In this study, we showed that early AsA administration compared to late AsA administration may improve the survival rate of a mouse model of sepsis by reducing vascular endothelial cell damage. Despite reports that administration of AsA to septic mice improves prognosis, there are no reports of differences in prognosis depending on the timing of administration [13, 16]. Here, we discuss the mechanism underlying the effectiveness of early AsA administration in reducing vascular endothelial cell damage and the appropriate timing of AsA administration.
The severity of illness and death in septic patients is related to organ damage caused by microcirculatory disturbances and increased vascular permeability due to vascular endothelial dysfunction . The BH2/BH4 ratio correlates with vascular endothelial function [8, 17]. Although AsA has antioxidant properties and inhibits BH4 oxidation, it is ineffective in reducing the already oxidized BH2. In animal experiments using guinea pigs, increased BH2/BH4 ratio has been shown in AsA-deficient models. AsA levels in the body are decreased in critically ill patients, who often experience difficulties in taking AsA orally, and, therefore, require supplemental therapy . In our septic mice, we also confirmed a decrease in AsA plasma concentration in preliminary experiments (data not shown). In this septic mouse, the BH2/BH4 ratio increased, suggesting that the lack of AsA could not inhibit BH4 oxidation. In case of absolute or relative lack of BH4, eNOS undergoes an uncoupling reaction, which produces superoxide, instead of NO, that reacts to form peroxynitrite (ONOO−), a powerful oxidant [7, 18, 19]. Our study showed that the BH2/BH4 ratio increased at 6 h after CLP. Early AsA administration immediately after operation significantly suppressed BH2/BH4 ratio increase. AsA administration has been reported to inhibit BH4 oxidation . Although the optimal AsA dose is unknown, the antioxidant capacity of vitamin C is dose-dependent, and a plasma vitamin C concentration of 175 mg/l (1000 µmol/l) or higher is required to demonstrate radical scavenging capacity. Vitamin C (10 g) is required to achieve a plasma concentration of more than 1000 µmol/l . In humans, the AsA dosage for sepsis is 200 mg/kg/day in most cases, but a very high volume of AsA (66 mg/kg/hour) was shown to be effective in the very early stage of burn injury . In addition, another study reported that vitamin C therapy at doses as high as 10 g within 2 days of admission reduced mortality in patients with severe burns , and no major side effects were observed, even with such a very high dose. Therefore, the optimal dosage of AsA should be investigated. Thus, although the effective dosage of vitamin C for critically ill patients varies according to the disease state, 200 mg/kg/day of AsA was considered the optimal dosage for this experiment as its effectiveness has been demonstrated previously in septic mice [14, 18, 24, 25]. As a result, early AsA administration may improve ONOO− generation, suggesting a protective effect on endothelial cells. In the AsA-non-treated group, increased serum syndecan-1 levels, an indicator of endothelial cell damage, and decreased eNOS expression, an indicator of endothelial cell protection, were observed 12 h after the operation. By contrast, in the early AsA administration group, both serum syndecan-1 levels and eNOS expression showed protective effect on the vascular endothelium. Increased serum syndecan-1 levels in sepsis are associated with vascular permeability and organ damage . Serum syndecan-1 levels are likely to be useful in diagnosing sepsis and may be related to its severity . In our single-center study, syndecan-1 was a predictor of fatal outcome in septic patients . Moreover, we examined liver tissues for organ damage. Indeed, organ damage occurred after 12 h in the group with elevated syndecan-1, but it was reduced in the group with suppressed syndecan-1 following early AsA administration. The difference in survival may be due to the protection of vascular endothelial cells by early AsA administration, thus suppressing organ damage.
In recent years, the effects of AsA administration in patients with septic shock have received much attention [29, 30]. The administration of AsA alone, as well as the simultaneous administration of vitamin B1 and hydrocortisone, have been studied widely. In particular, the simultaneous administration of vitamin B1 and hydrocortisone is known as HAT therapy [31-34]. Given that several recent studies have shown no positive effect of AsA administration in septic shock patients, it remains controversial whether AsA should be administered to these patients [35-37]. Some studies have cited delayed administration as a limiting factor to obtaining a good effect of AsA in septic shock . In our septic mouse, an increase in the BH2/BH4 ratio, which causes ONOO−, had already occurred after 6 h. In addition, organ damage and vascular endothelial cell damage also occurred; thus, administration of AsA at this time would not have been effective. Therefore, in studies reporting no effect of AsA therapy, it is possible that AsA was administered after the BH2/BH4 ratio had already increased, as we have shown in this study. Early high-dose AsA administration is effective in patients with sepsis , including those with hypoalbuminemia or severe organ failure . In this study, we only mentioned the protective effect of AsA on vascular endothelial cells by suppressing the increase in BH2/BH4 ratio, but AsA has additional effects, such as catecholamine production , adrenocorticotropic hormone production , and direct scavenging of free radicals , which may improve the prognosis of sepsis through various pathways .
This study has several limitations. First, it is unclear whether the results of this mouse sepsis model would be similar to those of human sepsis. Changes in BH4 and BH2 over time may differ between humans and mice. Second, because mice can synthesize AsA in their bodies, the dynamics of AsA concentration in their bodies may be different from those of humans. The appropriate dosage needs to be discussed in both human and animal studies. However, our present findings indicate that the timing of AsA administration affects prognosis and that the BH2/BH4 ratio is related to the mechanism of septic shock.
In the CLP mouse model, an increase in the BH2/BH4 ratio, which causes vascular endothelial cell damage, occurred 6 h after the disease onset. In the present study, administration of AsA at an earlier time before the increase in the BH2/BH4 ratio improved the prognosis of the CLP mouse model by protecting the vascular endothelium. In the future, it will be necessary to evaluate the time course of the BH2/BH4 ratio and the post-onset dynamics of syndecan-1 in humans, as well as study the appropriate timing for AsA administration.