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| LUO Hongbo,ZHOU Mingjian,CHEN Yuxin.Study of the roles and mechanism of NGR1 in regulating mitochondrial function and neuroinflammation after spinal cord injury in rats[J].Chinese Journal of Spine and Spinal Cord,2022,(9):823-833. |
| Study of the roles and mechanism of NGR1 in regulating mitochondrial function and neuroinflammation after spinal cord injury in rats |
| Received:November 17, 2021 Revised:August 03, 2022 |
| English Keywords:Spinal cord injury Notoginsenoside R1 Nuclear factor E2-related factor 2 Heme oxygenase-1 Mitochondrial function Neuroinflammation Rat |
| Fund:福建医科大学启航基金项目（2019QH1305） |
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| English Abstract: |
| 【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. |
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