied group was 88.9% and 76.9%, respectively. There was a significant difference between the two methods (χ(2) = 9.031, P < 0.01). CONCLUSION: DCUS is more accurate than oral contrast-enhanced ultrasonography as a useful diagnostic method for preoperative T staging of gastric cancer.

Development and Validation of UHPLC-MS/MS Assay for Therapeutic Drug Monitoring of High-dose Methotrexate in Children with Acute Lymphoblastic Leukemia.

PURPOSE: Precise and timely detection of methotrexate (MTX) concentration played a key role in high-dose MTX individualization therapy in acute lymphoblastic leukemia (ALL) children to avoid serious adverse effects or nonresponse. This report described a sensibility and validation of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for therapeutic drug monitoring (TDM) of methotrexate concentration in children's plasma. METHODS: One-step protein precipitation of samples was accomplished by adding 200 µL of acetonitrile to 100 µL of plasma sample. The separation of plasma samples was carried out on a ZORBAX Eclipse Plus C18 Rapid Resolution HD column with gradient elution using a mobile phase constituted of acetonitrile and 1% formic acid. The detection was executed by electrospray ionization (ESI) of triple quadrupole tandem mass spectrometer (TQMS) in the multiple reaction monitoring (MRM) mode with the transitions m/z 455.2 → 307.9 for methotrexate and m/z 458.2 → 311.2 for IS, separately. Linear concentration range of the calibration curve was 44-11,000 nmol/L and 44 nmol/L was the lower limit of quantification. RESULTS: The methotrexate elution time was at 1.577 min, and the overall running time was only 3.3 min. The intra- and interday precision for all the analysis results was within 11.24%, and mean recoveries rate of methotrexate exceeded 87.98%. CONCLUSION: The described and fully validated UHPLC-MS/MS method was successfully applied in clinical TDM after infusion of high-dose methotrexate 1-5 g/m2 to 41 childpatients.

Effect of Wenxia Changfu Formula Combined With Cisplatin Reversing Non-Small Cell Lung Cancer Cell Adhesion-Mediated Drug Resistance.

Non-small cell lung cancer (NSCLC), the major form of primary lung cancer, is a common cause of cancer-related death worldwide. Cell adhesion-mediated drug resistance (CAM-DR), a form of chemotherapy resistance, has been reported to confer resistance to various chemotherapeutic agents. Integrin ß1 signaling plays an important role in CAM-DR and has been proposed as a potential target for NSCLC. Wenxia Changfu Formula (WCF) is a Traditional Chinese Compound Prescription for the intervention treatment of NSCLC combined with cisplatin (DDP). This study aims to investigate the effect and mechanism of WCF combined with DDP in reversing CAM-DR. Firstly, the chemical profile of WCF was characterized by UPLC/Q-TOF-MS analysis. A total of 237 compounds with mzCloud Best Match of greater than 70 were identified by using the online database mzCloud. Secondly, we established A549 three-dimensional(3D) cells cultured in vitro and nude mice xenografts models of the A549 cell line with Integrin ß1 overexpression. In vitro, the cell viability, migration and adhesion were measured though MTT Assay, Wound Healing Assay and Cell Adhesion Assay, the Integrin ß1 expression of the A549 cells was assessed through immunocytochemistry; in vitro, the transplanted tumor morphology and the colocalization of Integrin ß1 and its ligands were tested by HE staining and immunofluorescence. As a result, we found that the combination effectively reduced cell viability, suppressed migration and adhesion, and downregulated the protein level of Integrin ß1 in three-dimensional cultured A549 cells. And the combination of WCF with DDP significantly inhibited tumor growth, increased organelle vacuolations and decreased colocalization of Integrin ß1 and its ligands including fibulin-2 and laminin. Taken together, our results confirm that the combination of WCF with DDP could reverse the lung cancer CAM-DR through the Integrin ß1 signaling pathway.

A UHPLC-MS/MS method coupled with liquid-liquid extraction for the quantitation of phenacetin, omeprazole, metoprolol, midazolam and their metabolites in rat plasma and its application to the study of four CYP450 activities.

Drug-drug interactions (DDIs) are thought to be associated with the inhibition of cytochrome P450 activities. The cocktail method with analysis of the metabolism of two or more probe drugs is used to determine CYP450 activities. In this study, we established a UHPLC-MS/MS method for simultaneous quantitation of four CYP450 probe drugs (phenacetin, omeprazole, metoprolol and midazolam) and their metabolites (acetaminophen, 5'-hydroxy omeprazole, α-hydroxy metoprolol and 1'-hydroxy midazolam) in rat plasma. Sample preparation by plasma protein precipitation was combined with a liquid-liquid extraction method. The separation was carried out on a ZORBAX Eclipse Plus C18 Rapid Resolution High Definition column with a gradient elution, using water containing 0.1% formic acid (A) and acetonitrile (B) in a run time of only 3.0 min. Detection was conducted with a 6420 series triple-quadrupole tandem mass spectrometer, using ESI in positive ion mode with multiple reaction monitoring (MRM). The calibration curves were linear over the concentration range 10-5000 ng/mL for phenacetin, omeprazole, metoprolol and midazolam, and 1-500 ng/mL for their metabolites. Intra- and inter-day precisions were within 15%, and the accuracies were in the range of 87-112%. The method was successfully applied to the pharmacokinetic study of probe drugs/metabolites and DDIs with 3-n-butylphthalide (NBP) after administration of a single oral dose of phenacetin, omeprazole, metoprolol and midazolam in rats.

Determination of Anlotinib, a Tyrosine Kinase Inhibitor, in Rat Plasma by UHPLC-MS/MS and Its Application to a Pharmacokinetic Study.

Anlotinib is a novel inhibitor of receptor kinase tyrosine with multitargets and has a broad spectrum of inhibitory action on tumor angiogenesis and growth. A simple and rapid UHPLC-MS/MS bioanalytical method was validated for the determination of anlotinib in rat plasma, using imatinib as an internal standard. An Acquity BEH C18 column was used to separate analytes. The eluents consisted of formic acid/water (0.1 : 100, v/v) and acetonitrile with a mobile phase. A triple quadrupole mass spectrometer was operated for the quantification with multiple reaction monitoring (MRM) to determine transitions: 408.2 ⟶ 339.1 for anlotinib, and 494.3 ⟶ 394.1 for imatinib. The validated range was 0.1-50 ng/mL for anlotinib. Mean recovery rate of anlotinib in plasma was ≥99.32% and reproducible. Also, the intra- and interday precisions were both below 15%. This robust method was successfully applied to support the pharmacokinetic study of anlotinib in rats.

Determination and pharmacokinetic study of pirfenidone in rat plasma by UPLC-MS/MS.

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of pirfenidone in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0 min and the elution of pirfenidone was at 1.39 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 186.2→92.1 for pirfenidone and m/z 237.1→194.2 for carbamazepine (IS), respectively. The calibration curve was linear over the range of 5-2000 ng/mL with a lower limit of quantitation (LLOQ) of 5 ng/mL. Mean recovery of pirfenidone in plasma was in the range of 80.4-84.3%. Intra-day and inter-day precision were both <12.1%. This method was successfully applied in pharmacokinetic study after oral administration of 10.0mg/kg pirfenidone in rats.

NGF accelerates cutaneous wound healing by promoting the migration of dermal fibroblasts via the PI3K/Akt-Rac1-JNK and ERK pathways.

As a well-known neurotrophic factor, nerve growth factor (NGF) has also been extensively recognized for its acceleration of healing in cutaneous wounds in both animal models and randomized clinical trials. However, the underlying mechanisms accounting for the therapeutic effect of NGF on skin wounds are not fully understood. NGF treatment significantly accelerated the rate of wound healing by promoting wound reepithelialization, the formation of granulation tissue, and collagen production. To explore the possible mechanisms of this process, the expression levels of CD68, VEGF, PCNA, and TGF-ß1 in wounds were detected by immunohistochemical staining. The levels of these proteins were all significantly raised in NGF-treated wounds compared to untreated controls. NGF also significantly promoted the migration, but not the proliferation, of dermal fibroblasts. NGF induced a remarkable increase in the activity of PI3K/Akt, JNK, ERK, and Rac1, and blockade with their specific inhibitors significantly impaired the NGF-induced migration. In conclusion, NGF significantly accelerated the healing of skin excisional wounds in rats and the fibroblast migration induced by NGF may contribute to this healing process. The activation of PI3K/Akt, Rac1, JNK, and ERK were all involved in the regulation of NGF-induced fibroblast migration.

Biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate by NADH-dependent reductase from E. coli CCZU-Y10 discovered by genome data mining using mannitol as cosubstrate.

The reductase (PgCR) from recombinant Escherichia coli CCZU-Y10 displayed high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). To efficiently synthesize (S)-CHBE (>99 % enantiomeric excess (ee)), the highly stereoselective bioreduction of COBE into (S)-CHBE with the whole cells of E. coli CCZU-Y10 was successfully demonstrated in a dibutyl phthalate-water biphasic system. The appropriate ratio of the organic phase to water phase was 1:1 (v/v). The optimum reaction temperature, reaction pH, cosubstrate, NAD(+), and cell dosage of the biotransformation of 100 mM COBE in this biphasic system were 30 °C, 7.0, mannitol (2.5 mmol/mmol COBE), 0.1 µmol/(mmol COBE), and 0.1 g (wet weight)/mL, respectively. Moreover, COBE at a high concentration of (1,000 mM) could be asymmetrically reduced to (S)-CHBE in a high yield (99.0 %) and high enantiometric excess value (>99 % ee). Significantly, E. coli CCZU-Y10 shows high potential in the industrial production of (S)-CHBE (>99 % ee).

[Effect of ligustrazine on expression of Fas/FasL in pulmonary injury induced by ischemia/reperfusion in rabbits].

AIM: To investigate the effect of ligustrazine (LGT) on expression of Fas/FasL mRNA during pulmonary ischemia/reperfusion injury (PI/RI) in the rabbits. METHODS: Single lung ischemia/reperfusion animal model was used in this study. The rabbits were randomly divided into three groups (n = 30, in each): sham operated group (Sham), I/R group (I/R) and I/R + LGT group (I/R + LGT). Changes of several parameters which included apoptotic index (AI), wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA) were measured at 1h, 3h, 5h after reperfusion in lung tissue. Meanwhile the location and expression of Fas/FasL mRNA were observed. Lung tissue was prepared for light microscopic and electron microscopic ob servation at 1 h, 3 h, 5 h after reperfusion. RESULTS: As compared with group I/R, Fas/FasL mRNA slightly expressed in intima and extima of small pulmonary artery, alveoli, and bronchiole epithelia in group LGT. The values of AI, W/D and IQA showed significantly lower in group I/R + LGT than that in group I/R at 1 h, 3 h, 5 h after reperfusion in lung tissue (P < 0.01 and P < 0.05). Meanwhile, abnormal changes of the lung tissue in morphologically were lessen markedly in group I/R + LGT. CONCLUSION: Ligustrazine has notable protective effects on PI/RI in rabbits by inhibiting Fas/FasL mRNA express in lung tissue and decreasing apoptosis.