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Background: It is well known that patients with chronic kidney disease, including diabetic nephropathy, often develop cardiovascular diseases. In case of radiographic procedures, reduced renal function may be deteriorated by the use of iodinated contrast medium (CM). This is known as CM-induced nephropathy. In this study, we have focused on the mechanisms of this type of injury in diabetic nephropathy and the preventive effects of serofendic acid., Methods: We evaluated the cytotoxicity of CM and high glucose on tubular epithelial cells using an LLC-PK1 cell line, and measured cell viability with an alamarBlue assay. We further evaluated superoxide production levels measured by dihydroethidium. We also examined the protective effects of serofendic acid on cytotoxicity with superoxide production of CM and high glucose., Results: CM reduced cell numbers in a dose-dependent and time-dependent manner in LLC-PK1 cells. Furthermore, cytotoxicity of CM in diluted concentration was additively influenced by high glucose. CM and high glucose increased superoxide production, which was evaluated by the response to dihydroethidium, and was suppressed by serofendic acid. Cytotoxicity of CM, high glucose, and H(2)O(2) was suppressed by serofendic acid, as well as the suppression by N-acetylcysteine on CM toxicity. Interestingly, the recovery by serofendic acid in H(2)O(2)- and high glucose-induced cellular injury was to the basal level, in contrast with the partial recovery from CM-induced injury. Finally, serofendic acid suppressed CM-induced injury and high glucose-induced apoptosis., Conclusions: These results suggest that CM and high glucose induce cytotoxicity and oxidative stress in LLC-PK1 cells and that serofendic acid protects the injury probably from superoxide generation.

We investigated the effect of serofendic acid, a neuroprotective substance derived from fetal calf serum, on the morphological changes in cultured cortical astrocytes. Cultured astrocytes developed a stellate morphology with several processes following exposure to dibutylyl cAMP (dbcAMP), a membrane-permeable cAMP analog; 8-Br-cGMP, a membrane-permeable cGMP analog; or phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator. Serofendic acid significantly accelerated the stellation induced by dbcAMP- and 8-Br-cGMP. In contrast, the PMA-induced stellation was not affected by serofendic acid. Next, we attempted to elucidate the mechanism underlying the dbcAMP-induced stellation and explore the site of action of serofendic acid. Both the stellation induced by dbcAMP and the promotional effect of serofendic acid were partially inhibited by KT5720, a specific protein kinase A (PKA) inhibitor. Furthermore, serofendic acid failed to facilitate the stellation induced by Y-27632, an inhibitor of Rho-associated kinase (ROCK). These results indicate that serofendic acid promotes dbcAMP- and 8-Br-cGMP-induced stellation and the promotional effect on dbcAMP-induced stellation is mediated at least partly by the regulation of PKA activity and not by controlling ROCK activity.

We previously reported that a sulfur-containing neuroprotective substance named serofendic acid purified and isolated from fetal calf serum prevented glutamate neurotoxicity in rat cortical cultured neurons. In the present study, we investigated the effect of serofendic acid on ischemic injury induced by a transient occlusion of the middle cerebral artery in rats. Serofendic acid was intracerebroventricularly administered 30 min after the onset of the occlusion. Serofendic acid (30 nmol) significantly reduced total infarct volume, similar to edaravone (30 nmol), a free radical scavenger. Treatment with serofendic acid (1-30 nmol) reduced the infarct volume in a dose-dependent manner. Moreover, serofendic acid (30 nmol) improved neurological deficit scores. These results suggest that intracerebroventricular administration of serofendic acid prevents the neurodegeneration induced by a transient focal cerebral ischemia and reperfusion.

We present efficient syntheses of serofendic acids A and B (SA-A and SA-B), novel neuroprotective substances isolated from fetal calf serum. Biological and pharmacological evaluation showed that SA-A and SA-B have potent protective action against glutamate-induced neurotoxicity, but do not interact directly with glutamate receptors. A pharmacokinetic study showed that they have good oral bioavailability in rats. The results indicate that SA-A and SA-B are potential lead compounds for candidate drugs to treat various neurological disorders.

Analogues of serofendic acid were prepared and their protective effects against L-glutamate (Glu)-induced neurotoxicity were examined using primary cultures of rat cortical neurons. Some analogues exhibited similar neuroprotective activity to that of serofendic acid.

We have previously reported that a neuroprotective substance, serofendic acid, was purified and isolated from fetal calf serum. Here, we investigated the effect of serofendic acid on glutamate-induced apoptosis using rat primary cultures of cortical neurons. Exposure of the cortical cultures to relatively low concentration of glutamate (100 microM) induced neuronal death and nuclear fragmentation. Glutamate exposure also induced a transient increase in caspase-3 activity. A membrane-permeable inhibitor of caspase-3 (DEVD-CHO) prevented the glutamate neurotoxicity. Serofendic acid (0.01-10 microM) markedly prevented glutamate-induced apoptotic neuronal death and nuclear fragmentation. To elucidate the protective mechanism of serofendic acid, we first examined the effect on the glutamate-induced increase in intracellular Ca2+ concentration. Glutamate-induced increase in intracellular Ca2+ concentration was significantly inhibited by MK-801, a NMDA receptor antagonist, but not by serofendic acid. Next, we investigated the effect of serofendic acid on the loss of mitochondrial membrane potential induced by glutamate by using a fluorescence indicator, tetramethylrhodamine methyl ester (TMRM). Glutamate exposure resulted in a rapid reduction of TMRM fluorescence, indicating that mitochondrial membrane was depolarized by glutamate. Serofendic acid prevented the loss of mitochondrial membrane potential following glutamate exposure. Moreover, serofendic acid reduced the activation of caspase-3 induced by glutamate. Finally, serofendic acid directly inhibited the activity of recombinant human caspase-3, -7 and -8 at higher concentrations. These results indicate that serofendic acid prevents glutamate-induced apoptosis in cultured cortical neurons by the prevention of loss of mitochondrial membrane potential and the reduction of the process of caspase-3 activation.

Serofendic acid was recently identified as a neuroprotective factor from fetal calf serum. This study was designed to evaluate the neuroprotective effects of an intranigral microinjection of serofendic acid based on behavioral, neurochemical and histochemical studies in hemi-parkinsonian rats using 6-hydroxydopamine (6-OHDA). Rats were injected with 6-OHDA in the presence or absence of serofendic acid, or were treated with serofendic acid on the same lateral side, at 12, 24 or 72 h after 6-OHDA lesion. Intranigral injection of 6-OHDA alone induced a massive loss of tyrosine hydroxylase (TH)-immunopositive neurons in the substantia nigra pars compacta (SNpc). Either simultaneous or 12 h post-administration of serofendic acid significantly prevented both dopaminergic neurodegeneration and drug-induced rotational asymmetry. Immunoreactivities for oxidative stress markers, such as 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (4-HNE), were markedly detected in the SNpc of rats injected with 6-OHDA alone. These immunoreactivities were markedly suppressed by the co-administration of serofendic acid, similar to the results in vehicle-treated control rats. In addition, serofendic acid inhibited 6-OHDA-induced alpha-synuclein expression and glial activation in the SNpc. These results suggest that serofendic acid protects against 6-OHDA-induced SNpc dopaminergic neurodegeneration in a rat model of Parkinson's disease.

We have previously reported that a sulfur-containing neuroprotective substance named serofendic acid was purified and isolated from lipophilic extract of fetal calf serum (FCS). In the present study, we investigated the effect of serofendic acid on glutamate neurotoxicity using embryonic rat spinal cord culture. When cultures were exposed to glutamate (20 microM) with a glutamate transporter inhibitor L-trans-pyrrolidine-2,4-decarboxylate (PDC; 40 microM) for 24 h, motor neurons were injured through both N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole/kainate receptors. This glutamate neurotoxicity was attenuated by nitric oxide (NO) synthase inhibitors. Serofendic acid (0.1-5 microM) prevented glutamate neurotoxicity in a concentration-dependent manner. S-Nitrosocysteine (SNOC; 10 microM), an NO donor, induced motor neuronal death. Serofendic acid (5 microM) also prevented SNOC-induced neurotoxicity. These results indicate that serofendic acid protects cultured motor neurons from glutamate neurotoxicity by reducing the cytotoxic action of NO.

We previously identified a novel endogenous substance, serofendic acid, from a lipophilic extract of fetal calf serum. Serofendic acid protects cultured cortical neurons against the cytotoxicity of glutamate and nitric oxide. Here, we reported the protective effect of serofendic acid on reactive oxygen species-induced oxidative stress using primary rat striatal cultures. In addition, we compared the neuroprotective effect and the radical-scavenging activity of serofendic acid with those of dimethyl sulfoxide (DMSO), because serofendic acid possesses a DMSO structure. Paraquat caused neuronal death, which was inhibited by a cell-permeable superoxide dismutase (SOD) mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (Mn-TBAP); a cell-permeable SOD/catalase mimetic, EUK-134 [manganese 3-methoxy N,N'-bis(salicylidene)ethylenediamine chloride]; and a ferrous ion chelator, 2,2'-dipyridyl, in rat striatal cultures. Serofendic acid (10-100 microM) suppressed the neurotoxicity of paraquat, whereas DMSO (10-100 microM) did not. By contrast, higher concentrations (30-300 mM) of DMSO ameliorated the paraquat-induced cell death. Furthermore, H(2)O(2) induced neurotoxicity, which was prevented by EUK-134 and 2,2'-dipyridyl. Serofendic acid (10-100 microM) also protected striatal neurons against the H(2)O(2)-induced toxicity. Higher concentrations (30-300 mM) of DMSO ameliorated H(2)O(2)-induced neuronal death, whereas lower concentrations (10-100 microM) did not. Electron spin resonance spectrometry with a spin-trapping technique revealed that serofendic acid and DMSO had approximately the same ability to inhibit the formation of the hydroxyl radical (.OH). These results suggest that the.OH-scavenging activity of serofendic acid is attributable to its DMSO structure and that the remaining components such as the atisane structure play an important role in eliciting neuroprotection at a concentration range of 10 to 100 microM.

(-)-Methyl 7 beta-hydroxykaurenoate (3) and its 4-demethyl acetate (-)-4 were both synthesized via methods that contained radical cyclization and intramolecular Diels-Alder reactions as key steps. Both compounds displayed potent neuroprotective activity against N-methyl-D-aspartate toxicity in cultured cortical neurons.

We have previously reported that a novel neuroprotective substance named serofendic acid was purified and isolated from ether extract of fetal calf serum. In the present study, we investigated the effect of serofendic acid on acute neurotoxicity induced by L-glutamate (Glu) using primary cultures of rat cortical neurons. Exposure of cortical cultures to Glu for 1 h caused a marked decrease in cell viability, as determined by trypan blue exclusion. This acute Glu neurotoxicity was prevented by N-methyl-D-aspartate (NMDA) receptor antagonists, extracellular Ca(2+) removal, nitric oxide (NO) synthase inhibitor and NO scavenger. Serofendic acid prevented acute Glu neurotoxicity in a concentration-dependent manner. Acute neurotoxicity was induced by ionomycin, a Ca(2+) ionophore, and S-nitroso-L-cysteine, an NO donor. Serofendic acid also prevented both ionomycin- and S-nitroso-L-cysteine-induced neurotoxicity. Moreover, the protective effect of serofendic acid on acute Glu neurotoxicity was not affected by cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor. These results indicate that serofendic acid protects cultured cortical neurons from acute Glu neurotoxicity by reducing the cytotoxic action of NO and de novo protein synthesis is not required for this neuroprotection.

Excess activation of glutamate receptors and production of free radicals including nitric oxide may result in severe and irreversible damage to the mammalian central nervous system (CNS), but endogenous defense systems that protect neurons from these insults are poorly understood. Here, we purified and isolated a neuroprotective substance, which has been named "serofendic acid," from a lipophilic fraction of FCS based on the ability to protect rat primary cortical neurons against nitric oxide cytotoxicity. Mass spectrometry and NMR spectroscopy revealed the chemical structure of serofendic acid (15-hydroxy-17-methylsulfinylatisan-19-oic acid) as a sulfur-containing atisane-type diterpenoid, which is unique among known endogenous substances. Synthetic serofendic acid exhibited potent protective actions on cortical neurons against cytotoxicity of a nitric oxide donor as well as of glutamate, although it did not show appreciable influences on glutamate receptor-mediated responses in these neurons. Electron spin resonance analysis demonstrated that serofendic acid had no direct scavenging activity on nitric oxide radicals but was capable of inhibiting the generation of hydroxyl radical, a presumed "executor" radical in the nitric oxide-mediated neurotoxic cascade. These findings suggest that serofendic acid is a low-molecular-weight bioactive factor that promotes survival of CNS neurons, probably through the attenuation of free radical-mediated insults.