三七皂苷R1对大鼠脊髓损伤后线粒体功能和神经炎症的作用及相关机制研究

Study of the roles and mechanism of NGR1 in regulating mitochondrial function and neuroinflammation after spinal cord injury in rats

中文关键词:  脊髓损伤  三七皂苷R1  核因子E2相关因子2  血红素加氧酶-1  线粒体功能  神经炎症  大鼠

英文关键词:Spinal cord injury  Notoginsenoside R1  Nuclear factor E2-related factor 2  Heme oxygenase-1  Mitochondrial function  Neuroinflammation  Rat

中文摘要:

  【摘要】 目的：探究三七皂苷R1（notoginsenoside R1，NGR1）对大鼠脊髓损伤（spinal cord injury，SCI）后线粒体功能和神经炎症的作用及相关机制。方法：SPF级2月龄SD雄性大鼠80只，体质量为250～280g，按随机数字表法分为A1组、B1组、C1组和D1组，每组各20只。A1组仅暴露脊髓，B1组、C1组和D1组大鼠采用改良的Allen′s打击法建立急性SCI模型。A1组和B1组腹腔注射生理盐水，C1组、D1组腹腔注射对应剂量的NGR1和ML385[核因子E2 相关因子2（nuclear factor E2 related factor2，Nrf2）抑制剂]。给药1d、2d、3d采用BBB评分法评估大鼠后肢运动功能恢复情况；给药结束后，各取3只大鼠脊髓样本，铬化青染色与TUNEL染色观察大鼠脱髓鞘及细胞凋亡；利用试剂盒评估脊髓组织线粒体功能；ELISA检测脊髓组织炎症因子的表达；免疫荧光双染色检测脊髓组织离子钙结合衔接分子1（ionized calcium binding adapter molecule 1，Iba1）+诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）+细胞数；RT-qPCR检测脊髓组织Nrf2、血红素加氧酶-1（heme oxygenase-1，HO-1）mRNA的表达；Western-blot检测脊髓组织Nrf2、HO-1、Bcl2 相关X（BCL2-associated X，Bax）蛋白、细胞淋巴瘤-2（B cell lymphoma-2，Bcl-2）蛋白表达。将购买的PC12细胞分为A2组、B2组、C2组和D2组。A2组正常培养，B2组、C2组和D2组细胞在含H2O2的培养基中培养，C2组和D2组分别加入的NGR1和ML385。各组细胞培养24h后采用CCK8法检测PC12细胞活性；Annexin V-FITC/PI染色检测PC12细胞凋亡；ELISA检测PC12细胞中炎症因子的表达；RT-qPCR检测PC12细胞Nrf2、HO-1 mRNA的表达；Western-blot检测PC12细胞Nrf2、HO-1、Bax蛋白、Bcl-2蛋白表达。结果：与A1组相比，B1组大鼠BBB评分降低，脱髓鞘加重，细胞凋亡增加，超氧化物歧化酶（superoxide dismutase，SOD）活性、Ca2+-Mg2+-ATP酶活性下降，丙二醛（malondialdehyde，MDA）浓度、磷脂酶A2（phospholipase A2，PLA2）活性升高，肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白介素-8 （interleukin-8，IL-8）、白介素-6（interleukin-6，IL-6）水平升高，Iba1+iNOS+细胞数增加，Bax蛋白、Nrf2、HO-1 mRNA和蛋白表达增加，Bcl-2蛋白表达减少（P＜0.05）；与B1组和D1组相比，C1组大鼠BBB评分升高（P＜0.05），脱髓鞘减轻，细胞凋亡减少，SOD活性、Ca2+-Mg2+-ATP酶活性升高，MDA浓度、PLA2活性降低，TNF-α、IL-8和IL-6水平降低，Iba1+iNOS+细胞数减少，Bax蛋白表达减少，Nrf2、HO-1 mRNA和蛋白、Bcl-2蛋白表达增加（P＜0.05）。与A2组相比，B2组细胞活性降低，细胞凋亡增加，TNF-α、IL-8和IL-6水平升高，Bax蛋白、Nrf2、HO-1 mRNA和蛋白表达增加，Bcl-2蛋白表达减少（P＜0.05）；与B2组和D2组相比，C2组细胞活性升高，细胞凋亡减少，TNF-α、IL-8和IL-6水平降低，Bax蛋白表达减少，Nrf2、HO-1 mRNA和蛋白、Bcl-2蛋白表达增加（P＜0.05）。结论：NGR1能够减轻SCI大鼠脊髓细胞凋亡及氧化应激，促进大鼠运动功能恢复，改善大鼠SCI后线粒体功能和神经炎症；NGR1能够提高PC12细胞活性，抑制其凋亡和炎症水平，其通过激活Nrf2/HO-1信号通路发挥作用。

英文摘要:

  【Abstract】 Objectives: To investigate the roles and related mechanism of notoginsenoside R1(NGR1) in regulating the mitochondrial function and neuroinflammation after spinal cord injury (SCI) in rats. Methods: 80 SPF 2-month old SD male rats, weighted 250-280g, were divided into A1 group, B1 group, C1 group, and D1 group with 20 rats in each group. A1 group only exposed the spinal cord, and the other three groups established acute spinal cord injury model with modified Allen′s percussion method. C1 group and D1 group were intraperitoneally injected with NGR1 and Nrf2 ML385, the nuclear factor E2 related factor2(Nrf2 inhibitor), and A1 group and B1 group were intraperitoneally injected with the same amount of normal saline. BBB score was used to evaluate the recovery of hind limb motor function. After administration, spinal cord samples were taken from 3 rats of each group respectively. Chromic blue and TUNEL were used to observe demyelination and apoptosis. Kit was used to evaluated mitochondrial function of spinal cord tissue. ELISA was used to detect the expression of inflammatory factors in spinal cord tissue. Immunofluorescence double staining was used to detect the number of positive ionized calcium binding adapter molecule 1(Iba1)+, inducible nitric oxide synthase(iNOS)+ cells in spinal cord tissue. RT-qPCR was used to detect the expressions of Nrf2 and heme oxygenase(HO)-1 mRNA in spinal cord tissue, Western-blot was used to detect the expressions of Nrf2, HO-1, BCL2-associated X(Bax), and B cell lymphoma-2(Bcl-2) protein. PC12 cell was divided into A2 group, B2 group, C2 group and D2 group. A2 group were cultured normally, B2 group, C2 group and D2 group were cultured in the medium containing H2O2, and C2 group and D2 group were added with NGR1 and ML385. After 24h of cell culture in each group CCK8 assay was used to detect the activity of PC12 cells. Annexin V-FITC/PI staining was used to detect the apoptosis of PC12 cells. ELISA was used to detect the expression of inflammatory factors in PC12 cells. RT-qPCR was used to detect the expressions of Nrf2 and HO-1 mRNA in PC12 cells, Western-blot was used to detect the expressions of Nrf2, HO-1, Bax, and Bcl-2 protein in PC12 cells. Results: Comparing with A1 group, the BBB score of rats in B1 group decreased, demyelination increased, apoptosis increased(P<0.05), superoxide dismutase(SOD) activity and Ca2+-Mg2+-ATPase activity decreased, malondialdehyde(MDA) concentration and phospholipase A2（PLA2） activity increased, tumor necrosis factor-α(TNF-α), interleukin(IL)-8 and IL-6 levels increased, and Iba1+iNOS+ cell number increased, the expression of Bax protein, Nrf2, HO-1 mRNA and protein increased, while the expression of Bcl-2 protein decreased(P<0.05). Comparing with B1 group and D1 group, the BBB score of rats in C1 group increased, demyelination decreased, apoptosis increased, SOD activity and Ca2+-Mg2+-ATPase activity increased, MDA concentration and PLA2 activity decreased, TNF-α, IL-8 and IL-6 levels decreased, and Iba1+iNOS+ cell number decreased, the expression of Bax protein decreased, while the expression of Nrf2, HO-1 mRNA and protein and Bcl-2 protein increased(P<0.05). Comparing with A2 group, the cell activity of B2 group decreased, apoptosis increased, the levels of TNF-α, IL-8, IL-6 increased, the expressions of Bax protein, Nrf2, HO-1 mRNA and protein increased, and the expression of Bcl-2 protein decreased(P<0.05). Comparing with B2 group and D2 group, the cell activity of C2 group increased, apoptosis decreased, the levels of TNF-α, IL-8 and IL-6 decreased, the expression of Bax protein decreased, and the expression of Nrf2, HO-1 mRNA and protein and Bcl-2 protein increased(P<0.05). Conclusions: NGR1 can alleviate the apoptosis and oxidative stress in spinal cord tissue of SCI rats, promote the recovery of rat motor function, and improve mitochondrial function and neuroinflammation in rats after SCI. At the same time, NGR1 can increase the activity of PC12 cells and inhibit their apoptosis and inflammation through activating the Nrf2/HO-1 signaling pathway.