OBJECTIVETo explore the neuroprotective effect and mechanism of picroside II on extracellular regulated protein kinases1/2 (ERK1/2) signal transduction pathway in cerebral ischemia injuryrats. METHODS The middle cerebral artery occlusion (MCAO) model was established by inserting a monofilament into middle cerebral artery. Totally 96 successfully modeled Wistar rats were divided into the modelgroup, the treatment (picroside II) group, the Lipopolysachcaride (LPS) group, and the U0126 group according to random digit table. Each group was further divided into 3 subgroups, i.e. 6, 12, and 24 h sub-groups. Picroside II (20 mg/kg) was peritoneally injected to rats in the treatment group 2 h after ischemia.LPS (20 mg/kg) and Picroside II (20 mg/kg) were peritoneally injected to rats in the LPS group 2 h after ischemia. U0126-EtOH (20 mg/kg)and Picroside II (20 mg/kg) were peritoneally injected to rats in the U0126group 2 h after ischemia. Equal volume of normal saline was peritoneally injected to rats in the control groupand the model group. The neurobehavioral function was evaluated by modified neurological severity score(mNSS) test. The structure of neurons was observed using hematoxylin-eosinstaining (HE) staining. Theapoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression of phosphorylated extracellular signal-regulated protein kinase1,2 (pERK1,2) in cortex was detected using immunohistochemistry (IHC) and Western blot.

RESULTSAfter cerebral ischemia injury neurological impairment score increased, the damage of neuron in the cortical area was aggravated, apoptotic cells increased in the model group as time went by. The expression of pERK1/2 increased more significantly in the model group than in the control group (P <0.05). The damage of neuron in the cortical area was milder, while apoptotic cells decreased, the expression of pERK1f2 obviously decreased more in the treatment group and the U0126 group (P < 0.05). The early damage of neuron in the cortical area was more severe, apoptotic cells and the expression of pERK12 were comparatively higher in early stage of the LPS group, but the expression of pERK1/2 was somewhat decreased in late stage.

CONCLUSIONSActivating ERK12 pathway could mediate apoptosis and inflammatory reactions of neurons after cerebral ischemia injury. Picroside II could protect the nerve system possibly through reducing activation of ERKI2 pathway, inhibiting apoptosis of neurons and inflammation reaction.

Animals ; Apoptosis ; Brain Ischemia ; drug therapy ; Cinnamates ; pharmacology ; Infarction, Middle Cerebral Artery ; drug therapy ; Iridoid Glucosides ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Neurons ; pathology ; Neuroprotective Agents ; pharmacology ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effects of berberine (BBR) and cinnamic acid (CA), the main active components in Jiaotai Pill (, JTP), on palmitic acid (PA)-induced intracellular triglyceride (TG) accumulation in NIT-1 pancreatic β cells.

METHODSCells were incubated in culture medium containing PA (0.25 mmol/L) for 24 h. Then treatments with BBR (10 μmol/L), CA (100 μmol/L) and the combination of BBR and CA (BBR+CA) were performed respectively. Intracellular lipid accumulation was assessed by Oil Red O staining and TG content was measured by colorimetric assay. The expression of adenosine monophosphate-activated protein kinase (AMPK) protein and its downstream lipogenic and fatty acid oxidation genes, including fatty acid synthase (FAS), acetyl-coA carboxylase (ACC), phosphorylation acetyl-coA carboxylase (pACC), carnitine acyl transferase 1 (CPT-1) and sterol regulating element binding protein 1c (SREBP-1c) were determined by Western blot or real time polymerase chain reaction.

RESULTSPA induced an obvious lipid accumulation and a significant increase in intracellular TG content in NIT-1 cells. PA also induced a remarkable decrease in AMPK protein expression and its downstream targets such as pACC and CPT-1. Meanwhile, AMPK downstream lipogenic genes including SREBP-1c mRNA, FAS and ACC protein expressions were increased. Treatments with BBR and BBR+CA, superior to CA, significantly reversed the above genes changes in NIT-1 pancreatic β cells. However, the synergistic effect of BBR and CA on intracellular TG content was not observed in the present study.

CONCLUSIONIt can be concluded that in vitro, BBR and BBR+CA could inhibit PA-induced lipid accumulation by decreasing lipogenesis and increasing lipid oxidation in NIT-1 pancreatic β cells.

AMP-Activated Protein Kinases ; metabolism ; Animals ; Berberine ; chemistry ; pharmacology ; Cell Line ; Cinnamates ; chemistry ; pharmacology ; Fatty Acids ; metabolism ; Gene Expression Regulation ; drug effects ; Insulin-Secreting Cells ; drug effects ; metabolism ; Intracellular Space ; metabolism ; Lipogenesis ; drug effects ; genetics ; Mice ; Oxidation-Reduction ; drug effects ; Palmitic Acid ; toxicity ; Triglycerides ; metabolism

The difference between three representative components of total salvianolic acids in pharmacodynamic activity were compared by three different pharmacological experiments: HUVECs oxidative damage experiment, 4 items of blood coagulation in vitro experiment in rabbits and experimental myocardial ischemia in rats. And the effects of contribution rate of each component were calculated by multi index comprehensive evaluation method based on CRITIC weights. The contribution rates of salvianolic acid B, rosmarinic acid and Danshensu were 28.85%, 30.11%, 41.04%. Apparent oil/water partition coefficient of each representative components of total salvianolic acids in n-octyl alcohol-buffer was tested and the total salvianolic acid components were characterized based on a combination of the approach of self-defined weighting coefficient with effects of contribution rate. Apparent oil/water partition coefficient of total salvianolic acids was 0.32, 1.06, 0.89, 0.98, 0.90, 0.13, 0.02, 0.20, 0.56 when in octanol-water/pH 1.2 dilute hydrochloric acid solution/ pH 2.0, 2.5, 5.0, 5.8, 6.8, 7.4, 7.8 phosphate buffer solution. It provides a certain reference for the characterization of components.
Animals ; Benzofurans ; chemistry ; pharmacology ; Cinnamates ; chemistry ; pharmacology ; Depsides ; chemistry ; pharmacology ; Lactates ; chemistry ; pharmacology ; Male ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Solubility

OBJECTIVETo study the protective effect of rosmarinic acid (Ros A) on the primary cardiomyocyte hypoxia/reoxygenation (H/R) injury.

METHODPrimary cardiomyocytes of rats were cultured in vitro to establish the H/R injury of cardiomyocytes and observe the changes in the cell viability and LDH leakage. The changes in ATP content and ROS in cardiomyocytes were measured by using chemiluminescence and fluorescent probe technique. The effects of rosmarinic acid on the apoptosis of cardiomyocytes, cleaved-caspase 3, Akt and p-Akt protein expression were further detected by flow cytometry and western blot analysis.

RESULTAccording to the experimental results, Ros A at doses of 25, 50, 100 mg x L(-1) could inhibit the decrease in H/R-induced cell viability, LDH leakage and excessive ROS generation, and maintain the ATP level in cells. Ros A at doses of 50, 100 mg x L(-1) could remarkably inhibit the H/R-induced cardiomyocyte apoptosis and down-regulate the expression of cleaved caspase-3. Moreover, Ros A at doses of 100 mg x L(-1) could significantly up-regulate the expression of p-Akt.

CONCLUSIONRos A has the significant effect in resisting the cardiomyocyte H/R injury, improve cardiomyocyte energy metabolism and reduce cell apoptosis, which is related to the activation of Akt pathway.

Adenosine Triphosphate ; metabolism ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Hypoxia ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Cinnamates ; pharmacology ; Depsides ; pharmacology ; Humans ; Hypoxia ; genetics ; metabolism ; physiopathology ; prevention & control ; Male ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Oxygen ; metabolism ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Rosmarinus ; chemistry

A new flavonone, named as (2R, 3S)-pinobanksin-3-cinnamate(1), together with six known compounds, pinocem-brin (2), pinobanksin (3), 3-O-acetylpinobanksin (4), galangin (5), kumatakenin(6), and 3-methylkaempferol (7), were isolated from a 95% ethanol extract of seeds of Alpinia katsumadai through a combination of various chromatographic techniques, including silica gel and Sephadex LH-20. The structure of compound 1 was elucidated by spectroscopic data analysis. Compound 1 exhibits a potent neuroprotective effect against the corticosterone-damaged PC12 cells, which may be underlying the effect by scavenging intracellular ROS.
Alpinia ; chemistry ; Animals ; Cell Death ; drug effects ; Cholestenones ; chemistry ; isolation & purification ; pharmacology ; Cinnamates ; chemistry ; isolation & purification ; pharmacology ; DNA Fragmentation ; drug effects ; Neuroprotective Agents ; chemistry ; isolation & purification ; pharmacology ; Oxidative Stress ; drug effects ; PC12 Cells ; Rats ; Reactive Oxygen Species ; metabolism ; Seeds ; chemistry

OBJECTIVETo construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system.

METHODThe 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI.

RESULTPlant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%.

CONCLUSIONGenetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

Anti-Bacterial Agents ; pharmacology ; Biomarkers ; Cinnamates ; pharmacology ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hygromycin B ; analogs & derivatives ; pharmacology ; Mannose-6-Phosphate Isomerase ; genetics ; metabolism ; Plants, Genetically Modified ; drug effects ; genetics ; metabolism ; Salvia miltiorrhiza ; drug effects ; genetics ; metabolism ; Transformation, Genetic

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.
Acetylation ; Animals ; Blastocyst ; cytology ; Cell Nucleus ; metabolism ; Cinnamates ; pharmacology ; Embryo, Mammalian ; drug effects ; metabolism ; Embryonic Development ; drug effects ; Epigenesis, Genetic ; Female ; Gene Expression ; Histone Deacetylase Inhibitors ; pharmacology ; Histones ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; In Vitro Techniques ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Nuclear Transfer Techniques ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Swine

A significant number of organic carboxylic acids have been shown to influence the absorption and distribution of drugs mediated by organic anion transporters (OATs). In this study, uptake experiments were performed to assess the inhibitory effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1, hOAT3, hOATP1B1, and hOATP2B1. After a drug-drug interaction (DDI) investigation, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were found and validated to inhibit hOAT1 in a competitive manner, and deoxycholic acid was found to be an inhibitor of all four transporters. The apparent 50% inhibitory concentrations of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were estimated to be 133.87, 3.69, 90.03 and 6.03 μmol·L(-1) for hOAT1, respectively. The apparent 50% inhibitory concentrations of deoxycholic acid were estimated to be 9.57 μmol·L(-1) for hOAT3, 70.54 μmol·L(-1) for hOATP1B1, and 168.27 μmol·L(-1) for hOATP2B1. Because cinnamic acid, ferulic acid, and cynarin are ingredients of food or food additives, the present study suggests there are new food-drug interactions to be disclosed. In addition, deoxycholic acid may be used as a probe for studying the correlation of OATs and OATPs.
Carboxylic Acids ; pharmacology ; Cinnamates ; pharmacology ; Coumaric Acids ; pharmacology ; Deoxycholic Acid ; pharmacology ; Diet ; Drug Interactions ; HEK293 Cells ; Humans ; Organic Anion Transport Protein 1 ; antagonists & inhibitors ; Organic Anion Transporters ; antagonists & inhibitors ; Plant Extracts ; pharmacology ; Plants, Medicinal ; chemistry

We studied the influence of the concentration of Ca2+ (0-50 mmol/L) in culture medium on the synthesis of rosmarinic acid (RA) and related enzymes in Salvia miltiorrhiza suspension cultures. Using verpamil (VP, a calcium channel antagonist) and ionophore A23187, we studied the mechanism of secondary metabolites of Salvia miltiorrhiza suspension cultures influenced by the concentration of Ca2+ in the culture medium. The synthesis of intracellular RA in 6-day incubation was significantly dependent on the medium Ca2+ concentration. At the optimal Ca2+ concentration of 10 mmol/L, a maximal RA content of 20.149 mg/g biomass dry weight was reached, which was about 37.3% and 20.4% higher than that at Ca2+ concentrations of 1 and 3 mmol/L, respectively. The variation of the activity of PAL and TAT, two key enzymes of the two branches of RA, could be affected by the concentration of Ca2+ in culture medium. The change of their activity occurred prior to the accumulation of RA, which suggested both of the key enzymes be involved in the synthesis of RA. Meanwhile, the enzymatic action of PAL was more distinct than TAT. The treatment of VP and A23187, respectively, indicated that the influence of RA affected by the concentration of Ca2+ in the culture medium was accomplished by the intracellular Ca2+, and the flow of Ca2+ from the extracellular to the intracellular environment could also participate in this process.
Calcium ; pharmacology ; Cinnamates ; metabolism ; Culture Media ; Culture Techniques ; methods ; Depsides ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Salvia miltiorrhiza ; chemistry ; enzymology ; growth & development ; Tyrosine Transaminase ; metabolism

Rosmarinic acid (RA), a phenolic acid, is one of the important secondary metabolites produced in Salvia miltiorrhiza. To observe the influence of salicylic acid (SA), an elicitor, on the synthesis of RA and related enzymes, we treated the cell suspension cultures of S. miltiorrhiza with SA and L-a-aminooxy-beta-phenylpropionic acid (AOPP), a competitive inhibitor of tyrosine aminotransferase (TAT). Under this condition, the activities of related enzymes, such as phenylalanine ammonia-lyase and TAT were traced and assayed; the accumulative amount of RA was measured. The results showed that the PAL activity reached the peak at 4 h, 124% higher than that of the control, and the content of RA reached its maximum ((5.914 +/- 0.296) mg/g dry weight) at 8 h, after treated by 6.25 mg/L SA on day 6 of the suspension culture. The results of treatment with 0.1 micromol/L AOPP showed that AOPP affected little on the TAT activity, while the PAL activity was significantly influenced, with 44% lower than that of the control at 6 h. Meanwhile, the reduced accumulation of RA ((4.709 +/- 0.204) mg/g dry weight) paralleled with the decrease in PAL activity. The co-treatment by 0.1 micromol/L AOPP and 6.25 mg/L SA relieved the restriction imposed by AOPP on PAL, and made the cell cultures accumulate more RA than sole treatment with AOPP, indicated that SA induced the accumulation of RA in suspension cell culture of S. miltiorrhiza, and the rate-limiting effect of PAL was stronger than TAT.
Cell Culture Techniques ; methods ; Cinnamates ; metabolism ; Depsides ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Plant Cells ; metabolism ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; cytology ; growth & development ; metabolism ; Suspensions ; Tyrosine Transaminase ; metabolism