PMSF Antagonizes TOCP-induced Changes Of NFs In Central Nerves Of Hens And Corresponding Mechanisms

ObjectiveOrganophosphorus compounds (OPs) are widely used as defoliants, fungicides, herbicides, insecticides, industrial fluids, flame retardants, nerve agents, and therapeutic agents. Tri-ortho-cresyl phosphate (TOCP) is one of organophosphorus esters. OPs are capable of producing organophosphorus esters induced delayed neurotoxicity (OPIDN) in human being and sensitive animals. OPIDN occurs in 2-3weeks after a single exposure when signs of both the acute cholinergic and the intermediate syndromes have subsided. Signs and symptoms of OPIDN include tingling of hands and feet, followed by sensory loss, progressive muscle weakness and flaccidity of the distal skeletal muscles of the lower and upper extremities, and ataxia. No treatment exists for OPIDN, and recovery from OPIDN is considered poor. Although the OPIDN was studied for a long time, its mechanism is unclear. Ultrastructural studies show axonal swelling containing aggregations of neurofilaments, microtubules, multivesicular vesicles, and proliferation of smooth endoplasmic reticulum soon after the appearance of clinical signs of OPIDN. In our previous study, Zhao et al. had observed significant changes of NFs in the central nervous system of hens treated with 750mg/kg TOCP after 21 days of treatment. However, one time-point assay could not identify the peak effect of NFs changes. The time course of NFs expression in the central nerve system of hens administrated by TOCP has not reported. In addition, phenylmethylsulfonyl fluoride (PMSF) is a neuropathy target esterase (NTE) and serine proteinase inhibitor, and it protects hens from OPIDN if injected before the administration of TOCP. On the other hand, it also potentiates OPIDN if injected after the administration of TOCP. So in present study, we have developed OPIDN hen model with TOCP and protected model with PMSF pre-treated, and investigated the alternations of NFs in central nerve tissues at different time points (0d, 1d,5d, 10d and 21d) by western blotting. Calpain, Ca2+-activated neutral protease responsible for the degradation of cytoskeleton, was investigated to explore mechanisms underlying neuropathy induced by OPs. In addition, neurofilament proteins especially NF-M and NF-H are highly phosphorylated post-translationally after NFs are transported into axons. So we also determined the changes of calpain pathway and cyclin dependent kinase5 (CDK5) pathway in the central nerve tissues of hens to compare model treated TOCP alone and model protection by PMSF. We hope that this study may provide useful data for exposure the mechanism of OPIDN and the protection or cure of OPIDN.Methods1. Adult Roman hens, which were kept in animal facility for 7days for acclimatization, were randomly divided into two groups. One group was hens treated with TOCP at a single dosage of 750 mg/kg TOCP. Another group was used to determine the protective effect of PMSF on TOCP-induced delayed neurotoxicity in hens. Hens in this set were injected with PMSF (60mg/kg, DMSO, sc) 24hr before the administration of TOCP (750 mg/kg, ig). Hens were weighed twice a week and examined daily for clinical signs. OPIDN was assessed by an 8-point graded scale (0, normal ambulation; 1-2, slight and infrequent hindlimb in coordination; 3-4, moderate but definite hindlmb incoordination; 5-6, severe and frequent difficulty in walking and standing erect; 7-8, virtual to complete hindlimb paralysis). Then all hens were sacrificed at the corresponding time points of 0(control),1,5,10, and 21 days respectively after a dosage of 750 mg/kg TOCP. Cerebrum and spinal cord were dissected quickly and frozen in liquid nitrogen before being stored at-80℃.2. Cerebrum and spinal cord were homogenized in 4 volumes of homogenizing buffer, which contained 1% Triton X-100,50mM Tris (pH 7.5),25mM KCl,2mM MgCl2, 5mM EGTA,5mM dithiothreitol, Protease Inhibitor Cocktail (50μl/g) and phosphatase inhibitors (5mM Na3VO4, 10mM Na4P2O7 and 1mM iodoacetic acid), and centrifuged at 100,000g for 60 min at 4℃to yield a high-speed pellet and a high-speed supernatant fraction. The pellets were again suspended in the original volume of homogenizing buffer, and the particulate fractions were analyzed by SDS-PAGE and western-blotting to determine the levels of high molecular neurofilament (NF-H), medium molecular neurofilament (NF-M) and low molecular neurofilament (NF-L).3. Calpain activity was measured using 500μM Suc-Leu-Tyr-7-amino-4-methylcoum-arin (Suc-Leu-Tyr-AMC) as substrate. First, the tissues were homogenized in the buffer, which contained lOmM NaHCO3,5mM NaN3,15mM Tris-HCl, pH6.8. Then 150μl supernatant was added to 1000μl reaction buffer (145mM NaCl, 100mM Tris-HCl, pH7.3) and incubated with shaking for 10min at 37℃. Second, 150μl,500μM calpain substrateⅡwas added into above solution and incubated with shaking for 1hr at 37℃. AMC released was measured by fluorimetry using 360nm excitation and 430nm emission filters. Standard curves were generated for each experiment using known concentrations of AMC.4. The tissues were homogenized in 4 volumes of homogenizing buffer, which contained 50mM Tris-HCl, pH7.4,150mM NaCl, 1mM MgCl2,5mM EGTA,2mM EDTA, 1mM PMSF, Protease Inhibitor Cocktail(0.5μl/mg protein), and centrifuged at 14,000r/min for 30 min at 4℃. Then levels of CDK5 and p35 in the supernatant were determined by SDS-PAGE and Western-blotting.Results1. SignsHens did not show any acute cholinergic toxicity after a single dose of 750 mg/kg TOCP, but delayed neurotoxicitic symptoms were observed. TOCP-treated hens began to show leg weakness after 5days, exhibited mild ataxia after 10 days and most of them showed sever ataxia or paralysis after 15 days of treatment. All hens showed complete paralysis in the end of 21-day experiment. The animals showed a slight decrease in body weight. In contrast, hens pretreated with PMSF (60 mg/kg, sc) did not show significant signs of delayed neurotoxicity following the treatment with a single dose of TOCP. Besides, the mean body weight of hens in PMSF+TOCP group did not show significant changes compared with the control group.2. Alterations of NF subunits in the supernatant and pellet fractions of nerve tissues2.1 Alterations of NFs in the supernatant and pellet fractions of cerebrumsHens in TOCP group show severe symptoms of OPIDN. The levels of NF-L, NF-M and NF-H in pellet changed more obviously than that in supernatant, which began to decrease after 1 day of treatment, sharply decreased 86.2%(P＜0.01), 87.0%(P＜0.01),54.1%(P＜0.05) respectively after 10 days, and came back little after 21 days of treatment. The OPIDN symptoms were not seen in PMSF+TOCP group. Compared with the control, NF-L and NF-M in PMSF+TOCP group remained no changes, and NF-H was increased, mostly 109.1%(P＜0.01) in pellet after 5 days. The levels of NFs in pellet of PMSF+TOCP group were decreased at different degree in contrast with that in TOCP group.2.2 Alterations of NFs in the supernatant and pellet fractions of spinal cordsThe levels of NFs proteins in spinal cords changed obviously. Compared with the control, NFs in pellet showed a dramatic decrease after 10 days of treatment and were followed by a recovery. In the supernatant, NFs showed similar changes, which decreased significantly after 10 days and almost recovered after 21 days. The levels of NF-L, NF-M and NF-H decreased by 50.9%,86.4% and 37.9% after 10 days of treatment, respectively. Signs of OPIDN were not observed in PMSF+TOCP group, and imbalances of NFs were obviously alleviated. Compared with the control, NF-M in pellet increased by 21.9%(P＜0.05) after 21 days, others remained no changes. In addition, the levels of NF-H and NF-M in supernatant increased by 19.1% and 35.2% after 21 days of treatment. Others were not found significant statistical differences.3. Alterations of calpain in the central nerve tissuesIn comparison with control group, the calpain activity of cerebrum in TOCP-treated group increased by 26.4%(P＜0.05),29.0%(P＜0.01),38.5%(P＜0.01) and 23.0%(P＜0.05) after 1 day,5 days,10 days and 21 days of treatment respectively. Furthermore, the calpain activity in spinal cords increased by 11.2%,52.3%(P＜0.01), 85.8%(P＜0.01) and 103.3%(P＜0.01) at above time points. PMSF pretreatment can make calpain in spinal cords normal in the progress of OPIDN expect after 10 days of treatment, however, the activities of calpain were potentated by PMSF-pretreated, by 104.4%(P＜0.01),70.9%(P＜0.05),149.1%(P＜0.01) and 112.5%(P＜0.01).4. Alterations of CDK5 and p35 in the central nerve tissuesCompared with control, the level of CDK5 in cerebrum decreased and then recovered, especially which decreased 28.9%(P＜0.05) after 5 days of treatment. Differently, p35 showed uptrend, which began to increase after 1 day. The level of CDK5 in spinal cord increased obviously, by 46.6%(P＜0.01),42.9%(P＜0.05) after 10 days and 21 days of treatment, respectively. The changes of p35 were similar to that in cerebrum, which significantly increased by 54.2%(P＜0.01) and 52.9% (P＜0.01) after 10 days and 21 days of treatment. However, the anomalies of CDK5 and p35 in PMSF+TOCP group were improved apparently. Compared with the control, CDK5 did not change in cerebrum and spinal cord. Yet, p35 in cerebrum increased obviously by 42.6% and 51.0%(P＜0.05) after 10 days and 21 days respectively. Others had no significantly statistical differences.Conclusions1. The hens administrated with TOCP showed paralysis gait at the end of 21-day experimental period. However, hens pre-treated with PMSF were not observed obviously signs of OPIDN. In a conclusion, PMSF, which was injected 24h before the administration of TOCP, could protect hens from OPIDN.2. TOCP caused the imbalance of NFs contents in OPIDN, and PMSF pretreatment could protect animals from OPIDN by reducing above changes, which, explained that TOCP-induced imbalance of NFs was associated with the occurrence and development of OPIDN.3. TOCP induced significant alternations on activity of calpain in hen central nervous tissues. The activity of calpain in spinal cords decreased near to normal by PMSF pretreated, but which in cerebrums changed in verse. Therefore, calpain activation might be responsible for the declining of NFs in spinal cords induced by TOCP, and it was not the reason for NF degradation in cerebrums.4. TOCP induced disorders of CDK5/p35 in central nerve tissues in progress of OPIDN, and PMSF pretreatment could protect animals from OPIDN by reducing abnormalities, which explains that TOCP-induced abnormalities of CDK5/p35 may be associated with the phosphorylation of NF subunits.