Study On Effect Of TNFR1-RIP1/RIP3 Signaling Pathway And Its Downstream MAPKs,MLKL And Ca MKⅡ In Learning And Memory Impairment Induced By Aluminum In Rats

Objective:To investigate the role of TNFR1-RIP1/RIP3 signaling pathway and its downstream MAPKs,MLKL and CaMKⅡ in learning and memory impairment induced by aluminum in rats through aluminum exposure in the lateral ventricle of rats,and add scientific data to the neurotoxicity mechanism of aluminum.Methods:Forty healthy adult male Sprague-Dawley rats were randomly divided into surgical control group,solvent control group,Nec-1 group,Nec-1+Al（mal）₃ group and Al（mal）₃group,with 8 rats in each group.Rats were exposed to intracerebroventricular injection,Nec-1 was given 5μL of Nec-1,Al（mal）₃ group was given 50 mmol/L Al（mal）₃ 5μL,Nec-1+Al（mal）₃ group 5μL Nec-1 was injected 1 h before 5μL 50mmol/L Al（mal）₃,and the solvent control group was given an equal volume of normal saline.The surgical control group was treated with the same lateral ventricle cannula without any reagent treatment.It is regularly exposed to poison every two days for 10 days.After the end of the treatment,the Y-maze was used to determine the learning and memory ability of rats.HE staining and Nissl staining were used to observe the pathological morphology of hippocampus.The ultrastructure of hippocampal neurons was observed by electron microscopy.The expression of TNFR1,RIP1 and RIP3 mRNA in hippocampal neurons was detected by PCR.The relative expression levels of TNFR1,RIP1,RIP3,p-P38,p-ERK,p-JNK,p-MLKL and p-CaMKⅡ proteins in hippocampal neurons were detected by Western-blot.Results:1.Rat behavioral test results:There was no statistically significant difference in the rate of spontaneous response in the surgical control group compared with the solvent control group.Compared with the solvent control group,there was no significant difference in the rate of spontaneous reaction among the Nec-1 group,the Nec-1+Al（mal）₃ group and Nec-1+Al（mal）₃ group.Compared with the solvent control group,the rate of spontaneous reaction in the Al（mal）₃ group decreased,and the difference was statistically significant（P<0.05）.2.HE staining results:The hippocampal neurons of the surgical control group,the solvent control group,the Nec-1 group and the Nec-1+Al（mal）₃ group were structurally intact and clear,the nuclei were clearly visible,the nucleoplasm ratio was large,and the coloration was uniform.The nucleolus was obvious and the cells distributed neatly and orderly.The Al（mal）₃ group of hippocampal neurons showed different degrees of cytoplasmic eosinophilic enhancement,the cell body shank,the cytoplasm was dark red,the cells distributed disorderly,and the number of neurons reduced.3.Nissl staining results:The hippocampal neurons of the surgical control group,the solvent control group,the Nec-1 group and the Nec-1+Al（mal）₃ group distributed neatly,and the Nissl bodies were observed in the neurons.In the Al（mal）₃ group,the hippocampal neurons disorderly,and the cell number reduced apparently,and the number of intracellular Nissl bodies was decreased.4.Electron microscopy results:Hippocampal neurons in surgical control group,solvent control group,Nec-1 group and Nec-1+Al（mal）₃ group were intact,and the organelles such as mitochondria and endoplasmic reticulum were clearly visible,and the nuclear membrane was intact.Al（mal）group₃ hippocampal neurons have irregular nuclear membrane,mitochondrial swelling,and decreased number of mitochondria,severe vacuolization,decreased organelles,and vacuolization-like disintegration of cytoplasm5.Expression of TNFR1,RIP1 and RIP3 genes in hippocampal neurons:（1）There was significant difference in the expression of TNFR1 mRNA between the groups after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,the expression of TNFR1 mRNA in hippocampal neurons of surgical control group,Nec-1 group and Nec-1+Al（mal）₃ group was not statistically significant.Compared with the solvent control group,the expression of TNFR1 mRNA in hippocampal neurons of Al（mal）₃ group was increased,the difference was statistically significant（P<0.05）.（2）There was significant difference in the expression of RIP1 mRNA between the groups after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,there was no significant difference in RIP1 mRNA expression of hippocampal neurons among surgical control group,Nec-1group and Nec-1+Al（mal）₃ group.Compared with the solvent control group,the expression of RIP1 mRNA in hippocampal neurons of Al（mal）₃ group increased,the difference was statistically significant（P<0.05）.（3）There was significant difference in the expression of RIP3 mRNA between the groups after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,the expression of RIP3 mRNA in hippocampal neurons of surgical control group,Nec-1 group and Nec-1+Al（mal）₃ group was not statistically significant.Compared with the solvent control group,the expression of RIP3 mRNA in hippocampal neurons of Al（mal）₃ group increased,the difference was statistically significant（P<0.05）.6.Expression of TNFR1,RIP1 and RIP3 proteins in hippocampal neurons:（1）There was significant difference in the expression of TNFR1 protein between the groups after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,the expression of TNFR1 protein in hippocampal neurons of surgical control group,Nec-1group and Nec-1+Al（mal）₃ group was not statistically significant.Compared with the solvent control group,the expression of TNFR1 protein in hippocampal neurons of Al（mal）₃group was increased（P<0.05）.（2）There was significant difference in the expression of RIP1 protein between the groups after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,the expression of RIP1 protein in hippocampal neurons of surgical control group,Nec-1 group and Nec-1+Al（mal）₃ group was not statistically significant.Compared with the solvent control group,the expression of RIP1 protein in hippocampal neurons of Al（mal）₃ group increased,the difference was statistically significant（P<0.05）.（3）There was significant difference in the expression of RIP3 protein between the groups after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,there was no significant difference in the expression of RIP3 protein in hippocampal neurons among surgical control group,Nec-1 group and Nec-1+Al（mal）₃ group.Compared with the solvent control group,the expression of RIP3 protein in hippocampal neurons of Al（mal）₃group increased,the difference was statistically significant（P<0.05）.7.Expression of p-P38,p-ERK and p-JNK proteins in rat hippocampal neurons:（1）There was significant difference in the expression of p-P38 protein between the groups after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,the expression of p-P38 protein in hippocampal neurons of surgical control group,Nec-1 group and Nec-1+Al（mal）₃ group was not statistically significant.Compared with the solvent control group,the expression of p-P38 protein in hippocampal neurons of Al（mal）₃ group increased,the difference was statistically significant（P<0.05）.（2）The expression of p-ERK protein in each group was significantly different after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,the expression of p-ERK protein in hippocampal neurons of surgical control group,Nec-1 group and Nec-1+Al（mal）₃ group was not statistically significant.Compared with the solvent control group,the expression of p-ERK protein in hippocampal neurons of Al（mal）₃ group decreased,the difference was statistically significant（P<0.05）.（3）There was significant difference in the expression of p-JNK protein between the groups after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,the expression of p-JNK protein in hippocampal neurons of surgical control group,Nec-1 group and Nec-1+Al（mal）₃ group was not statistically significant.Compared with the solvent control group,the expression of p-JNK protein in hippocampal neurons of Al（mal）₃ group increased,the difference was statistically significant（P<0.05）.8.Expression of p-MLKL and p-CaMKⅡ proteins in rat hippocampal neurons:（1）The expression of p-MLKL protein in each group was significantly different after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,the expression of p-MLKL protein in hippocampal neurons of surgical control group,Nec-1 group and Nec-1+Al（mal）₃ group was not statistically significant.Compared with the solvent control group,the expression of p-MLKL protein in hippocampal neurons of Al（mal）₃ group was increased,the difference was statistically significant（P<0.05）.（2）The expression of p-CaMKⅡ protein in each group was significantly different after maltol aluminum exposure（P<0.05）.Compared with the solvent control group,the expression of p-CaMKⅡ protein in hippocampal neurons of surgical control group,Nec-1 group and Nec-1+Al（mal）₃ group was not statistically significant.Compared with the solvent control group,the expression of p-CaMKⅡ protein in hippocampal neurons of Al（mal）₃ group decreased,and the difference was statistically significant（P<0.05）.Conclusions:1.Maltol aluminum can cause a decline in learning and memory in rats.2.Maltol aluminum can induce necroptosis of rat hippocampal neurons.3.The signal pathway of maltol aluminum induced necroptosis of rat hippocampal neurons is through TNFR1-RIP1/RIP3 and its downstream MAPKs,MLKL and CaMKⅡ.