消退素D1在大鼠烧伤疼痛中的作用及机制

发布时间：2018-06-14 10:49

本文选题：消退素D1 + 烧伤痛　；参考：《郑州大学》2016年硕士论文

【摘要】：背景消退素(resolvins,Rvs)是来源于ω-3多不饱和脂肪酸的内源性促炎症消退介质,包括D系和E系消退素,在多种炎性动物模型中具有抗炎和促炎症消退作用。最近的研究发现Rvs还能通过调节各种瞬时受体电位通道的活性、促炎因子和抗炎介质的表达及中枢敏化的形成等有效减轻炎性疼痛、术后疼痛和神经病理性痛。脊髓胶质细胞、小胶质细胞内的p-p38 MAPK和脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)及其受体原肌球蛋白相关激酶B(tropomyosin-related kinase B,Trk B)在神经病理性痛的形成中发挥重要作用,而烧伤疼痛含有神经病理性痛的成分。因此,它们可能也参与烧伤疼痛的形成。体内外研究表明Rvs可以抑制小胶质细胞和星形胶质细胞的活化,抑制小胶质细胞内p38 MAPK磷酸化,减少其分泌炎性介质。因此,我们提出假说消退素D1(resolvin D1,Rv D1)可能通过抑制脊髓背角内胶质细胞活化、小胶质细胞内p38 MAPK磷酸化、下调BDNF/Trk B受体信号,从而减轻烧伤疼痛。目的本研究通过建立大鼠烧伤模型、腹腔注射Rv D1探索Rv D1在烧伤疼痛中的作用及其机制;通过鞘内注射SB203580抑制p38 MAPK活化,观察烧伤大鼠的痛行为学变化,检测脊髓背角Iba-1、BDNF和Trk B的表达水平的变化,从而探讨p38 MAPK活化在Rv D1对于烧伤疼痛作用机制中所发挥的作用;通过鞘内注射Trk B-Fc抑制BDNF/Trk B信号,观察烧伤大鼠的痛行为学变化,检测脊髓背角Iba-1和p-p38 MAPK的表达水平的变化,从而探讨BDNF/Trk B信号在Rv D1对于烧伤疼痛作用机制中所发挥的作用。方法1.140~150g清洁级雄性SD大鼠用随机数字表法分为5组(n=6):假伤组(Sham+Veh1组),烧伤组(Burn+Veh1组),烧伤+Rv D1低剂量组(Burn+R(S)组),烧伤+Rv D1高剂量组(Burn+R(L)组),假伤+Rv D1高剂量组(Sham+R(L)组)。制备烧伤模型,于烧伤前30min及烧伤后1-7d分别腹腔注射100ng、300ng Rv D1或对照溶剂Veh1。分别于烧伤前1d和烧伤后1、3、5、7、14d测定大鼠机械缩足反应阈(MWT)。于烧伤后7d MWT测定结束后处死3只大鼠,用荧光免疫组化检测脊髓小胶质细胞和星形胶质细胞标记物Iba-1和GFAP、p-p38MAPK、BDNF/Trk B的表达水平。用免疫荧光双标检测p-p38 MAPK分别与Iba-1、GFAP和Neu N的共标情况。2.140~150g清洁级雄性SD大鼠用随机数字表法分为5组(n=6):Sham+Veh2组,Burn+Veh2组,Burn+SB203580组,Sham+SB203580组,Burn+R(L)组。制备烧伤模型,于造模后7d分别鞘内注射SB203580或对照溶剂Veh2,Burn+R(L)组腹腔注射Rv D1方法同前。分别于鞘内注药前、鞘内注药后15min、30min、1h和2h测定大鼠MWT。大鼠MWT测定结束后处死3只大鼠,用荧光免疫组化检测脊髓小胶质细胞标记物Iba-1和BDNF/Trk B的表达水平。3.140~150g清洁级雄性SD大鼠用随机数字表法分为5组(n=6):Sham+Veh3组,Burn+Veh3组,Burn+Trk B-Fc组,Sham+Trk B-Fc组,Burn+R(L)组。制备烧伤模型,于造模前1h和造模后1-7d用微量注射器通过鞘内置管分别给予Trk B-Fc或对照溶剂Veh3,Burn+R(L)组腹腔注射Rv D1方法同前。分别于烧伤前1d和烧伤后1、3、5、7、14d测定大鼠MWT。于烧伤后7d MWT测定结束后处死3只大鼠,用荧光免疫组化检测脊髓小胶质细胞标记物Iba-1和p-p38 MAPK的表达水平。结果1.与Sham+Veh1组比较,Burn+Veh1组烧伤后各时点MWT降低(P0.05);与Burn+Veh1组比较,Burn+R(S)组和Burn+R(L)组烧伤后各时点MWT均升高(P0.05);与Burn+R(S)组比较,Burn+R(L)组烧伤后各时点MWT升高(P0.05)。免疫荧光显示,与Sham+Veh1组比较,Burn+Veh1组脊髓背角Iba-1、GFAP、p-p38 MAPK、BDNF和Trk B受体表达水平均升高(P0.05);与Burn+Veh1组比较,Burn+R(L)组脊髓背角Iba-1、GFAP、p-p38 MAPK、BDNF和Trk B受体表达水平均降低(P0.05)。免疫荧光双标显示,p-p38 MAPK只与Iba-1共标,而不与GFAP和Neu N共标。2.与Sham+Veh2组相比,Burn+Veh2组MWT降低(P0.05);与Burn+Veh2组相比,Burn+SB203580组MWT升高(P0.05);与Burn+SB203580组注药后各时点相比,Burn+R(L)组MWT差异无统计学意义(P0.05)。免疫荧光显示,与Sham+Veh2组相比,Burn+Veh2组和Burn+SB203580组Iba-1、BDNF和Trk B受体表达水平均升高(P0.05);与Burn+Veh2组相比,Burn+SB203580组Iba-1、BDNF和Trk B受体表达水平均降低(P0.05)。3.与Sham+Veh3组相比,Burn+Veh3组烧伤后各时点MWT降低(P0.05);与Burn+Veh3组相比,Burn+Trk B-Fc组注药后MWT升高(P0.05);与Burn+Trk B-Fc组相比,Burn+R(L)组MWT差异无统计学意义(P0.05)。免疫荧光显示,与Sham+Veh3组相比,Burn+Veh3组和Burn+Trk B-Fc组Iba-1和p-p38MAPK表达水平均升高(P0.05);与Burn+Veh3组相比,Burn+Trk B-Fc组Iba-1和p-p38 MAPK表达水平均降低(P0.05)。结论消退素D1(resolvin D1,Rv D1)可通过抑制脊髓背角内胶质细胞活化、小胶质细胞内p38 MAPK磷酸化、下调BDNF/Trk B受体信号的表达,从而减轻烧伤疼痛。
[Abstract]:Resolvins (Rvs) is an endogenous pro-inflammatory regression medium derived from Omega -3 polyunsaturated fatty acids, including the D and E system antiinflammatory agents, which have anti-inflammatory and proinflammatory response in a variety of inflammatory animal models. Recent studies have found that Rvs can also regulate the activity of various transient receptor potential channels, proinflammatory factors and anti-inflammatory factors. The expression of medium and the formation of central sensitization effectively alleviated inflammatory pain, postoperative pain and neuropathic pain. Spinal glial cells, p-p38 MAPK in microglia and brain-derived neurotrophic factor (BDNF) and their receptor promyocal egg white related kinase B (tropomyosin-related kinase B, Trk B) It plays an important role in the formation of neuropathic pain, and burn pain contains neuropathic pain components. Therefore, they may also participate in the formation of burn pain. In vivo and in vivo studies have shown that Rvs can inhibit the activation of microglia and astrocytes, inhibit the phosphorylation of p38 MAPK in microglia, and reduce the secretion of inflammatory mediates Therefore, we suggest that the hypothesis D1 (resolvin D1, Rv D1) may inhibit the activation of glial cells in the dorsal horn of the spinal cord, the phosphorylation of p38 MAPK in microglia, down regulation of the B receptor signal of BDNF/Trk, and thus reduce the pain of the BDNF/Trk, thus the purpose of this study was to establish a rat burn model by intraperitoneal injection of Rv D1 to explore the pain of Rv in the burn. To observe the changes in pain behavior in burned rats by intrathecal injection of SB203580 and to inhibit the activation of p38 MAPK, the changes in the expression of Iba-1, BDNF and Trk B in the dorsal horn of the spinal cord were detected, and the effect of p38 MAPK activation on the mechanism of the action of Rv D1 on the mechanism of burn pain was investigated. The changes in the pain behavior of the burned rats were observed and the changes of the expression level of Iba-1 and p-p38 MAPK in the dorsal horn of the spinal cord were detected, and the role of BDNF/Trk B signal in the mechanism of Rv D1 on the action of burn pain was explored. Method 1.140~150g clean grade male SD rats were divided into 5 groups (n=6) with random number table: false injury group (Sham+Veh1 group) and burn group ( Burn+Veh1 group, +Rv D1 low dose group (Burn+R (S) group), +Rv D1 high dose group (Burn+R (L) group), false wound +Rv D1 high dose group (Sham+R (Sham+R) group). The threshold of contraction reaction (MWT). After the end of 7D MWT, 3 rats were killed and the expression level of Iba-1, GFAP, p-p38MAPK, BDNF/Trk B of spinal microglia and astrocytes was detected by immunofluorescence. The male SD rats were divided into 5 groups (n=6): group Sham+Veh2, group Burn+Veh2, group Burn+SB203580, group Sham+SB203580, Burn+R (L). The model of burn was prepared by injection of SB203580 or controlled solvent Veh2 in the sheath. 3 rats were killed after MWT determination in MWT. rats and 3 rats were killed. The expression level of microglia markers Iba-1 and BDNF/Trk B in the male SD rats was detected by immunofluorescence. The random number table method was used to determine the level of.3.140~150g clean male SD rats into 5 groups (n=6): Sham+Veh3, Burn+Veh3, Burn+Trk. To prepare the burn model, 1-7d was given Trk B-Fc or Veh3, Burn+R (L) group by intraperitoneal injection of Rv D1 (Rv D1) before the 1H and the model of the model after the model of the model. 3 rats were killed and the rats were killed after the burn before and after the burn, respectively. The expression level of the microglia markers Iba-1 and p-p38 MAPK was measured. Results 1. compared with the Sham+Veh1 group, MWT decreased at each time point in Burn+Veh1 group (P0.05). Compared with the Burn+Veh1 group, both Burn+R (S) and Burn+R (L) groups increased at every point after burn. Immunofluorescence showed that the expression level of Iba-1, GFAP, p-p38 MAPK, BDNF and Trk B receptor in the spinal dorsal horn of group Burn+Veh1 increased (P0.05), compared with the Sham+Veh1 group, and the expression level of the spinal dorsal horn of the group was lower than that in the Burn+Veh1 group. Compared with the Sham+Veh2 group, the MWT decreased (P0.05) in the Burn+Veh2 group without the co labeling of GFAP and Neu N, and the MWT increased (P0.05) in the Burn+SB203580 group compared with the Burn+Veh2 group. The expression level of Iba-1, BDNF and Trk B receptors in the 0 groups increased (P0.05). Compared with the Burn+Veh2 group, the expression level of Iba-1, BDNF and Trk B receptors in the Burn+SB203580 group decreased (P0.05) was lower than that in the group. Compared with group Burn+R (L), there was no significant difference in MWT (P0.05). Immunofluorescence showed that the expression level of Iba-1 and p-p38MAPK increased in both Burn+Veh3 and Burn+Trk B-Fc groups (P0.05). By inhibiting the activation of glial cells in the dorsal horn of the spinal cord, the phosphorylation of p38 MAPK in the microglia and the down regulation of BDNF/Trk B receptor signaling can reduce the burn pain.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R644

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