To our knowledge, this is the first study to evaluate the effects of GRS on cerebral oedema formation and clarify whether its underlying mechanism involves AQP4 function. The brain water content in a well-established mouse model of acute cerebral oedema was reduced by GRS and the symptoms related cerebral oedema (e.g., loss of righting reflexes and survival) were improved. We examined H217O dynamics through MRI and observed that GRS reduced the AUC of H217O in the LV and cortex. These data indicate that GRS inhibits cerebral oedema formation by reducing water influx into the brain. As the mechanism of action, we found that GRS inhibited AQP4 function. In Xenopus laevis oocytes expressing AQP4, GRS suppressed water permeability. As discussed below, AQP4 is known to be involved in cerebral oedema formation. Thus, the inhibitory effect on AQP4 function may be one of the mechanisms of the anti-cerebral oedema effect of GRS.
Cerebral oedema has been associated with headaches due to the physical pressure caused by brain tissue swelling [1–4]. It can be induced by trauma or haematoma, causing serious symptoms, such as impaired consciousness and abnormal breathing [32]. Headache was difficult to detect in the water intoxication mouse model used in this study. Thus, we analysed phenotypes that mimicked symptoms of severe cerebral oedema, such as tremor, loss of righting reflexes, decreased respiratory rate, and increased brain water content. These phenotypes suggest that the water intoxication model partially mimicked the clinical features of cerebral oedema.
Mild cerebral oedema due to decreased air pressure causes weather-related illnesses, including headaches [8–11]. Although such illnesses affect several individuals, limited treatment drugs are available. Therefore, the discovery of novel therapeutic agents is an important research topic.
AQP4, a water channel expressed in astrocytes in the brain, is involved in water transfer across the blood–brain barrier, regulation of cerebrospinal fluid volume, and hormone secretion [12, 13]. AQP4-mediated water transfer from blood vessels to the brain is accelerated during cerebral oedema formation [14–19]. Therefore, inhibiting AQP4 function is expected to suppress cerebral oedema formation and its related symptoms. Several compounds reportedly inhibit AQP4 function [33–35]. Acetazolamide and bumetanide, which are clinically used diuretics, inhibit AQP4 activity in rats [33–35]. However, these compounds have side effects, such as dehydration, and their long-term use for headache prevention is risky. Some phenylbenzamide compounds have also been reported to inhibit AQP4 in vitro and reduce cerebral oedema in vivo. However, these compounds have not yet been approved for clinical use. GRS is a drug clinically used for oedema and diarrhoea with only few reports of side effects related to electrolyte abnormalities. Therefore, clarifying the effect of GRS on cerebral oedema and elucidating its mechanism of action are important.
In previous studies, MRI has been used to analyse brain water dynamics in humans and animals [36–38]. Igarashi et al. [36] found that intravenous administration of H217O in AQP1- and AQP4-knockout mice induces signal changes. In their study, AQP1-knockout mice showed no signal change when compared with wild-type mice, whereas AQP4-knockout mice demonstrated reduced signal changes in the LV. These results indicate that AQP4 is involved in water transfer to the brain. In the present study, model-induced signal changes were observed in the LV and cortex, but the Cmax and AUC of the signal changes were reduced after GRS administration. Therefore, MRI analysis supported that GRS inhibited water entry into the brain, and the involvement of AQP4 was also predicted.
Yano et al. [26] reported that GRS improves brain damage after rat cerebral infarction and ameliorates the increase in brain water content after mouse cerebral ischaemia. They hypothesised that GRS suppresses the increase in AQP4 expression in these models. Nakano et al. [27] reported similar results in a mouse model of cerebral ischaemia. However, whether GRS directly inhibits the water permeability of AQP4 remains unclear. In the present study, we examined the effects of GRS on brain water content and symptom expression in a model of acute water intoxication, in which AQP4 expression was not expected to fluctuate. Quantitative reverse transcription polymerase chain reaction and western blot analyses were performed to measure the mRNA and protein expression levels of AQP4, respectively. Their results are provided in Additional files 2 and 3. No significant changes in the mRNA and protein expression levels of AQP4 were observed between the control and GRS. However, GRS ameliorated the increase in brain water content and inhibited symptoms, suggesting that GRS improved cerebral oedema through other mechanisms aside from modulating AQP4 expression. In addition, GRS inhibited AQP4 function in vitro, which may be one of its mechanisms of action.
This study has three potential limitations. First, the complete mechanism of GRS in a mouse model of water intoxication remains unclear. Because AQP4 was well known to improve the pathophysiology of this model, we focused on the effect of GRS on AQP4 function and did not examine other mechanisms of action. The other mechanism of GRS in this model warrants further investigation.
Second, GRS inhibited AQP4 function, but the ingredients that contributed to its action remain unclear. GRS contains a variety of ingredients derived from five crude drugs (e.g., (E)-cinnamic acid, cinnamaldehyde, atractylodin, and alisol C-23-O-acetate). Unlike the GRS mixture, which was dissolved in water, each of its ingredients was dissolved in dimethyl sulfoxide and then included in the assay system. However, the inhibitory activity of the ingredients is difficult to interpret accurately because the effect of dimethyl sulfoxide could not be ruled out. Hence, future studies should investigate the AQP4 inhibitory activity of GRS components in an assay system that is not easily affected by dimethyl sulfoxide, such as in mammalian cultured cells expressing AQP4.
Third, headache itself has not been evaluated. Because no general method to evaluate headache in vivo has been reported, we conducted using related indices (loss of righting reflexes, decreased respiratory rate, and brain water content) in a mouse model of water intoxication. When a method to evaluate headache is established in the future, the effects of GRS should be examined.
To the best of our knowledge, this study is the first to report the inhibitory effect of GRS on AQP4 function. When considering that GRS is used in treating headaches, the results of this study would have clinical relevance.