Protective Effects Of FK506 On Acrylamide-induced Neuropathy Of Rats And Its Mechanism Research

ObjectivesAcrylamide (ACR) is a water-soluble, vinyl monomer that is used primarily to produce polyacrylamides with different physical and chemical properties, e.g. waste water management, ore processing and dye synthesis. In addition, ACR is also used extensively in molecular laboratories for gel chromatography. Exposure of humans and laboratory animals to monomeric ACR produces ataxia, skeletal muscle weakness, and weight loss. However, the mechanism of neurological defects has not been understood. FK506, also named tacrolimus, has been marked for as an immunosuppressive agent for preventing or treating graft rejection in transplant patients that also has potent neuroregenerative and neuroprotective properties independent of its immunosuppressant action. Here, we show that FK506 dramatically protects against the development of ACR neuropathy in adult rats, as shown by the reduction in behavioral alterations. Furthermore, we explored the mechanism underlying neuroprotection. The intriguing possibility is the involvement of calcineurin (CaN), heat-shock protein 70 (HSP70), Bcl-2 and Bax. The aim of this study was to investigate the effects of FK506 on acrylamide-induced neuropathy and provide more effective therapeutic approaches to the neurodegenerative disorders.Methods1. treatment of animalsMale Wistar rats, weighing 200±20 g, purchased from Laboratory Animal Center of Shandong University were used in this study. Rats were housed individually in polycarbonate boxes. The rats were housed lived in the animal room for 1 week for acclimation (called 0 week exposure) and then were randomly assigned to four groups (n=15 rats per group). The rats in group 1, served as control, received normal saline, while the animals in group 2,3 and 4 were administered with ACR (30 mg/kg, i.p., dissolved in 0.9% saline) and subcutaneous injections of FK506 (0.5 and 1.0 mg/kg) in the back of the neck for consecutive 4 weeks with 5 times a week. The animals were sacrificed by decapitation after 4 weeks of ACR treatment. The cerebral cortex, cerebellum, spinal cord and sciatic nerves were dissected from fifteen animals of each group on ice, snap-frozen in liquid nitrogen and then stored at-80℃until analyzed. Neurobehavioral indexes and body weight were measured every week.2. measurement of indexesNeurobehavioral indexes including spontaneous locomotion in an open field, foot splays in hindlimb landing, thermal and mechanical stimuli, performance on the rotating rod were measured every week. Samples of the cerebral cortex, cerebellum, spinal cord and sciatic nerves were collected and used for the examination of HSP70, Bcl-2 and Bax protein levels using Western blotting.Results1. Body weight and gait evaluationThe body weight of the rats in the control group increased steadily within 4 weeks, and the velocity of increase in the intoxicated group was smaller than that in the control group. The bodyweight on 2nd week show significant difference between the intoxicated and the control group (P<0.01, the followed is same).The intoxicated rats'symptoms get severer with the administration time. The body weight of the rats in the FK506-treated groups was decreased than that in the intoxicated group from 1st week (P<0.01).2. The changes of the neurobehavioral indexes 2.1 Gait ObservationsBeginning in the 2nd week, ACR-treated rats developed classic signs of ACR neuropathy, and with the time passing by, intoxication of ACR caused progressive development of gait abnormalities, which were less apparent in the FK506-treated rats. In the last week of the test, ACR-treated rats exhibited a pronounced functional impairment consisting hindlimb paralysis and dragging of the feet along the floor. Compared with those of ACR group, FK506-treated rats with the low dose and the high dose profoundly reduced these motor deficits by 19.1% and 43.4%, respectively (P<0.01).2.2 Tail immersion testFor non-noxious thermal stimulus, ACR-treated rats showed a significant decrease by 20.4% from the 2nd week (P<0.01) and with the time passing by, intoxication of ACR caused progressive reduction of thermal stimulus. Compared with those of ACR group in the last week of the test, FK506 significantly prolong the duration of immersion, by 20.4% in the low dose and by 23.3% in the high dose, respectively (P<0.05, P<0.01).2.3 Paw pressure testCompared with control rats, a significant decrease for the paw pressure was observed in the ACR-treated and FK506-treated rats (P<0.01); FK506 in the low dose and the high dose group significantly increase the vocalization threshold of the pressure, by 23.1% from the 3rd and by 11.1% from the 1st week, and in the last week, increased by 33.3% and 38.5% in the FK506-treated groups (P<0.01).2.4 rotarodLatencies to fall in the four groups were tested. Performances were significantly reduced for ACR-treated rats. The latency to fall in FK506-treated groups was only slightly reduced and the rats in the high dose of FK506 group were able to dramatically stay on the rod at the last week (P<0.01). 2.5 Landing foot splay (LFS)Compared with control group, the distance between the two hind limbs of ACR-intoxicated rats was gradually increased from the 1st week while FK506 administration profoundly reduced these motor deficits. On the 4th week exposure, with respect to control group, the distance between the two hind limbs in ACR-treated group significantly increased by 101.2% (P<0.01). Compared with ACR-treated group, FK506-treated groups significantly decrease the distance by 8.6%(P<0.01) and 31.3% (P<0.01), respectively.3.1 The protein levels of CaN in the cerebral cortexCompared to control group, densitometric analysis showed that there was no significant change in the expression of CaN in the spinal cord, but increased by 24% and 32% in the cerebral cortex and cerebellum in ACR-treated rats (P<0.01); Compared to ACR-treated group, there were no significant changes in the expression of CaN in the spinal cord in the FK506-treated rats. In the low dose of FK506-treated rats, the relative amount of CaN was markedly decreased by 3.2% and 9.8%; In the high dose of FK506-treated rats, the relative amount of CaN was markedly decreased by 13.7% and 26.5%(P<0.01). There was no significant change in the expression in the sciatic nerves.3.2 The protein levels of HSP70, Bcl-2 and Bax in the cerebral cortexCompared to control group, densitometric analysis showed that there was no significant change in the expression of HSP70 and Bax in ACR-treated rats, and the relative amount of Bcl-2 expression was markedly increased (34%, P<0.01). Compared to ACR-treated group, there were no significant changes in the expression of Bcl-2 and Bax in the low dose of FK506-treated rats while the relative amount of HSP70 expression was markedly increased (11.6%, P<0.05); And in high dose group there was a up-regulation of HSP70 (33.3%, P<0.01), Bcl-2 (16.3%, P<0.01) and a down-regulation of Bax (16.4%, P<0.01).3.3 The protein levels of HSP70, Bcl-2 and Bax in the cerebellum We used western boltting to investigate HSP70, Bcl-2 and Bax protein content in the cerebellum of ACR-intoxicated and FK506-treated rats. Further semi-quantitative analysis revealed that compared to control, there were no significant changes in the expression of Bcl-2 and Bax while the relative amount of HSP70 expression was markedly decreased (15%, P<0.01); Compared to ACR-treated group, there was no significant change in the expression of Bax in the low dose of FK506-treated rats while the relative amount of HSP70 and Bcl-2 expression were markedly increased (29.4%,68.8%, P<0.01, P<0.01); And in high dose group there was a up-expression of HSP70 (41.2%, P<0.01), Bcl-2 (174.2%, P<0.01) and Bax (47.8%, P<0.01).3.4 The protein levels of HSP70, Bcl-2 and Bax in the spinal cordCompared to control group, densitometric analysis showed that there was no significant change in the expression of HSP70 and Bcl-2 while the relative amount of Bax expression was markedly increased (22.3%, P<0.01); Compared to ACR-treated group, there was no significant change in the expression of HSP70 and Bcl-2 in the low dose of FK506-treated rats while the relative amount of and Bax expression were markedly decreased (46.8%, P<0.01); And in high dose group there was no change of HSP70, a up-regulation of Bcl-2 (14.8%, P<0.01) and a down-regulation of Bax (40.2%, P<0.01).3.5 The protein levels of HSP70, Bcl-2 and Bax in the sciatic nervesSemi-quantitative analysis revealed that compared to control, there were no significant changes in the expression of Bcl-2 and Bax while the relative amount of HSP70 expression was markedly decreased (41%, P<0.01); Compared to ACR-treated group, there were no significant changes in the expression of Bcl-2 and Bax in the low dose of FK506-treated rats while the relative amount of HSP70 expression were markedly increased (56.3%, P<0.01); And in high dose group there was a up-regulation of HSP70 (58.5%, P<0.01), Bcl-2 (56%, P<0.01) and no change of Bax expression. Conclusions1. We further examined the sensory and motor functions including thermal and mechanical stimuli, hindlimb splay and the performance on the rotarod, and the data showed that FK506 not only improved the response of sensory but also induced changes in muscle properties. FK506 dramatically protects against the development of ACR neuropathy, and a dose-response relationship was observed.2. FK506 suppressed the expression of protein CaN, which may be associated with T-cell proliferation, neuronal apoptosis and calcium increase mediated by CaN that may account for FK506's neuroprotection.3. FK506 significantly attenuated ACR-induced neuropathy by enhancing the level of HSP70 in nervous system, which may be associated with the regulation of apoptotic proteins as well as the HSP70 induction.