The Regulation Of Necroptosis On Manganese Induced Neuron Death

Manganese is recognized as an acute and chronic accumulative neurotoxin.Manganese poisoning can produce symptoms similar to Parkinson injury in central nervous system and persisting after detoxification.Manganese accumulation in brain induces nerve cell death and dysfunctional state of the central nervous system,which leads to degeneration of the central nervous system.Apoptosis,autophagy and necrosis are the main forms of neural death induced by manganese.At present,the study of the mechanism on the manganese-induced neurotoxicity is mainly focused on apoptosis.Moreover,recent reports have been shown that cells necrosis also has special cell signaling transduction system.The RIP3 has been considered as a key factor of the transformation between apoptosis and necrosis which depends on the RIP1 mediated cell death.The pattern of the sequential activation of RIP3 and MLKL in regulatory cell death is called the programmed necrosis(Necroptosis).Our study demonstrated that the manganese exposed induced neuronal necrosis,which the ultrastructural morphology was tend to the characteristics of apoptosis cell under a certain conditions.This indicated that the manganese-induced neuronal necrosis might have specific regulatory mechanism.Whether manganese-induced cell necrosis was associated with necroptosis remained unknown.Here,we conducted a series of experiments including vivo and vitro experiments to investigate the mechanism of RIP1-mediated necroptosis pathway,which participate in death of neurneuron induced by manganese exposure.Through the RNA interference and the cell specific antagonist identify the role of RIP3-mediated necroptosis signaling pathway in the death of neurons induced by manganese.Methods I.Effect of zVAD-fmk and Nec-1 on manganese-exposed SK-N-SH cells(1)The SK-N-SH cells in the logarithmic growth phase were exposed to 400 ?mol/L manganese and treated with zVAD-fmk(a caspase inhibitors)and necrostatin-1(Nec-1)(a specific inhibitors of necroptosis)for 24 h in the logarithmic growth phase.Set up 10 group as follows: control group,400 ?mol/L Mn group,0.5 ?mol/L zVAD-fmk group,2 ?mol/L zVAD-fmk group,60 ?mol/L Nec-1 group,120 ?mol/L Nec-1 group,Mn + 0.5 ?mol/L zVAD-fmk group,Mn + 2 ?mol/L zVAD-fmk group,Mn + 60 ?mol/L Nec-1 group,Mn + 120 ?mol/L Nec-1 group.Morphologic change was assessed after treatment.MTT and flow cytometry assay was used to assess cell viability,the apoptosis rate and necrosis rate of the cell.(2)Cells were treated with manganese and inhibitor for 24 h in the logarithmic growth phase.Set up 5 group as follows: control group,200 ?mol/L Mn group,400 ?mol/L Mn group,Mn + 0.5 ?mol/L zVAD-fmk group,Mn + 120 ?mol/L Nec-1 group.ROS production were detected by DCFH-DA probe and RIP3 protein expression were detected by Western blot.II.Effect of lentivirus-mediated RIP3 RNAi on manganese-exposed SK-N-SH cells(1)A lentivirus RIP-3 RNAi SK-N-SH cell line was constructed.(2)RIP3 Knock down(KD)cells were treated with manganese and inhibitor for 24 h in the logarithmic growth phase.Set up blank SK-N-SH cell group(B,Control group),400 ?mol/L Mn group,RIP3 Scrambled group(NC),RIP3 RNAi group,RIP3 RNAi+400 ?mol/L Mn group,RIP3 RNAi+Mn+ 120?mol/L Nec-1 group.MTT and flow cytometry assay was used to assess cell viability,the apoptosis rate and necrosis rate of the cell.Western blot was used to detect the protein expression of RIP-3 and phosphorylated MLKL.III.The effect of Nec-1 on the subacute manganese exposure in the substantia nigra and the hippocampus of rats(1)Sprague Dawley(SD)rats were divided into Control group,Mn-exposed group,Nec-1 group and Mn+Nec-1 group,and the rats in all groups received lateral ventricle implantation.Subacute manganese poisoning model was established by intraperitoneal injection of manganese solution(15 mg/kg/day,5 day/w,4w)and the intervention group was given Nec-1 ventricle injection(19.28 mmol/L,5 ?L/time,3 times/w,4w).Cannulas were removed from the ventricle under anesthesia at the end of exposure and intervention.Rats were continued fed for 2 weeks.(2)After perfusion fixation in rats,the expression of glial fibrillary acidic protein(GFAP)positive cells in hippocampal CA1 region and tyrosine hydroxylase(TH)positive cells in substantia nigra were observed by immunohistochemistry.The ultrastructural changes of neurons in the hippocampus were observed by transmission electron microscopy.(3)Rats were sacrificed by cervical dislocation and the hippocampal tissues were harvested.The expression of RIP3,NOX2,NOX4,Bcl-2,and Caspase 3 genes were detected by fluorescent quantitation.ResultsI.The Mn-induced death of SK-N-SH cells was decreased by using Nec-1(1)Mn-exposed SK-N-SH cells became shrinkage and round.Protuberant retract and cytoplasmic vacuolar changes in the cytoplasm could be observed.0.5 ?mol/L zVAD-fmk and Nec-1 intervention were help to maintain the normal morphology and number.The neurites regenerated into a rich network and the vesicle-like altered cells decreased in 0.5 ?mol/L zVAD-fmk and Nec-1 intervention group.(2)The cell viability decreased with the increase of manganese concentration.Compared with the control group,the survival rate of manganese exposed group and zVAD-fmk 2?m ol / l intervention group was significantly lower(P<0.05).0.5?mol / L zVAD-fmk and nec-1 intervention can improve the viability of manganese exposed cells(P<0.05).(3)the apoptosis and necrosis rate of Mn-exposed cells were significantly increased(P<0.05).zVAD-fmk 0.5?mol / l could decrease the apoptosis rate on Mn-exposed cells(P<0.05),but the necrosis rate was higher than that of the control group(P< 0.05).Nec-1 intervention could reduce the necrosis rate of Mn-exposed cells(P<0.05),but the apoptosis rate was higher than that of the control group(P<0.05).(4)The expression of RIP3 protein was up-regulated in the Mn-treated group.The expression of RIP3 protein was slightly decreased after zVAD-fmk and Nec-1 intervention,but there was no significant difference among the groups.(5)A large number of strong fluorescent cells were observed indicated that ROS was increased in Mn-exposed group.The number of strong fluorescent cells was no significant difference between the Mn+zVAD-fmk group and the Mn-exposed group,while the number was decreased in the Nec-1 intervention group(P<0.05).II.The necrosis rate was decreased in manganese-exposed SK-N-SH cells by Lentivirus-mediated RIP3 RNAi(1)The lentivirus RIP3 RNAi SK-N-SH cell line was successfully constructed.(2)The survival rate of RIP-3-KD SK-N-SH cells exposed to manganese was higher than that of SK-N-SH cells exposed to manganese(P<0.05).The necrosis rate of Mn-exposed RIP-3-KD cells was significantly lower than that of Mn-exposed SK-N-SH cells(P<0.05),and there was no significant difference between nec-1 and Mn-exposed RIP-3-KD cells.(3)RIP3 protein expression and MLKL phosphorylation were increased after manganese exposed SK-N-SH cells(P<0.05);RIP3-KD cells showed decreased expression of RIP3 protein and decreased MLKL phosphorylation(P<0.05)and there was no significant difference in the protein expression after manganese exposed.III.Neuron in the substantia nigra and hippocampus of subacute manganese exposure rat was protected by Nec-1(1)The number of TH positive cells in the substantia nigra of manganese treatment group was not significantly decreased.GFAP positive cells in the hippocampal CA1 region of manganese treatment group was increased significantly(P<0.05).Nec-1 could reduce GFAP positive cells in the hippocampus in manganese-exposed rats,and there was statistical difference between Mn group and Mn+Nec-1 groups(P<0.05).(2)Apoptotic neurons(cell body contraction,increased electron density,cytoplasmic foamy changes,chromatin condensation,edge collection,nuclear membrane disintegration)and necrotic neurons(the cell body is swollen and translucent,the organelles are sparse,the chromatin of the nucleus is concentrated,the edge sets,and the nuclear membrane is dissolved)were observed under electron microscope in the substantia nigra of subacute Mn exposure rats.The mitochondrial space was widened and disintegrated,the neurites were edematous,and the synaptic space was blurred.Nec-1 intervention can reduce the degree of cell swelling and the degree of neurite edema.Necrotic neurons had more intracellular organelles remaining and the mitochondrion was intact but the space between the iliac crest was slightly enlarged.(3)Compared with control group,the Caspase3 and NOX4 gene expression were significantly higher(P<0.05)but Bcl-2 reduced in manganese exposed rats(P<0.05).Compared with manganese exposed group,NOX4 gene expression of Nec-1 intervention group is significantly reduced(P<0.05).Conclusion(1)The necroptosis was involved in the death of manganese exposed SK-N-SH cells.The signal transduction mechanism mediated by RIP3 was closely related to the necroptosis of SK-N-SH cells.(2)The inhibitory mechanism of Nec-1 on Mn-exposure neurons of rats might be related to the protective effect against the necroptosis in neurons,inhibiting astrogliosis,and the reduced inflammatory response in the central nervous system.