Research On Protective Effect And Mechanism Of JiSuiKang On Rats’ Neuron Of Spinal Cord Injury Model

Background:Spinal cord injury (SCI) is a serious complication of spinal disease and spins injury. With the advantages of multi-factors and multi-target effect, traditional Chinese medicin (TCM) has radually become the research hot spot to promote nerve regeneration after SCI "Jisuikang", the clinical effective Chinese prescription based on the characteristics of the SC pathogenesis:"kidney and Du deficiency, blood stasis in Du, and cardinal command derelictio; of duty", and the guidance theory of "warming kidney and mediate Du", bas been proved th function of promoting repair function after SCI by a large number of previous studies, but it intervention mechanism has not been clearfied.Objective:To construct the apoptosis model of rat spinal cord neurons in vitro, observe th effects of serum containing"Jisuikang" on survival and apoptosis of neuronal injury, and explor the possible mechanism of "Jisuikang" to anti spinal cord injury.Methods:Isolated, cultured and identificated rat spinal cord neurons in vitro and repare fo neuronal apoptosis model. serum containing different doses of "Jisuikang" and prednisone. wer prepared and intervened the model of SCI. Cell morphology, CCK-8 method, TUNEL was use to observe the effect on cell survival and apoptosis, and Western blot, qRT-PCR were applied t detect apoptosis index of Bcl-2, Bax and Caspase 3.Results:(1) Neuronal survival:Spinal cord neurons cultured in vitro after Glu injury, most cynaps fractured and retracted, some of which with karyopyknosis, cytoplasmic vacuoles, an disintegrated soma.48h after prednisone and different concentrations of Jisuikang containin serum added, necrosis rate slowed down in different degree. In the prednisone group most cell survived, with clear and complete outline, and the regenerative synapses interwoven into network. Cells in Jisuikang low dose group (JSK-L) disintegrated most, and survival cells wit fuzzy contour and sparse regenerative synapses, Cells in Jisuikang medium and high dose group (JSK-M & JSK-H) survived more, especially in JSK-H, with high refractivity and the survive cells growed in aggregation obviously. CCK-8 colorimetric analysis indicated that cell survive inter groups are not all the same (P<0.01), despite JSK-L, other groups had more cells tha model group (P<0.01), and there was no statistically significant difference between the thre groups (P>0.05).(2) Apoptosis:Cultured 48h with serum to neuron after damage, model group showed large number of apoptotic neurons, and only a small amount of neuronal survived with irregula shape. Prednisone group with more survival neurons, and a small number of apoptotic cell(?) JSK-L had a large number of neurons in nucleoplasm deep staining, and the cell body ha unclear outline. JSK-M had more apoptosis positive neurons, and survived neurons arranged i disorder with visible contour; JSK-H had some apoptosis positive cells, and survival cell with part of synapses interwoven into a network. JSK-H had lower apoptosis rate than JSK-L and positive control group, with statistical significance compared with the model group (P<0.05); there was no significant difference between the JSK-L and the model group (P> 0.05).(3) Western blot results showed:Bcl-2, bax and caspase 3 protein expression in each group were not all the same, the expression level was as follows:bcl-2:JSK-H>prednisone group> JSK-M>JSK-L> model group (P<0.01); Bax:model group> JSK-L> JSK-M> JSK-H> prednisone group (P<0.01). bcl-2/bax:prednisone group>JSK-H>JSK-M>JSK-L>model group (P<0.01). Caspase 3:model group>JSK-L>JSK-M>prednisone group>JSK-H (P< 0.001).(4) fluorescence quantitative PCR results were basically consistent with the protein expression, mRNA relative expression level were as follows:bcl-2:prednisone group>JSK-H> JSK-M>JSK-L (P<0.01); Bax:JSK-L>JSK-M>JSK-H>prednisone group (P<0.01). To calculate the relative expression of Bcl-2 and Bax, prednisone group was significantly up-regulated, JSK-L and JSK-H had higher ratio than that JSK-M, but with no statistical difference between the two groups (P=0.825>0.05). Caspase3:prednisone group and JSK-H were lower than that JSK-L and JSK-M(P<0.01), and no statistical difference between prednisone group and JSK-H, or JSK-L and JSK-M (P>0.05).Conclusion:(1) To a certain extent, 100μM Glu could simulate the micro environment of survived neurons after SCI, which could induce apoptosis of cultured spinal cord neurons, and be used for SCI cell model.(2) prednisone and medium and high dose of Jisuikang in rats serum were conducive to the survival of neurons after injury, the number of survived cells between the three groups had no significant difference, while high dose of Jisuikang reduced the apoptosis rate more than the other groups.(3) Prednisone and 3 doses of Jisuikang could all increase the expression of Bcl-2, and inhibit the expression of Bax and Caspase 3 on protein and mRNA, and increase the ratio of bcl-2/bax; Among 3 doses, the high dose of Jisuikang had more obvious effect in the up regulation of Bcl-2 protein and down-regulation of Caspase 3 protein than Prednisone, while on other indexes JSK-H were inferior to the prednisone group but better than JSK-L and JSK-M. Combined with the neuron survival rate and apoptosis analysis, we speculated that Jisuikang may influent neuronal survival through other ways derectly or inderectly.