| ObjectiveTri-ortho-cresyl phosphate(TOCP)is widely used in industrial production,which can lead to the occurrence of delayed neuropathy,but the exact mechanism is not clear.Previous studies have shown that the pathological changes of axons in OPIDN are similar to those of the wallerian degeneration caused by physical truncation,progressively the axons are degenerated and disintegrated from the end to the cell body.Autophagy is one of the main mechanisms of eukaryotic cells responsible for clearance,which plays an important role in maintaining their own homeostasis.Numerous studies have shown that neuronal autophagy is associated with axonal degeneration.In this study,we used the autophagy-related gene Atg7 knockout of autophagic defective N2a cells to establish a poisoning model to search the role of autophagy in TOCP-induced Neuro-2a(N2a)axonal injury,finanlly we can explore the mechanism of TOCP inducing delayed neuropathy.Methods1.Cytotoxicity test:CCK8 method was used to test the effect of different concentrations of TOCP on N2a cell proliferation and determine the dose concentration of the subsequent TOCP.2.Cell differentiation experiments:The autophagy defective N2a cells of wild type and Atg7 knockout were cultured by cell culture standard method.Two kinds of cells were induced by low serum and retinoic acid for 24h to 48h showing neuronal,respectively.3.Cell differentiation ability and axonal injury detection test:At the same time.RA and 0,1.25,2.5,5,10,20 ?M TOCP were used to treat wild-type cells and the effects of toxicants on cell differentiation were observed.Two differentiated cells were induced by TOCP exposure at 0,5,and 10 ?M,respectively.The changes of axons were observed and compared between them.4.Cell immunofluorescence assay:Immunofluorescence assay was used to detect the changes of axiotransporter dynaction and autophagic activity marker protein LC3B in the two cells after treating with 0,5,10 ?M TOCP.5.Western blotting test:The levels of LC3,p62,p-p62,beclin-1,k48-Ub,k63-Ub,kinesin and dynactin were detected by Western blotting and the effects of axon injury-related proteins SARM1,p38,p42 and jnk changes levels.6.Quantitative Real-time PCR test:The levels of autophagic activity-related genes LC3B,beclinl and the expression of autophagy-related transcription factor EB were detected by real-time quantitative PCR.The changes of calpain-1 and calpain-2,which may be related to autophagy disorders,were also detected.The expression of nmnat2,sarm1,DLK and scg10,etc.,which promote or inhibit axonal injury,were detected at the gene level.Results1.CCK8 test results show that TOCP exposure dose of 20?M,with the dose increases,the inhibition of cell proliferation is more and more obvious until more than 1000?M cell activity is zero.So we choose the experiment more appropriate TOCP concentration 0,5(low dose),10?M(high dose).2.Both low serum and RA two methods can induce the differentiation of two N2a cells after 48h,which means the protrusive length is more than twice the cell body.The use of RA after the axon differentiation length is more obvious.3.?After exposure to TOCP in wild-type N2a cells,the differentiation ability of the wild-type N2a cells was significantly affected,and with the increase of the dose.the inhibiting differentiation ability was more obvious.?After TOCP exposure,the axons of the two cells were significantly shortened,and with the TOCP dose increased,axonal injury gradually increased.At the same dose,the degree of axon shortening of the gene knockout was smaller than that of the wild type.4.The results of immunofluorescence showed that the expression of Dynactin in the axon microtubule was decreased and self-bite bubble green fluorescent highlights increased with the increase of the dose of TOCP.There was no difference between the two cells.5.Western blotting showes that The content of beclin-1 in Atg7+/+N2a cells decreased and the content of LC3-? increased significantly,and beclin-1 in Atg7-/-N2a cells showed a decreasing trend,while LC3-? was not expressed.P-p62 levels were up-regulated in both cells,and the expression of p-p62 in Atg7-/-N2a cells was higher.The expression of dynactin and kinesin in axon transport motor protein was decreased in both groups.The expression of K48-site ubiquitin in both cells increased and the expression of Atg7-/-N2a cells was higher.The expression of p-p38,p-p42/p44 phosphorylation,p-jnk expression and the expression of protein SARM1 were increased in Atg7+/+N2a cells.While no significant difference was found in Atg7-/-N2a cells except that the kinase p-p38 showed an increasing trend,p-p42/p44 had statistically significant differences between the two cells,and Atg7+/+N2a cells had higher levels of phosphorylation.6.Fluorescence quantitative PCR showed that the mRNA levels of autophagy-related genes LC3B,beclin1 and transcription factor EB increased gradually with the increase of drug dose in both cells.Calpain-1 was activated in high-dose Atg7+/+N2a cells,while calpain-1 and calpain-2 were activated in Atg7-/-N2a cells.The expression of nmnat2 show that Atg7+/+N2a cells increased gradually,while Atg7-/-N2a cells decreased,and the expression of Atg7-/-N2a cells was higher in 0,5 ?M TOCP group.The sarm1 associated with cochlear injury was significantly increased in both cells exposed to high doses.The content of DLK in the high dose Atg7+/+N2a cells increased,while Atg7-/-N2a cells showed the opposite trend.Conclusions1.TOCP exposure affects the differentiation ability of Atg7+/+N2a and Atg7-/-N2a cells and can induce the axonal injury of N2a cells.Atg7+/+N2a cells have been damaged more seriously than Atg7-/-N2a cells.2.TOCP exposure resulted in autophagy activation of N2a cells,while Atg7-/-N2a gene knockout resulted in loss of autophagy activity and affects the cells in the promotion of axonal survival and cell death signal key factors NMNAT2,Sarm1,DLK and MAPK kinase and so on.3.Autophagy gene Atg7 knockout can reduce the axonal damage of Neuro-2a cells by tri-ortho-cresylphosphate. |
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