The Research On The Change Of Autophagy-related Proteins In PC12Cells Induced By Tri-ortho-cresyl Phosphate (TOCP)

ObjectivesOrganophosphorus compounds (OPs) are a great variety of chemicals widely used as pesticide, plasticizers, lubricants, additives and chemical warfare agents, etc. Besides the acute organophosphorus compounds poisoning, part of OPs can also cause severe and irreversible delayed neuropathy named as organophosphorus ester-induced delayed neuropathy (OPIDN) in sensitive animals and humans. When a numerous of OPIDN incidents were first reported in the1890s in France, people began to realize tri-ortho-cresyl phosphate (TOCP) had the ability to cause the delayed neuropathy. Nearly a century of researches, however, there is no clear pathogenesis of OPIDN, the current clinical treatments are no specific effects, the prognosis is poorer.In this study, we use the tumor cells of rat adrenal pheochromocytoma (PC12) exposed to TOCP as a model in order to examine the changes of autophagy activity and autophagy-related proteins, and further explore the mechanism of OPIDN for providing theoretical thinking and experimental basis.Methods1The inhibiting effect of TOCP-induced proliferation of PC12cells were examined by MTT assay.2TOCP infected PC12cells, and inhibitors of intervention Conventional method to culture PC12cells to the logarithmic phase, then the cell model was built by infecting different dose of TOCP into the medium, to research separately the dose-response relationship and time-response relationship between the impact of TOCP and the autophagy activity.The study of dose-response relationship was devided into five groups:0,0.05mM,0.10mM,0.25mM, and0.50mM concentration of TOCP, then contaminate for24hours.The specific groups of the time-response relationship were:using the concentration of TOCP at0.05mM to infect cells,setting0,6h,12h,24h,48h five observation points,then collecting all the cells.In the drug intervention experiments,three kinds of inhibitors: phenylmethylsulfonyl fluoride (PMSF), calpain specific inhibitor (ALLN) and caspase inhibitor (Ac-DEVD-CHO) were given to block the effect of TOCP twenty minutes early before infecting TOCP. Then investigate the drug intervention’s influence on the change of autophagy activity caused by TOCP((including dose response and time-response relationship).3. Morphological observationUsing the fluorescence microscope, to observe the level of cell autophagy level by MDC method, and to test the expression of autophagy-related proteins.4. Determination of autophagy-related proteinsCells were collected after exposure or intervention, and were disposed at4℃12000*g centrifuge for10minutes after cell lysis solution treatment. Then take the supernatant to quantify the related protein levels through BCA method. Western blotting analysis was used to detect the changes of autophagy related protein expression in cells.Results1The results of MTT method showed that TOCP markedly inhibited the proliferation of PC12cells with the growing time and dose.2Observation of MDC staining found that there was a negative correlation between amount of autophagy body with concentration, and a negative relationship with infected time.3Immunofluorescence observations after TOCP infected cells significantly affected the Beclin1and p62protein expression.With the increase of dose and time, the red fluorescent of Beclin-1protein was gradually weakened, and the green fluorescence of p62protein was gradually strengthened. Application of PMSF and ALLN inhibitors respectively for intervention before TOCP infected, we found that the expression of fluorescence of Beclin-1was enhanced, and p62protein fluorescence intensity was weakened.4Western blot analysis indicated that with the increased exposure dose of TOCP, the amount of autophagy-related protein Beclin-1,UVRAG,LC3were declined, and the content of p62,μ-calpain,caspase-3were obviously up-regulated (P<0.05). With infected time growing, compared with normal group, expression of Beclin-1、UVRAG、LC3were significantly restrained, while the protein level of p62,μ-calpain,caspase-3were increased, which presented time-effect relationship (P<0.05).5Giving ahead PMSF, calpain inhibitor ALLN and caspase inhibitor (Ac-DEVD-CHO) to cell for intervention respectively, and then infected TOCP for different concentration doses or different times. Western blot results show that three different kinds of inhibitors have differently inhibit the degradation of autophagy-related proteins Beclin-1, UVRAG, LC3and p62and the activation of caspase.Conclusions1. Exposure of TOCP could cause change of autophagy in PC12cells.2. TOCP disturbed the expression of auotophagy related proteins Beclin-1, UVRAG, LC3, p62,μ-calpain and caspase-3in PC12cells.3Giving cells PMSF, calpain inhibitor ALLN and caspase inhibitor (Ac-DEVD-CHO) ahead of infected TOCP, the decline of autophagy activity and the change of autophagy related proteins were significantly prevented, which suggest that calpain and caspase-3may be degraded by autophagy-related proteins resulting in autophagy dysfunction.4. The change of autophagy activity caused by the activation of calpain and caspase-3might involve in the pathogenesis of OPIDN.