The Study Of Neurotrophin NT3 And Its Receptor TrkC In Mn-induced Neurotoxicity

[Objective] To explore the potential mechanism of NT3 and its receptor TrkC in Mn-induced neurotoxicity,we detected the level of neurotransmitter 3 and its receptor TrkC as well as the downstream signal transduction pathway-related proteins in the different concentrations of manganese treatment group in vitro and the mice in vivo.[Methods] The primary mouse neurons cultured in vitro were treated with different concentrations of MnCl2 · 4H2 O solution,and the morphology of the cells was observed at various time points under inverted phase contrast microscope.The survival rate was determined by MTT assay to determine the concentrations of manganese for the following experiment.The primary neurons were stained with different concentrations of manganese,and the expression levels of TrkC and NT3 were analyzed by PCR.Western Blot was used to determine the intracellular NT3 and TrkC and Expression levels of their downstream signal transduction pathway-associated proteins(p-Akt,Akt,p-Erk,Erk,P-CREB and CREB)and their downstream changes in apoptosis-related proteins(Caspase3,Bax and Bcl2)with different doses of manganese;changes of intracellular NT3 and TrkC were observed by immunofluorescence;After staining with TMRM dye,the changes of mitochondrial membrane potential were detected by fluorescence microscope and flow cytometry;apoptosis wasdetected by Annexin V-FITC apoptosis kit,Tunel apoptosis kit and DAPI staining.To verify whether TrkC receptors are cleaved by caspase 3 in the absence of NT3 after tread wieh manganese,the primary neurons were pre-treated with NT3 and Z-VAD-FAM,a antagonists of Caspase,respectively,and then treated with manganese at a dose of 200 uM to observe the cell morphology.The change of protein mentioned above were all determined again.In addition,24 male C57 mice of 6 weeks of age were randomly divided into control group and low,middle and high exposure groups.Each group was injected intraperitoneally with physiological saline and 5,20,and 50 mg/(kg*d)of Mncl2·4H2O solution for 2weeks.Additionaly,36 12-week-old male C57BL/6 mice were randomly divided into control group,Mn2+ 100 mg/L group,Mn2+ 200 mg/L group,and Mn2+ 400 mg/L group.The manganese was added in drinking water according to the corresponding dose,and the control group received ordinary drinking water for continuous exposure for 32 weeks.The cortex and hippocampus proteins were extracted after the end of manganese staining.The expression levels of NT3 and TrkC and their downstream signal transduction pathway related proteins(p-Akt,Akt,p-Erk,Erk,P-CREB,CREB)and Its downstream apoptosis-related protein(Bax and Bcl2)were detected by Western Blot.[Results]:1.Compared with the control group,when the manganese concentration was equal to or higher than 200 ?M,the survival rate of the primary mouse neurons was significantly decreased(P<0.05)as the concentration of manganese was increased.Different concentrations of manganese can cause different degrees of damage and morphological changes in mouse neuronal cells.2.With the increase of the concentration of manganese,the number of nuclearfission and nuclear condensation of mouse neurons increase,the number of labeled DNA breaks increases,and the rate of apoptosis increases.3.With the increase of the concentration of manganese,the mRNA levels of NT3 and TrkC in mouse neurons decreased,and the level of protein and the fluorescence intensity of NT3 and TrkC were decreased.4.With the increase of Mn concentration,the expression of NT3 and TrkC and its downstream signal transduction pathway-associated proteins and downstream apoptotic proteins in mouse neurons were significantly changed.Among them,the contents of pAkt,pErk1/2,pCREB,and Bcl 2 were significantly decreased with the increase of the concentration of manganese(P<0.05).In contrast,the pro-apoptotic proteins Bax and Cleav-Caspase 3 were detected with the manganese concentration.Increased significantly(P<0.05).5.With the increase of Mn concentration,the fluorescence intensity of mouse neuronal cells labed by TMRM decreased,indicating that the mitochondrial membrane potential changes significantly.6.Compared with 0 hours,neurons cell damage and morphological changes were observed at 12 hours after 200?M manganese treatment.The expression levels of NT3 and TrkC and their downstream signal transduction pathway-associated proteins and their downstream apoptotic proteins in mouse neurons changed over time,but the time at which each protein began to change was different.7.After pretreated with NT3 and Z-VAD-FAM for two hours,the level of TrkC and its activated downstream signaling pathway-associated proteins and their downstream apoptotic proteins in neuro cell were significantly altered.Among them,the levels of pAkt,pErk1/2,pCREB,and Bcl 2 proteins were significantly higher than those treated with 200 ?M manganese(P<0.05).In contrast,thepro-apoptotic proteins Bax and Cleav-Caspase 3 were significantly decreased(P<0.05).).8.After pretreated with NT3 and Z-VAD-FAM for two hours,the fluorescence intensity of TMRM-labeled neurons was higher than that of the 200 ?M manganese-treated group,indicating that the mitochondrial membrane potential damage was alleviated.9.After pretreated with NT3 and Z-VAD-FAM for two hours,the number of nuclear fission and nuclear condensation,and rate of apoptosis were decreased compared with the 200 ?M manganese treatment group,.10.After acute peritoneal injection of manganese in mice,the level of NT3 and TrkC,and their downstream signal transduction pathway-associated proteins and their downstream apoptotic proteins in the cortex and hippocampus were significantly altered.Among them,the protein contents of pAkt,pErk1/2,pCREB,and Bcl 2 decreased significantly with the increase of the concentration of manganese(P<0.05).In contrast,the pro-apoptotic proteins Bax and Cleav-Caspase 3 were detected with the manganese concentration.Increased significantly(P<0.05).11.After chronic exposure of mice to drinking water,the level of NT3 and TrkC and its downstream signaling pathway-associated proteins and their downstream apoptotic proteins in the hippocampus were significantly altered.Among them,the protein contents of pAkt,pErk1/2,pCREB,and Bcl 2 decreased significantly with the increase of the concentration of manganese(P<0.05).In contrast,the pro-apoptotic proteins Bax and Cleav-Caspase 3 were detected with the manganese concentration.Increased significantly(P<0.05).[Conclusion]:1.Manganese staining can cause the morphological changes of primary mouseneurons cultured in vitro,causing various degrees of cell damage,and reduce the cell survival rate.Manganese staining can also cause changes in the mitochondrial membrane potential of mouse neurons cultured in vitro and cause apoptosis.NT3 and Z-VAD-FAM can reduce the neuronal apoptosis in mice.2.Manganese can affect the level of NT3 and TrkC and its activated downstream signaling pathway-associated proteins,and alter the expression level of its downstream apoptotic proteins.3.NT3 and Z-VAD-FAM can reduce the neurotoxicity induced by manganese by improving the expression of NT3 and TrkC and their activated downstream signal transduction pathway related proteins and apoptotic protein content.