The Expression Of Autophagy Related Proteins And Its Phosphorylation Level In Nerve Tissue Of Hens Treated With Tri-ortho-cresyl Phosphate (TOCP)

ObjectiveOrganophosphorus compounds (OPs), the phosphoric acid derivatives wich contain organic groups, are widely used in pesticide, flame retardants, plasticizer and war gas. Besides can cause acute toxic poisoning, lots of organophosphorus compounds can induced a kind of irreversible neuropathy diseases known as organophosphate-induced of neuropathy (OPIDN) in human and sensitive animals. But, the pathogenesis of OPIDN is still not clear. Autophagy is an important mechanism to maintain its steady state. In rcent years, many studies have demonstrated that many neurodegenerative diseases are related with disrupted autophagy in neurons. OPIDN belongs to one of neurodegenerative diseases, so we speculated that the happen of OPIDN may also be related to the dysfunctional autophagy in neurons.Methods1. To build the OPIDN animals models and the OPIDN intervention animal model.Select 60 adult Roman hens 60, after 3 days,30 hens were randomly divided into five froups in OPIDN animals models, one is control group(n=6) and four experiental group (1d,5d, 10d,21d; n=6 each group), TOCP was dissolved in core oil, and treated hens in experimental group with TOCP by gavage at a single dose of 750 mg/kg and treated the control group with an equivalent volume of corn oil. The hens were sacrificed on the corresponding time points of Id,5d, 10d and 21d after TOCP exposure respectively and removed the nerves into liquid nitrogen and preserved at -80℃ for used. The other 30 hens were used for OPIDN intervention animal model, 30 hens were randomly divided into six groups, one control group(n=5) and five experimental group (0d, Id,5d, 10d,21d; n=5 each group), Hens in experimental groups were pretreated with PMSF and the control group were given equivalent volume of corn oil. PMSF was dissolved in DMSO and injected from subcutaneously with a dose of 90 mg/kg, After 24 h of PMSF pretreat, hens in five experimental groups were treated with a single dose of 750 mg/kg TOCP by gavage. The hens were sacrificed on the corresponding time points of Od, Id,5d, 10d and 21d after TOCP exposure respectively and removed the nerves into liquid nitrogen and preserved at-80℃ for used. Besides, we observed the symptom of hens every day, and evaluated the score of hens according the eight point graded scale.2. The level of autophagy-related protein in spinal cord and tibial nerve tissue were determined.Apply adequate the sponal cord and tibial nerve tissue, and put in precooling homogenate buffer accordance to the volume ratio of 1:5, respectively. Fist homogenate nerves in ice water bath, and then let the buffer stand in the ice for 30 min, and finally centrifugal at 14000 r/min、4℃ for 30 min. At the last, we used the BCATM protein Assay Kit for the protein content in supernatant quantitatively and test the levels of Beclin1、 p-Beclin1(T119)、ULK1、p-ULK1(Ser556)、P62、 p-P62(Thr/Ser272) in spinal cord and tibial nerves, respectively.Results1.Clinical signs:In TOCP group:Hens began to show slightly uncoordinated movements on day 5 after a single dose of 750mg/kg TOCP. As the time went on, the symptoms of OPIDN progressed very fast. The lower limbs began to show weaken and feeble, severe and frequent difficulty in walking and standing erect. All hens can’t stand, dropped to the ground, and showed complete paralysis on day 15, and this symptom continued to the end of experiment. But in the control group, there were no clinical signs of OPIDN.In PMSF group:the hens in PMSF group were like the control group, and there were no clinical signs of OPIDN.2. The alternation of P62 and ULK1 in nerve tissue in PMSF group:In PMSF group, the levels of P62 and ULK1 were not changed in the whole experimental period (P>0.05); in tibial nerve tissue the levels of P62 and ULK1 were increased only in 0 days statistic (p<0.05), there were no significant changes in other time points when compared with the control group.3. The alternation of p-P62Ser272/(Thr), p-ULK1 (Ser556), p-Beclinl (Thr119) in nerve tissue in TOCP group:TOCP affected the expression of p-P62(Thr/Ser272), p-ULKl(Ser556), p-Beclinl (Thr 119) in nerve tissue, the level of p-P62 (Thr/Ser272) were higher than the control group in spinal cord and tibial nerves; the level of p-ULK1 (Ser556) were decreased in the spinal cord and tibial nerve when compared with control group; the level of p-Beclinl (Thr119) were decreased in spinal cord and tibial nerves, but recoved to normal levels in the end of experimental.4. The alternation of p-P62(Thr/Ser272), p-ULK1(Ser556),p-Beclinl (Thr119) in nerve tissue in PMSF group:The level of p-P62(Thr/Ser272)、p-ULK1 (Ser556)、p-Beclinl(Thr119) were not decreased in the whole experimental period,the alternation of p-P62(Thr/Ser272) was not changed significantly in the spinal cord in experimental, but was higher than the control group in tibial nerve; the alternation of p-ULK1(Ser556) was not changed significantly in the spinal cord in experimental, in the tibial nerve it just increased on day 0; the alternation of p-Beclinl(Thr119) were higher than the control group in spinal cord and tibial nerve.Conclusions1. TOCP significantly affceted the expression of Beclin1、ULK1、P62 and their phospharylation state.2. PMSF can block the occurance of OPIDN,and reverse the changes of autophagy-related proteins.3.The pathogenesis of OPIDN may be associated with dysfunctional autophagy in neurons.