Hydrogen rich water ameliorate Trimethyltin induced spatial learning and memory impairment by regulation of Siah-1

Background: As an compounds, Trimethyltin (TMT) exists widely in industrial and agricultural productions. TMT can induce significant neurodegeneration in the limbic system, particular in hippocampus. However, the molecular mechanisms in TMT-induced learning and memory impairment are not fully clear. Thus, this research was to explore the role of Synaptophysin (SYP) and Ubiquitin-ligating enzyme Siah-1 in TMT-induced nerve impairment and the protective effect of hydrogen rich water (HRW). Results: The TMT-treated mice showed significant learning and memory disability. Moreover, TMT increased the level of Siah-1, reduced the expression of SYP in hippocampus and cortex. After 7 days of continuous pretreatment with HRW, the mice showed better memory ability, the expression of Siah-1 and tin content decreased accompanied with the increase of SYP. Conclusions: These results showed that HRW ameliorated the decrease of SYP and the nerve impairment induced by TMT, reduced tin content and up-regulated the expressions of Siah-1, and it might be an important role in the protection of neurodegenerative diseases induced by TMT.


Background
Trimethyltin (TMT) is a colorless crystal with odor of carrion grass under normal temperature and widely exists in paint, synthetic rubber, rodenticide, etc. It also can be completely absorbed by humans through the respiratory, skin, digestive tract and so on [1][2][3] . Besides, TMT has good lipophilicity and high affinity with hemoglobin in blood, and can be accumulated in human bodies [2]. After TMT treatment, mice can show agitation, aggressive behavior, learning and memory dysfunction [4][5][6][7]. According to reports, TMT can selectively damage the limbic system, especially in the prefrontal cortex, hippocampus and other brain regions and lead to the dysfunction and apoptosis in these regions, which are closely related to learning and memory function, suggesting that TMT-induced learning and memory dysfunction have a special correlation with such selective effect [8][9].
The mechanisms of nervous system damage induced by TMT include apoptosis, inflammatory response, oxidative damage, glutamate excitability, etc. [6,[10][11]. However, the exact mechanisms of TMT-induced widely nervous system damage are not fully clear. Synaptophysin (SYP) is a glycoprotein abundant in the membrane of synaptic vesicles; it exists in almost all neurons and neuroendocrine cells. Moreover, SYP is closely related to the release of neurotransmitters as well as the density and plasticity of synapses. Therefore, the immune activity of SYP can reflect the change in the number of the synaptic vesicles, and can be regarded as a marker of the synaptic density [12]. Our previous studies had demonstrated the connection between the decrease of SYP in the prefrontal cortex and hippocampus area and TMT-induced learning and memory impairment [13].
Above all, we believed that it was of great significance to explore how TMT 6 reduced the expression of SYP in the prefrontal cortex and hippocampus area, thus causing the learning and memory impairment.
Ubiquitin Proteasome System (UPS) is the main pathway for protein degradation in cells [14] . As a number of ubiquitin proteasome, Siah-1 can recognize and bind the target proteins, and degrade them through 26S proteasome pathway. In Anderson VE's report, theytreated hippocampal neurons with TMT and found that the immune activity of ubiquitin in hippocampal cell was enhanced, suggesting that TMT can affect the UPS function and enhance the degradation of related proteins [15]. Currently, some researchers have used TMT to induce hippocampal neuron damage to study the occurrence of neurodegenerative diseases [16]. Moreover, researchers have found that SYP has a binding domain of Siah-1, and over expression of Siah-1 can promote the degradation of SYP by the UPS [17-18].
Hydrogen is the simplest and most widely spread element in the nature, in recent years, the anti-oxidation effect and the neuroprotective effect of hydrogen has been proved [19]. The dissolved hydrogen in water can selectively neutralized OHand ONOOwhich cause the oxidative damage to cells [20]. Moreover, hydrogen has therapeutic effects on brain injury after cerebral ischemia and hypoxia, Parkinson's disease, Alzheimer's disease and so on [21][22][23][24][25][26]. Therefore, in this study, we aim to explore whether Siah-1 involves in the TMT-induced neurodegeneration disease by reducing SYP, and the protective effect of HRW in this approach.

Ethical statement
This experiment was approved by the Institutional Animal care and use Committee of the Chongqing Medical University. In this experiment, the research design has scientific basis and the selected animal species, quantity 7 and specification are appropriate. In this experiment, the animals were treated with anesthesia and analgesia and this experiment did not bring corresponding harm to the environment.

Study design
Male BALB/c mice were randomly divided into 4 groups: control group (saline, 4 ml/d, i.p.for seven days), HRW group (4 ml/d, i.p.for seven days), TMT group (2.25mg/kg, i.p. at the seventh day), and HRW plus TMT group.
The spatial learning and memory was tested by Morris water maze, the protein level of SYP and Siah-1 were determined by western blot and immunocytochemical analysis, and the tin contents were detected by ICP-MS.
The specific experimental design steps please refer to the Graphical Abstract.

Reagents and experimental drugs
Trimethyltin was purchased from Sigma, USA. Primary antibodies (SYP&Siah-1, β-actin) were respectively purchased from Abcam, England and Sigma, USA. The secondary antibodies were purchased from LI-COR Abcam, England. Materials for Western blot, such as enhanced chemiluminescent (ECL) and Polyvinylidene difluoride (PVDF, 0.2μm) membrane were purchased from Elabscience Biotechnology, Wuhan, China. Zinc (Beyotime Biotechnology, Shanghai, China) reacts with dilute sulfuric acid to form hydrogen, passed the hydrogen into normal saline water placed in ice water for four to six hours to prepare for hydrogen rich water. All other chemical reagents and medicines are purchased through regular channels and are available. Animals in control group received normal saline (4 ml/d, i.p.2 ml at 9:00 AM and 5:00 PM respectively) for seven days as vehicle. The HRW group was given hydrogen rich water intraperitoneally (4 ml/d, i.p.2ml at 9:00 AM and 5:00 PM respectively) for seven days which served as HRW control. The TMT group was administrated with TMT single i.p. injection (based as control group, 2.25 mg/kg, i.p. at 12:00 AM) at the seventh day at 12:00 AM. The dose of TMT and used in this study was based on our previous study [13]. In this process, the mice did not show any significant toxicity, including tremor, fighting and apparent damage. The TMT + HRW group animals was injected with TMT on the basis of the pretreatment of HRW (based as HRW group, treated TMT <2.25mg/kg, i.p.> at 12:00 AM at the seventh day), the processing method of this group was similar to the HRW group and TMT group, respectively. All the treatments were performed gently and all efforts were made to minimize animal suffering [27].

Morris water maze task
The Morris water maze task was designed to detect the spatial learning and memory impairment induced by TMT exposure and the protective effect of HRW. The tank (Tai Meng Technology, Chengdu, China) was divided into four equal quadrants, and this tank was 120 cm in diameter, 50 cm in height, the platform was 10 cm in diameter, the temperature of the water was about platform and the searching time would be recorded as sixty seconds. After the mice fond the platform, they could stay on the platform for 15 seconds to get familiar with the position of the platform. Completing all the tasks, the mice were dried and put back into the cage.

Immunohistochemical
At the end of the last Morris water maze task, about 0.4ml of 20% Urethane was intraperitoneally injected to anesthetize the mice, and then decapitated, and an appropriate amount of colorless formalin was infused through the cardia. After killing the mice, the brain tissue was quickly removed and fixed in 4% paraformaldehyde for use, or dehydrated by 75%-100% graded ethanol, cleared in xylene and embedded in paraffin. The thickness of the paraffin section was 5μm; HE staining was performed on every five sections.
Before dewaxing, the paraffin sections were placed at room temperature for 60 minutes, then the sections were immersed in xylene for 10 minutes and dehydrated in 100% -75% graded ethanol. About 40 -50ul polyclonal antibodies against SYP (1:1000) and Siah-1 (1:1000) were added to the tissue sample and placed them overnight at 4 °C. After placing overnight, rinsed the tissue sample three times with PBS, then added about 40 -50ul secondary antibody at room temperature for 1 hour. After completing all the processes mentioned above, rinsed with PBS three times again.

Western blot
The cortex and hippocampus of mice were separated and frozen at -

ICP-MS analysis
The mice were executed and the blood was removed by rinsing the tissues of the mice with ultra-pure water, 0.2mL blood and tissues samples were placed in Teflon digestion tank, 5.0mL digestion solution was added,  [29].

Statistical analysis
The average escape latency of mice in Morris water maze was compared by repeated measurements of variance analysis. Western Blot results analysis: the gray values of total protein Siah-1 and SYP were compared with the gray values of β-actin, respectively. The rest of the data were analyzed by independent sample T test. All the data were processed by SPSS 22.0 software, expressed as mean±standard deviation, and P<0.05 suggesting that the difference was significant.

TMT exposure induces the learning and memory impairment
We used Morris water maze to show mean group latencies to reach to hidden platform for all groups, which could reflect the learning and memory dysfunction induced by TMT and the protective effect of HRW. (Fig.1. A) The results showed TMT group had a worse performance, and TMT group mice spent a significant longer time in searching than Control group, especially from the fourth acquisition test. In the subsequent several experiences, the TMT group mice were still unable to locate the hidden platform, and their locating ability did not improve with the number of experiences. After the HRW treatment, the time for mice to search platform gradually reduced and the locating ability enhanced with the experiments, and there was no significant difference between TMT group and HRW group. Moreover, it was obvious that the searching strategies of TMT group mice were more disordered than other groups and spent more time to reach the previous platform region before reaching the target quadrant. Other three group mice adopted a relatively simple linear searching strategy, and the location was more accurate.

TMT exposure increases tin content in organs.
ICP-MS analysis allowed us to get the tin content in the organs of mice after the treatment of TMT. According to the Tab.1, the tin content in kidney reduced most obviously after the pretreatment of HRW compared to TMT group. Meanwhile, tin content in each brain region also reduced: left cortex was reduced to 0.57, right cortex to 0.61, left hippocampus to 0.76, right hippocampus to 0.64, Cerebellum to 0.87. In brain tissue, the tin content in left cortex decreased most significantly (P<0.05), while cerebellum had the most tin residues in the brain region.

TMT exposure affects the distribution of Siah-1 and SYP in cortex and hippocampus
In order to evaluate the effects of TMT on the expressions of Siah-1 and SYP, we used Immunohistochemical method for detection. (Fig.2. A) In the TMT group, the expressions of Siah-1 in CA3 and Cortex region obviously increased, especially in the CA3 region, but the DG and CA-1 regions did not change significantly compared to the Control group. At the meantime, the expressions of SYP in CA1, CA3, cortex regions also decreased obviously.
( Fig.2. B).After a further observation on the CA3 region, the distribution of SYP had an obvious increase and the expression of Siah-1 was similar to the control group after the pretreatment of HRW.

TMT exposure affects the protein levels of Siah-1 and SYP in hippocampus.
In the Western Blot analysis, we respectively detected the expressions of Siah-1 and synaptophysin in hippocampus ( Fig.3. A, B). In TMT group mice, the expressions of Siah-1 increased than other three groups, meanwhile, the expression of SYP also deduced, however, there were not statistical difference in the expression of Siah-1 and SYP between TMT+HRW group and HRW group. Therefore, it was obviously that the enhancement of Siah-1 and the decline of SYP were synchronized in the TMT-induced neurodegenerative disease.

Discussion
The neurotoxin effects of TMT have been widely proved by the researchers [30], but these studies have not well clarified the underlying molecular mechanisms of TMT-induced SYP decline and correlated the toxicity of TMT with tin content and distribution in the brain tissue.
Previously, we had reported that TMT could lead to the impairment of spatial memory in mice, and induced the decline of SYP but not GAP-43 in brain which rutin could effectively antagonize [13]. Therefore, in this study, we focused on the spatial impairment of TMT following a single i.p. does of TMT and speculated that TMT could induce the increase of Siah-1 which leading to the decrease of SYP, and finally caused the occurrence of a variety of neurodegenerative diseases. At the meantime, we also proved that hydrogen rich water could protect against TMT induced learning and memory impaired via Siah-1.
Our study illustrated that TMT could simultaneously cause the increase of Siah-1 and the decrease of SYP in TMT-induced neurodegenerative diseases, which suggested that TMT could induce the increase of Siah-1 to promote the ubiquitination degradation of SYP. In our present study, we mainly adopted four methods to explore the neural injury induced by TMT in mice. Firstly, we confirmed the impairment of spatial memory of TMT to mice through Morris water maze task, and the final result was consistent with previous conclusions [13]. In this study, TMT group mice showed the decline in localization ability and search ability, which was inflected by much longer time spent in TMT group compared to Control group. Moreover, the platform searching strategies adopted by TMT group were much more discarded. Especially since the fourth acquisition test, this difference was gradually obvious. However, in Hyun's report, the mice's searching ability has decreased in the first acquisition task; the difference might be caused by the injection dose and the type of mice. Finally, we detected the tin content in the organs of TMT group mice through ICP-MAS analysis. In the past, it has been reported that tin exists in cerebrospinal fluid of nonneoplastic neurological diseases patients [31] .
Therefore, we speculated that the tin content in the brain also played an important toxic role in TMT-induced neurodegenerative diseases. Our results confirmed that the tin content of many organs of mice in TMT group was higher than TMT+HRW group, and the left cortex was the most obvious.
However, the underlying metabolic mechanisms still need to be further explored.
The hydrogen in the HRW has the selective antioxidant and  and specification are appropriate. In this experiment, the animals were treated with anesthesia and analgesia and this experiment did not bring corresponding harm to the environment.

Consent for publication
All authors have read and agreed to submit this version for publication, and it is not currently submitted to other journals for consideration

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request

Competing interests
We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.