芸香苷抑制脊髓损伤大鼠炎症反应及相关机制的实验研究

发布时间：2018-06-02 06:15

本文选题：脊髓损伤 + 芸香苷　；参考：《山西医科大学》2017年博士论文

【摘要】：目的:在改良ALLEN法建立的大鼠脊髓损伤动物模型上,检测芸香苷是否能促进脊髓损伤后大鼠的神经功能恢复,改善脊髓损伤后炎症因子浸润,减轻炎症反应,并检测芸香苷对脊髓损伤后PI3K/AKT信号通路的影响,来探讨芸香苷发挥脊髓损伤后神经保护作用的作用机制。方法:1.动物模型和分组:120只雄性成年SD大鼠,随机分成4组,每组30只。假手术组(CON组):切除椎板,不损伤脊髓,注射1ml的DMSO;脊髓损伤组(SCI组):根据ALLEN法建立脊髓损伤大鼠模型(T10节段),注射1ml的DMSO;甲强龙治疗组(MP组):脊髓损伤同SCI组,腹腔内注射甲强龙30mg/kg;芸香苷治疗组(RT100组):脊髓损伤SCI组,腹腔内注射芸香苷100mg/kg。2.每组10只大鼠,在脊髓损伤后的第1、2、3周,采用体感诱发电位对大鼠脊髓功能进行客观检测,观察指标选用潜伏期和波幅。在脊髓损伤后的第1、7、14、21、28天,采用BBB评分、斜坡实验和改良Tarlov评分等行为学方法评价大鼠后肢运动功能和身体平衡能力。3.取材:在大鼠脊髓损伤后24小时处死动物,每组5只大鼠取新鲜脊髓用于含水量检测;每组5只大鼠取新鲜脊髓用于ELISA、明胶酶谱法和光谱分析法;每组5只大鼠取新鲜脊髓用Western Blot实验;每组5只大鼠经心脏灌注4%多聚甲醛内固定后取脊髓,放在相同固定液再固定24小时后,石蜡包埋切片用于免疫组织化学染色和HE染色。4.采用免疫组化染色法测定脊髓组织中IL-1β、TNF-α、MCP-1、IL-10、TGF-β、GFAP、IBA-1、Bax、Bcl-2的表达,采用HE染色观察脊髓组织形态和病理表现。5.采用Hsu公式测定脊髓损伤后脊髓组织含水量,采用酶联免疫吸附法(ELISA)测定脊髓组织中MIP-2的含量,明胶酶谱法测定脊髓组织中MMP-9的含量,光谱分析法测定IKKβ激酶活性。6.采用Western blot法检测脊髓组织中的P-AKT、NF-κB P65、Bax、Bcl-2的表达。结果:1.体感诱发电位结果:SCI组在第1、2、3周的SEP潜伏期与CON组比较明显延长,SEP的波幅均明显下降;与SCI组相比,MP组和RT100组在第1、2、3周潜伏期均呈现不同程度的缩短,波幅均呈现不同程度的升高,差异有统计学意义(p0.05)。2.BBB、斜坡实验、改良Tarlov功能评分的结果:在脊髓损伤后的每个检测时间点,SCI组大鼠的评分均明显低于CON组;与SCI组相比,MP组和RT100组大鼠的评分虽然在第1和第7天无明显差异,但在第14、21、28天均明显提高,差异有统计学意义(p0.05)。3.脊髓含水量的结果:与CON组相比,SCI组的含水量明显升高,差异有统计学意义(p0.05);与SCI组相比,RT100组脊髓含水量明显降低,差异有统计学意义(p0.01);MP组和RT100组相比,治疗效果相当,在脊髓组织含水量上没有差异,无统计学意义(p0.05)。4.HE染色结果:与CON组相比,SCI组中的神经元细胞数量明显减少,差异有统计学意义(p0.05);与SCI组相比,MP组和RT100组神经元细胞数目减少受到抑制,差异有统计学意义(p0.05);MP组和RT100组相比,在抑制神经元数目减少方面,效果相当,没有统计学意义(p0.05)。5.免疫组化染色炎症因子表达的结果:与CON组相比,SCI组中的IL-1β、TNF-α、MCP-1水平显著升高,差异有统计学意义(p0.05);与SCI组相比,RT100组和MP组IL-1β、TNF-α、MCP-1表达水平明显降低,差异有统计学意义(p0.05);与CON组相比,SCI组中的IL-10、TGF-β水平明显升高,差异有统计学意义(p0.05);与SCI组相比,RT100组和MP组中IL-10、TGF-β表达水平明显增高,差异有统计学意义(p0.05)。6.ELISA检测MIP-2含量的结果:与CON组相比,SCI组中MIP-2的水平明显增强,差异有统计学意义(p0.05);与SCI组相比,MP组和RT100组中MIP-2水平明显降低,差异有统计学意义(p0.05,p0.01);MP组和RT100组中MIP-2表达水平没有明显差异(p0.05)。7.明胶酶谱法测定MMP-9活性的结果:SCI组和CON组相比,MMP-9的活性和生成量都明显升高,差异有统计学意义(p0.05);与SCI组相比,MP组和RT100组中MMP-9的生成量和活性明显降低,差异有统计学意义(p0.01);MP组和RT100组相比,脊髓组织中MMP-9的活性和生成量没有差异,无统计学意义(p0.05)。8.免疫组化染色检测GFAP和IBA-1表达的结果:SCI组与CON组相比,GFAP、IBA-1表达水平明显增高,差异有统计学意义(p0.05);与SCI组相比,RT100组和MP组GFAP和IBA-1表达降低,差异有统计学意义(p0.05)。9.光谱分析检测IKKβ激酶活性结果:在CON组中,脊髓组织的IKKβ激酶活性较低,SCI组相比CON组中IKKβ激酶活性明显增高,差异有统计学意义(p0.05);MP组和RT100组IKKβ激酶活性相比SCI组明显降低,差异有统计学意义(P0.05);MP组和RT100组相比在抑制IKKβ激酶活性上差异无统计学意义(P0.05)。10.免疫组化染色和Western blot检测Bax和Bcl-2表达的结果:SCI组与CON组相比,Bax表达水平明显增高,而Bcl-2表达明显降低,差异有统计学意义(p0.05);与SCI组相比,RT100组和MP组Bax的表达降低,Bcl-2的表达升高,差异有统计学意义(p0.05);RT100组和MP组相比,差异没有统计学意义(p0.05)。11.Western blot p-Akt和NF-κB P65表达的结果:CON组中p-Akt、NF-κB P65蛋白的表达较少,SCI组和CON组相比,p-Akt、NF-κB P65蛋白表达明显增高,差异有统计学意义(p0.05);与SCI组相比,药物治疗组在p-Akt、NF-κB P65蛋白的生成量上都有不同程度的抑制作用,RT100组和MP组能够调节p-Akt、NF-κB P65蛋白的表达,差异有统计学意义(p0.05);MP组和RT100组在表达p-Akt、NF-κB P65蛋白方面无显著差异,没有统计学意义(p0.05)。结论:1.芸香苷能够缩短脊髓损伤大鼠SEP潜伏期,增加波幅,改善脊髓损伤后大鼠的后肢运动能力和身体平衡能力,对脊髓损伤后大鼠的神经功能有保护作用。2.芸香苷能够减轻脊髓损伤后的组织水肿,减少神经元细胞的损伤,抑制促炎因子IL-1β和TNF-α的表达,减少趋化因子MCP-1、MIP-2和MMP-9的产生,促进抗炎因子IL-10和TGF-β因子的表达,抑制星形胶质细胞和小胶质细胞的激活,减轻炎症反应。3.芸香苷能够抑制脊髓损伤后PI3K/AKT信号通路,降低IKKβ激酶活性,减少p-Akt和NF-κB P65蛋白的表达,下调Bax的表达,上调Bcl-2的表达,降低Bax/Bcl-2的比例,抑制细胞凋亡。芸香苷可能是通过PI3K/AKT实现对脊髓损伤大鼠的神经保护作用。
[Abstract]:Objective: to determine whether rutin can promote the recovery of nerve function in rats after spinal cord injury, improve the infiltration of inflammatory factors after spinal cord injury, reduce the inflammatory response, and detect the effect of rutin on the PI3K/AKT signaling pathway after spinal cord injury to explore the effect of rutin on the spinal cord injury in the rat model of spinal cord injury established by the improved ALLEN method. Methods: 1. animal models and groups: 1. animal models and groups: 120 adult male adult SD rats were randomly divided into 4 groups, 30 in each group. The sham operation group (group CON): the laminectomy, the spinal cord injury, the injection of 1ml DMSO, the spinal cord injury group (group SCI): the ALLEN method was used to establish the rat model of spinal cord injury (T10 segment), the DMSO of 1ml and a strength of 1ml Dragon treatment group (group MP): spinal cord injury and SCI group, intraperitoneal injection of methylprednisolone 30mg/kg, rutin treatment group (group RT100): spinal cord injury SCI group, intraperitoneal injection of rutin 100mg/kg.2. in each group of 10 rats, after spinal cord injury week 1,2,3, using somatosensory evoked potential to detect the function of spinal cord in rats, observe the incubation period of the incubation period And amplitude. On day 1,7,14,21,28 after spinal cord injury, the BBB score, slope experiment and improved Tarlov score were used to evaluate the motor function and body balance of the rats. The animals were killed 24 hours after the spinal cord injury in rats. 5 rats in each group were used for the detection of water content, and 5 rats in each group were taken out. The fresh spinal cord was used for ELISA, gelatin Zymogram and spectral analysis. 5 rats in each group were taken Western Blot experiment with fresh spinal cord. 5 rats in each group were fixed in the same fixed solution for 24 hours after perfusion of 4% polyformaldehyde, and the paraffin embedded sections were used for immunohistochemical staining and HE staining for.4. immunization. The expression of IL-1 beta, TNF- a, MCP-1, IL-10, TGF- beta, GFAP, IBA-1, Bax, Bcl-2 in spinal cord tissue was determined by staining, and HE staining was used to observe the morphological and pathological manifestations of spinal cord tissue and pathology. The content of spinal cord tissue was measured by the Hsu formula. The content of spinal cord tissue was determined by enzyme linked immunosorbent assay, and gelatinase assay was used to determine the content of spinal cord tissue. The content of MMP-9 in spinal cord tissue, the determination of the activity of IKK beta kinase activity by spectral analysis and Western blot method to detect P-AKT in spinal cord tissue, NF- kappa B P65, Bax, Bcl-2 expression. Results: 1. somatosensory evoked potential results: the incubation period in the SCI group was significantly longer than that of the group, and the amplitude of NF- kappa was obviously decreased; compared with the group The latency of the RT100 group in the 1,2,3 week showed a different degree of shortening, and the amplitude of the wave increased in varying degrees. The difference was statistically significant (P0.05).2.BBB, the slope experiment, and the result of improving the Tarlov function score: the score of the SCI group was significantly lower than that of the CON group at each detection time point after spinal cord injury, and the MP group and RT were compared with the SCI group. There was no significant difference between the 100 groups in the first and seventh days, but the difference was significantly higher in day 14,21,28. The difference was statistically significant (P0.05) the result of the water content in the.3. spinal cord. Compared with the CON group, the water content of the SCI group was significantly higher, the difference was statistically significant (P0.05). Compared with the SCI group, the water content of the RT100 group was significantly lower than that in the RT100 group, and the difference was unified. Study significance (P0.01); MP group and RT100 group compared with the treatment effect, there was no difference in the water content of the spinal cord, no statistical significance (P0.05).4.HE staining results: compared with the CON group, the number of neurons in the SCI group decreased significantly (P0.05), and the number of neurons in MP and RT100 groups decreased compared with the SCI group. The difference was statistically significant (P0.05), and in group MP and group RT100, the effect was similar in reducing the number of neurons, and there was no statistically significant (P0.05).5. immunohistochemical staining of the expression of inflammatory factors: compared with the CON group, the level of IL-1 beta, TNF- alpha, MCP-1 in the SCI group was significantly higher, and the difference was statistically significant (P0.05); and SCI with SCI. Compared with group RT100 and group MP, the expression level of IL-1 beta, TNF- alpha and MCP-1 was significantly lower, and the difference was statistically significant (P0.05). Compared with the CON group, the IL-10 and TGF- beta levels in SCI group were significantly higher, and the difference was statistically significant (P0.05). The results of ELISA detection of MIP-2 content: compared with the CON group, the level of MIP-2 in the SCI group was obviously enhanced, and the difference was statistically significant (P0.05). Compared with the SCI group, the MIP-2 level of MP and RT100 groups was significantly decreased, the difference was statistically significant (P0.05, P0.01). The results of MP-9 activity: the activity and production of MMP-9 increased significantly in the group SCI and the CON group, and the difference was statistically significant (P0.05). Compared with the SCI group, the amount and activity of MMP-9 in the MP group and RT100 group decreased significantly (P0.01), and there was no difference in the activity and production of the spinal cord between the MP group and the RT100 group. There was no statistical significance (P0.05).8. immunohistochemical staining to detect the expression of GFAP and IBA-1: the expression level of GFAP, IBA-1 in SCI group was significantly higher than that in CON group, and the difference was statistically significant (P0.05). The difference was statistically significant compared with the SCI group. Results: in the group CON, the activity of IKK beta kinase in the spinal cord was lower, and the activity of IKK beta kinase in the group SCI was significantly higher than that in the group CON (P0.05), and the IKK beta kinase activity in the MP group and RT100 group was significantly lower than that in the SCI group, and the difference was statistically significant (P0.05), and there was no statistical difference between the MP group and the group in the MP group. P0.05.10. immunohistochemical staining and Western blot were used to detect the expression of Bax and Bcl-2: the Bax expression level was significantly higher in the SCI group than that in the CON group, but the Bcl-2 expression was significantly lower, and the difference was statistically significant (P0.05). Compared with the MP group, the difference was not statistically significant (P0.05).11.Western blot p-Akt and NF- kappa B P65 expression results: CON group p-Akt, NF- kappa B protein expression was less. RT100 and MP groups could regulate the expression of p-Akt, NF- kappa B P65 protein, and the difference was statistically significant (P0.05). There was no significant difference in the expression of p-Akt, NF- kappa B protein in MP and RT100 groups. Conclusion: 1. rutin can shorten the potential of spinal cord injury rats. .2. rutin can reduce the tissue edema after spinal cord injury, reduce the damage of neuron cells, inhibit the expression of IL-1 beta and TNF- alpha, and reduce the chemokine MCP-1, MI, and reduce the chemokine MCP-1, MI The production of P-2 and MMP-9 promotes the expression of anti-inflammatory factors IL-10 and TGF- beta factors, inhibits the activation of astrocytes and microglia, and reduces the inflammatory reaction.3. rutin can inhibit the PI3K/AKT signaling pathway after spinal cord injury, reduces the activity of IKK beta kinase, reduces the expression of p-Akt and NF- kappa B P65 protein, down regulate the expression of Bax, and up regulation of the expression of Bax. Rutin could reduce the proportion of Bax/Bcl-2 and inhibit cell apoptosis. Rutin may be a neuroprotective effect of PI3K/AKT on spinal cord injury in rats.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R651.2

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