| Manganese (Mn), a trace metal element, is necessary to human body, but excessive Mn exposure can cause accumulation of Mn in the brain, leading to syndromes of neurodegenerative dysfunction, which was called manganism. Mn neurotoxicity can cause metabolism disorder of iron and glutamate in body and cells. Transferrin and ferritin play an important role on the transportation and storage of iron. Glutamine, glutamine synthetase, phosphate-activated glutaminase, glutamate transporters (GLAST and GLT-1) and transforming growth factor alpha participate in the regulating process of manganese on glutamate. Sodium para-aminosalicylic acid is effective drug for Mn-exposed hippocampal neurons in vitro and rats in vivo and manganese poisoning patients. However, mechanism of PAS-Na treatment of manganese is unclear. Therefore, the purpose of this study was to investigate whether PAS-Na is effective for metabolism disorder of iron and glutamate caused by Mn exposure, and provide scientific basis for the prevention and treatment of manganese neurotoxicity.ObjectiveThe objective of this study is to investigate whether PAS-Na is effective for metabolism disorder of iron and glutamate caused by Mn exposure.Methods and MaterialsAfter adaptive feeding,100 adult male Sprague-Dawley rats were randomly divided into five groups (20 rats/group):negative control group (Ctrl), PAS control group (P-Ctrl), manganese exposed group (Mn), the low dose of PAS-Na intervention group (L-PAS) and the high dose of PAS-Na intervention group (H-PAS). Rats of Mn, L-, H-PAS group were intraperitoneally injected (ip) MnCl2·4H20 15mg/kg/day, and rats of Ctrl and P-Ctrl group were ip equal volume/kg normal saline(N.S) once/day,5 days/week, for 4 weeks. Then, rats of L-and H-PAS group were subcutaneously injected on the back (sc) PAS-Na 100 and 200mg/kg/day, respectively,while rats of Ctrl,P-Ctrl and Mn group were sc equal volume/kg normal saline (N.S) once/day, for 3 or 6 weeks continuously.At the end of 7th (Mn exposure for 4weeks and PAS-Na treatment for 3 weeks) and 10th (Mn exposure for 4weeks and PAS-Na treatment for 6 weeks) week in observation period,10 rats were dissected and the indicators were mesured.(1) The indicators of growth and development of rats ndicators were determined, including:weight of rats at the intial time, end of each week, fasting body weight at the end of 7th and 10th week, weight of liver, spleen, kidney and testis, and the ratio of organ weight/body weight. (2) Chemical methods were used to examine the total iron binding capacity (TIBC) in serum, ELISA methods were used to examine the content of transferring (Tf) and ferritin (Fr) in serum and basal ganglia of rats. (3) Chemical methods were used to determine the content of glutamate(Glu),glutamine (Gln) and activity of glutamine synthetase(GS),and ELISA method was used to determine the content of phosphate activated glutaminase (PAG) in the striatum (ST), globus pallidus (GP), hippocampus (HP) and thalamus (TH) of rats. Real-time PCR was used to determine the mRNA expression of glutamate transporter GLAST and GLT-1 and transforming growth factor alpha (TGF-a) in the ST, GP, HP and TH of rats.Results1. The influence of PAS-Na on growth and development of Mn-exposed rats(1) From the 1st to 5th week in the observation period (Mn exposure for 4 weeks and PAS-Na treatment for 1 week), weight of Mn group was significantly lower than the Ctrl group (P<0.05). From the 6th to 10th week, weight of Mn group had no significant difference, compared with the Ctrl group. In the 7-week and 10-week observation period, weight of P-Ctrl group had no significant difference, compared with the Ctrl group (P>0.05). In the process of subcutaneous injection of PAS-Na 100 and 200 mg/kg/day for 3 or 6 weeks, weight of L-PAS and H-PAS group both had no significant difference, compared with the Mn group (P>0.05).(2) At the end of the 7th and 10th week, weight of organs and ratio of organ weight/body weight of P-Ctrl group had no significant difference, compared with the Ctrl group (P>0.05). At the end of the 7th week, weight of testis and ratio of testis weight/body weight of Mn group was significantly lower than the Mn group (P<0.05), and weight of testis and ratio of testis weight/body weight of L-PAS and H-PAS group was significantly higher than the Ctrl group (P<0.05).2. The influence of PAS-Na on Fe metabolism in serum and basal ganglia of Mn-exposed ratsAt the end of the 7th week, the content of Fr in basal ganglia of P-Ctrl group was higher, and content of Fr in basal ganglia of Mn group is lower,compared with the Ctrl group (P<0.05). At the end of the 10th week, the contents of TIBC and Tf of Mn group were significantly lower than the Ctrl group, and low and high dose of PAS could increase the contents of TIBC and Tf of Mn-exposed rats(P<0.05).3. The influence of PAS-Na on Glu related amino acids and enzymes of Mn-exposed rats(1) The contents of Glu and PAG of normal SD rats were both highest in the ST, and the content of Gln and activity of GS were both highest in the HP.(2) At the end of the 7th and 10th week, in the ST, the content of Glu of Mn group was both increased and the content of Gln and activity of GS of Mn group was both decreased (P<0.05),while low and high dose of PAS-Na could inhibit Mn-induced increase of Glu and decrease of GS activity (P<0.05).(3) At the end of the 7th week, in the GP of rats, compared with the Ctrl group, Glu content of P-Ctrl group significantly decreased, and Glu and PAG content of Mn group increased, with the decrease of Gln content and GS activity (P<0.05). High dose of PAS-Na inhibited Mn-induced decrease of Glu and PAG content. At the end of the 10th week, in the GP of rats, compared with the Ctrl group, Glu content of P-Ctrl group significantly decreased, and Glu content of Mn group increased, with the decrease of Gln content and GS activity (P<0.05), while L-PAS and H-PAS treatment decreased the Glu content of Mn-exposed rats, and H-PAS treatment increased the Gln content of Mn-exposed rats (P<0.05).(4) At the end of the 7th and 10th week, in the HP of rats, compared with the Ctrl group, Glu and PAG content of Mn group increased, with the decrease of Gln content (P<0.05). At the end of the 7th week, H-PAS treatment decreased the Glu content of Mn-exposed rats, while L-PAS and H-PAS treatment both decreased the PAG content of Mn-exposed rats (P<0.05). At the end of the 10th week, H-PAS decreased the Glu and PAG content and increased the Gln content in HP of Mn-exposed rats (P<0.05).(5) At the end of the 7th and 10th week, in the TH of rats, compared with the Ctrl group, Glu and PAG content of Mn group increased, with the decrease of Gln content and GS activity (P<0.05). At the end of the 7th week, L-PAS and H-PAS treatment both decreased the PAG content of Mn-exposed rats, and increased the Gln content and GS activity of Mn-exposed rats. At the end of the 10th week, L-PAS and H-PAS treatment both decreased the Glu and PAG content in the TH of Mn-exposed rats, and increased the Gln content and GS activity of Mn-exposed rats.4. The influence of PAS-Na on mRNA expression of GLAST, GLT-1 and TGF-a of Mn-exposed ratsAt the end of the 7th and 10th week, mRNA expression of GLAST, GLT-1 and TGF-α in ST, GP, HP and TH of Mn-exposed rats was significantly lower than that of Ctrl group (P<0.05), and mRNA expression of GLAST, GLT-1 and TGF-α in ST and GP of P-Ctrl group rats was significantly higher than that of Ctrl group (P<0.05), while mRNA expression of GLAST, GLT-1 and TGF-α in ST and GP of L-PAS and H-PAS group was higher than that of Mn group (P<0.05). At the end of the 10th week, mRNA expression of GLAST, GLT-1 and TGF-α in HP and TH of P-Ctrl group was higher than that of Ctrl group (P<0.05), while mRNA expression of GLAST, GLT-1 and TGF-α in HP and TH of L-PAS and H-PAS group was higher than that of Mn group (P<0.05).Conclusions:(1) Mn exposure slowed down the increase of rat weight, and led to decrease of testis weight and the ratio of testis weight/body weight, but PAS-Na inhibited the decrease of testis weight and the ratio of testis weight/body weight, with no effect for body weight of Mn-exposed rats.(2) Mn exposure caused contents of TIBC and Tf in serum of rats decreasing, and PAS-Na inhibited the decrease of Tf level in serum of Mn-exposed rats.(3) Mn exposure caused increase of Glu and decrease of Gln in ST, GP, HP and TH of rats, decrease of GS activity in ST, GP and TH, and increase of PAG in GP, HP and TH. PAS-Na had therapeutic effect for disorder of contents of Glu and related amino acid and activity of enzymes induced by Mn.(4) Mn exposure caused decreasing mRNA expression of GLAST, GLT-1 and TGF-α in ST, GP, HP and TH of rats, which were effectively inhibited by PAS-Na. |
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