| Background: Spinal cord injury is one of neurosurgery common diseases,and there was no good method to restore nerve function. Based on newunderstanding of the pharmacological effects of piperine and the pathologicalprocess of spinal cord injury, this study was designed to observe whetherpiperine contains functional protective effect and investigate the possiblemechanism, in order to find a new method of recovering the nerve functionafter spinal cord injury.Part one:The effect of piperine on motor function recovery of spinal cordinjury in ratsObjective: To observe the effect of piperine on motor function recovery ofspinal cord injury in rats.Methods:(1)Animal origin and grouping:36adult male SD rats wererandomized into four groups(n=9), and divided into sham group, saline group,piperine solvent group and piperine group.(2)Modeling of animal with spinalcord injury: The rats of all groups were removed of the spinous process of thedorsal spine at about the T10level, and saline group, the piperine solventgroup and the piperine group were enrolled to establish a spinal cord injurymodel by Allen's method. But the sham group was given operation withoutspinal cord injury.(3)Mode of administration: The rats in piperine group wereintragastric administrated of piperine suspension with50mg/kg (containing7ml/kg piperine solvent), the rats in piperine solvent group were given onlypiperine solvent (containing sterile water for injection, anhydrous ethanol andpolyethylene glycol-400) with7ml/kg, and the rats in sham group and salinegroup were given saline with7ml/kg. Each rat was administered at1d, once a day until14d.(4) The rat spinal cord function test: Each rat was scored byBBB behavior score at1d,7d and14d. The rats were included in theexperiment, when their scores were0at1d. All the rats were sacrificed at14d,for taking out5mm spinal cord of the damage parts. The5mm spinal cord wasparaffin-embedded, and reserved for the second part of the study.(5)Statistics:the experimental data was statistical analyzed by SPSS13.0, the data werestatistical measurement data, it was expressed by mean±standard deviation orthe median. Multiple comparisons of the data were analyzed by multi-sampleOne-Way ANOVA and LSD-t, or by Mann-Whitney U method ofnon-parametric test. It was set for significant difference when P<0.05.Result:1The rats in sham group at1d was no paraplegia(points=21), the rats insolvent group, saline group and piperine group at1d were lower limbparalysis(points=0). Scores of piperine group at7d and14d were respectively(6.33±1.58,11.00±0.87). Scores of piperine solvent group at7d and14dwere respectively (2.89±0.78,6.78±1.09). Scores of saline group at7d and14d were respectively(3.00±1.32,6.44±1.24). Except the sham group, thescores all increased in other groups.2There were significant differences between sham group and piperinegroup(Z=-3.841,P=0.000), between sham group and piperine solventgroup(Z=-3.855,P=0.000), and between sham group and salinegroup(Z=-3.839,P=0.000) at7d. There were significant differences betweensham group and piperine group(Z=-3.848,P=0.000), between sham group andpiperine solvent group(Z=-3.841,P=0.000), and between sham group andsaline group(Z=-3.848,P=0.000)at14d. There were no significant differencesbetween solvent group and saline group at7d and14d(Z=-0.184P=0.854,Z=-0.509P=0.611). But there were significant differences between piperinegroup and saline group at7d and14d(Z=-3.485P=0.000,Z=-3.621P=0.000),and there were significant differences between piperine group and piperinesolvent group at7d and14d(Z=-3.621P=0.000,Z=-3.616P=0.000).Conclusion: Piperine possess the pharmacological effect of motor nerve function recovery on spinal cord injury in rats.Part two: The mechanism of motor never functional recovery of piperineon spinal cord injury in ratsObjective: To investigate the mechanism of motor never functionalrecovery of piperine on spinal cord injury in rats.Methods: Animal origin and grouping,Modeling of animal with spinal cordinjury,Mode of administration and Statistics were the same as the part one.The rats were included in the experiment, when their scores were0at1d. Eachrat was sacrificed at7d for taking out5mm spinal cord of the damage parts.The5mm spinal cords at7d and14d were paraffin-embedded, and observedfor spinal cord injury pathological changes by HE staining, detection of spinalcord neuronal apoptosis changes by TUNEL staining, detection of caspase-3protein expression changes by immunohistochemical staining.Result:1Observe spinal cord pathological changes by HE stainingSpinal cord tissue was complete in sham operation group, the cells werearranged in neat rows, and there was no significant necrosis and defects.Saline group, piperine solvent group and piperine group had different levels oftissue destruction, hemorrhage, edema, and cavity at7d. The piperine groupspinal cord tissue was more continuous at7d. Each group was significantlyrestored at14d. Compared with the saline group and piperine solvent group,piperine group was more continuous with only mild vacuolar changes, and theedema was significantly reduced.2Measure apoptotic cells by TUNEL2.1TUNEL staining positive cells were found more in serious region ofspinal cord injury in rats. There were very few positive cells in Sham group at7d and14d in spinal cord tissue, and were respectively(7.54±2.51,6.27±2.27). In piperine group, piperine solvent group and saline group, TUNELapoptotic cells were respectively(29.80±6.30,42.58±10.46,41.50±7.97) at 7d, and respectively(20.32±6.64,30.71±7.10,33.59±6.04) at14d.2.2There were significant differences between sham group and piperinegroup(Z=-3.576,P=0.000), between sham group and piperine solventgroup(Z=-3.576,P=0.000), and between sham group and salinegroup(Z=-3.576,P=0.000) at7d. There were significant differences betweensham group and piperine group(Z=-3.576,P=0.000), between sham group andpiperine solvent group(Z=-3.576,P=0.000), and between sham group andsaline group(Z=-3.576,P=0.000)at14d. There was no significant differencebetween saline group and piperine solvent group at7d and14d(Z=-0.486,P=0.627,Z=-0.927, P=0.354). There were significant differences betweenpiperine group and the piperine solvent group at7d and the14d(Z=-2.517P=0.012,Z=-2.693P=0.007), and there were significant differences betweenpiperine group and saline group at7d and14d(Z=-2.958P=0.003,Z=-3.223P=0.001).3Observe caspase-3positive expression3.1In the region of spinal cord injury, caspase-3positive cells expressedmore. In sham group, caspase-3positive cells were rarely found at7d and14d,average optical density was respectively(0.1323±0.0149,0.1371±0.0122).Average optical density in piperine group, solvent group and saline groupwere respectively(0.2824±0.0464,0.3452±0.0497,0.3411±0.0449) at7dand respectively(0.1936±0.0378,0.2551±0.0367,0.2456±0.0262) at the14d.3.2There were significant differences between sham group and piperinegroup(P=0.000), between sham group and piperine solvent group(P=0.000),and between sham group and saline group(P=0.000) at7d. There weresignificant differences between sham group and piperine group(P=0.000),between sham group and piperine solvent group(P=0.000), and between shamgroup and saline group(P=0.000)at14d. There was no significant differencebetween saline group and piperine solvent group at7d and14d (P=0.835,P=0.505). There were significant differences between piperine group and thepiperine solvent group at7d and14d(P=0.003,P=0.000), and there were significant differences between piperine group and saline group at7d and14d(P=0.005,P=0.001).Conclusion: Piperine can reduce apoptosis in the nerve cells of the ratswith spinal cord injury, and its mechanism may be related to inhibition ofCaspase-3protein expression in rat with spinal cord injury. |
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