Sites and Regulation of L-Type Ca2+ Channel Cav1.2 Phosphorylation in Brain.

Cav1.2 channel phosphorylation plays an important role in regulating neuronal plasticity by action potential-dependent Ca2+ entry. Most studies of Cav1.2 regulation by phosphorylation have been reported in heart and muscles. Here, we identified phosphorylation sites of neuronal Cav1.2 channel protein purified from rat brain using mass spectrometry. The functional characterization of these phosphorylation sites showed altered voltage-dependent biophysical properties of the channel, without affecting current density. These results show that neuronal Cav1.2 channel is regulated by phosphorylation in a complex mechanism involving multiple phosphorylation sites.

Melatonin modulates nitric oxide-regulated WNK-SPAK/OSR1-NKCC1 signaling in dorsal raphe nucleus of rats.

The sleep-wake cycle is regulated by the alternating activity of sleep- and wake-promoting neurons. The dorsal raphe nucleus (DRN) secretes 5-hydroxytryptamine (5-HT, serotonin), promoting wakefulness. Melatonin secreted from the pineal gland also promotes wakefulness in rats. Our laboratory recently demonstrated that daily changes in nitric oxide (NO) production regulates a signaling pathway involving with-no-lysine kinase (WNK), Ste20-related proline alanine rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1), and cation-chloride co-transporters (CCC) in rat DRN serotonergic neurons. This study was designed to investigate the effect of melatonin on NO-regulated WNK-SPAK/OSR1-CCC signaling in wake-inducing DRN neurons to elucidate the mechanism underlying melatonin's wake-promoting actions in rats. Ex vivo treatment of DRN slices with melatonin suppressed neuronal nitric oxide synthase (nNOS) expression and increased WNK4 expression without altering WNK1, 2, or 3. Melatonin increased phosphorylation of OSR1 and the expression of sodium-potassium-chloride co-transporter 1 (NKCC1), while potassium-chloride cotransporter 2 (KCC2) remained unchanged. Melatonin increased the expression of tryptophan hydroxylase 2 (TPH2, serotonin-synthesizing enzyme). The present study suggests that melatonin may promote its wakefulness by modulating NO-regulated WNK-SPAK/OSR1-KNCC1 signaling in rat DRN serotonergic neurons.

Role of nitric oxide and WNK-SPAK/OSR1-KCC2 signaling in daily changes in GABAergic inhibition in the rat dorsal raphe neurons.

Serotonergic neurons in the dorsal raphe nucleus (DRN) act as wake-inducing neurons in the sleep-wake cycle and are controlled by gamma-aminobutyric acid (GABA) synaptic inputs. We investigated daily changes in GABAergic inhibition of the rat DRN neurons and the role of nitric oxide (NO) and cation-chloride co-transporters in the GABAergic action. Neuronal NO synthase (nNOS) was co-expressed in 74% of serotonergic DRN neurons and nNOS expression was higher during daytime (the sleep cycle) than that during nighttime (the wake cycle). GABAergic hyperpolarization of DRN neurons produced by GABAA receptor agonist muscimol was greater and the equilibrium potential of muscimol showed a hyperpolarizing shift during daytime compared to that during nighttime. Expression levels of potassium-chloride co-transporter 2 (KCC2) were higher during daytime than that during nighttime, whereas there were no changes in sodium-potassium-chloride co-transporter 1 (NKCC1) expression. With-no-lysine kinase (WNK) isoform 1 was more highly expressed during daytime than that during nighttime. Total Ste20-related proline alanine rich kinase (SPAK) and oxidative stress response kinase 1 (OSR1) were also higher during daytime than during nighttime, while there were no changes in phosphorylated SPAK and OSR1. Consistent with the findings during the sleep-wake cycle, ex vivo treatment of DRN slices with a NO donor sodium nitroprusside (SNP) increased the expression of KCC2, WNK1, WNK2, WNK3, SPAK, and OSR1, whilst decreasing phosphorylated SPAK. These results suggest that GABAergic synaptic inhibition of DRN serotonergic neurons shows daily changes during the sleep-wake cycle, which might be regulated by daily changes in nNOS-derived NO and WNK-SPAK/OSR1-KCC2 signaling.

Fulgidic Acid Isolated from the Rhizomes of Cyperus rotundus Suppresses LPS-Induced iNOS, COX-2, TNF-α, and IL-6 Expression by AP-1 Inactivation in RAW264.7 Macrophages.

To identify bioactive natural products possessing anti-inflammatory activity, the potential of fulgidic acid from the rhizomes of Cyperus rotundus and the underlying mechanisms involved in its anti-inflammatory activity were evaluated in this study. Fulgidic acid reduced the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Consistent with these findings, fulgidic acid suppressed the LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein level, as well as iNOS, COX-2, TNF-α, and IL-6 at mRNA levels. Fulgidic acid suppressed the LPS-induced transcriptional activity of activator protein-1 (AP-1) as well as the phosphorylation of c-Fos and c-Jun. On the other hand, fulgidic acid did not show any effect on LPS-induced nuclear factor κB (NF-κB) activity. Taken together, these results suggest that the anti-inflammatory effect of fulgidic acid is associated with the suppression of iNOS, COX-2, TNF-α, and IL-6 expression through down-regulating AP-1 activation in LPS-induced RAW264.7 macrophages.

GABAergic inhibition is weakened or converted into excitation in the oxytocin and vasopressin neurons of the lactating rat.

BACKGROUND: Increased secretion of oxytocin and arginine vasopressin (AVP) from hypothalamic magnocellular neurosecretory cells (MNCs) is a key physiological response to lactation. In the current study, we sought to test the hypothesis that the GABAA receptor-mediated inhibition of MNCs is altered in lactating rats. RESULTS: Gramicidin-perforated recordings in the rat supraoptic nucleus (SON) slices revealed that the reversal potential of GABAA receptor-mediated response (EGABA) of MNCs was significantly depolarized in the lactating rats as compared to virgin animals. The depolarizing EGABA shift was much larger in rats in third, than first, lactation such that GABA exerted an excitatory, instead of inhibitory, effect in most of the MNCs of these multiparous rats. Immunohistochemical analyses confirmed that GABAergic excitation was found in both AVP and oxytocin neurons within the MNC population. Pharmacological experiments indicated that the up-regulation of the Cl(-) importer Na(+)-K(+)-2Cl(-) cotransporter isotype 1 and the down-regulation of the Cl(-) extruder K(+)-Cl(-) cotransporter isotype 2 were responsible for the depolarizing shift of EGABA and the resultant emergence of GABAergic excitation in the MNCs of the multiparous rats. CONCLUSION: We conclude that, in primiparous rats, the GABAergic inhibition of MNCs is weakened during the period of lactation while, in multiparous females, GABA becomes excitatory in a majority of the cells. This reproductive experience-dependent alteration of GABAergic transmission may help to increase the secretion of oxytocin and AVP during the period of lactation.

Chemopreventive effects of standardized ethanol extract from the aerial parts of Artemisia princeps Pampanini cv. Sajabal via NF-κB inactivation on colitis-associated colon tumorigenesis in mice.

Chronic inflammation is an underlying risk factor of colon cancer, and NF-κB plays a critical role in the development of inflammation-associated colon cancer in an AOM/DSS mouse model. The aim of this study was to determine whether the standardized ethanol extract obtained from the aerial parts of Artemisia princeps Pampanini cv. Sajabal (EAPP) is effective at preventing inflammation-associated colon cancer, and if so, to identify the signaling pathways involved. In the present study, protective efficacy of EAPP on tumor formation and the infiltrations of monocytes and macrophages in colons of an AOM/DSS mouse model were evaluated. It was found that colitis and tumor burdens showed statistically meaningful improvements after EAPP administration. Furthermore, these improvements were accompanied by a reduction in NF-κB activity and in the levels of NF-κB-dependent pro-survival proteins, that is, survivin, cFLIP, XIAP, and Bcl-2. In vitro, EAPP significantly reduced NF-κB activation and the levels of IL-1ß and IL-8 mRNA and pro-survival proteins in HT-29 and HCT-116 colon cancer cells. Furthermore, EAPP caused caspase-dependent apoptosis. Based on these results, the authors suggest EAPP suppresses inflammatory responses and induces apoptosis partly via NF-κB inactivation, and that EAPP could be useful for the prevention of colitis-associated tumorigenesis.

Amomum tsao-ko fruit extract suppresses lipopolysaccharide-induced inducible nitric oxide synthase by inducing heme oxygenase-1 in macrophages and in septic mice.

Amomum tsao-ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti-inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao-ko (AOM) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS-induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase-1 (HO-1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO-1) and silencing HO-1 using siRNA prevented the AOM-mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF-E2-related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N-acetyl-l-cysteine (NAC; a ROS scavenger) diminished the AOM-induced phosphorylation of ERK and JNK and AOM-induced HO-1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM-induced signalling of HO-1 expression. In LPS-induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO-1 expression and survival rates. These results indicate that AOM strongly inhibits LPS-induced NO production by activating the ROS/MAPKs/Nrf2-mediated HO-1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.

Anti-inflammatory effects of 7-hydroxyl-1-methylindole-3-acetonitrile, a synthetic arvelexin derivative, on the macrophages through destabilizing mPGES-1 mRNA and suppressing NF-κB activation.

We previously demonstrated that 7-hydroxyl-1-methylindole-3-acetonitrile (7-HMIA), a synthesized analog of arvelexin, showed the strong inhibitory effects on LPS-induced NO and PGE2 production in macrophages. In this study, we focused on elucidating the anti-inflammatory properties of 7-HMIA and the mechanisms involved using in vitro and in vivo experimental models. In LPS-induced RAW 264.7 macrophages, 7-HMIA significantly inhibited the release of proinflammatory mediators such as prostaglandin E2 (PGE2), nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). We also found that 7-HMIA suppressed PGE2 production not by inhibiting cyclooxygenase-2 (COX-2) expression or activity, but rather by suppressing the mRNA stability of microsomal prostaglandin E synthase (mPGES-1). Furthermore, 7-HMIA mediated attenuation of inducible NO synthase (iNOS), TNF-α, and IL-6 was closely associated with suppression of transcriptional activities of nuclear factor-kappa B (NF-κB), by decreasing p65 nuclear translocation and Akt phosphorylation. Animal studies revealed that 7-HMIA potently suppressed the carrageenan-induced paw edema and myeloperoxidase (MPO) activity in paw tissues. Taken together, our data indicated that the molecular basis for the anti-inflammatory properties of 7-HMIA involved the inhibition of mRNA stability of mPGES-1 and PI3K/Akt-mediated NF-κB pathways.

Anti-inflammatory effect of a standardized triterpenoid-rich fraction isolated from Rubus coreanus on dextran sodium sulfate-induced acute colitis in mice and LPS-induced macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubus coreanus Miquel (Rosaceae), the Korean black raspberry, has traditionally been used to treat inflammatory diseases including diarrhea, asthma, stomach ailment, and cancer. Although previous studies showed that the 19α-hydroxyursane-type triterpenoids isolated from Rubus coreanus exerted anti-inflammatory activities, their effects on ulcerative colitis and mode of action have not been explored. This study was designed to assess the anti-inflammatory effects and the molecular mechanisms involving19α-hydroxyursane-type triterpenoid-rich fraction from Rubus coreanus (TFRC) on a mice model of colitis and lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. MATERIALS AND METHODS: Experimental colitis was induced by DSS for 7 days in ICR mice. Disease activity indices (DAI) took into account body weight, stool consistency, and gross bleeding. Histological changes and macrophage accumulation were observed by immunohistochemical analysis. Pro-inflammatory markers were determined using immunoassays, RT-PCR, and real time PCR. Signaling pathway involving nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) activation was determined by luciferase assay and Western blotting. RESULTS: In DSS-induced colitis mice, TFRC improved DAIs and pathological characteristics including colon shortening and colonic epithelium injury. TFRC suppressed tissue levels of pro-inflammatory cytokines and reduced macrophage infiltration into colonic tissues. In LPS-induced RAW 264.7 macrophages, TFRC inhibited the production of NO, PGE2, and pro-inflammatory cytokines by down-regulating the activation of NF-κB and p38 MAPK signaling. CONCLUSION: The study demonstrates that TFRC has potent anti-inflammatory effects on DSS-induced colonic injury and LPS-induced macrophage activation, and supports its possible therapeutic and preventive roles in colitis.

Regulatory Effect of 25-hydroxyvitamin D3 on Nitric Oxide Production in Activated Microglia.

Microglia are activated by inflammatory and pathophysiological stimuli in neurodegenerative diseases, and activated microglia induce neuronal damage by releasing cytotoxic factors like nitric oxide (NO). Activated microglia synthesize a significant amount of vitamin D3 in the rat brain, and vitamin D3 has an inhibitory effect on activated microglia. To investigate the possible role of vitamin D3 as a negative regulator of activated microglia, we examined the effect of 25-hydroxyvitamin D3 on NO production of lipopolysaccharide (LPS)-stimulated microglia. Treatment with LPS increased the production of NO in primary cultured and BV2 microglial cells. Treatment with 25-hydroxyvitamin D3 inhibited the generation of NO in LPS-activated primary microglia and BV2 cells. In addition to NO production, expression of 1-α-hydroxylase and the vitamin D receptor (VDR) was also upregulated in LPS-stimulated primary and BV2 microglia. When BV2 cells were transfected with 1-α-hydroxylase siRNA or VDR siRNA, the inhibitory effect of 25-hydroxyvitamin D3 on activated BV2 cells was suppressed. 25-Hydroxyvitamin D3 also inhibited the increased phosphorylation of p38 seen in LPS-activated BV2 cells, and this inhibition was blocked by VDR siRNA. The present study shows that 25-hydroxyvitamin D3 inhibits NO production in LPS-activated microglia through the mediation of LPS-induced 1-α-hydroxylase. This study also shows that the inhibitory effect of 25-hydroxyvitamin D3 on NO production might be exerted by inhibiting LPS-induced phosphorylation of p38 through the mediation of VDR signaling. These results suggest that vitamin D3 might have an important role in the negative regulation of microglial activation.

T-type Ca2+ channel blocker, KYS05090 induces autophagy and apoptosis in A549 cells through inhibiting glucose uptake.

It has been reported that [3-(1,1'-biphenyl-4-yl)-2-(1-methyl-5-dimethylamino-pentylamino)-3,4-dihydroquinazolin-4-yl]-N-benzylacetamide 2hydrochloride (KYS05090), a selective T-type Ca2+ channel blocker, reduces tumor volume and weight in the A549 xenograft model, but the molecular mechanism of cell death has not yet been elucidated. In this study, KYS05090 induced autophagy- and apoptosis-mediated cell death in human lung adenocarcinoma A549 cells. Although KYS05090 decreased intracellular Ca2+ levels, it was not directly related with KYS05090-induced cell death. In addition, KYS05090 generated intracellular reactive oxygen species (ROS) and reduced glucose uptake, and catalase and methyl pyruvate prevented KYS05090-induced cell death. These results indicate that KYS05090 can lead to autophagy and apoptosis in A549 cells through ROS generation by inhibiting glucose uptake. Our findings suggest that KYS05090 has potential chemotherapeutic value for the treatment of lung cancer.

Inhibitory effects of ß-chamigrenal, isolated from the fruits of Schisandra chinensis, on lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages [corrected].

Much is known about the bioactive properties of lignans from the fruits of Schisandra chinensis. However, very little work has been done to determine the properties of sesquiterpenes in the fruits of S. chinensis. The aim of the present study was to investigate the anti-inflammatory potential of new sesquiterpenes (ß-chamigrenal, ß-chamigrenic acid, α-ylangenol, and α-ylangenyl acetate) isolated from the fruits of S. chinensis and to explore their effect on macrophages stimulated with lipopolysaccharide. Of these four sesquiterpenes, ß-chamigrenal most significantly suppressed lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages (47.21 ± 4.54 % and 51.61 ± 3.95 % at 50 µM, respectively). Molecularly, the inhibitory activity of ß-chamigrenal on nitric oxide production was mediated by suppressing inducible nitric oxide synthase activity but not its expression. In the prostaglandin E2 synthesis pathway, ß-chamigrenal prevented the upregulation of inducible microsomal prostaglandin E synthase-1 expression after stimulation with lipopolysaccharide. Conversely, ß-chamigrenal had no effect on the expression and enzyme activity of cyclooxygenase-2. In addition, the expression of early growth response factor-1, a key transcription factor of microsomal prostaglandin E synthase-1 expression, was inhibited by ß-chamigrenal. These results may suggest a possible anti-inflammatory activity of ß-chamigrenal which has to be proven in in vivo experiments.

Standardized ethyl acetate fraction from the roots of Brassica rapa attenuates the experimental arthritis by down regulating inflammatory responses and inhibiting NF-κB activation.

This study was undertaken to investigate the anti-arthritic potential of a standardized ethyl acetate fraction from the roots of Brassica rapa (EABR) and to explore the molecular mechanisms in adjuvant-induced arthritic rats and macrophages. In AIA-induced arthritic rats, EABR significantly reduced paw swelling, an arthritic index, serum rheumatoid factor, and tissue expression ratio of RANKL/OPG versus vehicle-administered group. This was found to be well correlated with significant suppressions in productions of PGE2, NO, and pro-inflammatory cytokines and in activations of NF-κB in AIA-induced paw tissues and LPS-induced macrophages. EABR attenuated NF-κB activation by reducing the nuclear translocation and phosphorylation of the p65 NF-κB, which were accompanied by parallel reductions in the degradation and phosphorylation of IκBα after blocking the phosphorylation mediated IKK activation. The findings suggest EABR exerts its anti-arthritic and anti-inflammatory properties via NF-κB inactivation in vitro and in vivo, and that EABR is a potential therapeutic for the treatment of arthritis and inflammation-associated disorders.

Chloroform fraction of Solanum tuberosum L. cv Jayoung epidermis suppresses LPS-induced inflammatory responses in macrophages and DSS-induced colitis in mice.

In this study, the authors investigated the molecular mechanism underlying the antiinflammatory effects of the chloroform fraction of the peel of 'Jayoung' (CFPJ), a color-fleshed potato, on lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in mice with dextran sulfate sodium (DSS)-induced colitis. CFPJ inhibited the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the transcription level, and attenuated the transcriptional activity of nuclear factor-κB (NF-κB) by reducing the translocation of NF-κB depending on degradation of inhibitory κB-α (IκB-α). Furthermore, CFPJ attenuated the phosphorylations of mitogen-activated protein kinase kinases3/6 (MKK3/6) and of p38. In colitis model, CFPJ significantly reduced the severity of colitis and the productions and protein levels of pro-inflammatory mediators in colonic tissue. These results suggest that the anti-inflammatory effects of CFPJ are associated with the suppression of NF-κB and p38 activation in macrophages, and support its possible therapeutic role for the treatment of colitis.

5,6,7-trimethoxyflavone suppresses pro-inflammatory mediators in lipopolysaccharide-induced RAW 264.7 macrophages and protects mice from lethal endotoxin shock.

5,6,7-Trimethoxyflavone (TMF), methylations of the hydroxyl groups of oroxylin A or baicalein, was found to significantly inhibit the productions of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. However, no report has been issued on the anti-inflammatory potential of TMF and the underlying molecular mechanism. In the present study, we investigated the anti-inflammatory effects of TMF in LPS-induced RAW 264.7 macrophages and LPS-induced septic shock in mice. TMF dose-dependently inhibits iNOS and COX-2 at the protein, mRNA, and promoter binding levels and that these inhibitions cause attendant decreases in the productions of NO and PGE2. TMF inhibits the productions and mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 induced by LPS. Furthermore, TMF suppress the transcriptional activity of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), and nuclear translocations of NF-κB, AP-1, and signal transducer and activator of transcription 1/3 (STAT1/3). Pretreatment with TMF increase the survival rate of mice with LPS-induced endotoxemia and reduced the serum levels of cytokines. Taken together, these findings suggest that TMF down-regulates the expressions of the pro-inflammatory iNOS, COX-2, TNF-α, IL-1ß, and IL-6 genes in macrophages by interfering with the activation of NF-κB, AP-1, and STAT1/3.

Expression and activity of the na-k ATPase in ischemic injury of primary cultured astrocytes.

Astrocytes are reported to have critical functions in ischemic brain injury including protective effects against ischemia-induced neuronal dysfunction. Na-K ATPase maintains ionic gradients in astrocytes and is suggested as an indicator of ischemic injury in glial cells. Here, we examined the role of the Na-K ATPase in the pathologic process of ischemic injury of primary cultured astrocytes. Chemical ischemia was induced by sodium azide and glucose deprivation. Lactate dehydrogenase assays showed that the cytotoxic effect of chemical ischemia on astrocytes began to appear at 2 h of ischemia. The expression of Na-K ATPase α1 subunit protein was increased at 2 h of chemical ischemia and was decreased at 6 h of ischemia, whereas the expression of α1 subunit mRNA was not changed by chemical ischemia. Na-K ATPase activity was time-dependently decreased at 1, 3, and 6 h of chemical ischemia, whereas the enzyme activity was temporarily recovered to the control value at 2 h of chemical ischemia. Cytotoxicity at 2 h of chemical ischemia was significantly blocked by reoxygenation for 24 h following ischemia. Reoxygenation following chemical ischemia for 1 h significantly increased the activity of the Na-K ATPase, while reoxygenation following ischemia for 2 h slightly decreased the enzyme activity. These results suggest that the critical time for ischemia-induced cytotoxicity of astrocytes might be 2 h after the initiation of ischemic insult and that the increase in the expression and activity of the Na-K ATPase might play a protective role during ischemic injury of astrocytes.

Protective effects of 6-hydroxy-1-methylindole-3-acetonitrile on cisplatin-induced oxidative nephrotoxicity via Nrf2 inactivation.

We previously demonstrated the ethanol extract of the roots of Brassica rapa protects against cisplatin-induced nephrotoxicity by attenuating oxidative stress. Here, we investigated the nephroprotective effects of 6-hydroxy-1-methylindole-3-acetonitrile (6-HMA), which was isolated from the roots of B. rapa, on cisplatin-induced toxicity in renal epithelial LLC-PK1 cells and in rats with acute renal injury. Pretreatment of LLC-PK1 cells with 6-HMA ameliorated cisplatin-induced cytotoxicity caused by oxidative stress, as was demonstrated by reductions in the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and increased levels of glutathione (GSH). In addition, 6-HMA inhibited cisplatin-induced heme oxygenase-1 (HO-1) expression, possibly due to the suppression of the nuclear translocation and binding activity of NF-E2-related factor 2 (Nrf2). Furthermore, 6-HMA administered rats showed lower levels of blood urea nitrogen (BUN), creatinine, and urinary lactate dehydrogenase (LDH) than cisplatin alone-treated rats in cisplatin-induced renal injury model. Moreover, 6-HMA inhibited the cisplatin-induced formation of MDA and GSH depletion and increased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR). Taken together, these findings indicate 6-HMA is a major active constituent from the roots of B. rapa to have a protective effect against cisplatin-induced nephrotoxicity by attenuating oxidative stress.

Echinocystic acid isolated from Eclipta prostrata suppresses lipopolysaccharide-induced iNOS, TNF-α, and IL-6 expressions via NF-κB inactivation in RAW 264.7 macrophages.

In this study, we aimed to identify the compounds in Eclipta prostrata responsible for its anti-inflammatory effects using an in vitro bioassay. Three triterpenoids, eclalbasaponin I, eclalbasaponin II, and echinocystic acid, were isolated from an EtOAc fraction of the 70 % EtOH extract of E. prostrata by activity-guided fractionation based on the inhibition of nitric oxide release from lipopolysaccharide-induced RAW 264.7 macrophages. Of these three triterpenoids, echinocystic acid inhibited lipopolysaccharide-induced production of nitric oxide and cytokines such as tumor necrosis factor-α and interleukin-6. Consistent with these observations, echinocystic acid concentration-dependently inhibited lipopolysaccharide-induced inducible nitric oxide synthase expression at the protein level and inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-6 expression at the mRNA level, and inhibited lipopolysaccharide-induced iNOS promoter binding activity. In addition, echinocystic acid suppressed the lipopolysaccharide-induced transcriptional activity of nuclear factor-κB by blocking the nuclear translocation of p65.

Proteomic analysis of primary cultured rat cortical neurons in chemical ischemia.

To elucidate the molecular events involved in early ischemic neuronal death, we performed two-dimensional proteome profiling of primary cultures of rat cortical neurons following chemical ischemia induced by the administration of sodium azide under glucose-free conditions. Using a lactic dehydrogenase assay and Western blot analysis of dephosporylation of the voltage-gated potassium channel Kv2.1, we determined duration of chemical ischemia of 2 h to be the relevant time-point for early ischemic neuronal death. Sixty-one proteins were differentially expressed, and 26 different proteins were identified by MALDI-TOF with Mascot database searching. The proteome data indicated that chemical ischemia altered the expression of 20 proteins that are involved in stress response/chaperone, brain development, cytoskeletal/structural proteins, metabolic enzymes, and calcium ion homeostasis. Western blotting and immunocytochemical studies of the 6-most functionally significant proteins showed that, in the ischemia-treated group, the expression of glucose-related protein 78, heat shock protein 90 alpha, and α-enolase was significantly increased, while the expression of inositol triphosphate receptor 1 and ATP synthase beta subunit was decreased. In addition, the expression of dihydropyrimidinase-like 3 showed a truncated pattern in the ischemia group. The changes in the expression of these proteins might be significant indicators of early ischemic neuronal death.

6,7-Dimethoxy-3-(3-methoxyphenyl)isoquinolin-1-amine induces mitotic arrest and apoptotic cell death through the activation of spindle assembly checkpoint in human cervical cancer cells.

Previously, we reported that 6,7-dimethoxy-3-(3-methoxyphenyl)isoquinolin-1-amine (CWJ-082) has potent cytotoxic effects on various cancer cells, but the underlying molecular mechanism responsible was not determined. In the present study, CWJ-082 caused cervical cancer cell cycle arrest at the G2/M phase and subsequent caspase-dependent apoptosis. The mitotic arrest caused by CWJ-082 found to be due to increases in the activation of cyclin-dependent kinase 1/cyclin B1 complex and the phosphorylation of histone H3. In addition, CWJ-082 induced the phosphorylation of BubR1 and the association between mitotic arrest deficient 2 (Mad2) and cell division cycle protein 20. These findings suggested that CWJ-082 activated the mitotic spindle checkpoint. Furthermore, knockdown of the spindle checkpoint proteins BubR1 or Mad2 using specific small interfering RNAs significantly reduced CWJ-082-induced mitotic cell accumulation and apoptosis. In addition, CWJ-082 induced the appearance of spindle abnormalities by inducing α-tubulin polymerization. In BALB/c(nu/nu) mice bearing a HeLa xenograft, CWJ-082 significantly inhibited tumor growth. Taken together, these results suggest that CWJ-082 inhibits cell growth via mitotic arrest by activating the mitotic spindle checkpoint and by inducing α-tubulin polymerization and that these events ultimately lead to the apoptosis of human cervical cancer cells and inhibit tumor growth in HeLa xenograft mice.