Protective Role of the Toll-Like Receptor 5 Agonist KMRC011 against Murine Colitis Induced by Citrobacter rodentium and Dextran Sulfate Sodium.

This study aimed to identify the therapeutic ability of a novel toll-like receptor (TLR) 5 agonist, KMRC011, on ulcerative colitis induced by Citrobacter rodentium and dextran sulfate sodium in a C57BL/6N mouse model. Ulcerative colitis was induced in the mice by the oral administration of 1% dextran sulfate sodium in sterile drinking water for seven days ad libitum, followed by C. rodentium infection on the seventh day by intra-gastric administration (DSS-CT group). KMRC011 was administered intramuscularly at both 24 h and 15 min before (Treatment 1 group), and at both 15 min and 24 h after (Treatment 2 group) the C. rodentium infection. The length of the large intestine and histopathological counts were significantly greater and mucosal thickness was significantly thinner in the Treatment 1 group compared to the DSS-CT and Treatment 2 groups. Il-6 and Il-10 mRNA expression levels were upregulated, while Ifn-γ and Tnf-α mRNA expression levels were significantly downregulated in the Treatment 1 group, compared to the DSS-CT group. NF-κB p65 expression level was elevated due to ulcerative colitis in the DSS-CT group, but was significantly downregulated in the Treatment 1 group. Overall, KMRC011 showed protective effects against murine colitis by inhibiting NF-κB signaling.

A first-in-human study of KMRC011, a potential treatment for acute radiation syndrome, to explore tolerability, pharmacokinetics, and pharmacodynamics.

KMRC011 is a novel Toll-like receptor 5 agonist under development as a treatment for acute radiation syndrome (ARS). The aim of this first-in-human study was to investigate the tolerability, pharmacokinetics, and pharmacodynamics of a single intramuscular dose of KMRC011 in healthy subjects. A randomized, single-blind, placebo-controlled, single dose-escalation study was conducted with the starting dose of 5 µg. Eight (4 only for 5 µg cohort) subjects per cohort were randomly assigned to KMRC011 or placebo in a 3:1 ratio. Dose-limiting toxicity (DLT) was assessed throughout the study. Serum concentrations of KMRC011, granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6) were measured up to 48 h postdose. Based on safety review, the dose of KMRC011 escalated up to 20 µg, and consequently, a total of 4 dose levels (5, 10, 15, and 20 µg) were explored. The most common adverse event was injection site reaction, showing no dose-related trend. Three DLTs (2 cases of hepatic enzyme increased and 1 of pyrexia) were observed; 1 in the 15 µg cohort and 2 in the 20 µg cohort. A developed method could not detect any KMRC011 in serum. KMRC011 15 µg and 20 µg showed significant increases of G-CSF, IL-6, and absolute neutrophil counts, compared with the placebo. A single intramuscular administration of KMRC011 ranging from 5 to 15 µg was tolerated in healthy subjects. Doses of KMRC011 equal to or greater than 15 µg exerted TLR5 agonist-like activities by increasing serum G-CSF and IL-6. It suggests that KMRC011 has the potential for a treatment for ARS.

IgLON5 Regulates the Adhesion and Differentiation of Myoblasts.

IgLON5 is a cell adhesion protein belonging to the immunoglobulin superfamily and has important cellular functions. The objective of this study was to determine the role played by IgLON5 during myogenesis. We found IgLON5 expression progressively increased in C2C12 myoblasts during transition from the adhesion to differentiation stage. IgLON5 knockdown (IgLON5kd) cells exhibited reduced cell adhesion, myotube formation, and maturation and reduced expressions of different types of genes, including those coding for extracellular matrix (ECM) components (COL1a1, FMOD, DPT, THBS1), cell membrane proteins (ITM2a, CDH15), and cytoskeletal protein (WASP). Furthermore, decreased IgLON5 expression in FMODkd, DPTkd, COL1a1kd, and ITM2akd cells suggested that IgLON5 and these genes mutually control gene expression during myogenesis. IgLON5 immunoneutralization resulted in significant reduction in the protein level of myogenic markers (MYOD, MYOG, MYL2). IgLON5 expression was higher in the CTX-treated gastrocnemius mice muscles (day 7), which confirmed increase expression of IgLON5 during muscle. Collectively, these results suggest IgLON5 plays an important role in myogenesis, muscle regeneration, and that proteins in ECM and myoblast membranes form an interactive network that establishes an essential microenvironment that ensures muscle stem cell survival.

KMRC011, an agonist of toll-like receptor 5, mitigates irradiation-induced tissue damage and mortality in cynomolgus monkeys.

In the study here, the potential applicability of KMRC011 - an agonist of toll-like receptor-5 - as a countermeasure for radiation toxicities was evaluated. Following a single 5.5 Gy total body irradiation (TBI, surface absorbed dose = 7 Gy) of Co60 γ-rays, mortality rates and degrees of pathological lesions that developed over 80 days were compared in monkeys that received TBI only and a group that was injected once with KMRC011 (10 µg/kg) after TBI. Compared to the TBI-only hosts (80%), the death rate was significantly improved by the use of KMRC011 (40%), all deaths in both groups occurred in the period from Days 19-24 post-TBI. Further analysis of monkeys that survived until the end of the experiment showed that AST and ALT levels were elevated only in the TBI group, and that radiation-induced tissue damage was alleviated by the KMRC011 injection. Additionally, expression of cell death-related proteins was lower in tissues from the KMRC011-treated hosts than in those in the TBI-only group. Other measured parameters, including body weight, food uptake, and hematological values did not significantly differ between the two groups over the entire period. The results of this study, thus demonstrate that KMRC011 could potentially be used as a medical countermeasure for the treatment of acute radiation exposure.

Radioprotective effect of newly synthesized toll-like receptor 5 agonist, KMRC011, in mice exposed to total-body irradiation.

Exposure to ionizing radiation leads to severe damages in radiosensitive organs and induces acute radiation syndrome, including effects on the hematopoietic system and gastrointestinal system. In this study, the radioprotective ability of KMRC011, a novel toll-like receptor 5 (TLR5) agonist, was investigated in C57BL6/N mice exposed to lethal total-body gamma-irradiation. In a 30-day survival study, KMRC011-treated mice had a significantly improved survival rate compared with control after 11 Gy total-body irradiation (TBI), and it was found that the radioprotective activity of KMRC011 depended on its dosage and repeated treatment. In a 5-day short-term study, we demonstrated that KMRC011 treatment stimulated cell proliferation and had an anti-apoptotic effect. Furthermore, KMRC011 increased the expressions of genes related to DNA repair, such as Rad21, Gadd45b, Sod2 and Irg1, in the small intestine of lethally irradiated mice. Interestingly, downregulation of NF-κB p65 in the mouse intestine by KMRC011 treatment was observed. This data indicated that KMRC011 exerted a radioprotective activity partially by regulating NF-κB signaling. Finally, peak expression levels of G-CSF, IL-6, IFN-γ, TNF-α and IP-10 induced by KMRC011 treatment were different depending on the route of administration and type of cytokine. These cytokines could be used as candidate biomarkers for the evaluation of KMRC011 clinical efficacy. Our data indicated that KMRC011 has radioprotective activity in lethally irradiated mice and may be developed as a therapeutic agent for radioprotection.

TLR5 binding and activation by KMRC011, a flagellin-derived radiation countermeasure.

Entolimod (CBLB502) is a flagellin-derived radiation countermeasure currently under clinical trial. Entolimod exerts radioprotective activity by directly interacting with TLR5, an innate immune receptor, using the conserved domains of flagellin. Entolimod was designed to contain an artificially introduced N-terminal region that is not related to drug effects and might trigger unexpected toxic immunogenic reactions in humans. To refine the entolimod drug design, we engineered entolimod into KMRC011 by removing its ancillary region. The TLR5 binding and activating capacities of KMRC011 were assessed through biophysical and cellular analyses. KMRC011 forms an exceptionally stable complex with TLR5 at a 1:1 molar ratio with an equilibrium dissociation constant of ∼100 pM and potently activates TLR5. Moreover, alanine scanning mutagenesis identified the R90 and E114 residues of KMRC011 as a TLR5 activation hotspot. Further comparative analysis demonstrated that KMRC011 binds and activates TLR5 in a mode similar to that of entolimod. Thus, we propose that KMRC011 can be used in place of entolimod as a second-generation radiation countermeasure that shows none of the immunogenic side effects derived from the entolimod ancillary region.

Repeated injection of KMRC011, a medical countermeasure for radiation, can cause adverse health effects in cynomolgus monkeys.

High-dose radiation-induced tissue damage is a major limiting factor in the medical application of nuclear technology. Herein, we tested 28-day repeated-dose toxicity of KMRC011, an agonist of toll-like receptor (TLR) 5, which is being developed as a medical countermeasure for radiation, using cynomolgus monkeys. KMRC011 (0.01, 0.02 or 0.04 mg/kg/day) was intramuscularly injected once daily for 4 weeks, and each two monkeys in both control and 0.04 mg/kg/day group were observed for an additional 2-week recovery period. There were no dose-related toxicological changes in mortality, clinical observations, body weight, food consumption, ophthalmological findings, electrocardiographs, coagulation, serum chemistry, organ weights, or urinalysis and urine chemistry. Although treatment-related changes, such as increased white blood cells, increased absolute and relative neutrophils, decreased relative lymphocytes and inflammatory lesions, were noted in the maximum dose group, these findings were not observed after the 2-week recovery period. Further, we considered that the kidneys and heart may be target organs of TLR5 agonists, as well as the spleen, and that autophagic signals can be triggered in tissue damage and the repair process. Importantly, accumulation of p62 protein, an indicator of autophagy, and a decrease of caveolin-1 protein, a regulator of TLR5 protein half-life, were found in both tissues from the highest dose group. Therefore, we conclude that the no-observed-adverse-effect level for KMRC011 may be greater than 0.04 mg/kg/day in male and female monkeys. Additionally, we propose that further studies are needed to identify the molecular signals, which are related to KMRC011-induced adverse effects.

Optimization of recombinant human platelet-derived growth factor-BB encapsulated in Poly (lactic-co-glycolic acid) microspheres for applications in wound healing.

Growth factors play multiple and critical roles in wound repair processes. Platelet-derived growth factor (PDGF) is a potent growth factor that is particularly important in the early inflammatory phase of wound healing. In order to extend the half-life of PDGF, polymeric encapsulation is used. In the current study, Poly (lactic-co-glycolic acid) (PLGA) microspheres containing recombinant human (rh) PDGF-BB were prepared to prolong the effectiveness of this growth factor. PLGA microspheres were optimized using a modified w/o/w-double-emulsion/solvent evaporation method by changing the processing conditions of stirring speed and emulsifier (polyvinyl alcohol) concentration. Microspheres prepared using the optimized method released rhPDGF-BB for up to three weeks. An in vitro migration assay showed a significant decrease in the wound area in cells treated with rhPDGF-BB microspheres compared to control cells. These findings demonstrate the potential of rhPDGF-BB encapsulated in microspheres to enhance wound healing.

Comparative proteomic tissue analysis in patients with ossification of the posterior longitudinal ligament.

OBJECTIVE: The ossification of the posterior longitudinal ligament (OPLL) involves the ligament that lines the posterior surface of the spinal vertebral bodies. Hormonal and metabolic factors as well as hereditary factors have been proposed to be involved in pathologic ligamentous OPLL. However, there are currently no definitive serological biomarkers for OPLL that might be used to achieve a more convenient and economic diagnosis. To find an easier and simpler diagnostic method and to identify pathogenic proteins associated with OPLL, we assessed PLL tissues from patients with OPLL for proteomic alterations. METHODS: OPLL tissues were collected from 12 patients with OPLL, and non-OPLL tissues were collected from 12 healthy subjects without OPLL. To minimize individual variations, we matched the sex and age of the patients in the healthy and OPLL groups. The two-dimensional electrophoresis patterns of tissue from 12 OPLL patients and 12 healthy subjects were compared. RESULTS: We found 25 proteins that were significantly and consistently different on the two-dimensional electrophoresis gels between the group of ossified PLL tissues from the patients with OPLL and the group of nonossified PLL tissues from the healthy subjects. Among them, 21 proteins were up-regulated in the patients with OPLL, whereas the remaining four proteins were down-regulated. CONCLUSIONS: The information obtained via this proteomic analysis will be very useful in understanding the pathophysiology of OPLL as well as in finding protein candidates to serve as new diagnostic biomarkers of OPLL.

Antibacterial properties of a pre-formulated recombinant phage endolysin, SAL-1.

To evaluate the phage endolysin SAL-1 as a therapeutic agent for Staphylococcus aureus infections, the in vitro and in vivo antibacterial properties of a pre-formulation containing recombinant SAL-1 as an active pharmaceutical ingredient were investigated. The stable pre-formulation (designated SAL200) uniquely included calcium ions and Poloxamer 188 as enhancing and stabilising ingredients, respectively. SAL-1 was successfully produced with no extraneous amino acids by decreasing the culture temperature and was highly purified using a two-step chromatography procedure consisting of ion exchange and hydrophobic interaction chromatography. SAL200 exhibited rapid and effective bactericidal activity against encapsulated and biofilm-forming S. aureus as well as against planktonic S. aureus cells. In addition, SAL200 demonstrated increased effectiveness in the serum environment, with a significantly reduced minimum bactericidal concentration compared with that determined in culture medium. In in vitro antibacterial tests performed against 425 clinical isolates [including 336 meticillin-resistant S. aureus (MRSA) isolates and 1 vancomycin-intermediate S. aureus isolate], collected from 421 patients and four animals, SAL200 exhibited obvious antibacterial activity against all S. aureus isolates tested. Intravenous injection of SAL200 in a mouse model of MRSA infection prolonged the viability of mice and significantly reduced bacterial counts in the bloodstream and splenic tissue. The results presented in this article strongly support SAL200 as a highly potent bactericidal agent against MRSA with an adequate pharmaceutical formulation.

Differential regulation of matrix metalloproteinase-9 and tissue plasminogen activator activity by the cyclic-AMP system in lipopolysaccharide-stimulated rat primary astrocytes.

We investigated the effect of the cAMP system on lipopolysaccharide (LPS)-induced changes in the activity of matrix metalloproteinases (MMPs) and tissue plasminogen activator (tPA) in rat primary astrocytes. LPS stimulation increased MMP-9 and decreased tPA activity in rat primary astrocytes. Co-treatment with a cAMP analog, dibutyryl-cAMP (db-cAMP), or the cAMP elevating beta-adrenergic agonist, isoproterenol, concentration-dependently inhibited LPS-induced MMP-9 activity. In contrast, db-cAMP concentration-dependently increased tPA activity in both basal and LPS-stimulated rat primary astrocytes. To confirm the effect of cAMP on MMP-9 and tPA activity, we treated LPS-stimulated astrocytes with cAMP phosphodiesterase inhibitors, IBMX or rolipram, and they exhibited similar effects to db-cAMP, namely decreasing MMP-9 activity and increasing tPA activity. RT-PCR analysis of MMP-9 mRNA expression and MMP-9 promoter luciferase reporter assays revealed transcriptional upregulation by LPS stimulation and downregulation by db-cAMP. In contrast, the level of tPA mRNA expression was increased both by LPS and by cAMP treatment. Consistent with RT-PCR analysis, tPA promoter reporter assays showed increased activity by both LPS and cAMP stimulation. Interestingly, the level of mRNA encoding plasminogen activator inhibitor-1 (PAI-1) was increased by LPS stimulation and decreased back to control level after co-treatment with db-cAMP, suggesting that PAI-1 expression plays a major role in the regulation of tPA activity. To examine PKA involvement in the effects of db-cAMP on MMP-9 and tPA activity, we added the PKA inhibitors, H89 or rp-cAMP, along with db-cAMP, and they inhibited db-cAMP-mediated changes in tPA activity without affecting MMP-9 activity. These data suggest that cAMP differentially modulates MMP-9 and tPA activity through a mechanism related to PKA activation. The differential regulation of MMP-9 and tPA by the cAMP system may confer more sophisticated regulation of physiological processes, such as extracellular matrix remodeling and cell migration, by activated astrocytes.

Activation of protease-activated receptor1 mediates induction of matrix metalloproteinase-9 by thrombin in rat primary astrocytes.

Thrombin plays an important role in diverse neurological processes such as proliferation, cell migration, differentiation and neuroinflammation. In this study, we investigated the effect of thrombin on matrix metalloprotease-9 (MMP-9) expression in rat primary astrocytes. Thrombin (1-10U/ml) induced a significant increase in MMP-9 activity as measured by gelatin zymography. Thrombin also increased MMP-9 mRNA expression. Among three isotypes of thrombin receptor, i.e. protease-activated receptor (PAR)-1, -3 and -4, PAR1 agonist (1-100muM) but not PAR3 and PAR4 agonist induced MMP-9 expression. Inhibition of thrombin-induced MMP-9 production by SCH 79797 (10-50nM), a selective PAR1 receptor antagonist, confirmed that PAR1 is a main receptor for thrombin-induced MMP-9 expression. In astrocytes, thrombin activated Erk1/2, and it was inhibited by PD98059. In this study, thrombin-induced MMP-9 expression was inhibited by PD98059. PAR1 agonist activated Erk1/2 and PD98059 inhibited PAR1 agonist-induced MMP-9 expression. MMP-9 promoter reporter assay confirmed the positive effect of ERK1/2 on MMP-9 expression. These results suggest that the activation of PAR1 mediates thrombin-induced MMP-9 expression through the regulation of Erk1/2.

Characterization of ubiquitin C-terminal hydrolase 1 (YUH1) from Saccharomyces cerevisiae expressed in recombinant Escherichia coli.

The YUH1 gene coding for ubiquitin C-terminal hydrolase 1, a deubiquitinating enzyme, was cloned from the Saccharomyces cerevisiae genomic DNA and expressed in Escherichia coli. YUH1 was fused with the 6 histidine tag at the N-terminus (H6YUH1) or C-terminus (YUH1H6) and purified by an immobilized metal affinity chromatography with high purity. By using a fluorogenic substrate, Z-Arg-Leu-Arg-Gly-Gly-AMC, the deubiquitinating activities for H6YUH1 (1.72U/mg) and YUH1H6 (1.61U/mg) were about 18 times higher than 0.092U/mg for H6UBP1, ubiquitin specific protease 1 of S. cerevisiae containing the 6 histidine residue at the N-terminus which is normally used in protein engineering. YUH1 had the optimal temperature of 27 degrees C and acidity of pH 8.5. Analysis of thermal deactivation kinetics of H6YUH1 estimated 3.2 and 1.4h of half lives at 4 and 52 degrees C, respectively. Immobilization onto the Ni-NTA affinity resin and environmental modulation were carried out to improve the stability of YUH1. Incubation of the immobilized YUH1 in 50% glycerol solution at -20 degrees C resulted in 52% of decrease in specific activity for 7days, corresponding to a 2.7-fold increase compared with that of the free YUH1 incubated in the same solution at 4 degrees C.

Role of p38 MAPK on the down-regulation of matrix metalloproteinase-9 expression in rat astrocytes.

In spite of their pathophysiological importance in neuro-inflammatory diseases, little is known about the signal transduction pathways that lead to the induction of matrix metalloproteinases (MMPs) in the central nervous system. We reported previously that lipopolysaccharide (LPS) induced MMP-9 expression through ERK1/2 pathway in rat primary astrocytes (Glia 41:15-24, 2003). Here, we investigated the role of other MAPK pathways, including p38 and JNK/SAPK, on the regulation of MMP-9 expression in LPS-stimulated rat primary astrocytes. LPS activated both p38 and JNK in astrocytes. Treatment with a specific p38 MAPK inhibitor SB203580, but not JNK inhibitor SP600125, increased the LPS-stimulated MMP-9 expression in a concentration-dependent manner. Anti-inflammatory cytokines, including IFN-gamma and IL-4, activated p38 MAPK and decreased MMP-9 production in LPS-stimulated astrocytes. When p38 MAPK activation was blocked by SB203580, the inhibitory effects of these cytokines on MMP-9 induction were abolished. Finally, direct injection of SB203580 into the lateral ventricle of rat brain increased the LPS-induced MMP-9 activity in cerebral cortex. Altogether, these results suggest that p38 activation down-regulates the inflammatory stimulation-induced overexpression of MMP-9, both in primary astrocytes and in cerebral cortex. The elaborate interplay between ERK1/2 and p38 pathways provides a more sophisticated mechanism for regulating MMP-9 activity in neuroinflammatory diseases.

Comparative analysis of serum proteomes to discover biomarkers for ossification of the posterior longitudinal ligament.

STUDY DESIGN: Serum proteomes from normal subjects and the patients with ossification of the posterior longitudinal ligament (OPLL) were analyzed by using proteomics. OBJECTIVES: To identify novel serologic biomarkers for diagnosing OPLL. SUMMARY OF BACKGROUND DATA: OPLL can compress the spinal cord, and special planning is required for surgeries that are done from the front of the cervical spine. However, the definitive serologic biomarkers for OPLL are still unclear. METHODS: The 2-dimensional electrophoresis patterns of sera from OPLL patients and normal subjects were compared. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization quadruple time-of-flight mass spectrometry. RESULTS: Nine spots that were differentially expressed in the sera of OPLL patients were found and were identified. PRO2675, human serum albumin in a complex with myristic acid and tri-iodobenzoic acid, an unknown protein, chain B of the crystal structure of deoxy-human hemoglobin beta6, pro-apolipoprotein, ALB protein, retinol binding protein and chain A of human serum albumin mutant R218h complexed with thyroxine (3,3',5,5', tetraiodo-L-thyronine), were up-regulated in the sera of OPLL patients, whereas alpha1-microglobulin/bikunin precursor was down-regulated. CONCLUSIONS: These proteins could be used as diagnostic biomarkers of OPLL.

Down-regulation of matrix metalloproteinase-9 expression by nitric oxide in lipopolysaccharide-stimulated rat primary astrocytes.

Immunologically activated astrocytes over-express matrix metalloproteinase-9 (MMP-9) and nitric oxide (NO). Because they have both beneficial and detrimental effects on the pathophyiological outcomes of several neurological diseases, their expression should be tightly regulated in the CNS. NO can modify the activity of other proteins either by directly modifying protein structure or regulating the expression of target proteins. In this study, we investigated the role of NO on the expression of MMPs in rat primary astrocytes. Rat primary astrocytes were stimulated with lipopolysaccharide (LPS), resulting in the over-expression of both MMP-9 and NO. Inhibition of NO production using nitric oxide synthase inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME), further increased MMP-9 expression, suggesting NO inhibits MMP-9 expression. In line with this observation, exogenous addition of NO donor, sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP), inhibited MMP-9 expression in astrocytes. The inhibitory effect of NO was mediated by the down-regulation of mRNA and protein levels of MMP-9 but not by the direct modification of the enzymatic activity of MMP-9. The effect of NO on MMP-9 expression was mimicked by dibutyryl-cGMP and inhibited by PKG inhibitor KT5823, suggesting NO regulates MMP-9 expression via guanylate cyclase-PKG pathway. Finally, SNP or dibutyryl-cGMP inhibited the activation of ERK1/2 in LPS-stimulated astrocytes, which is an essential regulator of MMP-9 expression in astrocytes. The regulation of MMP-9 expression by NO may confer additional levels of fine-tuning of the level of MMP-9 during brain inflammatory conditions.

Glucose deprivation increases hydrogen peroxide level in immunostimulated rat primary astrocytes.

Activated astrocytes produce a large amount of bioactive molecules, including reactive oxygen and nitrogen species. Astrocytes are in general resistant to those reactive species. However, we previously reported that immunostimulated astrocytes became highly vulnerable to metabolic insults, such as glucose deprivation. In this study, we investigated whether H(2)O(2) production was associated with the increased vulnerability. Glucose deprivation for up to 8 hr did not change the intracellular level of H(2)O(2) in astrocytes. Treatment with lipopolysaccharide plus interferon-gamma for 48 hr evoked astroglial H(2)O(2) production; however, no apparent death or injury was observed in immunostimulated astrocytes. Glucose deprivation after 48 hr of immunostimulation markedly increased H(2)O(2) level, depleted adenosine triphosphate (ATP), and enhanced lactate dehydrogenase (LDH) release. The ATP depletion and LDH release were in part prevented by catalase, mannitol, and N-acetyl-L-cysteine. The enhanced level of H(2)O(2) in glucose-deprived immunostimulated astrocytes appeared to be secondary to the depletion of reduced glutathione. 4-(2-Aminoethyl)bebzenesulfonyl fluoride (AEBSF), an inhibitor of NADPH oxidase, reduced H(2)O(2) level and LDH release in glucose-deprived immunostimulated astrocytes. H(2)O(2), either endogenously produced or exogenously added, depolarized mitochondrial transmembrane potential in glucose-deprived astrocytes, leading to their ATP depletion and death. The present results strongly indicate that glucose deprivation causes deterioration of immunostimulated astrocytes by increasing the intracellular concentration of H(2)O(2).

Induction of matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide-stimulated primary astrocytes is mediated by extracellular signal-regulated protein kinase 1/2 (Erk1/2).

In the present study, we investigated whether the activation of protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (Erk1/2) are involved in the induction of MMP-9 in lipopolysaccharide (LPS)-stimulated primary astrocytes. The expression of MMP-9 but not MMP-2 was increased by LPS. LPS treatment induced activation of Erk1/2 within 30 min, which was dose-dependently inhibited by PD98059, a specific inhibitor of the Erk kinase (MEK). In this condition, PD98059 blocked the increase in MMP-9 protein and mRNA level as well as gelatin-digesting activity. Inhibition of PKC activity blocked the LPS-induced activation of Erk1/2 as well as MMP-9 expression. In addition, activation of PKC by phorbol myristoyl acetate (PMA) activated Erk1/2 with concomitant increase in MMP-9 production. Moreover, treatment of PD98059 dose-dependently decreased the PMA-induced MMP-9 expression. The results from the present study suggest that induction of MMP-9 by LPS in rat primary astrocytes is mediated, at least in part, by the sequential activation of PKC and Erk1/2. The Erk1/2-mediated MMP-9 induction may provide insights into the regulation of MMP-9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation.

Platycodin D and D3 increase airway mucin release in vivo and in vitro in rats and hamsters.

The root of Platycodon grandiflorum has been widely used for the treatment of various chronic inflammatory diseases including airway disease in oriental medicine. The root extract of the plant has been known to be effective in the expectoration of sputum or mucus, thereby improving airway respiratory function and preventing secondary airway inflammation. In this study, we investigated the effect of platycodin D and D3, the saponin components that are anti-inflammatory components in Platycodon grandiflorum. Platycodin D and D3 increased mucin release from rat and hamster tracheal surface epithelial cell culture and also from intact rat trachea upon nebulization. The effect of platycodin D3 was stronger than that of ATP, a potent mucin secretagogue and also of ambroxole, a mucolytic drug. The results from the present study suggest that platycodin D and D3 are useful as expectorant agents in the treatment of various airway diseases.