Study On Dose-effect Relationship Of Neuroprotection And Mechanism Of Piperine After Acute Spinal Cord Injury In Rats

Background: Spinal cord injury is one of the common diseases of nervoussystem. Because of its pathological process so complex that the research ofdomestic and foreign scholars for many years, it is still lack of effectivetreatment, remains a medical problem. Based on the new understanding of thepathophysiological processes after spinal cord injury and piperine (Piperine)pharmacological effects, and we have previously shown that Piperine hasprotective effect on rat spinal cord injury animal model, but lack of whetherthere is a dose-effect relationship with piperine data on nerve functionrecovery after spinal cord injury in rats. Therefore, designing this study toobserve the protective effect of neurological function of piperine and itsdose-effect relationship after spinal cord injury, and further explore its possiblemechanism, that’s to provide a theoretical basis for the clinical development ofnew drugs.Part one: Observation on dose-effect relationship of piperine for hind-limbfunctional recovery after spinal cord injury in ratsObjective: To observe the relationship between piperine for the recovery ofhind-limb function after acute spinal cord injury in rats and the dose ofpiperine application.Methods:(1)Animal origin and grouping:72adult male Wistar rats (bodyweight250g-320g) were randomized into eight groups by random numbertable(n=9), and divided into sham operation group, piperine solvent group,piperine1st group(25mg/kg), piperine2nd group(50mg/kg), piperine3rdgroup(75mg/kg), piperine4th group(100mg/kg), piperine5thgroup(125mg/kg), piperine6th group(150mg/kg),all8groups set number A BC D E F G H respectively.(2)Modeling of animal with spinal cord injury: Allthe rats in the10level of posterior laminectomy fenestration, exposed the dural sac. Sham operation group only open laminectomy, do not hit the spinalcord, and other groups with the modified Allen ’ s method in10g×5cm forceimpact the spinal cord, making a model of acute spinal cord injury.(3)Modeof administration: After the modeling, the sham operation group rats weregiven7ml/kg normal saline by intragastric administration, piperine solventgroup rats were given7ml/kg orally administered piperine solvent, Piperinegroups rats were administered piperine suspension by the experimental grouprequirements (including7ml/kg piperine solvent) intragastric administration.The rats in each group were given intragastric administration on postoperative1d, once a day.(4) The rat spinal cord nerve function test: Each rat was scoredby BBB behavior score to evaluate hind-limb function at postoperative3d,7dand14d. Preoperative1d score was0points into the experiment, saidmodeling success, if not eliminate, the number of additional cullingreengineering mode.(5)Statistics: The experimental data using SPSS13.0statistical software for analysis. If they meet the normal distribution by themean±standard deviation (x±s), otherwise median. Multiple samples meanwere compared, if the data accord with normal distribution and homogeneityof variance using one-way ANOVA (F test), pairwise comparison with SNK-qmethod or Dunnet-t method test. Otherwise, use non-parametric testMann-Whitney U of rank transformation. P<0.05is significant difference.Results：1According to the BBB score, postoperative1d, except group A (Shamoperation group) no paraplegia, other groups were paralysis, score of0points.Postoperative3d,7d and14d, group A without paraplegia, scores are21points.The hind-limbs function have some recovery in group B with the time, BBBscores of group B are （1.444±0.527，2.333±0.500，7.111±1.054）.In the sametime, with the increase of piperine dosage, BBB score increase. BBB scores ofgroup C are（2.000±0.707，3.00±0.707，7.778±1.201），BBB scores of groupD are（3.333±0.866，4.111±0.781，9.889±0.781）, BBB scores of group E are（4.778±0.667，6.111±1.269，11.778±1.201）, BBB scores of group F are（6.000±0.707，8.111±0.781，13.778±0.971）, BBB scores of group G are （6.444±0.881，8.777±0.666，14.333±0.707）, BBB scores of group H are（7.000±1.118，9.555±1.013，15.111±0.781）.2Postoperative3d,7d and14d, comparison of group A with each group wasstatistically significant (P=0.000, P<0.05). Group B (Vehicle group)compared with each dose group show that low dose making no obvious effectand high dose making no obvious change. Postoperative3d,7d and14d, groupB (Vehicle Group) comparing with group C (25mg/kg) has no significantdifference(P=0.706,P=0.435,P=0.980，P>0.05). When the dose is higher than50mg/kg, with the increase of piperine dose, the differences is obviousgradually. While the dose was greater than125mg/kg, with the increase ofpiperine dose, the effect did not change significantly. The comparison betweengroup F(100mg/kg)and groupG(125mg/kg)(P=0.988,P=0.669,P=0.952,P>0.05)，group G (125mg/kg) andgroup H(150mg/kg)(P=0.989,P=0.684,P=0.490,P>0.05)，group F(100mg/kg)and group H(150mg/kg)(P=0.463,P=0.069,P=0.096,P>0.05) have nostatistical significance. In the same time, group B compare with group C,group D, group E and compared each other have statistical significance(P<0.05).3According to the observations in this experiment, with the increase of thepiperine dosage, the function of hind-limbs of rats restoring force increase, butalso increase the drug toxicity. Performance for the rat survival time isshortened. When the piperine dosage more than125mg/kg, the greater thedose, the shorter survival time. Synthesize the effect and toxicity of piperine,the optimum concentration to promoting the restoration of spinal cord injuryin rats was50~100mg/kg.Conclusion:(1) Piperine for the recovery of neural function of rats withspinal cord injury has a promotion function, and the effect and piperinecontent in a dose-effect relationship manner in a certain extent.(2)Large dosesof piperine has toxic effect, and the optimal concentration of promotingrecovery of spinal cord injury was50~100mg/kg.Part two：Study on the action mechanism of piperine on protecting nerve function after spinal cord injury in ratsObjective: To further explore the mechanism of action of piperine onneurologic protection after acute spinal cord injury in ratsMethods:（1）48adult male Wistar rats (weight250g-320g), groupingmethod with the first part of the experiment, but with6rats in each group.Making of animal model, the mode of administration and data statistics weresame with the first part. Postoperative3d and7d, three rats randomly selectedfrom each group respectively were killed and draw materials. Take part about5mm injured spinal cord tissue as pathological specimens, with4%paraformaldehyde fixed48hours, embedded in paraffin,4℃saved andreserved.（2）The spinal specimens of postoperative3d,7d,14d were routineHE staining to observe pathological changes, and admeasure the contentchange of inflammatory cell factor TNF-α、IL-6by immunohistochemistry.Results:1The results of HE staining of spinal cord pathology Postoperative3d groupA (sham operation group) spinal cord tissues was complete, cells arranged inneat, no obvious necrosis and cavity formation, spinal cord gray matter clearboundaries, normal neuron and myelin sheath structure, neurons have goodmorphology, numbers and whole cell membrane.Yet, other groups havedifferent levels of change, from the local hemorrhage and necrosis,inflammatory cell infiltration into neurons atrophy, neurons atrophy, astrocytesproliferate and white matter showed reticular demyelinating. Postoperative7dand14d, no obvious pathological changes in group A, other groups havedifferent degrees of improvement. With the increase of piperine dosage, thearea of spinal cord injury decreased gradually, normal tissue and tissuecontinuous increased.2The immunohistochemical results2.1The expression of TNF-α2.1.1The most obvious expression of TNF-α is in spinal cord injury center,group A expression at least, group B at most. Postoperative3d,7d and14d,almost no TNF-α expression and no significant staining in Group A, the number of positive cell classification were (7.83±1.16,5.83±0.75,4.83±1.47)respectively, staining intensity were （0,0,0）, and IHS were (0,0,0)respectively. Group B, the number of positive cell classificationwere(86.333±1.211,79.666±1.032,67.666±1.632), staining intensity were（3,3,3）, and IHS were （12,12,9） respectively. The effect is insignificant insmall doses. Group C, the number of positive cell classificationwere(85.166±1.169，78.166±0.752，66.333±1.366), staining intensitywere(3,3,3), and IHS were(12,12,9). The same time, the rest of the dosegroups, with the dose increase, the number of positive cells decrease andstaining intensity reduce. Group D, the number of positive cell classificationwere (82.166±1.169,74.00±0.894,62±1.549), staining intensity were (3,2.5,2), and IHS were (12,7.5,6). Group E, the number of positive cellclassification were (79.00±0.894,70.33±1.505,58.166±1.169), stainingintensity were (2.5,2.5,2), and IHS were (10,7.5,6). Group F, the number ofpositive cell classification were (75.00±0.894,65.666±1.632,54.5±1.516),staining intensity were (2,2,1), and IHS were (6,6,3). Group G, the numberof positive cell classification were (73.833±0.752,64.666±1.940,53.166+1.169), staining intensity were (2,2,1), and IHS were (6,6,3). GroupH, the number of positive cell classification were (72.666±0.333,62.666±1.751,52.00+1.414), staining intensity were (2,1,1), and IHS were (6,3,3).2.1.2Postoperative3d,7d,14d, there was significant difference group Acompare respectively with each groups(P<0.05).When group B compare withother dose groups, the results suggest that small dose application has nosignificant difference early. There was no significant difference between groupB and group C (P=0.813, P=0.224, P=0.889, P>0.05), while significantdifference between group B and group D, E, F, G, H (P=0.000, P<0.05).Comparison between each dose group(group C、D、E、F、G、H), group C, D,E, F were statistically significant (P <0.05), group F, G, H was not statisticallysignificant. Postoperative3d, Comparison between group F and group G(P=0.388, P>0.05), group G and group H (P=0.327, P>0.05), group F and group H (P=0.054, P>0.05) have no statistical significance. Postoperative7d,Comparison between group F and group G (P=0.922, P>0.05), group G andgroup H (P=0.936, P>0.05), group F and group H (P=0.162, P>0.05) have nostatistical significance. Postoperative14d, Comparison between group F andgroup G (P=0.807, P>0.05), group G and group H (P=0.879, P>0.05), group Fand group H (P=0.192, P>0.05) have no statistical significance.2.2The expression of IL-62.2.1The expression of IL-6positive cells mainly located in the central area ofinjury, and in peripheral zone the positive cells were gradually reducing.Postoperative3d,7d,14d, the group A had no or very rare positive cells,showed little change over time, the number of positive cell classification were（4.83±1.16，4.5±0.54，3.33±0.81）respectively, cell color intensity were (0,0,0) respectively, IHS were (0,0,0) respectively. Group B, positive cellsexpression and staining intensity are at most. Postoperative3d, the positivecell number of low dose groups at most, but the difference was not obvious,and the number of positive cells and cell color intensity decreased somewhatwith increasing dose. Group B,C,D,E,F, G, H positive cell numberclassification were (85.166±3.656，83.833±0.752,77.166±1.471,73.333±1.032,68.00±1.788,66.666±1.632,64.833+1.169),staining intensitywere (3,3,3,2.5,2.5,2,2), and IHS were(12,12,12,10,6,6,6) respectively.Postoperative7d, the number of positive cells decreased, with the doseincreased the number of positive cells decreased, cell staining intensity wasreduced gradually, and more obvious differences. Group B,C,D,E,F,G,Hpositive cell number classification were (81.833±1.471,80.333±1.505,73.166±2.228,67.166±1.940,60.666±1.211,59.000±1.141,57.833±0.983),staining intensity were (3,3,2.5,2.5,2,2,1), and IHSwere(12,12,12,7.57.5,6,6) respectively. Postoperative14d, the number ofpositive cells was significantly reduced, basically stable, is still higher thanthat of group A. Group B,C,D,E,F,G,H positive cell number classification were(74.50±0.836,73.833±0.752,69.833±1.602,63.666±1.211,57.00±3.033,55.333±1.366,53.5±1.378),staining intensity were (3,3,2,2,1,1,1), and IHS were(9,9,6,6,3,3,3) respectively.2.2.2Postoperative3d,7d and14d, group A compare respectively with eachgroups came out significantly different. Postoperative3d,7d and14d,comparison between group B and other dose groups, the difference was notobvious in low dose early. Group B and group C (P=0.998, P=0.787, P=0.921,P>0.05) have no significant difference, group B and groups C, D, E, F, G, Hwere significantly different (P=0.000, P<0.05). Comparison between eachdose group(group C、D、E、F、G、H), group C, D, E, F were statisticallysignificant (P<0.05), group F, G, H was not statistically significant.Postoperative3d, Comparison between group F and group G (P=0.952,P>0.05), group G and group H (P=0.501, P>0.05), group F and group H(P=0.083, P>0.05) have no statistical significance. Postoperative7d,Comparison between group F and group G (P=0.522, P>0.05), group G andgroup H (P=0.829, P>0.05), group F and group H (P=0.052, P>0.05) have nostatistical significance. Postoperative14d, Comparison between group F andgroup G (P=0.969, P>0.05), group G and group H (P=0.453, P>0.05), group Fand group H (P=0.361, P>0.05) have no statistical significance.Conclusion: Piperine put up protective effect on spinal cord injury may beassociated with it could down-regulate the expression of inflammatorymediators TNF-α and IL-6and reduce inflammation.