| Objective Manganese(Mn),as an essential trace element in human body,participates in various important physiological processes,however,excessive Mn intake may damage the extravertebral system and impair learning and memory.The hippocampus not only is the main brain area that dominates learning and memory but also manganese damage.Previous studies have shown that Mn exposure could activate microglia and at the same time it makes NOD-like receptor protein 3(Nlrp3)inflammasome overexpression,so the inflammatory response enhanced in hippocampus of mice.Cellular endoplasmic reticulum stress(ERS)may promote oxidative stress.As a results it induces overexpression of thioredoxin interacting protein(Txnip)to activate Nlrp3 inflammasome.Sodium Para-aminosalicylate(PAS-Na)is a non-steroidal anti-inflammatory drug,which has anti-inflammatory and anti-oxidative effects,and it may inhibit the activation of microglia and inflammation.Our preliminary study found that PAS-Na was effective in the therapeutic of manganism patients.Therefore,this study intended to explore the mechanism of ERS-Txnip/Nlrp3 inflammasome signaling pathway and the intervention effect of PAS-Na with vivo and vitro experiment.Methods 1.In vitro study:Rat microglia cell line-HAPI cell was selected as the research object in vitro study.(1)Firstly,the viability of HAPI cells exposed to varying concentrations of manganese and PAS-Na for 24h was measured to use a cytotoxicity assay(MTT).(2)Grouping and drug treatment were as the following:? Manganese damage model:including that control,low,middle and high manganese groups exposed to 0,50,100,200 ?mol/L(?M)Mn,respectively.?PAS-Na intervention groups:including that control group,Mn-exposed group,low,middle and high PAS-Na group(L-,M-,H-PAS-Nagroup)and PAS-Na group were exposed to medium,Mn 200 ?M,Mn 200+PAS-Na 100 ?M,Mn 200+PAS-Na 200 ?M,Mn 200+PAS-Na 400 ?M and PAS-Na 400 ?M,respectively.?Tauroursodeoxycholic acid(TUDCA)inhibition groups:including that control group,Mn-exposed group,dimethyl sulfoxide(DMSO)group,TUDCA group and TUDCA inhibition group were given medium,Mn 200 ?M,DMSO,TUDCA 100?M,TUDCA 100+Mn 200?M.?Resveratrol inhibition groups:including that control group,Mn-exposed group,DMSO group,Resveratrol group and Resveratrol inhibition group were exposed to medium,Mn 200 ?M,DMSO,Resveratrol 20 ?M,Resveratrol 20+Mn 200 ?M,respectively.(3)Reactive oxygen species(ROS)production levels in manganese damage groups,PAS-Na intervention groups and TUDCA inhibition groups were detected by ROS kit.(4)The protein expression of B-cell lymphoma-2(Bcl-2),BCL2-Associated X protein(Bax),Nlrp3,Caspase-1,Txnip and inositol-requiring enzyme-1(IRE1)were detected by Western blotting(WB).(5)The content of Malondialdehyde(MDA),protein carbonyl(PCO),interleukin(IL)-18,IL-1? and Txnip were detected by Enzyme-linked immunosorbent assay(Elisa).(6)The Messenger Ribonucleic Acid(mRNA)expression of Bcl-2,Bax,Nlrp3,Caspase-1,Txnip,IRE1,IL-18,IL-1? were detected by real-time fluorescence quantitative polymerase chain reaction(RT-PCR).2 In vi'vo study:(1)Animals groups:?Manganism groups:A total of 48 SD rats were randomly divided into control group,low,middle and high manganese group with 12 rats per group according to their bodies weight.The rats of low,middle and high manganese group were intraperitoneally injected with MnC12 4H2O at doses of 5,10 and 20 mg/kg,respectively.And the rats of control group were intraperitoneally injected with the same amount of saline,the regulars were that once a day,5 days per week for 8 weeks.?PAS-Na therapeutic groups:A total of 72 SD rats were randomly divided into control group,Mn-exposed group,L-,M-,H-PAS-Na group and PAS-Na group with 12 rats per group according to their bodies weight.During the first eight weeks,the rats of control group and PAS-Na group were intraperitoneally injected with sterilized saline.The rats of Mn-exposed group,L-,M-,H-PAS-Na group were intraperitoneally injected with MnCl2·4H2O at a dose of 20 mg/kg,once a day,5 days per week for 8 weeks.And then the rats of control and Mn-exposed group were intraperitoneal to inject with the same amount of saline,while the rats of L-,M-,H-PAS-Na group and PAS-Na group were subcutaneously injected with PAS-Na 100,200,300 and 300 mg/kg,once a day,5 days per week for 6 weeks,respectively.(2)Animal index detection:? Animal weight and performance were both recorded every day.?A test of learning and memory ability:A total of 9 rats in each group were selected for Morris water maze test one week before the end of the experiment.? Blood samples were collected and then brain tissues were separated.Other organs of rats were obtained and weighed,and organ coefficients were calculated after the experiment,either.? The concentration of whole blood Mn and hippocampus Mn was measured by graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry(ICP-MS),respectively.? The ultrastructural changes of hippocampal neurons were observed by transmission electron microscopy.?The expression of Bax and Bcl-2 positive cells in hippocampus was detected by immunohistochemistry.The other indexes detected in animal were the same as those of HAPI cells indexes detected by WB,Elisa and RT-PCR.Results 1 In vitro study:(1)The effect of PAS-Na and Mn on viability of HAPI cells:The viability of 86.6%,73.6%and 57.4%of HAPI cells after 200,300 and 400 ?M Mn exposure for 24 h,respectively.There was no significant difference on viability of HAPI cells exposed to 100,200,300,400 and 600 ?M PAS-Na exposure for 24 h(P>0.05).(2)The effect of PAS-Na on the expression of apoptosis-related gene in HAPI cells exposed to manganese:Comparison with contol group,increased expression of Bax mRNA and protein was observed in high manganese group(P<0.01)and the expression of Bcl-2 in middle and high manganese group also increased(P<0.05).In the PAS-Na intervention test,the expression of Bax mRNA and protein increased but Bcl-2 mRNA and protein declined in manganese-exposed group(P<0.01 or 0.05).However,comparison with the manganese-exposed group,the expression of Bax increased and Bcl-2 decreased in PAS-Na intervention groups,suggesting PAS-Na could antagonize the abnormal expression of Bax and Bcl-2 gene.(3)The effect of PAS-Na on the expression of inflammation in HAPI cells exposed to manganese:Comparison with the control group,the expression of N1rp3 mRNA and protein and Caspase-1 protein increased,and IL-18 content,IL-1? mRNA and content also increased after manganese exposure(P<0.01 or 0.05).In the PAS-Na intervention test,the mRNA and protein expression of Caspase-1 and Nlrp3,the expression of IL-1? mRNA were all lower than those in manganese-exposed group(P<0.01 or 0.05).(4)The effect of PAS-Na on oxidative stress of HAPI cells exposed to manganese:ROS-positive cells increased with the increase of manganese dosage.MDA content in high manganese group was also significantly higher than that in control group(P<0.05).In PAS-Na intervention test,ROS-positive cells increased significantly in manganese-exposed group,while L-,M-,H-PAS-Na treatment could antagonize this effect.(5)The effect of PAS-Na on the expression of Txnip in HAPI cells exposed to manganese:The relative expression of Txnip protein and concentration of Txnip in high manganese group were all higher than those in control group(P<0.01 or 0.05).After PAS-Na intervention,the expression of Txnip protein in L-,M-,H-PAS-Na group was lower than that in manganese-exposed group(P<0.01 or 0.05).(6)The effect of PAS-Na on the expression of IRE 1 in HAPI cells exposed to manganese:The expression of IRE1 mRNA and protein in high manganese group were all higher than those in control group(all P<0.01).The expression of IRE1 mRNA and protein in H-PAS-Na group were all lower than those in manganese-exposed group(P<0.01).(7)Inhibitory effect of TUDCA on Txnip/Nlrp3 signaling pathway:Comparison with the manganese-exposed group,ROS production level decreased,the expression of IRE 1 mRNA and protein,the relative expression of Txnip protein and concentration of Txnip,Caspase-1 mRNA,expression of IL-1? mRNA and content in TUDCA inhibition group all significantly decreased in varying degrees(P<0.01 or 0.05).(8)Inhibitory effect of Resveratrol on Txnip/Nlrp3 signaling pathway:Comparison with the manganese-exposed group,the expression of mRNA and protein in Txnip and Nlrp3 and the expression of mRNA in Caspase-1 and IL-1?in Resveratrol inhibition group all significantly decreased(P<0.01 or 0.05).2.In vivo study:(1)The effects of PAS-Na on the growth and development of manganese-exposed rats:Diarrhea,loss of appetite and irritability were observed in 8-weeks manganese-exposed rats and the body weight of rats decreased with the increase of manganese dosage(P<0.05 or 0.01).The liver and kidney coefficients in high manganese group were significantly higher than those in control group.After 6-weeks PAS-Na treatment,the poisoning symptoms in rats disappeared and there was no significant difference in body weight,liver and kidney coefficients between groups.(2)The effect of PAS-Na on manganese content in blood and hippocampus of manganese-exposed rats:The concentration of blood manganese in 8-weeks manganese-exposed rats increased with the increase of manganese dosage,and there were significant differences between groups(P<0.01).The concentration of manganese in hippocampus of low,middle and high manganese group was higher than that in control group(P<0.01).In L-,M-,H-PAS-Na groups,blood and brain manganese decreased with the increase of PAS-Na treatment dosage(P<0.01 or 0.05).(3)The effect of PAS-Na on learning and memory of manganese-exposed rats:The swimming distance and escape latency of rats in the third to fifth day of Morris water maze test in high manganese group were higher than those in control group(P<0.01 or 0.05),and the number of platform crossings in high manganese group was less than that in control group(P<0.05).After 6-weeks PAS-Na treatment,the swimming distance and escape latency in each group recovered and approached the level in control group,and there was no significant difference in number of platform crossings.(4)Intervention of PAS-Na on hippocampal injury in manganese-exposed rats:Manganese exposure increased Bax-positive cells in hippocampus,and the expression of Bax mRNA and protein increased(P<0.01 or 0.05),while the expression of Bcl-2 mRNA and protein in high manganese group was lower than that in control group(P<0.01).Hippocampal neurons in middle and high manganese groups degenerated in varying degrees.After 6-weeks PAS-Na treatment,Bax-positive cells were also observed in manganese-exposed group,only a small number of Bax-positive cells were found in PAS-Na treatment groups.The expression of Bax mRNA and protein increased while expression of Bcl-2 protein decreased in manganese-exposed group(P<0.01 or 0.05),however,PAS-Na treatment could antagonize the abnormal expression of Bax and Bcl-2 gene.Hippocampal neurons in manganese-exposed group remained slightly degenerated,while those in each PAS-Na intervention groups returned to normal.(5)The effect of PAS-Na on the expression of inflammation in hippocampus of rats exposed to manganese:The expression of Nlrp3 mRNA and protein in high manganese group was higher than those in control group,low and middle manganese group(P<0.01 or 0.05),and the expression of Caspase-1 mRNA and protein in middle and high manganese group was higher than those in control group(P<0.01 or 0.05).The mRNA expression and content of IL-18 in middle manganese group and the mRNA expression and content of IL-1? in middle and high manganese group were higher than those in control group(P<0.01 or 0.05).After PAS-Na treatment,the expression of Nlrp3 mRNA and Caspase-1 protein were all lower than manganese expousure group,the mRNA expression and content of IL-18 and IL-1? in M-,H-PAS-Na groups also decreased(all P<0.01).(6)The effects of PAS-Na on oxidative stress in hippocampus of manganese-exposed rats:Comparison with the control group,the content of MDA and PCO in high manganese group all increased(P<0.01 or 0.05).The content of MDA and PCO in H-PAS-Na group were lower than those in manganese-exposed group(P<0.05).(7)The effect of PAS-Na on the expression of Txnip in hippocampus of rats exposed to manganese:The relative expression protein and protein content of Txnip in high manganese group were all higher than those in control group(P<0.01 or 0.05).The expression of Txnip mRNA and protein in H-PAS-Na group was lower than those in manganese-exposed group(all P<0.01).(8)The effect of PAS-Na on the expression of IRE 1 in hippocampus of rats exposed to manganese:The expression of IRE 1 mRNA and protein in middle and high manganese group was higher than that in control group(P<0.01 or 0.05).The expression of IRE1 mRNA and protein in H-PAS-Na group was lower than that in manganese-exposed group(P<0.01).Conclusions(1)Manganese exposure can affect the growth and development of rats,resulting in weight loss and increased liver and kidney coefficients.PAS-Na has therapeutic effect on the growth and development of manganism rats.(2)Manganese exposure can increase the concentration of manganese in blood and hippocampus,impair hippocampus and then affect learning and memory ability in rats.PAS-Na can promote manganese excretion,and then alleviate hippocampus injury.(3)Manganese exposure may induce ERS in HAPI cells and in hippocampal microglia,which may cause oxidative damage and then activate Txnip/Nlrp3 signaling pathway to mediate inflammation,therefore,nerve cells were damaged.PAS-Na treatment may inhibit the activation of ERS-Txnip/Nlrp3 inflammasome signaling pathway and ameliorate manganese neuroinflammation. |
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