An Active Fraction Of Achyranthes Bidentata Polypeptides Promote Hippocampal Neuron Outgrowth And Peripheral Nerve Regeneration After Crush Injury

Objective1. To observe the protective effects of ABPPk(an active fraction k of Achyranthes bidentata polypeptides) on neurite growth in cultured hippocampal neuron of fetal rats.2. To observe the repair effects of ABPPk on the crushed sciatic nerve in rats.Methods1. To observe the protective effects of ABPPk on neurite growth in cultured hippocampal neuron of fetal rats.1) Primary hippocampal neurons were cultured under normal conditions. MTT assay was applied to detect cell viability after incubation with different concentrations(10 ng/ml?50 ng/ml?250 ng/ml) of ABPPk for 24 h.2) ?-tubulin fluorescent immunocytochemical staining was performed to detect the neurite growth of cultured hippocampal neurons treated with ABPPk(10 ng/ml?50 ng/ml?250 ng/ml). Phalloidin fluorescent immunocytochemical staining was performed to detect the growth cone area of cultured hippocampal neurons treated with ABPPk(10 ng/ml?50ng/ml ? 250 ng/ml). Synaptotagmin1+2 and PSD95 fluorescent immunocytochemical staining was performed to detect the formation of Synapses in cultured hippocampal neurons treated with ABPPk(10 ng/ml?50 ng/ml?250 ng/ml).3) Western blot analysis was performed to examine the expression of GAP43?PSD95 and the phosphorylation level of ERK1/2 after treating with ABPPk(10, 50 and 250 ng/ml) in the cultured hippocampal neurons.4) Living cells workstation was used to observe the effect of ABPPk on the growth speed of cultured hippocampal neurons.2. To observe the repair effects of ABPPk on the crushed sciatic nerve in rats.Fifty SD rats were randomly divided into five groups(n=10/group): high dose(10.0 mg/kg), medium dose(5.0 mg/kg), low dose(2.5mg/kg) group of ABPPk, mecobalamine group(0.13 mg/kg, positive control) and normal saline group(blank control). The left side of the sciatic nerve in rats was transectioned and sutured. After surgery, all animals were received Intraperitoneal injection of saline,mecobalamine and different concentration of ABPPk. Thermal pain threshold experiment was performed to measure the thermal pain threshold in rats at one day before surgery and 1, 7, 14, 21, 28 days after surgery. Footprinting assay was performed to measure sciatic nerve function index at 7, 14, 21, 28 days after surgery. Electrophysiological tests was performed to measure compound muscle action potential(CMAP) at 28 day after surgery. Transmission electron microscope was used to measure the myelin sheath thickness of myelinated nerve fibers and Myelin layer plate number. Masson trichrome stain was performed to measure muscle fiber cross section of gastrocnemius.Results1. Effects of ABPPk on the hippocampal neuron outgrowth in fetal rats.1) Effects of ABPPk on the neuronal activity of hippocampal neurons.The result of MTT showed that ABPPk(50 and 250 ng/ml) and BDNF could promote the neuronal activity of hippocampal neurons compared with the control group, and the neuronal activity was increased with increasing concentration of ABPPk(p<0.05).2) Effects of ABPPk on the neurite outgrowth of hippocampal neurons.The result of Western blot showed that the protein levels of GAP43 were up-regulated in cultured hippocampal neurons after treated with ABPPk(50 ng/ml, 250 ng/ml) compared with the control group(p<0.05).The result of ?-tubulin fluorescent immunocytochemistry showed that ABPPk(50 and 250 ng/ml) promoted the length and number of axon in cultured hippocampal neurons compared with the control group(p<0.05), and there is dose-effect relation.The result of Live Cell Station showed that the hippocampal neurons grow faster in ABPPk(250 ng/ml) group than that in control group.The result of phalloidin fluorescent immunocytochemistry showed that the growth cone area in ABPPk(250 ng/ml) group is larger than that in control group.3) Effects of ABPPk on the synapse formation of hippocampal neurons.The result of Synaptotagmin1+2 and PSD95 fluorescent immunocytochemistry showed that the number of Synaptics in ABPPk(250 ng/ml) group is larger than that in control group(p<0.05).The result of Western blot showed that the protein levels of PSD95 were up-regulated in cultured hippocampal neurons after treated with ABPPk(250 ng/ml) compared with the control group(p<0.05). 4) Mechanism of a preliminary study.Western blot analysis showed that the ratio of the phosphorylated ERK1/2(p-ERK1/2) to the total ERK1/2(t-ERK1/2) in cultured hippocampal neurons was significantly increased by treatment with 250 ng/ml ABPPk for 5 min to 24 h and reached the peak value at 15 min of treatment.2. Effects of ABPPk on the transectioned sciatic nerve of rats.Comparing with the saline group, thermal pain threshold, sciatic nerve function index, compound muscle action potential amplitude, regenerated myelinated nerve fiber layer thickness of myelin sheath, plate number and gastrocnemius muscle fiber cross-sectional area of high dose(10.0 mg/kg) and medium dose(5.0 mg/kg) were significantly higher in a dose-depedent manner.Conclusions1. ABPPk could promote the neuronal activity, synapse formation and neurite outgrowth of cultured hippocampal neurons.ABPPk may encouraged hippocampal neuron outgrowth through activation of ERK1/2.2. ABPPk could promote the regeneration of peripheral nerve and improve the recovery of nerve functions and morphology.