| ObjectiveAs the epidemiological investigations and zoology experiments revealed, biological hazards of Aluminum (Al) being identified and received widespread attention. Al is one of the most abundant elements in earth’s crust, people live through air, water, food and food additives etc. increasing the intake of Al, which then accumulates in the body and produces toxicity effects. Al can affect various functions in the nerve system, especially learning and memory ability. Many scholars believe that Al plays toxicity effects in neurodegenerative diseases such as Alzhermer’s Disease (AD), Parkinson’s Disease (PD) and Dialysis Encephalopathy (DE). Taurine (Tau), also named a-aminoethanesulfonic acid, is a simple sulfur-containing non-protein amino acids and commonly found in tissues and organs. As the active substance regulating normal physiological activity, Tau plays a wide range of biological effects in the body. In our experiment, Wistar rats were chosen to receive Aluminum Chloride Hexahydrate (AlC13-6H2O) via oral gavage to build poisoning model, then they were given Tau to detoxify. We select four point, i.e. behavioral experiments, biochemistry experiment, pathomorphological observation and determination of Al and other required elements, to study and discuss the mechanism of Tau on improving learning and memory against Al-induced impairment in rats.Methods1. Animal experiment Fifty-four male Wistar rats (180-220g) of healthy level were used as experimental animals with food and water freely in plastic cages. Rats were acclimatized to the laboratory conditions prior to the experimentation for1week, and then were randomized into6groups according to their weight. Rats in control group received1.0ml/d saline only; the other groups received281.4mg/kg daily Aluminum Chloride Hexahydrate (AlC13-6H2O); for Tau prevention group,400mg/kg/day tau were given4h after Al administration as previously, the exposure time lasts4weeks. After the success of modeling, rats in Tau low, medium and high dose groups received200,400and800mg/kg/day tau, respectively; Tau prevention group rats were given taurine400mg/kg/day; control group and Al exposure group were given1.0ml/d saline instead, the detoxing time lasts4weeks, too. All solutions above were administrated via oral gavage.Selected2rats from each group randomly,4%paraformaldehyde was used for perfusion to make pathologic slices. Rats left behind were fasted for24h after Morris experiment, then we sacrificed them, removed the brains immediately and saved them in-20℃.2. Effect of Tau on normal growth and development in rats exposed to AlObserved the common situation like mental state, eating and drinking everyday, weighed them twice per week. Body weights of rats were recorded and drawed. After the rats were sacrificed, dissected brain tissue, weighed and calculated the brain coefficient.3. Effect of Tau on learning and memory abilities in rats expose to AlAfter the animal experiment, rats were investigated for their spatial learning and memory abilities with the help of Morris water maze test. The test was conducted in two phases, the hidden platform test (first4days) and the probe trial (the fifth day). We recorded4indexes:the daily escape latency, the swimming distance, swimming time in the quadrant of platform and the number of crossings over the previous platform location, to evaluate the improvement of Tau on learning and memory.4. Effect of Tau on essential element in Al-exposed rats brainBrain tissues were digested by graphite digestion for following analysis. Content of element Al was detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and elements Ca, Mg, Cu, Fe, Zn were detected by Atomic Absorption Spectrometry (AAS). Results were used to study whether Tau had the effect on removing Al and protecting other elements.5. Effect of Tau on the activity of Nitric Oxide Synthase (NOS) and Acetylcholinesterase (AChE) in Al-exposed rats brainWeighed the brain tissue of rats and made them into homogenates, the activity of NOS and AChE were determined by NOS and T-ChE testing box separately, to investigate the improvement of Tau on rats exposed to Al.6. Effect of Tau on the content of Monoamine neurotransmitters in Al-exposed rats brainThe content of Norepinephrine (NE), Epinephrine (E), Dopamine (DA) and5-hydroxy tryptamine (5-HT) in brain tissue of rats were tested by high performance liquid chromatograph with a fluorescence detector.7. Effect of Tau on the content of Amino Acid neurotransmitters in Al-exposed rats brain7.1Methodological studyThe content of Aspartate (Asp), Glutamate (Glu), Glycine (Gly), Taurine (Tau) and y-Aminobutyric acid (y-GABA) in brain tissue of rats were detected by high performance liquid chromatograph with a diode array detector.Chromatographic column:Agilent C18(150mm×5mm,4.6μm). Mobile phase:A is0.05mol/L sodium acetate buffer (pH is6.0by acetic acid), B is acetonitrile, C is water. Flow rate:1.0ml/min. Column oven:40℃. Sample size:10μl. Detection wavelength:350nm. Gradient elution program:0-5min, A:B:C (V:V:V)=65:17.55:17.55,5-14min, A:B:C (V:V:V)=40:30:30.7.2Determination of the Amino Acid neurotransmitter content in rats brainWeighed the brain tissue of rats and used the method above to determine the content of Asp, Glu, Gly, Tau andy-GABA in rats brain.8. Effect of Tau on histopathologic injury of brain tissue in rats exposed to AlAfter fixed by4%paraformaldehyde, brain tissue were sliced and stained by hematoxylin-eosin (HE), after that, sections were observed under microscope, to study the recovery effect of Tau against brain damage induced by Al.Results1. Effect of Tau on normal growth and development in rats exposed to AlRats in Al exposure group appeared fur lustrous poorly, lifeless and listless. Others showed a good condition. Starting from the sixth week, the Al exposure group showed a significant decline in their body weight gain compared to control group (P <0.05), and at the8week, the gain in Tau prevention group was increased significantly compared to Al exposure group (P<0.05). The brain coefficient in Al exposure group was significantly gained compared to control group or Tau prevention group (P<0.05).2. Effect of Tau on learning and memory abilities in rats expose to AlEscape latencies and swimming distances in all groups manifested as a downward trend with time increasing, which in Al exposure group showed slower, no significant differences were found when compared (P>0.05).The swimming time in the quadrant of platform was significantly longer in the control group compared to those in Al exposure group, which in Tau medium, high dose and prevention group were longer than that in Al exposure group, too. All the differences had statistical significant (P<0.01). Compared to control group, the numbers of crossing the platform were reduced significantly in Al exposure group (P <0.01), which in Tau medium, high dose and prevention group was gained compared to Al exposure group (P<0.01).3. Effect of Tau on essential element in Al-exposed rats brainElement Al content in rat’s brain of Al exposure group was significantly higher than that in control, Tau medium and prevention group (P<0.05).Element Mg, Cu, Fe, Zn content in rat’s brain of Al exposure group was significantly lower than that in control group (P<0.05). Mg, Zn content in Tau medium, high dose group and prevention group were significantly higher than that in Al exposure group (P<0.05). Cu content in Tau prevention group was higher than that in Al exposure group (P<0.05). Fe content in Tau high dose group was higher than that in Al exposure group (P<0.05). No significant difference was found among groups in content of Ca (P>0.05).4. Effect of Tau on the activity of Nitric Oxide Synthase (NOS) and Acetylcholinesterase (AChE) in Al-exposed rats brainThe activity of brain NOS in Al exposure group was significantly lower than that in control, Tau medium, high dose and prevention group (P<0.01).The brain AChE activity in Al exposure group was higher than control group statistically (P<0.05), which in Tau medium and prevention group was significantly lower than that in Al exposure group (P<0.05).5. Effect of Tau on the content of Monoamine neurotransmitters in Al-exposed rats brainThere was a nice separation of4kinds of Monoamine neurotransmitters in brain tissues of rats, the retention time was1-NE (3.33min),2-E (3.83min),3-DA (5.52min) and4-5-HT (11.79min), respectively. Made linear regression using the concentration(C) to the corresponding peak area(A), good linear relationship was present between0.02and0.4μg/ml, and the correlation coefficient was more than0.999. NE, E, DA,5-HT content in brain of rats in Al exposure group was significantly lower than that in control and Tau medium group (P<0.05). DA and5-HT content in rat’s brain in Tau prevention group was higher than that in Al exposure group statistically (P<0.05).6. Effect of Tau on the content of Amino Acid neurotransmitters in Al-exposed rats brain6.1Methodological studyThere was a nice separation of5kinds of Amino Acid neurotransmitters in brain tissues of rats, the retention time was1-Asp (5.66min),2-Glu (7.39min),3-Gly (11.14min),4-Tau (11.85min) and5-γ-GABA (13.55min), respectively. Made linear regression using the concentration(C) to the corresponding peak area(A), good linear relationship was present between10and200μg/ml, and the correlation coefficient was more than0.999. Asp:,A=13506.29C+84213.45, r=0.9991. Detection limit was3.05×10-3μg/ml. Intra-day RSD under low, medium and high concentration were3.16%,2.13%,2.37%. Inter-day RSD were4.78%,3.77%,5.05%. Recoveries were97.15%,89.17%,95.61%.Glu:A=12632.94C+62248.02, r=0.9994. Detection limit was2.03×10-3μg/ml. Intra-day RSD under low, medium and high concentration were3.36%,2.32%,2.88%. Inter-day RSD were5.21%,4.20%,3.95%. Recoveries were93.33%,95.01%,94.67%.Gly:A=26456.88C+69370.37, r=0.9992. Detection limit was9.84×10-4μg/ml. Intra-day RSD under low, medium and high concentration were2.99%,3.35%,3.55%. Inter-day RSD were5.48%,3.69%,4.17%. Recoveries were91.93%,97.18%,101.76%.Tau:A=16750.69C+67084.01, r=0.9993. Detection limit was1.04×10-3μg/ml. Intra-day RSD under low, medium and high concentration were3.55%,1.98%,3.36%. Inter-day RSD were4.51%,4.54%,4.40%. Recoveries were104.93%,96.02%,88.73%.γ-GABA:A=19494.50C+67672.88, r-0.9993. Detection limit was6.50×104μg/ml. Intra-day RSD under low, medium and high concentration were2.70%,2.56%,3.09%. Inter-day RSD were3.80%,5.04%,4.03%. Recoveries were96.15%,93.15%,93.29%.6.2Determination of the Amino Acid neurotransmitter content in rats brainThe content of brain Asp and Glu in Al exposure group were significantly higher than that in control, whose Tau and y-GABA content were lower than that in control, results had significant difference (P<0.05).The content of brain Asp and Glu in Tau medium and prevention group were significantly lower compared to Al exposure group (P<0.05). Brain Tau and y-GABA content in Tau prevention group were higher than that in Al exposure group, significantly (P<0.05). No statistical signification was found in the content of Gly in brain of rats (P>0.05).7. Effect of Tau on histopathologic injury of brain tissue in rats exposed to Al Compared to control group, hippocampus of rats in Al exposure group appear many vacuoles, nerve cell lines seemed to blurred and indistinct, cells arranged sparsely and scattered, neurons in cerebral cortex appear gaps, membrane deformability and nucleoli smaller. No obvious improvement in Tau low dose group. Brain tissues in Tau medium, high dose and prevention group have significant improvement:numbers of neurons in hippocampus get more, nerve cell lines become clearer and tidier, cytoplasm of cerebral cortex get richer and the cells have clear nuclei.Conclusions1. Tau has significant improvement against slow weight gain in rats induced by Al, could promote growth and development of rats.2. Tau has significant improvement on learning and memory ability in rats exposed to Al.3. Tau has a certain effect in expelling element Al, exists some improvement on Al-induced disorders of element Mg, Cu, Fe and Zn, while it has no significant influence about Ca.4. Tau could protect against lower NOS and higher AChE activity in rats exposed to Al.5. Tau could alleviate the dysfunction of Monoamine and Amino Acid neurotransmitters induced by Al.6. Tau has significant recovering function on pathological damage caused by Al in brain tissues of rats.In conclusion, Tau can improve the damage on learning and memory ability in rats caused by Al and has protective effect on nervous system of rats. |
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