Research For The Intervention Mechanism Of Sodium Aminosalicylate For The Limbic System Damage Of Rats Induced By Manganese

The trace element manganese is essential for the normal human physiological function, however, high amounts of manganese will produce toxic damage to body.The long-term study of manganese neurotoxicity focused chiefly on extrapyramidal dopaminergic neurons. Clinical observation showed the victim of manganese poisoning can appear the dysfunction of learning, memory, cognition and the sense of direction, and emotional disorder including intension, anxiety, timidity and autism, which may be related to toxical damage of the limbic system induced by manganese.In recent years, it has been constantly reported that application of sodium aminosalicylate (PAS-Na) therapy for manganese–poisonging in clinical or in laboratory, in which obtained certain effect. Yet there were lacked report concerned with the damage of the limbic system induced by manganese, and mechanism of therapy for manganese–poisonging of PAS-Na still unknowed. This study intends to explore the intervention mechanism of PAS-Na for the limbic system damage of rats induced by manganese in vitro and in vivo.1 Effect of Sodium aminosalicylate (PAS-Na) to Apoptosis of Cultured Hippocampal Neurons of Manganese Exposure Rats[Objective]To explore the effect of PAS-Na to apoptosis of cultured hippocampal neurons of manganese exposure rats. [Methods and materials] (1) Hippocampal neurons of new-born SD rats were cultured to establish the hippocampal neurons culturing system; (2) Hippocampal neurons after purification were randomly divided into 6 groups, including control, 50μM Mn, 100μM Mn, PAS-Na 100μM, PAS-Na 500μM, 50μMMn+PAS-Na100μM , 50μM Mn+PAS-Na 500μM, 100μMMn+PAS-Na100μM, 100μM Mn+PAS-Na 500μM, respectively. And then, (1) MTT method was used to detect the cell viability; (2) single-celled gel electrophoresis(SCGE) was used to determine the DNA damage of neurons; (3) Annexin (FITC + V - double dye streaming cells PI was used to determine the apoptosis rate of neurons(ecxcept grouds of PAS-Na 100μM, 50μMMn+PAS-Na100μM and 100μMMn+PAS-Na100μM). [Results] (1) The cell viability of manganese exposed hippocampal neurons decreased significantly(P<0.05), and the cell viability of manganese exposed neurons increased after interfered by PAS-Na; (2) Manganese exposure increased the DNA damage of neurons(P<0.05), and the level of DNA damage of manganese exposed neurons degard after interfered by PAS-Na; (3) Manganese increased the apoptosis rate of neurons(P<0.05), and the apoptosis rate of manganese exposed neurons reduced after interfered by PAS-Na;2 Effect of PAS-Na to the Limbic System Damage of Manganese Exposed Rats in Vivo2.1 Effect of PAS-Na to the limbic system damage of subacute manganese exposed rat.[Objective] To explore the effect of PAS-Na to the limbic system damage of subacute manganese exposed rats, including learning and memory dysfunction, the changes of cells and ultrastructure of hippocampus. [Metholds and materials] 64 healthy male SD rats were randomly divided into manganese exposure group, low and high dosage of PAS-Na treatment group (L -, H - PAS group) and normal control group (control group). Rats expect for the normal control group were intraperitoneally injected (ip)MnCl2·4H2O 15mg/kg, and the normal control group was ip the same capacity of saline water per day, 5 days a week, for three weeks.Then L–PAS group and H - PAS group were given subcutaneous injection(sc) in back with PAS-Na 100mg/kg, 200mg/kg per day for 5 weeks, respectively. Morris water maze test was used to detect learning and memory of rats after 4-week treatment. Immunohistochemistry methods were used to determine the changes of number of choline acetyl transferase (ChAT) positive cells of basal forebrains bevel belt level (hDB )and oblique hits with vertical(vDB), The subgrandular zone of dentate gyrus (SGZ) neural microfilament protein (NF) positive cells and the hippocampus CA1 area glial fibrils acid protein (GFAP) positive cells.Transmission electron microscope was used to observe the hippocampal CA1 area ultrastructural changes of rats. [Results] (1) Manganese exposure significantly reduced learning and memory ability of rats(P<0.05), and decreased significantly the number of ChAT positive cells of basal forebrains nuclers of the hDB (P<0.05); After PAS-Na treatment, the learning and memory ability of the L-PAS group and H-PAS group was better than manganese exposure group, and ChAT positive cells of basal forebrains were more than those of manganese exposure group. (2) NF positive cells in SGZ were decreased in manganese exposure group(P<0.05), but were increased significantly in H-PAS treatment group(P<0.05). (3) Manganese lead to significant increase of the GFAP positive cells in hippocampal CA1 area (P<0.05), but L-PAS and H-PAS treatment groups decreased(P<0.05). (4) Manganese exposure lead to more serious neuronal degeneration, apoptosis, necrosis, neural protuberant edema and pith scabbard denaturation, astrocytes hyperplasia, hypertrophy and degeneration, microglia activation, blood brain barrier (BBB) permeability increase. H-PAS treatment could improve the pathological ultrastructure of mitochondria of manganese exposure rats, alleviate nerve protuberant edema and pith scabbard denaturation, reduce hypertrophy of glial cells, as well as increased the relic cellular organ in necrosis neurons , which showed non-typical microscopical of it.2.2 Effect of PAS-Na to the limbic system damage of chronic manganese exposed rats [Objective]To explore the effect of PAS-Na to chronic manganese exposed rats, including content of manganese in vivo tissues, learning and memory dysfunction, and the changes of ultrastructure of hippocampus and genes expression related apoptosis. [Methods and Materials] 144 healthy male SD rats were randomly divided into control group, manganese exposure group, PAS-Na prevention treatment group(prevention group) and PAS-Na treatment group(treatment group), and control group. In 12th week during experiment, The control group and the manganese exposure group were subdivided into two groud respectively, so 6 groud all together were builded . Rats expect for the control group were intraperitoneally injected (ip)MnCl2·4H2O 15mg/kg, and the control group was ip the same capacity of saline water per day, 5 days a week for 12 weeks. PAS-Na prevention treatment group(prevention group) were given subcutaneous injection(sc) in back with PAS-Na 200mg/kg during exposed to manganese. PAS-Na treatment group was given sc in back with PAS-Na 200mg/kg per day for 6 weeks after 12-week manganese exposure. Before the anatomy, Morris water maze test lasting for one week was used to detect learning and memory ability of rats, and escape incubation period and swimming distance were recorded. Then, 8 rats of each group were cervically dislocated, and blood was collected form heart, and fresh tissues was got after qucik craniotomy.These samples were used to determine (1) content of manganese in hippocampus, cortex, and red blood cells. (2)the activation of choline acetyl transferase (ChAT)in basal forebrains. (3) Bax,Bcl-2,Caspase-3,Caspase-9,COXI,COXIV mRNA expression of hippocampus with RT-PCR. The other 16 rats of each group were fixed by heart perfusion, and fixed brains were used to determine:(1) the changes of number of choline acetyl transferase (ChAT) positive cells of basal forebrains bevel belt level (hDB )and oblique hits with vertical(vDB), neural microfilament protein (NF) positive cells of the dentate gyrus granulosa cells (SGZ) and glial fibrils acid protein (GFAP) positive cells in the hippocampus CA1 area with immunohistochemistry methods; (2) the hippocampal CA1 area ultrastructural changes of rats by transmission electron microscope. [Results] (1)After 12-week manganese exposure, PAS-Na prevention treatment 12-week①the content of manganese in hippocampus, cortex and red blood cells of manganese exposure group was significantly higher than control group(P<0.05); the content of manganese in red blood cells decreased obviously in PAS-Na prevention treatment group(P<0.05);②Morris test showed escape incubation period and swimming distance of the fifth day's test of manganese exposure group was both longer than control group(P<0.05), and PAS-Na prevention treatment could improve the learning and memory ability (P<0.05);③ChAT positive cells of basal forebrains hDB and vDB of manganese exposure group were respectively less than control group(P<0.05), and those of PAS-Na prevention group were more than manganese exposure group(P<0.05);④the activation of ChAT in basal forebrains of manganese exposure group was significantly lower than control group(P<0.05), and that of PAS-Na prevention group was higher than manganese exposure group(P<0.05);⑤Pathological changes of hippocampal ultrastructure in chronic manganese exposed were similar to those of the subacute manganese exposed rats, while the nerve felt and pith scabbard and structural changes of mitochondria was more obviously. But the ultrastructure of mitochondria recovered to the normal level after PAS-Na prevention treatment, while increased the relic cellular organ in necrosis neurons, and lessen oedema of nervors process and apomorphosis of myelin, and lessen hypertrophia of gliacyte at the same time.⑥GFAP positive cells of manganese exposure group were significantly more than those of control group (P<0.05), PAS-Na prevention treatment inhibited the increase of GFAP positive cells(P<0.05).⑦TUNEL positive cells in hippocampal CA1 area of manganese exposure group were much more, compared with control group(P<0.05), and those of PAS-Na prevention group were less than those of manganese exposure group(P<0.05).⑧The result of RT-PCR show: COXI, COXIV, Bcl-2 gene expression in manganese exposure group was significantly lower than that in control group, as well as Caspase-3 gene expression higher than that in control group(P<0.05). and that of Bcl-2 gene expression higher than that in manganese exposure group, while COXI, COXIV, Caspase-3 in PAS-Na prevention group was similar to manganese exposure group.(2) 12-week manganese exposure and following 6-week PAS-Na treatment①Morris test was used to detect the learning and memory ability of rats, escape incubation period and swimming distance of the fifth day's test of manganese exposure group was longer than control group(P<0.05), and PAS-Na treatment could improve the learning and memory ability(P<0.05).②ChAT positive cells of basal forebrains vDB of manganese exposure group were less than control group (P<0.05), and ChAT positive cells of basal forebrains vDB of PAS-Na treatment group were more than manganese exposure group(P<0.05);③Pathological changes of hippocampal ultrastructure in chronic manganese exposed were similar to those of the subacute manganese exposed rats, while the pathological changes of myelin more serious than those in subacute manganese exposed rats. pathological changes of nervous process and myelin were recovered to the normal level after PAS-Na treatment, mast glial cells were not obvious.④NF positive cells of the SGZ of manganese exposure group were less than control group(P<0.05), and those of PAS-Na treatment group were more than manganese exposure group( P<0.05.⑤GFAP positive cells of manganese exposure group were significantly more than those of control group (P<0.05), and those of PAS-Na treatment group were more than manganese exposure group, with no significance between groups(P>0.05).⑥expression of COXI and Bcl-2 genes in manganese exposure group was less lower than that of control group(P<0.05), while the gene of Caspase-3 showed higher lever in manganese exposure group(P<0.05). Bcl-2 gene expression in PAS-Na treatment group was much higher than that of manganese exposure group(P<0.05).[conclusion]①The Sodium aminosalicylate, which concentration of 100μM or 500μM has not poisonous effect to the cultured hippocampal neurons.②The Sodium aminosalicylate may possess the ability of alleviate the damage degree of cultured hippocampal neurons, and prevents the damnification of DNA, and recedes the rate of apoptosis of maganese exposed hippocampal neurons in a certain extent.③The Sodium aminosalicylate can improve the learning and memory ability by elevate the survive of choline acetyl neurons, and enhance the activation of ChAT in basal forebrains, promoted the creating of acetylcholine.④Sodium aminosalicylate bring into neuro-protective via its ability of chelate excessive manganum, and promoted the evacuation of manganum in vivo. ⑤The Sodium aminosalicylate showed great intervention to the reactive hyperplasea and ultrastructure abnormity of gelatine.⑥Manganese exposed lead to ultrastructure pathologic change extersively of hippocampi tissue of rats, and the Intervention of Sodium aminosalicylate can reverse the pathologic morphous of mitochondria and myelin and meuropil, while lead to increase of remanetd cell organelle necrosis neurocyte, which showed non-typical in it's ultrastructure .⑦Manganese exposed lead to abnormal expressive of genes of COXI, COXIV, Caspase-3 and Bcl-2, The Intervention of Sodium aminosalicylate can reverse expressive of gene of Bcl-2 to normal level.