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The Protective Effects Of PAS-Na On The Inflammation Response In The Brain Of Mn-exposed Rats

Posted on:2018-11-07Degree:DoctorType:Dissertation
Country:ChinaCandidate:S J LiFull Text:PDF
GTID:1314330518452307Subject:Health Toxicology
Abstract/Summary:PDF Full Text Request
Objective Manganese(Mn)is an essential trace element in human body.However,chronic excessive Mn exposure may cause manganism.Manganism not only damages the extrapyramidal system,but also the other brain parts,including cortex,hippocampus and thalamus.Inflammation plays an important role in the pathogenesis process of neurodegenerative diseases.Furthermore,MAPK signaling pathway has been concerned as amain target pathway of Mn-induced neurotoxicity.A non-steroidal anti-inflammatory drug(NSAIDs)has neuroprotective effects by showed that NSAIDs inhibited the progressive degeneration of dopaminergic neurons via inhibiting the inflammatory responses and the activations of microglia cells in vivo and vitro studies.Clinical,our preliminary study found that sodium para-aminosalicylic acid(PAS-Na,a NSAIDs drug)has a good curative effect on manganism patients.Therefore,the present study aims to explore the inflammatory response in cortex,hippocampus and thalamus of rats induced by Mn,and the interventional mechanisms of PAS-Na byusing atomic absorption spectrometry,immunohistochemistry,enzyme-linkedimmunosorbent assay(ELISA),real-time fluorescent quantitative PCR(RT-PCR)and western blottingin vivo.Methods Experimental design: After specific pathogen free(SPF)male adult Sprague-dawley(SD)rats had acclimated for one week,sub-acute and sub-chronic experiments were performed.(1)Sub-acute experiment: Forty SPF male SD rats(184.5±10.7 g)were divided randomly into two groups(with 20 rats in each groups),including control and Mn-treated groups.The Mn-treated group received intraperitoneal(i.p.)injections of 15 mg/kg Mn Cl2 once aday,five days per week,for 4 weeks.While the control group received i.p.injections of sterile physiological saline in the meantime.(2)Sub-chronic experiment: SPF male SD rats(80±20 g)were divided randomly into fourteen groups,including control,PAS control,Mn-treated,PAS-Na(PAS)intervention,low,median and high doses PAS-Na treatment(L-,M-and H-)groups.The sub-chronic experiment contains three observation periods 8,12 and 18 weeks.The Mn-treated,PAS intervention and the three doses PAS-Na treatment groups received i.p.injections of 15 mg/kg Mn Cl2 once a day,five days per week,for 8or 12 weeks,while the control,PAS control group in 18 weeks’ observation period received i.p.injections of sterile physiological saline in the meantime.Besides,PAS intervention and PAS control groups in 8 weeks’ and 12weeks’ observation periods receiveddaily back subcutaneous(s.c.)injection of240 mg/kg PAS-Na,five days per week for 8 or 12 weeks.Subsequent,rats in the 18 weeks’ observation period received back s.c.injections with physiological saline(for the Mn-treated and control groups)and different doses of PAS-Na(80,160,240,240 mg/kg PAS-Na for L-,M-,H-PAS and PAS control groups)once a day,three days per week,for an additional six weeks.The PH value of the Mn Cl2 solution was between 5.5 and 5.9,and was thusadjusted to 6.3 by using 1 mol/L Na HCO3 solution before administration.The PH value of the PAS-Na solution was between 6.7 and 7.0.Determination of indicators:(1)The indicators of growth and development of rats were determined,includingweighing the weight of the experimental animals one time per weeks.Obtainedweights of the fasting body and the organs of the experimental animals at the end of the experiments,including heart,liver,spleen,lung,kidneyandtestis,and calculate the ratio of organ weight/body weight.(2)The spatiallearning and memory ability of the experimental animals were determined by using Morris water maze test(10 rats in each group).(3)Whole blood Mn levels and brain Mn levels(cortex,thalamus and hippocampus)of the experimental animals were measured by using graphite furnace atomic absorption spectrometry.(4)The positive expressions of glial fibrillary acidic protein(GFAP),bax,bcl-2,caspese-3 in cortex,hippocampus and thalamus were detected by using immunohistochemistry.After perfuse with the fixation liquid of 4% paraformaldehyde in 0.1M PB(five rats in each group)through the heart,the brains of rats were separated for the immunohistochemistry.(5)Detection of inflammatory cytokines: At the end of the experiment(15 rats in each group),the experimental animals were sacrificed to obtain the cortex,hippocampus and thalamus.The brain tissues were froze in liquid nitrogen and stored at-80℃.Inflammatory cytokines TNF-α,IL-1β,IL-6 and PGE2 contents in serum,cortex,hippocampus and thalamus were determined by using ELISA.The m RNA expressions of TNF-α,IL-1β,IL-6 and COX-2 in cortex,hippocampus and thalamus were measured by using RT-PCR.Protein expression of MAPK pathway relates proteins were determined by using western blot,including T-/p-JNK,ERK,p38 and COX-2.(1)The effect of PAS-Na on growth and development of Mn-exposed rats(1)The effect of PAS-Na on the weight of Mn-exposed ratsResults rom the 3rd or 4th week in every observation period,weight of Mn-treated group was lower than those of the control group(P<0.05).In the sub-chronic experiments(observation periods of 8 weeks or 12 weeks),the weight of PAS intervention group was higher than those in the Mn-treated group at the 5thand6thweek(P<0.05).While the weight of rats in PAS control group(observation periods of 8 weeks or 12 weeks)were lower than those of the control group at3 rd week.In observation period of 18 weeks,the weights of rats in M-and H-PAS groups were higher than those of the Mn-treated group at 5th and 6th week of the treatment period(P<0.05).Except the above-mentioned times,PAS-Na intervention or PAS-Na treatment has no effects on the weight of the Mn-exposed rats.(2)The effect of PAS-Na on the organ coefficient(the ratio of weight of organs/body weight)of Mn-exposed rats.In observation period of 4 weeks,the organ coefficient of spleen and kidney of Mn-treated group were higher than those in the control group(P <0.05).While the organ coefficients of heart,liver and testis of the Mn-treated group had no significant difference when compared with those of the control group.In observation period of 8 weeks,the organ coefficient of heart,liver,spleen and kidney of the Mn-treated group were higher than those of the control group.In contrast,the organ coefficient of spleen of PAS intervention group was lower than those of Mn-treated group(P<0.05).The organ coefficient of heart,liver and spleen of PAS intervention group had no significant difference when compared with those of the Mn-treated group.And the organ coefficient of heart,liver and spleen of PAS control group had no significant difference when compared with those of the control group.In observation period of 12 weeks,the organ coefficients of the heart,kidney,testis and lung in the Mn-treated group were lower than those in the control group.In contrast,the organ coefficient of testis in PAS intervention group was higher than those of the Mn-treated group(P < 0.05).While the organ coefficient of heart and spleen of PAS intervention group had no significant difference when compared with those of the Mn-treated group.And the organ coefficient of heart,liver and spleen of PAS control group had no significant difference when compared with those of the control group.In observation period of 18 weeks,the organ coefficients of the heart and testis in the Mn-treated group were lower and the liver were higher than those in the control group.In contrast,the organ coefficient of heart in H-PAS group was higher than those of the Mn-treated group(P<0.05).In contrast,the organ coefficient of liver in H-PAS group was lower than those of the Mn-treated group(P<0.05).And the organ coefficient of heart,liver and spleen of PAS control group had no significant difference when compared with those of the control group.(2)The effect of PAS-Na on the spitallearning and memory ability ofMn-exposed rats1 The effects of PAS-Na on the learning ability of Mn-exposed ratsAs shown in the spatial navigation test for assessing learning abilities,the average escape latency and swimming distance of rats in each group were decreased with the increasing training days.In observation period of 4 weeks,the escape latency and swimming distance of the Mn-treated group were longer than those of the control group at 1st,4th and 5th day(P<0.05 or 0.01).In observation period of 8 weeks,the escape latency and swimming distance of the Mn-treated group were longer than those of the control group at 1st,3rd,4th and5 th day(P<0.05 or 0.01).In contrast,the escape latency and swimming distance of PAS intervention group were shorter than those of the Mn-treated group at 3rd,4th and 5th day(P<0.05 or 0.01).In observation period of 12 weeks,the escape latency and swimming distance of the Mn-treated group were longer than those of the control group at 2nd,3rd,4th and 5th day(P<0.05 or 0.01).In contrast,the escape latency and swimming distance of PAS intervention group were shorter than those of the Mn-treated group at 4th and 5th day(P<0.05 or 0.01).In observation period of 18 weeks,the escape latency and swimming distance of the Mn-treated group were longer than those of the control group at 3rd,4th and5 th day(P<0.05 or 0.01).In contrast,the escape latency and swimming distance of M-and H-PAS groups were shorter than those of the Mn-treated group at 3rd,4th and 5th day(P<0.05 or 0.01).And the escape latency and swimming distance of PAS control group in each observation period had no significant difference when compared with those of the control group.2 The effects of PAS-Na on the memory ability of Mn-exposed ratsAfter the pace exploration experiment,the navigation experiment was measured.In observation period of 4 weeks,the ratio of platform quadrant time/total time and the number of platform crosses of the Mn-treated group were lower than those of the control group(P<0.05).In observation period of 8 weeks,the ratio of platform quadrant time or distance/total time or distance and the number of platform crosses of the Mn-treated group were lower than those of the control group(P<0.05 or 0.01).In contrast,the number of platform crosses of PAS intervention group were higher than those of the Mn-treated group(P<0.05 or 0.01).In observation period of 12 weeks,the ratio of platform quadrant time or distance/total time or distance and the number of platform crosses of the Mn-treated group were lower than those of the control group(P<0.05 or 0.01).In contrast,the ratio of platform quadrant time or distance/total time or distance and the number of platform crosses of PAS intervention group were higher than those of the Mn-treated group(P<0.05 or 0.01).In observation period of 18 weeks,the ratio of platform quadrant time or distance/total time or distance and the number of platform crosses of the Mn-treated group were lower than those of the control group(P<0.05 or 0.01).In contrast,the ratio of platform quadrant time or distance/total time or distance and the number of platform crosses of M-and H-PAS groups were higher than those of the Mn-treated group(P<0.05 or 0.01).There is no significant difference in the ratio of platform quadrant time or distance/total time or distance and the number of platform crosses between the PAS control and the control group in each observation period.And there is no significant difference in the swimming speed among the groups.(3)The effect of PAS-Na on the Mn levels in whole blood and brain(cortex, halamus and hippocampus)of Mn-exposed ratsIn sub-acute experiment,the Mn levels in whole blood and thalamus of the Mn-treated group was higher than those in the control group(P <0.05).In sub-chronic experiment,the Mn levels in whole blood and brain(cortex,thalamus and hippocampus)in the Mn-treated group was higher than that of the control group in all observation periods.In contrast,the Mn levels in whole blood and brain(cortex,thalamus and hippocampus)of PAS intervention group or PAS-Na treatment groups were lower than those of Mn-treated group.(4)The effect of PAS-Na on positive expression of GFAP in cortex,hippocampus and thalamus of Mn-exposed rats.In observation period of 4 weeks,the positive expression of GFAP in thalamus of the Mn-treated group were higher than those of the control group18(P<0.05).In observation period of 8 weeks,the positive expression of GFAP in thalamus of the Mn-treated group was higher than those of the control group(P<0.01).In contrast,the positive expression of GFAP in thalamus of PAS intervention group was lower than those of the Mn-treated group(P< 0.01).In observation period of 12 weeks,the positive expression of GFAP in thalamus,hippocampus and cortex of the Mn-treated group was higher than those of the control group(P< 0.01).In contrast,the positive expression of GFAP in thalamus and cortex of PAS intervention group was lower than those of the Mn-treated group(P< 0.01).While PAS-Na intervention had no effects on the positive expression changes of GFAP in hippocampus induced by Mn exposure.In observation period of 18 weeks,the positive expression of GFAP in thalamus,hippocampus and cortex of the Mn-treated group was higher than those of the control group(P<0.01).In contrast,the positive expression of GFAP in thalamus of M-and H-PAS,hippocampus of M-PAS groups was lower than those of the Mn-treated group(P< 0.01).While PAS-Na treatment had no effects on the positive expression changes of GFAP in cortex induced by Mn exposure.There is no significant difference in the positive expression of GFAP in the brain tissues between the PAS control and the control group in each observation period.(5)The effects of PAS-Na on the positive expression of bax,bcl-2 andcaspase-3 in cortex,hippocampus and thalamusIn observation period of 4 weeks,compared with the control group,the positive expression of caspase-3 in thalamus of the Mn-treated group were increased,the positive expression of bcl-2 and ratio of bcl-2/bax in thalamus were decreased(P<0.05).There is no significance difference on the positive expression of bax in thalamus between the Mn-treated and control groups.Inobservation period of 8 weeks,compared with the control group,the positive expression ofcaspase-3 in thalamus of the Mn-treated group were increased,the positive expression of bcl-2 and ratio of bcl-2/bax in thalamus were decreased(P<0.05).There is no significance difference on the positive expression of caspase-3,bcl-2 and ratio of bcl-2/bax in thalamus between the Mn-treated and PAS intervention groups(P>0.05).In observation period of 12 weeks,compared with the control group,the positive expression of caspase-3 in thalamus,hippocampus and cotex,and bax in the hippocampus of the Mn-treated group were increased,the positive expression of bcl-2 and ratio of bcl-2/bax in thalamus,hippocampus and cotex were decreased(P<0.05).There is no significance difference on the positive expression of bax in thalamus and cortex between the Mn-treated and control groups(P>0.05).PAS-Na intervention decreased the positive expression of caspase-3 in thalamus and hippocampus and bax in the hippocampus,increased the positive expression of bcl-2,ratio of bcl-2/bax in the thalamus and hippocampus of the Mn-exposed rats(P<0.05 or0.01).In observation period of 18 weeks,compared with the control group,the positive expression ofcaspase-3,bax in thalamus,hippocampus and cotex of the Mn-treated group were increased,the positive expression of bcl-2 and ratio of bcl-2/bax in thalamus,hippocampus and cotex of the Mn-treated group were decreased(P<0.01).Compared with Mn-treated group,the positive expression of caspase-3 and bax in the thalamus and cortex of M-and H-PAS groups were decreased,and the positive expression of bcl-2 and ratio of bcl-2/bax in the thalamus and cortex of M-and H-PAS groups were increased(P<0.05 or 0.01).Compared with Mn-treated group,the positive expression of caspase-3 in hippocampus of M-and H-PAS groups were decreased(P<0.05 or 0.01).PAS-Na treatment has no effects on the positive expression changes of bcl-2、bax and ratio of bcl-2/bax in the hippocampus of Mn-exposed rats.The positive expression of caspase-3 in cortex of the PAS control group was higher than those of the control group(P<0.01).(6)The effect of PAS-Na on inflammatory factors in serum,cortex, ippocampus and thalamus of Mn-exposed rats.In observation period of 4 weeks,IL-1β,IL-6,TNF-α levels in serum,IL-1βand PGE2 levels in thalamus of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01).There is no significance difference on the PGE2 level in serum,TNF-αand IL-6 in thalamus between the Mn-treated and control groups(P>0.05).In observation period of 8 weeks,IL-1β,IL-6,TNF-α,PGE2 levels in serum,IL-1βand PGE2 levels in thalamus of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01).There is no significance difference on the TNF-αand IL-6 levels in thalamus between the Mn-treated and control groups(P>0.05).Compared with the Mn-treated group,IL-1β,TNF-α levels in the serum,IL-1βand PGE2 levels in thalamus of PAS intervention group were decreased(P<0.05).PAS-Na intervention has no effects on the increase of IL-6 and PGE2 levels in serum of Mn-exposed rats(P>0.05).In observation period of 12 weeks,IL-1β,IL-6,TNF-α,PGE2 levels in serum,thalamus,hippocampus and cortex of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01).Compared with the Mn-treated group,IL-1β,TNF-α levels in the serum,IL-1 β,PGE2 levels in thalamus,TNF-α levels in hippocampus and IL-1βlevels in cortex of PAS intervention group were decreased(P<0.05).In observation period of 18 weeks,IL-1β,IL-6,TNF-α,PGE2 levels in serum,thalamus,hippocampus and cortex of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01).Compared with the Mn-treated group,the IL-6 levels in the thalamus of M-PAS,PGE2 and TNF-α levels in the thalamus of H-PAS group were decreased(P<0.05 or 0.01).IL-1β,IL-6,TNF-α,PGE2 levels in hippocampus of H-PAS group were lower than those of the Mn-treated group(P<0.05 or 0.01).Compared with the Mn-treated group,the IL-1β levels in the cortex of M-PAS,IIL-6,PGE2 and TNF-αlevels in the cortex of H-PAS group were decreased(P<0.05 or 0.01).There is no significant difference in the inflammatory factors in the brain tissues and serum between the PAS control and the control group in each observation period.(7)The effect of PAS-Na on the m RNA expression of IL-1β,IL-6,COX-2 andTNF-αin brain tissues of Mn-exposed rats.In observation period of 4 weeks,the m RNA expression of IL-1β and COX-2 in thalamus of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01).There is no significance difference on the m RNA expression of TNF-αand IL-6 in thalamus between the Mn-treated and control groups(P>0.05).In observation period of 8 weeks,the m RNA expression of IL-1β,COX-2 and TNF-α in thalamus of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01).There is no significance difference on the m RNA expression of IL-6 levels in thalamus between the Mn-treated and control groups(P>0.05).Compared with the Mn-treated group,the m RNA expression of IL-1β and COX-2 in thalamus of PAS intervention group were decreased(P<0.05).PAS-Na intervention has no effects on the increase of m RNA expression of TNF-α in thalamus of Mn-exposed rats(P>0.05).In observation period of 12 weeks,the m RNA expression of IL-1β,IL-6,TNF-α,PGE2 levels in thalamus,hippocampus and cortex of Mn-treated group were higher than those of the control group(P<0.05 or 0.01).Compared with the Mn-treated group,the m RNA expression of TNF-αin thalamus,COX-2 in hippocampus,IL-6,IL-1β,TNF-α in cortex of PAS intervention group were decreased(P<0.05).While PAS-Na intervention has no effects on the increase in m RNA expression of IL-6,IL-1βin thalamus,COX-2in hippocampus and cortex.In observation period of 18 weeks,the m RNA expression of IL-1β,IL-6,TNF-α,COX-2 in cortex,IL-1β,PGE2,TNF-αin thalamus,IL-6,IL-1 β and TNF-α in hippocampus of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01).Compared with the Mn-treated group,the m RNA expression of IL-1β,COX-2,TNF-αin thalamus,IL-1 β and IL-6 in hippocampus of H-PAS group were decreased(P<0.05 or0.01).High dose of PAS-Na treatment restored the above-mentioned changes of inflammatory factors induced by Mn.There is no significant difference in the m RNA expression of inflammatory factors in the brain tissues between the PAS control and the control group in each observation period.(8)The effect of PAS-Na on the protein expression of MAPK pathway relatedproteins in the brain tissues of Mn-exposed rats.In observation period of 4 weeks,the ratio of p-ERK/T-ERK and p-JNK/T-JNK in thalamus of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01),which indicated that Mn exposure increased the phosphorylation of ERK and JNK in thalamus.There is no significance difference on the ratio of p-P38/T-P38 and COX-2 protein expression in thalamus between the Mn-treated and control groups(P>0.05).In observation period of 8 weeks,the protein expression of COX-2,ratio of p-ERK/T-ERK,p-JNK/T-JNK and p-P38/T-P38 in thalamus of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01).Compared with the Mn-treated group,the ratio of p-JNK/T-JNK in thalamus of PAS intervention group was decreased(P<0.05).PAS-Na intervention has no effects on the increase of thephosphorylation of p38,ERK and the COX-2 protein expression in thalamus of Mn-exposed rats(P>0.05).In observation period of 12 weeks,the protein expression of COX-2,p-ERK,p-JNK,p-p38 in thalamus,COX-2,p-ERK,p-p38 in hippocampus,COX-2 and p-ERK cortex of Mn-treated group were higher than those of the control group(P<0.05 or 0.01).Compared with the Mn-treated group,the protein expression of COX-2 in thalamus,hippocampus and cortex of PAS intervention group were decreased(P<0.05).In observation period of 18 weeks,the protein expression of COX-2,p-ERK,p-JNK,p-p38 in thalamus,COX-2,p-ERK,p-p38 in hippocampus and COX-2 in cortex of the Mn-treated group were higher than those of the control group(P<0.05 or 0.01).PAS-Na treatment restored the above-mentioned changes of protein expression in the brain tissues of Mn-exposed rats.There is no significant difference in the protein expression of MAPK related protein and COX-2 in the brain tissues between the PAS control and the control group in each observation period.Conclusions(1)Mn exposure slows down the increase of body weight of the experimental animals and changes the organ coefficients of heart,kidney,spleen,testis and lung.PAS-Na intervention or high-dose PAS-Na treatment can inhibit the organ coefficient,but no effect on the body weight of Mn-exposed rats.Additionally,PAS-Na also has effects on d of rats by showing to slow down the increase of body weight at 3rd week.(2)Both Sub-acute and sub-chronic Mn exposure has affects on the learning ability of rats,and impairments of the memory ability are only found in sub-chronic Mn exposure.PAS-Na treatment or prevention has antagonized effects on the impairment of spatial learning and memory ability induced by Mn.(3)Mn exposure increases the Mn levels in whole blood and brain(cortex,thalamus and hippocampus)of rats.PAS-Na intervention and treatment decreases the Mn levels in whole bloodand brain(cortex,thalamus and hippocampus)of Mn-exposed rats.(4)Excessive Mn exposure increases the positive expression of GFAP,bax,caspese-3 and decreases the positive expression of bcl-2 in the brain tissues including cortex,hippocampus and thalamus.PAS-Na prevention and / or treatment have antagonistic effects on the above-mentioned changes in the brain tissues induced by Mn.(5)Excessive Mn exposure increase the inflammatory factors levels and m RNA expression in serum and brain tissues including cortex,hippocampus and thalamus.PAS-Na prevention and / or treatment may have an antagonistic effect on the inflammatory response in serum,cortex,hippocampus and thalamus induced by Mn.(6)Mn may induce inflammatory responses in the cortex,hippocampus and thalamus via activating the MAPK pathway and COX-2,especially activate the phosphorylation of ERK and p38.PAS-Na prevention and / or treatment may have antagonistic effect on the inflammatory response in cortex,hippocampus and thalamus of Mn-exposed rats’ via inhibit the MAPK pathway.
Keywords/Search Tags:Sodium para-aminosalicylic acid, Manganese, Inflammation factors, Cortex, Hippocampus, Thalamus
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