Carnosic acid protects neuronal HT22 Cells through activation of the antioxidant-responsive element in free carboxylic acid- and catechol hydroxyl moieties-dependent manners.

In a previous study, we found that carnosic acid (CA) protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1. J Neurochem., in press]. In the present study, we used HT22 cells, a neuronal cell line, to test CA derivatives that might be more suitable for in vivo use, as an electrophile like CA might react with other molecules prior to reaching its intended target. CA and carnosol protected the HT22 cells against oxidative glutamate toxicity. CA activated the transcriptional antioxidant-responsive element of phase-2 genes including hemeoxygenase-1, NADPH-dependent quinone oxidoreductase, and gamma-glutamyl cysteine ligase, all of which provide neuroprotection by regulating cellular redox. This finding was confirmed by the result that CA significantly increased the level of glutathione. We synthesized a series of its analogues in which CA was esterified at its catechol hydroxyl moieties to prevent the oxidation from the catechol to quinone form or esterified at those moieties and its carbonic acid to stop the conversion from CA to carnosol. In both cases, the conversion and oxidation cannot occur until the alkyl groups are removed by an intracellular esterase. Thus, the most potent active form as the activator of the Keap1/Nrf2 pathway, the quinone-type CA, will be produced inside the cells. However, neither chemical modulation potentiated the neuroprotective effects, possibly because of increased lipophilicity. These results suggest that the neuroprotective effects of CA critically require both free carboxylic acid and catechol hydroxyl moieties. Thus, the hydrophilicity of CA might be a critical feature for its neuroprotective effects.

Nitric oxide generation directly responds to ultrasound exposure.

Recently, several reports have been published on ultrasonic vascular dilation produced with relatively low-frequency ultrasound. It has been speculated that nitric oxide (NO) is an important factor for this ultrasonic vascular dilation. However, a quantitative relationship between the ultrasound intensity and NO generation was not clarified in these reports. We investigated the quantity of NO generated by various ultrasonic intensities by means of real-time measurement of NO concentration in the adductor muscles of the thigh of New Zealand white rabbits exposed to a continuous-wave ultrasound (490 kHz). In the quantitative relationship between NO generation and ultrasonic intensity, the percent increase in NO concentration was 1.25% +/- 1.25%, 10.6% +/- 2.9% and 20.1% +/- 3.5%, with the maximum muscle temperature increase 0.5 +/- 0.2 degrees C, 0.7 +/- 0.2 degrees C, and 0.8 +/- 0.3 degrees C at the ultrasonic intensity (SPTA) of 0.21, 0.35 and 0.48 W/cm(2), respectively. The effect of ultrasound on NO generation was intensity-dependent with a progressive increase from 0.21 W/cm(2) to 0.48 W/cm(2) without significant thermal effect. Ultrasonic NO generation was partially reduced by NOS inhibitor of L-NMMA, clarifying that ultrasound can activate both NOS-dependent and NOS-independent NO generation. These new findings provided scientific basis for ultrasonic vasodilatation and support the potentiality of a new ultrasonic technology for the treatment and prevention of the ischemic tissue based on the new concept of NO generated angiogenesis. (E-mail: furuhata@jikei.ac.jp).

Insonation facilitates plasmid DNA transfection into the central nervous system and microbubbles enhance the effect.

Many of the diseases which affect the central nervous system are intractable to conventional therapies and therefore require alternative treatments such as gene therapy. Therapy requires safety, since the central nervous system is a critical organ. Choice of nonviral vectors such as naked plasmid DNA may have merit. However, transfection efficiencies of these vectors are low. We have investigated the use of 210.4 kHz ultrasound and found that 5.0 W/cm(2) of insonation for 5 s most effectively transfected a plasmid DNA into culture slices of mouse brain (147.68-fold increase compared with 0 W/cm(2) of insonation for 5 s). The effect was reinforced by combination with echo contrast agent, Levovist. One hundred fifty mg/mL of Levovist significantly increased gene transfection by ultrasound (5.23-fold when insonated at 5.0 W/cm(2) for 5 s). When DNA was intracranially injected, Levovist also enhanced gene transfection in newborn mice (4.49-fold increase when insonated at 5.0 W/cm(2) for 5 s). Since ultrasound successfully transfected naked plasmid DNA into the neural tissue and Levovist enhanced the effect, this approach may have a significant role in gene transfer to the central nervous system.