Experimentall Study On Necroptosis Of Central Nernervous System Induced By Manganese Exposure In Rats

Our early studies have showed that the necrosis can be induce by manganese exposure in the central nervous cells of rats,and in vitro cultured of neuroblastoma cell(SK-N-SH cells)in manganese exposure model,we had found necrosis-like apoptosis-specific blockade.And the necrosis rate can be decreased by the intervention of Nec-1 in SK-N-SH cells manganese exposure,in which the mechanism might be related to the effect of nerve cells death induced by manganese exposure.Recent studies have reported the discovery of RIP3 inhibitors,including GSK’840,GSK’843 and GSK’872.To provide more evidence for the mechanism of RIP3-induced necrosis-like apoptosis in central nervous system injury of rats induce by manganese exposure,our study intends to make further study on the ultrastructure of necrotic cells and the intervention of GSK’872 in the central nervous system of rats and in which effect and mechanism was investigated.Objective: To study the ultrastructural changes of hippocampal necrotic neurons in rats exposed to subacute manganese;To study on the mechanism of RIP3 inhibitor GSK’872 in neuronal death induced by exposed to subacute manganese in rats.Methods:(1)A total of 36 SD rats were randomly divided into control group,manganese exposure group,with 18 groups in each group.Manganese exposure group rats were intraperitoneally injected(iP)with manganese chloride(15mg/kg,5 days a week,for 4 weeks).While exposure group animals were given the manganese,the control group were given the same amount of normal saline via the intraperitoneally injected(iP).Then we continue to raise animals for two weeks after manganese and drug intervention.And 2% paraformaldehyde and 2.5% glutaraldehyde mixed fixative were fixed in the animal body,and bilateral hippocampus tissues were taken.Ultrathin sections of transmission electron microscopy were made and the ultrastructural changes of hippocampal CA1 area in rats were observed by transmission electron microscope and photographed.(2)A total of 72 SD rats were randomly divided into control group,GSK’872 control group,manganese exposure group and GSK’872 intervention group.Then the manganese poisoning model were established via the method of subacute intraperitoneal injection(15mg/kg,5 days for a week,total 4weeks).GSK’872 solution(25mmol/L,10μL/time,1 time/w,total 3weeks)was injected into the cisterna magna in the second week after manganese exposure.While other animals were given the same dose of normal saline to the cerebellum.GSK’872 solution(25mmol/L,10μL/time,1time/w,total 3 weeks)was injected into the cisterna magna in the second week after manganese exposure,while other animals were given the same dose of normal saline to the cerebellum.Then continue to raise animals for two weeks after manganese exposure and drug intervention.(3)After perfusion fixation,the tyrosine hydroxylase(TH)positive cells in substantia nigra and Glial fibrillary acidic protein(GFAP)positive cells in the cortical and hippocampal CA1 region were detected by method of immunohistochemistry.(4)The rats were sacrificed by cervical dislocation.Then the parietc cortex,hippocampus and substantia nigra of the animals were harvested when sampling.The expression of GFAP and RIP3 protein in the cortex,hippocampus and the expression of TH protein in the substantia nigra were detected by method of immunoblotting.The expression of RIP3,RIP3,MLKL,NOX1 and NOX4 genes in cortex,hippocampus and substantia nigra were detected by method of RT-PCR.Results:(1)The ultrastructural characteristics of neurons in hippocampal of rats exposed to manganese showed the necrotic apoptotic cells,in which the cell body was swollen and the organelles were sparse.Nuclear condensation,edge collection,nuclear membrane dissolution and the mitochondrial space was changed and disintegration.The ultrastructure of these cells was distinct from apoptotic neurons.(2)Compared with the control group,the number of GFAP-positive cells in the manganese-exposed group was significantly increased,which was significantly different from the other groups.There was no significant difference in the nigral TH-positive cells in the manganese-exposed group.GSK’872 can reduce the number of GFAP-positive cells in rats exposed to manganese;GSK’872 has no significant effect on increasing the number of TH cells in manganeseexposed group.There is no significant difference between the two groups(P<0.05).(3)Compared with the control group,the expression of RIP3,NOX1,NOX4 and MLKL genes had increased in the manganese exposed group(P<0.05).Compared with the manganese exposed group,the expression of RIP3,NOX1,NOX4 and MLKL had decreased in the GSK’872 intervention group,and the difference between the groups was statistically significant(P<0.05)(4)Compared with the control group,the expression of GFAP and RIP3 protein in the manganese exposed group had increased(P<0.05);And the expression of TH protein in the substantia nigra had decreased;Compared with the manganese exposure group,the expression of GFAP and RIP3 proteins had decreased in hippocampus and cortical of the GSK’872 intervention group,(P<0.05);And the expression of TH protein in the substantia nigra had increased.The difference between the two groups was statistically significant(P<0.05).Conclusion : The hippocampal necrotic neurons exposed to manganese have the ultra-morphological characteristics of necrotic apoptotic cells.The necrotic apoptotic signaling pathway is involved in manganese exposure to induce cell necrosis.GSK’872 can antagonize the neurotoxicity of manganese by antagonizing the occurrence of neuronal necrotic apoptosis,down-regulating the activation level of astrocytes,and reducing the level of microglial inflammation.