Our data indicate that hemorrhage-induced injuries to small intestine were reduced by pretreatment with orally administered 17-DMAG. The protection we observed correlated with a 17-DMAG-induced reduction in iNOS and caspase-3 activity and an increase in Bcl-2 protein.
Hemorrhage can occur after a wide variety of injuries, ranging from wound trauma to stroke, aneurysm, birth labor, or blast impact. Hemorrhage lowers the levels of oxygen and nutrition available to tissues and results in an accumulation of carbon dioxide and metabolic waste, leading to activation of signal transduction pathways and tissue damage . In our experiments, hemorrhage activated the iNOS pathway (Figures 2 and 3), generated oxidative and nitrosative stress (Figure 4), and reduced cellular ATP (Figure 4). Subsequently, caspase-3 was activated (Figure 5) and cell death occurred (Figure 7). These observations are consistent with those previously described [4–7, 17]. The hemorrhage-induced increase in iNOS protein was due to increases in transcription factors NF-κB and KLF6 and a decrease in KLF4 (Figures 2,3). It is known that NF-κB and KLF6 stimulate iNOS gene expression whereas KLF4 inhibits it .
17-DMAG treatment inhibited activation of the iNOS pathway and caspase-3-mediated cell death mainly by inhibiting NF-κB and KLF6 and increasing KLF4. The interpretation that iNOS inhibition leads to reduced hemorrhage damage is reinforced by the results of studies employing iNOS gene knockout  and treatment with iNOS-specific inhibitors [6, 18]. How 17-DMAG exerts its action on these iNOS transcription factors is not completely clear and further study is required.
An increase in HSP-70 has been shown to inhibit the iNOS increase triggered by hemorrhage [4, 8]. However, it does not appear that the ability of 17-DMAG to inhibit hemorrhage damage in jejunum is due to its ability to increase HSP-70. 17-DMAG treatment at the EC50 concentration for inducing HSP-70 markedly elevated HSP-70 levels, but the combination of 17-DMAG treatment and hemorrhage resulted in a lower level of HSP-70 that was no different than that seen after hemorrhage alone.
Because 17-DMAG in our experiments was administered orally, the possibility that 17-DMAG was absorbed by small intestine, and locally protected it from hemorrhage injury cannot be excluded. It is known that hemorrhage induces a p38-MAPK increase  and a Bcl-2 decrease (Figure 6) . 17-DMAG may exert an effect on these proteins as well. More studies are needed in this area.
Hemorrhage increased ATP depletion in agreement with our previous report . 17-DMAG treatment prevented the depletion, probably due to its ability to upregulate production of pyruvate dehydrogenase protein .
Hemorrhage increased TNF-α and IL-10 (Figure 4D-E) and IL-6. Increased TNF-α has been observed in other experimental models such as those for ionizing radiation [21–25] and ischemia . Inhibition of TNF-α levels by drug treatment or antibody neutralization results in an improvement in bone marrow regeneration after radiation exposure . Yen et al.  reported that TNF-α is the key to the development of hemorrhage injury; Pfeifer et al.  showed that hemorrhage in fact triggers increased TNF-α. It is likely in our experiments that apoptosis was also caused by the extrinsic pathway involving TNF-α receptors, which probably contributed to the final caspase-3 activation. The fact that 17-DMAG inhibited the hemorrhage-induced increase in TNF-α is desirable, but the fact it also inhibited the beneficial IL-10 is undesirable in terms of 17-DMAG's use as a drug. The hemorrhage-induced increase in IL-10 we observed could be a self-defense response to injury, but its level is in the range of pg/ml, a level too low to combat the damage. We have shown that treatment with ng/ml levels of IL-10 are required to improve survival from a lethal dose of γ-irradiation .
Bcl-2 is anti-apoptotic and p53 is pro-apoptotic . Hemorrhage reduced Bcl-2 and increased p53 in our experiments, which probably partly contributed to the damage observed. However, 17-DMAG treatment increased not only Bcl-2 but surprisingly also p53 (Figure 6). The anti-apoptotic activity of Bcl-2 stimulated by 17-DMAG is apparently greater than the pro-apoptotic activity of p53.
To summarize, 17-DMAG is water-soluble, distributes to all tissues in the body, and is non-toxic in mice [9, 10]. Compared to other iNOS inhibitors, 17-DMAG is potentially superior because of its ability to increase HSP-70 and Bcl-2 proteins and decrease HSP-90 activity and TNF-α concentration as well as ATP depletion. Its water solubility and non-toxicity and the fact that 17-DMAG is effective when administered orally make it a promising candidate drug for ameliorating hemorrhage injury.