Trilinolein, a Natural Triacylglycerol, Protects Cerebral Ischemia through Inhibition of Neuronal Apoptosis and Ameliorates Intimal Hyperplasia via Attenuation of Migration and Modulation of Matrix Metalloproteinase-2 and RAS/MEK/ERK Signaling Pathway in VSMCs.

Cerebrovascular disease is one of the leading causes of disability and death worldwide, and seeking a potential treatment is essential. Trilinolein (TriL) is a natural triacylglycerol presented in several plants. The effects of TriL on cerebrovascular diseases such as cerebral ischemia and carotid stenosis have never been studied. Accordingly, we investigated the protection of TriL on cerebral ischemia/reperfusion (I/R) and vascular smooth muscle cell (VSMC) migration in vivo and in vitro. The cerebral infarction area, the intima to media area (I/M ratio), and proliferating cell nuclear antigen (PCNA)-staining of the carotid artery were measured. Platelet-derived growth factor (PDGF)-BB-stimulated A7r5 cell migration and potential mechanisms of TriL were investigated by wound healing, transwell, and Western blotting. TriL (50, 100, and 200 mg/kg, p.o.) reduced: the cerebral infarction area; neurological deficit; TUNEL-positive apoptosis; intimal hyperplasia; and PCNA-positive cells in rodents. TriL (5, 10, and 20 µM) significantly inhibited PDGF-BB-stimulated A7r5 cell migration and reduced matrix metalloproteinase-2 (MMP-2), Ras, MEK, and p-ERK protein levels in PDGF-BB-stimulated A7r5 cells. TriL is protective in models of I/R-induced brain injury, carotid artery ligation-induced intimal hyperplasia, and VSMC migration both in vivo and in vitro. TriL could be potentially efficacious in preventing cerebral ischemia and cerebrovascular diseases.

Perturbation of Akt Signaling, Mitochondrial Potential, and ADP/ATP Ratio in Acidosis-Challenged Rat Cortical Astrocytes.

Cells switch to anaerobic glycolysis when there is a lack of oxygen during brain ischemia. Extracellular pH thus drops and such acidosis causes neuronal cell death. The fate of astrocytes, mechanical, and functional partners of neurons, in acidosis is less studied. In this report, we investigated the signaling in acidosis-challenged rat cortical astrocytes and whether these signals were related to mitochondrial dysfunction and cell death. Exposure to acidic pH (6.8, 6.0) caused Ca2+ release and influx, p38 MAPK activation, and Akt inhibition. Mitochondrial membrane potential was hyperpolarized after astrocytes were exposed to acidic pH as soon as 1 h and lasted for 24 h. Such mitochondrial hyperpolarization was prevented by SC79 (an Akt activator) but not by SB203580 (a p38 inhibitor) nor by cytosolic Ca2+ chelation by BAPTA, suggesting that only the perturbation in Akt signaling was causally related to mitochondrial hyperpolarization. SC79, SB203580, and BAPTA did not prevent acidic pH-induced cell death. Acidic pH suppressed ROS production, thus ruling out the role of ROS in cytotoxicity. Interestingly, pH 6.8 caused an increase in ADP/ATP ratio and apoptosis; pH 6.0 caused a further increase in ADP/ATP ratio and necrosis. Therefore, astrocyte cell death in acidosis did not result from mitochondrial potential collapse; in case of acidosis at pH 6.0, necrosis might partly result from mitochondrial hyperpolarization and subsequent suppressed ATP production. J. Cell. Biochem. 118: 1108-1117, 2017. © 2016 Wiley Periodicals, Inc.

Paeoniflorin inhibits excitatory amino acid agonist-and high-dose morphine-induced nociceptive behavior in mice via modulation of N-methyl-D-aspartate receptors.

BACKGROUND: Pain, the most common reasons for physician consultation, is a major symptom in many medical conditions that can significantly interfere with a person's life quality and general functioning. Almost all painkillers have its untoward effects. Therefore, seeking for a safe medication for pain relieve is notable nowadays. Paeonia lactiflora is a well-known traditional Chinese medicine. Paeoniflorin is an active component found in Paeonia lactiflora, which has been reported to inhibit formalin-induced nociceptive behavior in mice. Aims of this present study were to investigate effects of paeoniflorin on excitatory amino acid agonist- or high-dose morphine-induced nociceptive behaviors in mice. RESULTS: Paeoniflorin (100, 200, 500 nmol, i.c.v.) alone and combined with glutamatergic antagonists (MK-801 14.8 pmol, or NBQX 5 nmol, i.t.) inhibited nociception. Those agents also inhibited the clonic seizure-like excitation induced by high-dose morphine (250 nmol, i.t) in mice. Antisense oligodeoxynucleotides of NMDA receptor subunits NR1, NR2A, NR2B significantly enhanced the inhibition of paeoniflorin on excitatory amino acid-and high-dose morphine-induced nociception. Docking energy data revealed that paeoniflorin had stronger binding activity in NR2A and NR2B than NR2C of NMDA receptors. CONCLUSIONS: Results of this study indicate that paeoniflorin-induced inhibition of excitatory amino acid agonist- and high-dose morphine-induced nociceptive behaviors might be due to modulation of NMDA receptors, specifically the NR2B subunit.

Protection of Gueichih-Fuling-Wan on cerebral ischemia-induced brain injury in rodents is mediated by trans-cinnamaldehyde via inhibition of neuroinflammation and apoptosis.

Background: Stroke is the leading cause of mortality and morbidity worldwide, and an effective therapeutic strategy for the prevention of patients with cerebral ischemia induced brain injury is lacking. Traditional Chinese medicine with neuroprotective activities might be beneficial and provide alternative therapeutic opportunities for cerebral ischemia. Purposes: This study aimed to evaluate the neuroprotection and possible mechanisms of Gueichih-Fuling-Wan (GFW), its' constitutive herbs, and their active compounds on cerebral ischemia/reperfusion (I/R)-induced brain injury in rodents. Methods: Various doses of extracts (0.25, 0.5, and 1.0 g/kg) of GFW and five constituent herbs (Cinnamomi Cortex, CC; Poria cocos, PC; Paeonia lactifloa, PL; Paeonia suffruticosa, PS and Prunus perisica, PP) were orally administered. Different doses of active compounds (0.5, 1.0, and 2.0 mg/kg) of GFW such as cinnamaldehyde, cinnamic acid (from CC), paeoniflorin (from PL), and paeonol (from PS) were intraperitoneally administered. Their effects on cerebral ischemia/ reperfusion (I/R)induced brain injury in rodents were evaluated. Results: GFW, its' constituent herbs, and the active compounds reduced the infarct area dose-dependently (***P < 0.001). Cinnamaldehyde showed the most significant reduction (***P < 0.001). Therefore, trans-cinnamaldehyde (TCA) was further used to evaluate the neuroprotective mechanism of the I/R-induced brain injury. TCA (10, 20, 30 mg/ kg, p.o.) showed an inhibitory effect of I/R-induced brain damage in mice in a dose-dependent manner. Besides, GFW and TCA dose-dependently reduced the COX-2 protein expression level, and TCA reduced the TUNEL (+) apoptosis. TCA dose-dependently increased the pro-survival NR2A and Bcl-2 protein expression level and decreased the pro-apoptotic NR2B and cytochrome c, caspase 9, and caspase 3 expression (***P < 0.001). Conclusion: The above data revealed that GFW, its' constituent herbs, and active compounds protected against I/R-induced brain injury in rodents. TCA from CC might participate in GFW protecting against cerebral ischemia-induced brain injury by inhibiting neuroinflammation and apoptosis.

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells.

BACKGROUND: Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells. RESULTS: In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G0/G1 and Sub-G1 cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G0/G1 phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment. CONCLUSIONS: The results demonstrated that HCT-induced G0/G1 phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells.

Guizhi-Fuling-Wan, a Traditional Chinese Herbal Medicine, Ameliorates Memory Deficits and Neuronal Apoptosis in the Streptozotocin-Induced Hyperglycemic Rodents via the Decrease of Bax/Bcl2 Ratio and Caspase-3 Expression.

Brain neuronal apoptosis and cognitive impairment are associated with hyperglycemia and diabetes mellitus. The present study determined if the Chinese herbal medicine Guizhi-Fuling-Wan (GFW) would reduce memory loss and neuronal apoptosis in streptozotocin- (STZ-) induced hyperglycemic rodents. Two weeks after STZ induction, GFW was orally administered once daily for 7 days. GFW significantly improved spatial memory deficits in STZ-induced hyperglycemic mice. GFW decreased TUNEL-positive cells and caspase-3 positive cells in STZ-induced hyperglycemic rats. It also was found that GFW treatment reduced caspase-3 protein levels and increased levels of the antiapoptotic protein Bcl-2 that were indicative of neuroprotection. The protective therapeutic effects of GFW on neuronal apoptosis and cognition deficits caused by STZ-induced hyperglycemia may be due in part to inhibition of the cellular apoptosis pathway. GFW may have therapeutic effects in patients with diabetes-mellitus-induced neuropathology.

Green Tea Extract Ameliorates Learning and Memory Deficits in Ischemic Rats via Its Active Component Polyphenol Epigallocatechin-3-gallate by Modulation of Oxidative Stress and Neuroinflammation.

Ischemic stroke results in brain damage and behavioral deficits including memory impairment. Protective effects of green tea extract (GTex) and its major functional polyphenol (-)-epigallocatechin gallate (EGCG) on memory were examined in cerebral ischemic rats. GTex and EGCG were administered 1 hr before middle cerebral artery ligation in rats. GTex, EGCG, and pentoxifylline (PTX) significantly improved ishemic-induced memory impairment in a Morris water maze test. Malondialdehyde (MDA) levels, glutathione (GSH), and superoxide dismutase (SOD) activity in the cerebral cortex and hippocampus were increased by long-term treatment with GTex and EGCG. Both compounds were also associated with reduced cerebral infraction breakdown of MDA and GSH in the hippocampus. In in vitro experiments, EGCG had anti-inflammatory effects in BV-2 microglia cells. EGCG inhibited lipopolysaccharide- (LPS-) induced nitric oxide production and reduced cyclooxygenase-2 and inducible nitric oxide synthase expression in BV-2 cells. GTex and its active polyphenol EGCG improved learning and memory deficits in a cerebral ischemia animal model and such protection may be due to the reduction of oxidative stress and neuroinflammation.

Balanophora spicata and Lupeol Acetate Possess Antinociceptive and Anti-Inflammatory Activities In Vivo and In Vitro.

Aims of the present study were to investigate effects of Balanophora spicata (BS) on antinociception and anti-inflammation both in vivo and in vitro. Crude extract of BS inhibited vascular permeability induced by histamine, serotonin, bradykinin, and PGE(2), but not by PAF. Furthermore, BS crude extract, different layers (n-hexane, ethyl acetate, n-butanol, and water layer), and lupeol acetate had significant antinociceptive and anti-inflammatory effects on acetic acid-induced abdominal writhing response, formalin-induced licking behavior, carrageenan-, and serotonin-induced paw edema. The n-hexane layer had the most effective potency among all layers (IC50: 67.33 mg/kg on writhing response; IC50s: 34.2 mg/kg and 21.29 mg/kg on the early phase and late phase of formalin test, resp.). Additionally, lupeol acetate which was isolated from the n-hexane layer of BS effectively inhibited the acetic acid-induced writhing response (IC50: 28.32 mg/kg), formalin-induced licking behavior (IC50: 20.95 mg/kg), NO production (IC50: 4.102 µM), iNOS expression (IC50: 5.35 µM), and COX2 expression (IC50: 5.13 µM) in LPS-stimulated RAW 264.7 cells. In conclusion, BS has antinociceptive and anti-inflammatory effects which may be partially due to the inhibition of changes in vascular permeability induced by histamine, serotonin, bradykinin, and PGE(2) and the attenuation of iNOS and COX-2 expression.

Crude extract of Desmodium gangeticum process anticancer activity via arresting cell cycle in G1 and modulating cell cycle-related protein expression in A549 human lung carcinoma cells.

Background: Desmodium gangeticum (L.)DC., which belongs to the Leguminosae family, has been used in Taiwan and other subtropical countries as an external medicine to remove blood stasis, activate blood circulation, and reduce inflammation. It has been reported to have antioxidant effects and improve inflammatory responses in rats stimulated by pro-inflammatory agents and induced gastric ulcers in experimental animals over the past few decades. This plant has also been used to treat parasitic infections, but there are no reports regarding its effects on lung cancer. Therefore, this study attempted to investigate its water crude extract (in abbreviation DG) on lung cancer cells. Methods: A549 human lung cancer cells were tested for survival using MTT, trypan blue, and propidium iodide. The effects of various concentrations of the crude extract of D. gangeticum (DG) (0.125~1 mg/ml) on the cell cycle and apoptosis of A549 cells were analyzed by flow cytometry and Western blotting methods. Results: DG can inhibit the growth of A549 human lung cancer cells in a concentration- and time-dependent manner. DG arrested A549 cells in the G1 phase by increasing the proteins expression of p21, p27, cyclin D1, and cyclin E. Additionally, DG decreased the expression of cyclin A, B1, and Cdc 2 (CDK1) proteins. Conclusions: DG demonstrated the anti-lung cancer activity by arresting the cell cycle in G1 via increasing the p21, p27, cyclin D1, cyclin E, and decreasing Cdc2, cyclin A, and B1 proteins expression in A549 human lung cancer cells.

Bioactivities and action mechanisms of active compounds from Hottuynia cordata Thunb on human lung cancer cells.

BACKGROUND: Lung cancer is the leading cause of death in Taiwan for years. Besides the currently used chemotherapy, herbal medicine may play a role in the treatment of lung cancer. Hottuynia cordata Thunb (HC), one of the frequently used herbal medicine in Taiwan, has been widely used in various diseases. Review from literatures, HC has many effects, including anti-inflammatory, anti-viral, anti-bacterial, anti-SARS, and anti-tumor activities. However, there is no literatures describe its active compounds on lung cancer. This present study aims to evaluate the possible effect and action mechanism of active compounds from HC (aristolactam BII, aristolactam AII, and noraristolodione) on lung cancer. A549 lung cancer cell line was used to evaluate the effects of HC on the cell viability and possible anti-tumor effects. METHODS: We used A549 cells in the evaluation of anticancer activity. Cell viability, cell cycle, cell apoptosis and apoptosis related protein expression were studied. RESULTS: Active compounds from HC significantly inhibited A549 cell viability and induced accumulation of cell cycle at S or G2/M phase on A549 cells in a concentration-dependent manner, and induced A549 arrest at S or G2/M phase via increasing p21, p27, p53 and reducing cyclin-E, -A, cyclin-dependent kinase 2 (CDK2), cdc-2 (CDK1) protein expression. Additionally, HC induced A549 cell late apoptosis by up-regulating caspase-3, -8, Bax and decreasing Bcl-2 protein expression. CONCLUSIONS: The anti-tumor effects of aristolactam BII, aristolactam AII, and noraristolodione on human lung carcinoma A549 cells were via cell cycle arrest and apoptosis.

Purinergic P2X receptor regulates N-methyl-D-aspartate receptor expression and synaptic excitatory amino acid concentration in morphine-tolerant rats.

BACKGROUND: The present study examined the effect of P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) on morphine tolerance in rats. METHODS: Male Wistar rats were implanted with two intrathecal catheters with or without a microdialysis probe, then received a continuous intrathecal infusion of saline (control) or morphine (tolerance induction) for 5 days. RESULTS: Long-term morphine infusion induced antinociceptive tolerance and up-regulated N-methyl-d-aspartate receptor subunits NR1 and NR2B expression in both total lysate and synaptosome fraction of the spinal cord dorsal horn. TNP-ATP (50 µg) treatment potentiated the antinociceptive effect of morphine, with a 5.5-fold leftward shift of the morphine dose-response curve in morphine-tolerant rats, and this was associated with reversal of the up-regulated NR1 and NR2B subunits in the synaptosome fraction. NR1/NR2B-specific antagonist ifenprodil treatment produced a similar effect as TNP-ATP; it also potentiated the antinociceptive effect of morphine. On day 5, morphine challenge resulted in a significant increase in aspartate and glutamate concentration in the cerebrospinal fluid dialysates of morphine-tolerant rats, and this effect was reversed by TNP-ATP treatment. Moreover, the amount of immunoprecipitated postsynaptic density-95/NR1/NR2B complex was increased in morphine-tolerant rats, and this was prevented by the TNP-ATP treatment. CONCLUSIONS: The findings suggest that attenuation of morphine tolerance by TNP-ATP is attributed to down-regulation of N-methyl-d-aspartate receptor subunits NR1 and NR2B expression in the synaptosomal membrane and inhibition of excitatory amino acids release in morphine-tolerant rats. The TNP-ATP regulation on the N-methyl-d-aspartate receptor expression may be involved in a loss of scaffolding proteins postsynaptic density-95.

ß-carboline alkaloids from Stellaria dichotoma var. lanceolata and their anti-inflammatory activity.

The present investigation on the chemical constituents of the roots of Stellaria dichotoma var. lanceolata has resulted in the isolation of 21 ß-carboline alkaloids, including 13 new compounds, dichotomides III-XIV (1-12) and dichotomine E (13), and eight known compounds. The structures of the new compounds were established on the basis of spectroscopic data analysis. Among these isolated alkaloids, five compounds were examined for their anti-inflammatory potential for the inhibition of NO production in LPS-treated RAW 264.7 cells. All compounds tested exhibited significant inhibition of NO production, with IC(50) values in the range of 11.3 to 19.3 µM.

Cyclohexylamine, an active compound from Toddalia asiatica, contracts epididymal vas deferens via serotonergic receptors.

BACKGROUND: Toddalia asiatica of Rutaceae, a Taiwan folk medicine, is used as an analgesic and anti-inflammatory herb. Cyclohexylamine (CHA) is an active compound from T. asiatica. Previous reports indicate CHA contracts rat vas deferens. However, the contractile mechanism of CHA on rat vas deferens was not yet reported. The purpose of this study was to investigate the contractile mechanism of CHA on rat epididymal portion of vas deferens. METHODS: Male S.D. rats weighting between 200 g to 250 g were used. The epididymal portion of vas deferens was isolated and was added with various concentrations of serotonin, serotonin antagonists and CHA. RESULTS: Serotonin elicited dose-dependent (1 × 10-7M~1 × 10-4M) contractions on rat epididymal vas deferens, which were inhibited by pretreatment with ketanserin (1 × 10-8 M ~ 1 × 10-6 M), methysergide (1 × 10-5 M) and propranolol (1 × 10-4 M), respectively. CHA elicited dose-dependent (1 × 10-8M~1 × 10-4M) contractions on rat epididymal vas deferens. The contractions of CHA (1 × 10-4M) on epididymal vas deferens were enhanced by serotonin in a dose-dependent manner. Methysergide (1 × 10-7 ~1 × 10-5 M) did not affect the contractions evoked by CHA. However, the CHA elicited contraction was almost completely inhibited by ketanserin (1 × 10-5 M) and was enhanced by propranolol. The effect of propranolol at the concentration of 1 × 10-4 M on CHA was likely as CHA at high concentration alone. CONCLUSIONS: From the above results, the contraction evoked by CHA on the isolated rat epididymal vas deferens might be mediated by serotonergic receptors through 5-HT2A subtype.

Hypoxia-induced matrix metalloproteinase-13 expression in astrocytes enhances permeability of brain endothelial cells.

Matrix metalloproteinase-13 (MMP-13) is involved in the degradation of extracellular matrix in many kinds of tissues. Here we found that hypoxia increased MMP-13 protein and mRNA levels in primary rat astrocyte cultures. Hypoxia stimulation also increased the secretion of MMP-13 from astrocytes, as shown by zymographic analysis. In addition, exposure to hypoxia up-regulated the expression of c-Fos and c-Jun time-dependently. Hypoxia-induced MMP-13 overexpression was antagonized by transfection with antisense oligodeoxynucleotides (AS-ODN) of c-Fos or c-Jun. Furthermore, hypoxic-conditioned medium (Hx-CM) collected from astrocytes exposed to hypoxia increased paracellular permeability of adult rat brain endothelial cells (ARBECs). Administration of MMP-13 neutralizing antibody antagonized Hx-CM-induced paracellular permeability of ARBECs. Furthermore, pre-transfection of astrocytes with AS-ODN of c-Fos, c-Jun or MMP-13-shRNA significantly decreased hyperpermeability of ARBECs induced by Hx-CM. The arrangement of tight junction protein (TJP) zonular occludens-1 (ZO-1) of ARBECs disorganized in response to Hx-CM. Administration of Hx-CM to ARBECs also resulted in the production of proteolytic fragments of ZO-1, which was antagonized by transfection of MMP-13-shRNA in primary astrocytes. Administration of MMP-13 recombinant protein to ARBECs led to the disorganization and fragmentation of ZO-1 protein and also increased paracellular permeability. These results suggest that hypoxia-induced MMP-13 expression in astrocytes is regulated by c-Fos and c-Jun. MMP-13 is an important factor leading to the disorganization of ZO-1 and hyperpermeablility of blood-brain barrier in response to hypoxia.

Houttuynia cordata Thunb extract induces apoptosis through mitochondrial-dependent pathway in HT-29 human colon adenocarcinoma cells.

The Houttuynia cordata Thunb (HCT) extract has been used as a traditional Chinese herb medicine and as well as an effective drug for treating allergic inflammation for thousands of years. In this study, we investigated the anti-cancer activity of HCT and its molecular mechanisms in the human colon adenocarcinoma cell line HT-29. HCT inhibited HT-29 cell viability in a dose- and time-dependent manner by MTT assay. Treatment with 450 microg/ml of HCT for 48 and 72 h led to DNA damage and apoptosis by DAPI staining and comet assay. HCT increased reactive oxygen species production and decreased the levels of mitochondria membrane potential (MMP) in HT-29 cells by flow cytometry analysis. HCT caused the release of cytochrome c, Apaf-1, pro-caspase-9 and AIF from mitochondria via a decrease of the MMP. The decrease of MMP was then associated with a decrease in the ratio of Bax/Bcl-2 and activation of caspase-9 and -3 by Western blotting and caspase activity assay. Caspase-9 and -3 inhibitors almost completely suppressed HCT-induced caspase-9 and -3 activities. Our results demonstrated that the HCT-induced apoptosis in human colon adenocarcinoma cell line HT-29 might be related to a mitochondrial-dependent pathway.

Association of increased pain threshold by noise with central opioid neurons.

Several studies indicated that stress would induce analgesia. Noise, one of the stressors, was assumed to be one of the elements to enhance the threshold of pain tolerance. Since noise might affect human's daily life, it is important to know the mechanism underlying this phenomenon. The objective of this study was to explore the possible mechanism which was trying to explain how the noise affects central nervous system and the possible relationship between this effect and the involvement of opioid neurons. In the preliminary study, the analgesic effect was corroborated in ICR mice in a formalin study. The results are as follows: [1] Naloxone (a micro-opioid receptor antagonist; 1 mg/kg, i.p.), beta-FNA (a delta-opioid receptor antagonist; 5, 10 mg, i.c.v.) and naltrindole (a delta-opioid receptor antagonist; 1, 5 mg/kg, i.p.) were found to reduce antinociceptive effect. [2] nor-BNI (a kappa-antagonist; 1 microg, i.c.v.) had much effect on noise induced analgesic. In conclusion, this study suggests that noise stress enhanced the threshold of analgesia, which might be related to micro- and delta-opioid receptors in the central nervous system.

In vivo microdialysis and in vitro HPLC analysis of the impact of paeoniflorin on the monoamine levels and their metabolites in the rodent brain.

BACKGROUND: Paeoniflorin (PF) possesses several effects such as analgesic, the anti-spasmodic effect on smooth muscle. It protects the cardiovascular system and reveals the neuroprotective effect on cerebral ischemia. Monoamine system has been identified to have complex regulatory effects in pain signaling. There are no reports regarding the impact of PF on monoamine levels in the rodent brain by microdialysis. In this study, the effects of PF on monoamines and their metabolites in the rodent brain using in vivo microdialysis and in vitro high performance liquid chromatography (HPLC) analysis. METHODS: Male S.D. rats were anesthetized, fixed onto the stereotaxic instrument to identify the positions of corpus striatum and cerebral cortex. Drilled a hole in the skull of anesthetic rats and proceeded microdialysis, and gave PF (100 µg, i.c.v.). Collected the dialysate and the concentration of monoamines and their metabolites in dialysate and analyzed with HPLC-ECD. Male ICR mice were administered with PF (96 µg, i.c.v.) and with Ringer solution as a control. After 20 mins of administration, the mice were cut off the brain immediately and separated into eight regions according to the method of Glowinski. Added extraction solution to each region, homogenized and extracted for further procedure. The extract was centrifuged, sucked the transparent layer and centrifuged once more. The transparent layer was filtered with a 0.22 µm nylon filter and analyzed with HPLC-ECD (electrochemical detection). RESULTS: PF increased the content of DOPAC and NE in the cortex, and increased the content of NE and decreased the content of 5-HT in the medulla of the homogenized mice brain tissue. By microdialysis, PF increased the content of DOPAC and 5-HIAA in anesthetic rat cortex and expanded the content of DOPAC, HVA, and 5-HIAA in anesthetic rat striatum. CONCLUSIONS: It reveals that PF could activate the release of monoamines and increase their metabolites in the rodent brain.

Caspase­dependent apoptotic death by gadolinium chloride (GdCl3) via reactive oxygen species production and MAPK signaling in rat C6 glioma cells.

Gadolinium (Gd) compounds serve as magnetic resonance imaging contrast agents and exert certain anticancer activities. Yet, the molecular signaling underlying the antitumor effect of Gd chloride (GdCl3) on glioma remains unclear. In the present study, we aimed to ascertain the apoptotic mechanisms of GdCl3 on rat glioma C6 cells. Our results demonstrated that GdCl3 significantly reduced cell viability and shrunk cell morphology of C6 cells in a concentration­dependent manner. GdCl3 led to apoptotic C6 cell death as detected by TUNEL staining. An increase in cleaved caspase­3, cleaved caspase­8 and cleaved caspase­9 occurred in GdCl3­treated C6 cells as detected by immunoblotting analysis. The activities of caspase­3, caspase­8 and caspase­9 were increased, and the specific inhibitors of caspase­3/­8/­9 individually reversed cell viability, which caused apoptotic death in C6 cells prior to GdCl3 exposure. GdCl3 also caused an elevation in the cytoplasmic Ca2+ level and reactive oxygen species (ROS) production, as well as the loss of mitochondrial membrane potential (ΔΨm) as shown by flow cytometric analysis in C6 cells. The results from the immunoblotting analysis demonstrated that there were upregulated protein levels of cytochrome c and Bax but a downregulated protein level of Bcl­2 in C6 cells after GdCl3 treatment. Additionally, GdCl3 decreased the protein levels of phosphorylated­extracellular signal­regulated kinases, phosphorylated­c­Jun N­terminal kinase and phosphorylated­p38 mitogen­activated protein kinases in C6 cells. In conclusion, ROS production and MAPKs signaling pathways contribute to GdCl3­induced caspase cascade­mediated apoptosis in C6 cells. Our findings provide a better understanding of the molecular mechanisms underlying the role of GdCl3 in rat glioma C6 cells.

Naringin-induced bone morphogenetic protein-2 expression via PI3K, Akt, c-Fos/c-Jun and AP-1 pathway in osteoblasts.

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone resorption and bone formation. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. Therefore, components involved in BMP activation are good targets for the development of anti-osteoporosis drugs. In this study, naringin a polymethoxylated flavonoid, was shown to enhance alkaline phosphatase activity, osteocalcin level, osteopontin synthesis and cell proliferation in primary cultured osteoblasts. Naringin increased mRNA and protein levels of BMP-2 using Western blot, ELISA and RT-PCR assay. In addition, naringin also prevented the decreasing of BMP-2 and bone loss inducing by ovariectomy in vivo. The transcriptional regulation of BMP-2 by naringin was mediated by phosphorylation of Akt and activation of the activator protein-1 (AP-1) components c-Fos and c-Jun. The binding of c-Fos and c-Jun to the AP-1 element on the BMP-2 promoter was enhanced by naringin. Transfection with dominant-negative mutant of p85 and Akt or c-Fos and c-Jun antisense oligonucleotide inhibited the potentiating action of naringin on BMP-2 production. Taken together, our results provide evidence that naringin increase BMP-2 expression and enhance osteogenic response via the phosphoinositide 3-kinase (PI3K), Akt, c-Fos/c-Jun and AP-1-dependent signaling pathway.

Paeonol inhibits RANKL-induced osteoclastogenesis by inhibiting ERK, p38 and NF-kappaB pathway.

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with osteoporosis. Paeonol (2'-hydroxy-4'-methoxyacetophenone), the main active compound of the traditionally used Chinese herb Paeonia lactiflora Pallas, has anti-inflammatory activity. Here we found that paeonol markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL) plus macrophage colony stimulating factor (M-CSF)-induced osteoclastic differentiation from bone marrow stromal cells and RAW264.7 macrophage cells. In addition, in an assay of osteoclast activity on substrate plates, paeonol significantly decreased the resorption activity of mature osteoclasts. Treatment of RAW264.7 macrophages with RANKL induced extracellular signal-regulated kinases (ERK), p38 and c-Jun N-terminal kinase (JNK) phosphorylation. However, RANKL-induced ERK, p38 but not JNK phosphorylation was attenuated by paeonol. Furthermore, RANKL-mediated increase of IkappaBalpha phosphorylation, p65 phosphorylation at Ser(536), kappaB-luciferase activity and NF-kappaB binding activity was inhibited by paeonol. In addition, paeonol also prevented the bone loss inducing by ovariectomy in vivo. Our data suggest that paeonol inhibits osteoclastogenesis from bone marrow stromal cells and macrophage cells via attenuated of RANKL-induced ERK, p38 and NF-kappaB activation, which in turn protect bone loss from ovariectomy.