The Mechanism Of DFP Protecting Against Nervous System, Liver And Kidney In Aluminum-Exposed Rats

Objectives:With the development of technology,aluminum biotoxicity was gradually being revealed.When people expose excess aluminum,it can accumulate in vivo and disturb the activity of central nervous system.It may cause digestive system disorder and hinder calcium and phosphorus metabolism.It also could inhibit the antioxidant system and cause various diseases.Chelators have been useful to treat aluminium-overload diseases in clinical practice.In the following experiments,DFP (1,2-dimethyl-3-hydroxy-4-pyridone)is used as a new type of aluminum chelating agent in animals.We investigate the mechanism of DFP protecting against nervous system,liver and kidney in aluminum-exposed rats to provide foundation for seeking a safe and efficacious chelator for aluminum overload.Methods1.The effect of DFP protecting against nervous system1.1 The effects of DFP on learning and memory impairment caused by aluminum in ratsHealthy male wistar rats in the weight range of 160-180g were randomly divided into 6 groups by body weight:Negative control group,low-dose group,medium-dose group,high-dose group,Al-loaded group,preventive group.All the agents were administered by gavage.The other rats were treated with AlCl₃ except negative control group to prepare the rats' model of aluminum intoxication.The rats of preventive group were served aluminum simultaneously with DFP.Negative control and Al-loaded group were treated with saline four weeks later.At the same time,the low,medium and high-dose groups were intragastrically administered with DFP for 1 week.Two days before the administration ended,maze test was performed to investigate the effects of DFP on learning and memory impairment caused by aluminum in mice.1.2 Effects of DFP removing aluminum and other essential elements in brainsThe rats were sacrificed by decapitation at the end of the treatment.The serum samples were obtained by collecting and centrifuging blood samples.The tissues of brain were digested by microwave digestion for later analysis of aluminum by graphite furnace atomic absorption spectrophotometry and zinc,copper,iron,calcium and magnesium elements concentration by flame atomic absorption spectrophotometry.1.3 Pathomorphological observation of the hippocampusThe hippocampuses of the rats were fixed with 4%paraformaldehyde,and then did hematoxylin-eosin staining.Pathomorphological observations were carried out to investigate the morphological changes.1.4 Effect of recovering the neurotransmitter in the central nervous systemThe activity of AchE in the homogenate of brain tissues of the rats was determined in every group to investigate the effect of DFP on recovering the neurotransmitter in the central nervous system.1.5 Effect of recovering the anti-oxidizing systemThe activities of SOD,GSH-PX and the content of MDA in the homogenate of brain tissues of the rats were determined in every group.1.6 Effect of recovering the adenosine triphosphatase(ATPase)in brainThe activities of Na⁺ K⁺-ATPase,Ca²⁺Mg²⁺-ATPase in the homogenate of brain tissues of the rats were determined in every group.2.The effect of DFP protecting against the liver tissues2.1 Effects of DFP removing aluminum and other essential elements in liversThe tissues of liver were digested by microwave digestion for later analysis of aluminum by graphite furnace atomic absorption spectrophotometry and zinc,copper, iron,calcium and magnesium elements concentration by flame atomic absorption spectrophotometry.2.2 Effect of recovering hepatic functionThe activity of ALT,AST and ALP,the contents of total protein(TP),albumin, total bilirubin(TB)and conjugated bilirubin in serum were detected separately to evaluate hepatic function in every group.2.3 Pathomorphological observation of the liver cellsThe livers of the rats were fixed with 4%paraformaldehyde,and then did hematoxylin-eosin staining.Pathomorphological observations were carried out to investigate the morphological changes.2.4 Effect of recovering the anti-oxidizing systemThe activities of SOD,GSH-PX and the content of MDA in the homogenate of liver tissues of the rats were determined in every group.2.5 Effect of recovering the adenosine triphosphatase(ATPase)in liverThe activities of Na⁺ K⁺-ATPase,Ca²⁺Mg²⁺-ATPase in the homogenate of liver tissues of the rats were determined in every group.3.The effect of DFP protecting against the kidney tissues3.1 Effects of DFP removing aluminum and other essential elements in kidneysThe tissues of kidney were digested by microwave digestion for later analysis of aluminum by graphite furnace atomic absorption spectrophotometry and zinc,copper, iron,calcium and magnesium elements concentration by flame atomic absorption spectrophotometry.3.2 Effect of recovering renal functionThe contents of creatinine,blood urea nitrogen and the uric acid in serum were detected separately to evaluate renal function in every group.3.3 Pathomorphological observation of the kidney tissuesThe kidneys of the rats were fixed with 4%paraformaldehyde,and then did hematoxylin-eosin staining.Pathomorphological observations were carried out to investigate the morphological changes.3.4 Effect of recovering the anti-oxidizing systemThe activities of SOD,GSH-PX and the content of MDA in the homogenate of kidney tissues of the rats were determined in every group.3.5 Effect of recovering the adenosine triphosphatase(ATPase)in kidney tissuesThe activities of Na⁺ K⁺-ATPase,Ca²⁺Mg²⁺-ATPase in the homogenate of kidney tissues of the rats were determined in every group.Results1.The effect of DFP protecting against nervous system1.1 The effects of DFP on learning and memory impairment caused by aluminum in ratsThe maze test showed that the total reaction time(TRT)of Al-loaded group was significantly more than that of negative control group(P<0.05).The TRT of high-dose group was less than Al-loaded group,and there was no statistical significance between three doses groups and the negative control group.The error numbers of Al-loaded group were more significantly more than that of negative control group(P<0.05). Three doses groups and preventive group were less than Al-loaded group and no significance could be observed between them and negative control rats.1.2 Effects of DFP removing aluminum and other essential elements in brainsAluminum loading produced significant elevation of Al level compared to control rats in serum and brains(P<0.01).Aluminum contents of high dose group in serum were lower than that of negative control whereas the same condition was not found in the low,medium dose group and preventive group.Aluminum contents of the three doses groups in brain were significantly lower Al-loaded group(P<0.01). There was no statistical significance between high dose group and the negative control group whereas low,medium doses group and preventive group were higher negative control(P<0.01).Zinc content of Al-loaded group were significantly higher negative control (P<0.05).There was no significant difference between all the DFP groups and negative control group.No significant differences existed between all the DFP groups and Al-loaded group.As far as copper contents,Al-loaded group were higher than negative control.The medium and high doses groups were significantly lower than Al-loaded group.Furthermore,there was no statistical significance between high dose group and the negative control group.There were no significant differences among various groups in the contents of iron,calcium and magnesium contents in brains.1.3 Pathomorphological observation of the hippocampusThe hippocampus damage had been found among Al-loaded rats in the pathological examination.Neurons of the DFP-treated groups recovered to normal as compared with Al-loaded rats.The preventive group and high dose group recovered better.1.4 Effect of recovering the neurotransmitter in the central nervous systemThe results showed that the activity of Ache in Al-loaded group was higher than that of the negative control group(P<0.01).In comparison with the Al-loaded group, significant differences existed in the medium and high doses groups(P<0.05, P<0.01).1.5 Effect of recovering the anti-oxidizing systemThe results showed that the activity of GSH-PX and SOD in Al-loaded group was lower than that of the negative control group(P<0.01).All the DFP-treated rats were higher than the Al-loaded group(P<0.01,P<0.05).In comparison with the negative control group,no significant differences existed in the medium,high doses groups and preventive group.The results showed that the content of MDA in brain of the Al-loaded group was higher than that of the negative control group(P<0.05).The high dose DFP group was significantly lower than the Al-loaded group.There was no statistical significance in the three DFP doses groups as compared with negative control group.1.6 Effect of recovering the adenosine triphosphatase(ATPase)in brainThe results showed that no significant differences existed among various groups in the activities of Na⁺ K⁺-ATPase in liver.The activity of Ca²⁺Mg²⁺-ATPase of Al-loaded was lower than that of the negative control group(P<0.05).In comparison with the Al-loaded group,significant differences existed in the high DFP dose group (P<0.05).At the same time,dose-response relation exists and there is no significance between four DFP-treated groups and the negative control group.2.The effect of DFP protecting against the liver tissues2.1 Effects of DFP removing aluminum and other essential elements in liversThe results showed that the aluminum content of Al-loaded group in liver was significantly higher than that of the negative control group(P<0.01).Aluminum contents of the three doses group were lower than that of Al-loaded group(P<0.01) and dose-response relations exist among three doses groups.There was no statistical significance between high DFP dose group and negative control group.The zinc content of the Al-loaded group was higher than that of negative control group,but no significant difference existed between them.The three doses groups were lower than Al-loaded group(P<0.01,P<0.05)and significant difference could not be observed between three doses groups and negative control group.The copper content of the low dose group was significantly lower than that of negative control group(P<0.05),yet the same condition could not be observed in other groups.The calcium content of the low dose group was significantly lower than that of Al-loaded group(P<0.05).In comparison with the negative control group,no significant differences existed in the four DFP-treated groups.The Mg content of Al-loaded group was significantly higher than that of negative control group(P<0.05)whereas the same condition could not be observed in the four DFP-treated groups.No significant differences existed among various groups in the contents of iron in liver.2.2 Effect of recovering hepatic function No significant differences existed among various groups in the activity of ALT in serum.The activity of AST of Al-loaded group was significantly higher than that of negative control(P<0.01).The three DFP doses groups were lower than Al-loaded group(P<0.01),yet the same condition could not be observed in negative control. Results showed that the activity of ALP of Al-loaded group was significantly higher than that of negative control(P<0.01).The low DFP dose group was lower than negative control(P<0.01).Significant differences could be observed between preventive,medium,high DFP doses groups and negative control(P<0.01,P<0.05). The total protein(TP)contents of the Al-loaded and low DFP dose groups were significantly lower than the negative control(P<0.01).The medium,high DFP doses and preventive group were higher than the Al-loaded group(P<0.01),while the same condition hasn't been exhibited as compared with negative control.The result of albumin contents demonstrated that there was no statistical significance among all the groups.Compared with the negative control,the TB contents of the other five groups raised obviously(P<0.05,P<0.01).The contents of the conjugated bilirubin in all groups were not significantly different.2.3 Pathomorphological observation of the liver cellsThe liver cells of Al-loaded group presented apomorphosis and the hepatic cords arranged disorderly.Significant recovering could be observed in the DFP-treated groups.Dose-response relations exist among three doses groups.2.4 Effect of recovering the anti-oxidizing systemThe activity of SOD of Al-loaded rats was significantly lower than that of negative control(P<0.01).All the DFP-treated groups were higher than Al-loaded rats (P<0.01,P<0.05).In comparison with negative control,no significant differences existed in medium,higher DFP doses groups and preventive group.No significant differences existed among various groups in the activity of GSH-PX and content of MDA in liver.2.5 Effect of recovering the adenosine triphosphatase(ATPase)in liverThe results showed that no significant differences existed among various groups in the activities of Na⁺ K⁺-ATPase in liver.The activities of Ca²⁺Mg²⁺-ATPase of Al-loaded and all the DFP-treated groups were lower than that of the negative control group(P<0.01,P<0.05).In comparison with the Al-loaded group,no significant differences existed in the four DFP-treated groups.3.The effect of DFP protecting against the kidney tissues 3.1 Effects of DFP removing aluminum and other essential elements in kidneysThe results showed that the aluminum content of Al-loaded group was significantly higher than that of negative control group(P<0.01).All the DFP-treated groups were lower than Al-loaded group(P<0.01)and there was no statistical significance between the medium,high DFP doses groups and negative control.The copper contents of Al-loaded and low DFP dose groups were lower than that of negative control(P<0.01),yet the same condition could not be observed in medium and high DFP dose groups.No significant differences existed among various groups in the content of zinc,iron,calcium and magnesium in kidney.3.2 Effect of recovering renal functionThe BUN content of the Al-loaded group was significantly higher than the negative control,whereas the same condition could not be observed in the four DFP-treated groups.No significant differences existed among various groups in the content of creatinine and uric acid in serum.3.3 Pathomorphological observation of the kidney tissuesThe kidney damage had been found among aluminum-loaded rats in the pathological examination,significant recovering could be observed in the DFP-treated groups.Dose-response relations exist among three doses groups.3.4 Effect of recovering the anti-oxidizing systemThe results showed that no significant differences existed among various groups in the activities of SOD,GSH-PX and the content of MDA in kidney.3.5 Effect of recovering the adenosine triphosphatase(ATPase)in kidneyThe results showed that no significant differences existed among various groups in the activities of Na⁺ K⁺-ATPase and Ca²⁺Mg²⁺-ATPase in kidney.Conclusions1.DFP could effectively eliminated Al without removing essential elements in brain. DFP could counter the aluminum-induced aging of the neurotransmitter in the central nervous system.It can also reduce lipid peroxidization and thus remarkably improve the Al-induced pathological damages of hippocampus.It is able to improve the activity of ATPase and the learning and memory ability induced by aluminum.2.DFP could chelate aluminum and reduce aluminum contents in liver and has no obvious effect on other essential elements.It can also enhance the activity of the antioxidant enzyme,maintain the integrity of the liver cellular membrane,reduce peroxidization of lipid,remarkably recover the Al-induced liver function,and inhibit pathological damages of liver.In short,DFP played an oral in protecting the liver in many sides.3.DFP could remarkably recover the Al-induced renal function by reducing aluminum contents without removing essential elements.So it produced a marked effect on protecting against kidney in aluminum-exposed rats.