The Effects Of Sodium Para-aminosalicylate On Mitophagy Disorder Of Manganese-exposed Rats

Objective To explore the effect of amino acid sodium?PAS-Na?on mitophagy disorder in striatum of manganese?Mn?-exposed rats.In order to provide scientific basis for further elucidating the mechanism of Mn neurotoxicity and the treatment of PAS-Na on Mn-induced poisoning.Methods To establish the model of manganese exposure and PAS-Na treatment:150 Sprague-dawley?SD?male rats were fed acclimately for one week and then divided into two groups:?1?Exposure model?8-week experimental group?:60 rats were randomly assigned to a normal control group,low-,medium-and high-dose Mn-treated?L-?M-and H-?groups,15 in each group.L-?M-and H-groups were respectively received intraperitoneal injected?i.p.?of 5,10,20 mg/kg MnCl₂·4H₂O once a day,five days per week,for 8weeks.While the normal control group was received i.p.of sterile physiological saline in the meantime.?2?PAS-Na treatment model?14-week experimental group?:90 rats were randomly divided into normal control group,Mn-treated group,PAS-Na control group,and PAS-Na-treated low-,medium-,and high-dose?L-?M-and H-PAS?groups,15 in each group.Mn-treated group?L-?M-and H-PAS groups were received i.p.20mg/kg MnCl₂·4H₂O and the normal control group?PAS-Na control group received i.p.isovolumic sterile physiological saline once a day,five days per week,for 8weeks.And then PAS-Na control group?L-?M-and H-PAS groups were respectively received subcutaneous injection?s.c.?of 300,100,200,300 mg/kg PAS-Na,the normal control group?Mn-treated group received s.c.isovolumic sterile physiological saline once a day,five days per week,for 6 weeks.Determination of experimental indicators:?1?The indicators of growth and development:the rats were weighed every working day.At the end of the model establishment,rats were sacrificed.Rat organs including liver,kidney and testicles were taken and weighed to calculate the organ coefficient ratio.?2?The spatial learning and memory ability:six days before the end of the model establishment,10 rats in each group were randomly selected for Morris water maze test to detect the changes of spatial learning and memory ability.?3?Whole blood Mn levels of the rats were measured by using graphite furnace atomic absorption spectrometry.?4?The ultrastructure of mitochondria autophagy of basal ganglia neurons was observed by transmission electron microscope.After the model was established,3 rats were randomly selected for cardiac perfusion fixation in each group,and the basal ganglia was isolated and made into ultrathin sections.Ultrastructural changes of mitochondria autophagy in basal ganglia neurons were observed by transmission electron microscope?TEM?.?5?The relative expression of P62 mRNA was detected by real-time quantitative PCR?RT-PCR?.Results?1?The growth and development of rats:In the 8-week experimental group,from the 2nd week to the 6th week,the weight of the L-?M-and H-groups was lighter than those of the control group?P<0.05?;At the7th week in observation period,the weight of the M-group was lower than those of the L-group?P<0.05?;At the 8th week,the weight of the L-?M-and H-groups was lighter than those of the control group,the weight of the M-group was lower than those of the L-group?P<0.05?.In the 14-week experimental group,at the 1st and 2nd week of treatment,the Mn-treated group was lighter than the normal control group?P<0.05?,and there was no statistical difference between the PAS-Na control group and the normal control group.From the 3rd to the 6th week of treatment,compared with the normal control group,the weight of the Mn-treated group and the L-?M-and H-PAS groups gradually increased and reached the level of the normal control group by the 6th week of treatment,but the difference was not statistically significant.?2?The liver?kidney and testis coefficient of the rats:In the 8-week experimental group,the liver and kidney coefficient of the H-group was higher than those of the control group and the L-?M-groups?P<0.05?.?3?Spatial learning and memory ability of rats:Spatial learning and memory ability of rats:In the 8-week experimental group,the average escape latency and swimming distance in each group decreased as the number of test days increased.The escape latency and swimming distance of the L-and M-groups onthe5thdayofthetestwerelongerthanthecontrol group?P<0.05?.Compared with the M-group,the escape latency and swimming distance of the H-group on the 5th day of the test were longer?P<0.05?.In the14-week experimental group,there was no significant difference between the average escape latency and swimming distance in each group?P>0.05?.On the2nd day after the end of the navigation experiment,space exploration experiments were conducted.In the 8-week experimental group,compared with the control group,the percentages of the distance and time of the quadrants,the average speed,and the number of crossing platforms of the L-?M-?H-groups decreased with the increase of the concentration of the rats exposed to manganese,but there was no statistical difference?P>0.05?.?4?Mn levels in whole blood of the rats:Compared with the control group,the Mn levels in whole blood of the rats was increased with the increase of manganese concentration?MnCl₂·4H₂O;5,10,20mg/kg?In the 8-week experimental group?P<0.05?.In the 14-week experimental group,compared with the control group,the Mn levels in whole blood increased significantly after exposed to manganese?P<0.05?;Compared with the Mn-treated group,the Mn levels in whole blood of the M-and H-PAS groups were significantly reduced,while that in the H-PAS group was significantly lower than that in the M-PAS group?P<0.05?.?5?Ultrastructural changes of mitochondrial autophagosomes in basal ganglia neurons:In the 8-week experimental group,normal subcellular organelle morphology of basal ganglion cells in normal control and L-group was observed under a transmission electron microscope,and the number of autophagosomes is minimal.The number of autophagosomes and lysosomes in the basal ganglion cells was increased in rats of the M-and H-groups.In the14-week experimental group,a large amount of lysosomes and a small amount of autophagosomes were formed in the Mn-treated group?20 mg/kg?.Some of the mitochondrial membranes were destroyed,the crest gap was widened and the disintegration was vacuolated.A large number of lysosomes were formed and a small amount of mitochondrial membrane was damaged in the L-PAS group,the crest gap was widened and disintegrated.The rough endoplasmic reticulum is active,encircling damaged mitochondria.A large amount of lysosomes were formed in H-PAS group,and the recovery of mitochondrial structure was close to normal,and the number of autophagosomes was very small.H-PAS group had a small amount of lysosomes,and the mitochondrial structure returned to normal.?6?The P62 mRNA expression of the rats:In the8-week experimental group,compared with the control group,P62 mRNA expression in the M-?H-group increased?P<0.05?;compared with the L-?M-group,H-group P62 mRNA expression Increased?P<0.05?.In the 14-week experimental group,P62 mRNA expression was increased in the Mn-treated group compared with the control group?P<0.05?.Compared with the Mn-treated group,P62 mRNA expression was decreased in the L-?M-and H-PAS groups?P<0.05?.Conclusion?1?Mn exposure can cause growth retardation and organ toxicity.While PAS-Na treatment has a certain degree of change in body weight,liver and kidney coefficient of rats exposed to Mn.?2?Mn exposure may affect the learning and memory ability of rats to a certain extent,But the effect of PAS-Na treatment on learning and memory ability of rats exposed to Mn is not obvious.?3?Mn exposure can increase the Mn levels in whole blood of rats and increase with increasing dose of Mn.While PAS-Na treatment can effectively promote Mn excretion and the residual Mn levels in whole blood will decrease as the dosage increases.?4?Mn exposure may affect the ultrastructure of mitochondrial autophagy in basal ganglia neurons of rats.PAS-Na treatment may have some antagonism to its pathological changes,and its alteration may be restored.?5?Excessive Mn exposure could induce the up-regulation of P62 mRNA expression in rats.PAS-Na treatment had a certain antagonistic effect on the up-regulation of P62 mRNA expression.