In Vitro And In Vivo Study Of The Mechanism Of Sodium P-aminosalicylate On Lead Induced Hippocampal Neuroinflammation In Rats

BackgroundLead is an environmental heavy metal pollutant that is mainly absorbed by the human body through the respiratory tract and digestive tract.Lead can damage nerve,hematopoietic,reproductive and other human systems,and its toxic effects may be irreversible.With the improvement of technology and environmental protection,environmental lead pollution is manifested as low-level chronic exposure,which is generally in sub-clinical manifestations or atypical,so that it is often difficult to arouse people’s vigilance or attention.Many studies have shown that the neurotoxicity of lead may be related to neurodegenerative diseases,such as Alzheimer’s disease（AD）.Developmental neurotoxicity can still be observed in lead at very low concentrations（blood lead levels less than 100μg/L）,which is negatively correlated with children’s IQ.Lead exposure in early life can promote the accumulation of Aβamyloid plaques in the prefrontal cortex of old monkeys,forming AD-like pathological changes.Neurons are the most basic structural and functional units of the nervous system.They can receive and send out information and regulate the life activities of the body.Pyrolysis is involved in neurodegenerative disease.The pyroptosis mediated by NLRP3/caspase-1 inflammasome can promote the occurrence of AD-like changes caused by lead.However,most studies only focus on the lead-induced pyroptosis of glial cells,and ignore the relationship between the neuron itself and the pyroptosis.Lead can disturb the homeostasis of calcium ions in neurons,affect the activation of calcium regulatory protein Ca MKII and its downstream learning and memory regulatory protein CREB,disorder learning and memory.In addition,Lead can activate Thr and Tyr at the phosphate sites of ERK1/2,and participate in the process of lead-induced learning and memory impairment.However,the mechanism of lead-induced neurotoxicity is not yet understood,and it is extremely important to deeply explore the relationship between lead exposure and AD-like changes.Sodium aminosalicylate（PAS-Na）has a non-specific anti-inflammatory effect.According to reports,PAS-Na is effective in treating patients with manganese poisoning.Therefore,we propose that PAS-Na may also have a curative effect on lead poisoning.Our earlier study found that PAS-Na can inhibit lead-induced apoptosis of PAS-NA can inhibit lead-induced apoptosis of nerve cells,improve the ultrastructure of the hippocampus and the learning and memory ability of lead-exposed rats.However,its mechanism is still unclear.Therefore,this study used in vivo and in vitro experiments to explore the efficacy of PAS-Na on lead-induced neurodegeneration.Objective1.To investigate whether lead exposure can induce NLRP3/caspase-1inflammasome-mediated pyrolysis of neurons.2.To clarify the relationship between NLRP3/Caspase-1 inflammasome mediated pyroptosis,Ca²⁺/Ca MKII/CREB pathway,ERK1/2 protein and lead-induced AD-like degeneration,so as to find a more clear and effective therapeutic target for lead-induced AD-like degeneration.3.To verify the therapeutic effect of PAS-Na on lead-induced hippocampal nerve injury,and explore the possible mechanism of its therapeutic effect.Method1.In vitro studies0,25,50,100μmol/L lead acetate was selected to treat primary hippocampal neurons,SH-SY5Y cells and N2a cells for 24 hours.（1）Use flow cytometry to detect the effect of lead on the N2a cell cycle;use LDH cytotoxicity detection method to detect the mortality of primary hippocampal neurons and SH-SY5Y cells to assess the cytotoxicity of lead.（2）Use WB to detect the expression of AD-related proteins in primary hippocampal neurons and SH-SY5Y cells:BACE,P-GSK-3α,GSK-3α/3β,and Tau;use immunofluorescence to detect the expression of Aβin SH-SY5Y cells treated with different concentrations of lead.（3）Use immunofluorescence method to detect the expression level of GSDMD protein in primary hippocampal neurons;use Western blot（WB）to detect the expression of pyroptosis-related proteins of primary hippocampal neurons and SH-SY5Y cells:GSDMD,NLRP3,cleaved caspase-1,IL-18 and IL-1β.（4）Use WB to detect the expression of ERK1/2 and P-ERK1/2 proteins in primary hippocampal neurons and SH-SY5Y cells to observe the effect of lead on the ERK1/2 MAPK pathway.（5）Use fluorescent probe method to detect the Ca²⁺level in primary hippocampal neurons;use WB to detect the protein expression of P-Ca MKII,Ca MKII,P-CREB and CREB in primary hippocampal neurons and SH-SY5Y cells.（6）The primary hippocampal neurons were pretreated with 50μmol/L caspase-1 inhibitor VX-765,10μmol/L Ca MKII inhibitor KN-93,and 10μmol/L ERK1/2 inhibitor SCH772984,and then lead acetate was added（final The concentration is 50μmol/L）;The caspase-1 activity detection kit was used to detect the caspase-1 activity in the primary hippocampal neurons.The protein of IL-18 and IL-1βin primary hippocampal neurons were detected by ELISA.Above proteins（in vitro study）in primary hippocampal neurons were detected by WB.And then use LPS and lead alone or together to treat primary hippocampal neurons;Use WB to detect the expression of the above proteins to clarify the pyrolysis mediated by NLRP3/Caspase-1 inflammasome,Ca2+/Ca MKII/CREB pathway and ERK1/2 The relationship between MAPK pathway and lead-induced AD-like degeneration.2.In vivo studies（1）Establishment of subchronic lead poisoning model:Weaned SD rats were selected and intraperitoneally injected 2 mg/kg lead acetate for 12 weeks,5 times a week,to establish a lead poisoning model.A group of lead-exposed rats was injected with 1 mg/kg VX-765 into the lateral ventricle,and after 48 hours of recovery,the follow-up test was continued.The water maze test was used to assess the learning and memory ability of rats.HE staining and Nissl staining were used to assess the damage effect of lead on the hippocampus of rats.ICP-MS was used to detect lead and calcium content in whole blood and hippocampus.Use immunofluorescence to detect the co-localization of cleaved caspase-1and neuron marker protein MAP2 or P-Ca MKII,Tau and cleaved caspase-1.Use WB to detect the protein expression of P-Ca MKII,Ca MKII,P-CREB,CREB,BACE,P-GSK-3α,GSK-3α/3βand Tau.ELISA was used to detect the protein expression of IL-1βand IL-18.To evaluate the effect of NLRP3/Caspase-1 mediated neuronal inflammatory necrosis on lead-induced cognitive dysfunction in rats.（2）The influence of PAS-Na on lead-induced inflammatory necrosis of hippocampal neurons and cognitive dysfunction:（1）The effect of PAS-Na on subchronic lead exposure-induced inflammatory necrosis of hippocampal neurons and cognitive dysfunction:Weaned SD rats were selected and intraperitoneally injected 2 mg/kg lead acetate for 12 weeks,5 times a week.Subsequently,lead exposure was stopped,and 80,160,and 240 mg/kg PAS-Na were injected subcutaneously on the back for 6 weeks,5 times a week.The water maze test was used to evaluate the learning and memory ability of rats.HE staining and Nissl staining were used to evaluate the damage effect of lead on the hippocampus of rats.The content of lead and calcium in whole blood and hippocampus was detected by ICP-MS.Above-mentioned proteins were detected by WB and ELISA.（2）Comparing the effects of PAS-Na and EDTA on lead-induced inflammatory necrosis of hippocampal neurons and cognitive dysfunction:Adult SD rats were intraperitoneally injected with 6mg/kg lead acetate for 4 weeks,5times a week.Subsequently,lead exposure was stopped,and 100,200,300 mg/kg PAS-Na and 100 mg/kg calcium disodium edetate（EDTA）were injected subcutaneously on the back for 3 weeks,5 times a week.The expression of GSDMD in rat hippocampus was detected by immunohistochemistry.Other methods are the same as in vivo study（1）.Results1.In vitro studies results（1）Cytotoxicity of lead:Morphological observation of cells,LDH cytotoxicity test and cell cycle results showed that lead could cause neuron damage of primary hippocampal neurons and SH-SY5Y cells.In addition,lead increased the mortality（P<0.01）,but did not cause cell cycle arrest in N2A cells.（2）The effect of lead on AD-related protein in nerve cells:WB results showed that lead exposure increased the expression of BACE and P-GSK-3protein and increased the ratio of P-GSK-3 to GSK-3 in primary hippocampal neurons and SH-SY5Y cells.Besides,lead exposure increased the expression of Tau protein in primary hippocampal neurons（P<0.05）.Immunofluorescence results showed that lead could increase the expression of Aβprotein in SH-SY5Y cells.It is suggested that lead exposure can cause AD-like neuropathy.（3）The effect of lead on the expression of pyroptosis-related protein in neuronal cells mediated by NLRP3/caspase-1 inflammasome:immunofluorescence results show that lead can promote the increase of GSDMD protein expression in primary hippocampal neurons.WB results showed that lead can promote the expression of pyrolysis-related proteins GSDMD,NLRP3,cleaved caspase-1,IL-18 and IL-1βin primary hippocampal neurons and SH-SY5Y cells（P<0.05）.（4）The effect of lead on the ERK1/2 protein of nerve cells:Lead can promote the increase of P-ERK1/2 expression in primary hippocampal neurons and SH-SY5Y cells,and the ratio of P-ERK1/2 to ERK1/2 increased（P<0.01）.It is suggested that lead exposure can activate the ERK1/2 MAPK pathway.（5）The effect of lead on the Ca MKII/CREB pathway of nerve cells:cell morphology observation results show that lead can damage primary hippocampal neurons,shrink the cell body,break synapses,damage the network structure,and accompanied by intracellular Ca²⁺levels increased.P-Ca MKII protein expression increased,P-CREB protein expression decreased in primary hippocampal neurons and SH-SY5Y cells（P<0.05）,suggesting that lead exposure can disrupt the Ca MKII/CREB pathway.（6）Role of NLRP3/Caspase-1 mediated neuronal inflammatory necrosis in lead-induced AD-like neuropathy:the results of cell morphology showed that Caspase-1 inhibitor VX-765 and Ca MK inhibitor KN-93 have a certain antagonistic effect on lead-induced neuronal damage,while ERK1/2 inhibitor SCH772984 has no obvious protective effect on it.ELISA results showed that VX-765 can reduce the expression of IL-1βand IL-18 protein in primary hippocampal neurons exposed to lead（P<0.01）;KN-93 can reduce the expression of IL-1βprotein in primary hippocampal neurons exposed to lead（P<0.05）,but has a negative effect on IL-Level 18 has no effect.WB results show that both VX-765 and KN-93 can reduce the protein expression of P-Ca MKII and P-GSK-3αin primary hippocampal neurons and increase the protein expression of P-CREB after lead treatment.It is suggested that inhibition of caspase-1 can alleviate NLRP3/Caspase-1 mediated neuronal inflammatory necrosis,correct lead-induced Ca MKII/CREB pathway disorder,and reduce GSK-3 enzyme activity.The expression of GSDMD,cleaved caspase-1,IL-18,IL-1β,P-ERK1/2,P-Ca MKII and BACE protein was increased in primary hippocampal neurons treated with LPS and lead,while the expression of P-CREB protein were decreased（P<0.05）.These results indicated that LPS promoting lead-induced inflammatory necrosis could further activate the ERK1/2 protein and disrupt the Ca MKII/CREB pathway,thus aggravating the lead-induced neurodegenerative diseases.2.In vivo studies results（1）The effect of NLRP3/Caspase-1 mediated neuronal inflammatory necrosis on lead-induced cognitive dysfunction in rats:The results of the Morris water maze test showed that compared with the control group,the escape latency was increased on the fourth and fifth days,and the swimming speed was slowed down on the first day of lead-exposed rats（P<0.05）.It shows that lead can impair learning and memory in rats.VX-765 has no effect on lead-induced increase of escape latency and slowing of swimming speed in rats.ICP-MS results showed that the lead content in the whole blood and hippocampal of rats in the lead-exposed group and Pb+VX-765 group were increased（P<0.001）.However,there is no difference between the two groups（P>0.05）.The immunofluorescence results showed that in the lead-exposed group red fluorescence decreased,MAP2expression decreased,neurons decreased,green fluorescence increased,and cleaved caspase-1 expression increased;after VX-765 treatment,red fluorescence increased,MAP2 expression increased,and the number of neurons increased;in addition,the green fluorescence decreases,and the expression of cleaved caspase-1 decreased.The protein test results showed that the expression of NLRP3,cleaved caspase-1,IL-1β,IL-18,P-ERK1/2,P-Ca MKII protein were increased,and the P-CREB decreased in hippocampus of the lead-exposed rats（P<0.05）,as compared to the control group.However,VX-765 treatment could reverse the above changes.In addition,immunofluorescence results showed that VX-765treatment cloud reduce the expression of cleaved caspase-1,P-Ca MKII and Tau,while they were increased in the hippocampus of lead-exposed rats.It shows that inhibition of caspase-1 can antagonize lead-induced AD-like degeneration.（2）The effect of PAS-Na on the inflammatory necrosis of hippocampal neurons and cognitive dysfunction of rats caused by subchronic lead exposure:The results of the Morris water maze test showed the PAS-Na treatment had no significant effect on the learning and memory impairment in lead-exposed rats（P>0.05）.ICP-MS test results shown that the whole blood lead concentration and hippocampal lead concentration were increased in the hippocampus of lead-exposed rats（P<0.05）.The lead content in the hippocampus of PAS-Na treatment group had a tendency to decrease,but there was no significant difference from the lead exposure group（P>0.05）.Protein results showed that PAS-NA treatment could reduce the expression of NLRP3,Cleaved caspase-1,IL-1β,P-ERK1/2protein correct the lead-induced Ca MKII/CREB pathway disorder,antagonize the lead-induced GSK-3 enzyme activation,thus decrease expression of Tau protein in hippocampus of lead-exposed rats.These results indicate that PAS-NA can alleviate lead induced AD-like degeneration to a certain extent.（3）The effect of PAS-Na and Ca Na₂EDTA on the inflammatory necrosis of hippocampal neurons and cognitive dysfunction of rats caused by subacute lead exposure.The results of the Morris water maze test showed the Pb-exposed group had longer escape latency on the first and second days and longer swimming distance on the second day as compared to the control（P<0.05）.The escape latency on the second day of the 300mg/kg PAS treatment group and the EDTA treatment group was shorter than that of the lead exposure group（P<0.05）.It shows that PAS-Na and Ca Na₂EDTA may improve the learning and memory impairment of rats caused by lead.ICP-MS results showed that the hippocampal Pb content of the PAS-Na treatment group was not significantly different from that of the lead-exposed group（P>0.05）,while the hippocampal Pb content of the Ca Na₂EDTA treatment group was lower than that of the lead-exposed group（P<0.05）.It shows that the effect of Ca Na₂EDTA in promoting Pb excretion is better than PAS-Na.However,the protein results showed that PAS-Na can antagonize the increase in the expression of NLRP3,Cleaved caspase-1,IL-1βand IL-18 in the hippocampus of rats caused by lead（P<0.05）,while Ca Na₂EDTA treatment can only reduce the expression of IL-18 protein without affecting other proteins.At the same time,PAS-Na treatment can antagonize the lead-induced ERK1/2 and P38 pathways activation in the hippocampus of rats,and has little effect on the JNK pathway.The Ca Na₂EDTA treatment can antagonize the lead-induced ERK1/2 pathways activation in the hippocampus of rats,which has a negative effect on the P38 and JNK pathways.In addition,PAS-Na could correct the disorder of the Ca MKII/CREB pathway and antagonize the activation of GSK-3 enzyme caused by lead,while Ca Na₂EDTA has no obvious effect on them.Conclusions1.Lead exposure can activate neuronal NLRP3/caspase-1 inflammasome,and then disrupt the Ca MKII/CREB pathway and induce neurodegenerative disease in hippocampal neurons.2.PAS-Na can inhibit the activity of NLRP3/Caspase-1 inflammasome,correct the Ca MKII/CREB pathway,and alleviate the lead-induced neurodegenerative disease,and the effect is better than Ca Na2EDTA.