Neuroprotective Effect of Catalpol via Anti-Oxidative, Anti-Inflammatory, and Anti-Apoptotic Mechanisms

Oxidative stress (OS) has been implicated in various degenerative diseases in aging. In an attempt to quantify OS in a cell model, we examined OS induced by incubating for 30 min with various free radical generators in PC12 cells by using the dichlorofluorescein (DCF) assay, modified for use by a fluorescent microplate reader. The nonfluorescent fluorescin derivatives (dichlorofluorescin, DCFH), after being oxidized by various oxidants, will become DCF and emit fluorescence. By quantifying the fluorescence, we were able to quantify the OS. Our results indicated that the fluorescence varied linearly with increasing concentrations (between 0.1 and 1 mM) of H2O2 and 2,2'-azobios(2-amidinopropane) dihydrochloride (AAPH; a peroxyl radical generator). By contrast, the fluorescence varied as a nonlinear response to increasing concentrations of 3-morpholinosydnonimine hydrochloride (SIN-1; a peroxynitrite generator), sodium nitroprusside (SNP; a nitric oxide generator), and dopamine. Dopamine had a biphasic effect; it decreased the DCF fluorescence, thus acting as an antioxidant, at concentrations <500 microM in cells, but acted as a pro-oxidant by increasing the fluorescence at 1 mM. While SNP was not a strong pro-oxidant, SIN-1 was the most potent pro-oxidant among those tested, inducing a 70 times increase of fluorescence at a concentration of 100 microM compared with control. Collectively, due to its indiscriminate nature to various free radicals, DCF can be very useful in quantifying overall OS in cells, especially when used in conjunction with a fluorescent microplate reader. This method is reliable and efficient for evaluating the potency of pro-oxidants and can be used to evaluate the efficacy of antioxidants against OS in cells.

Hippocampal long-term potentiation (LTP) is thought to serve as an elementary mechanism for the establishment of certain forms of explicit memory in the mammalian brain. As is the case with behavioral memory, LTP in the CA1 region has stages: a short-term early potentiation lasting 1 to 3 hours, which is independent of protein synthesis, precedes a later, longer lasting stage (L-LTP), which requires protein synthesis. Inhibitors of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) blocked L-LTP, and analogs of cAMP induced a potentiation that blocked naturally induced L-LTP. The action of the cAMP analog was blocked by inhibitors of protein synthesis. Thus, activation of PKA may be a component of the mechanism that generates L-LTP.

Chronic inflammation is associated with a broad spectrum of neurodegenerative diseases of aging. Included are such disorders as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, the Parkinson-dementia complex of Guam, all of the tauopathies, and age-related macular degeneration. Also included are such peripheral conditions as osteoarthritis, rheumatoid arthritis, atherosclerosis, and myocardial infarction. Inflammation is a two-edged sword. In acute situations, or at low levels, it deals with the abnormality and promotes healing. When chronically sustained at high levels, it can seriously damage viable host tissue. We describe this latter phenomenon as autotoxicity to distinguish it from autoimmunity. The latter involves a lymphocyte-directed attack against self proteins. Autotoxicity, on the other hand, is determined by the concentration and degree of activation of tissue-based monocytic phagocytes. Microglial cells are the brain representatives of the monocyte phagocytic system. Biochemically, the intensity of their activation is related to a spectrum of inflammatory mediators generated by a variety of local cells. The known spectrum includes, but is not limited to, prostaglandins, pentraxins, complement components, anaphylotoxins, cytokines, chemokines, proteases, protease inhibitors, adhesion molecules, and free radicals. This spectrum offers a huge variety of targets for new anti-inflammatory agents. It has been suggested, largely on the basis of transgenic mouse models, that stimulating inflammation rather than inhibiting it can be beneficial in such diseases as AD. If this were the case, administration of NSAIDs, or other anti-inflammatory drugs, would be expected to exacerbate conditions such as AD, PD, and atherosclerosis. However, epidemiological evidence overwhelmingly demonstrates that the reverse is true. This indicates that, at least in these diseases, the inflammation is harmful. So far, advantage has not been taken of opportunities indicated by these epidemiological studies to treat AD and PD with appropriate anti-inflammatory agents. Based on this evidence, classical NSAIDs are the most logical choice. Dosage, though, must be sufficient to combat the inflammation. Analysis of mRNA levels of inflammatory mediators indicates that the intensity of inflammation is considerably higher in AD hippocampus and in PD substantia nigra than in osteoarthritic joints. Thus, full therapeutic doses of NSAIDs, or combinations of anti-inflammatory agents, are needed to achieve the suggested neurological benefits.