胡椒叶提取物干预可减轻急性脊髓损伤模型大鼠的氧化应激及炎症反应
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摘要
背景:研究表明胡椒叶提取物对链脲佐菌素诱导糖尿病大鼠有明确的保护作用,作用机制与抗氧化损伤有关,但其是否能通过抗氧化应激减轻急性脊髓损伤还未见报道。目的:探究胡椒叶提取物对急性脊髓损伤大鼠氧化应激、炎症反应及神经损伤的影响。方法:将100只SD大鼠(购自成都达硕实验动物公司)随机分为假手术组、脊髓损伤组及胡椒叶提取物低、中、高剂量组,每组20只。除假手术组外,其余组大鼠采用改良Allen’s法复制大鼠急性脊髓损伤模型,造模后,胡椒叶提取物低、中、高剂量组灌胃给予100,200,400mg/(kg·d)的胡椒叶提取物,连续给药28 d。给药后3,7,14,28 d后进行BBB评分和斜板实验;给药后28 d,检测血清超氧化物歧化酶、谷胱甘肽过氧化物酶、丙二醛、白细胞介素1β、白细胞介素6和肿瘤坏死因子α水平,免疫印迹检测Bcl-2、Bax、Caspase-3、脑源性神经营养因子和神经/胶质细胞抗原、IκBα、p-IκBα和NF-κB p65蛋白表达,同时进行脊髓组织苏木精-伊红染色与TUNEL染色。实验方案经海南大学热带农林学院实验动物伦理委员会批准(NR001803)。结果与结论:(1)与假手术组比较,脊髓损伤组给药后不同时间点的BBB评分和斜板实验最大倾斜角度降低(P <0.01);与脊髓损伤组比较,胡椒叶提取物中、高剂量组给药后7,14,28 d的BBB评分和斜板实验最大倾斜角度升高(P <0.05,P <0.01);(2)与假手术组比较,脊髓损伤组脊髓组织损伤严重,细胞凋亡率、Bax、Caspase-3、丙二醛、白细胞介素1β、白细胞介素6、肿瘤坏死因子α、p-IκBα、NF-κB p65、脑源性神经营养因子和神经/胶质细胞抗原表达升高(P <0.05,P <0.01),Bcl-2、IκBα、超氧化物歧化酶、谷胱甘肽过氧化物酶表达降低(P <0.05,P <0.01);与脊髓损伤组比较,胡椒叶提取物低、中、高剂量组脊髓组织损伤减轻,细胞凋亡率、Bax、Caspase-3、丙二醛、白细胞介素1β、白细胞介素6、肿瘤坏死因子α、p-IκBα、NF-κB p65、脑源性神经营养因子和神经/胶质细胞抗原表达降低(P <0.05,P <0.01),Bcl-2、IκBα、超氧化物歧化酶、谷胱甘肽过氧化物酶表达升高(P <0.05,P <0.01);(3)结果表明,胡椒叶提取物可通过抑制急性脊髓损伤大鼠炎症反应及氧化应激减轻脊髓损伤并发挥神经保护作用,作用机制可能与抑制NF-κB信号通路激活有关。
BACKGROUND: Extract of piper auritum plays a protective role in streptozotocin-induced diabetic rats, and the mechanism is associated with anti-oxidative damage. However, whether it can alleviate acute spinal cord injury by anti-oxidative stress is unknown.OBJECTIVE: To investigate the effects of extract of piper auritum on oxidative stress, inflammation and nerve injury of acute spinal cord injury rats.METHODS: One hundred rats(provided by Chengdu Dashuo Experimental Animal Company) were randomly divided into sham group, spinal cord injury group, and low-, moderate-, and high-dose extract of piper auritum groups. Except for sham group, the rats in the other groups were used to establish acute spinal cord injury model by Allen's method. Afterwards, the rats in the extract of piper auritum groups were given100, 200 and 400 mg/(kg·d) extract of piper auritum via gavage, respectively, for 28 consecutive days. Basso, Beattie Bresnahan scores and tilt board experiment were carried out at 3, 7, 14 and 28 days after administration. The concentrations of superoxide dismutase, glutathione peroxidase, malondialdehyde, interleukin-1β, interleukin-6 and tumor necrosis factor-α in serum were measured by ELISA. The protein levels of Bcl-2, Bax, Caspase-3, brain-derived neurotrophic factor, neuronal/glial cell antigen, IκBα, p-IκBα and NF-κB p65 were detected by western blot assay. Spinal cord tissues underwent hematoxylin-eosin staining and TUNEL staining. The study was approved by the Laboratory Animal Ethics Committee of Institute of Tropical Agriculture and Forestry, Hainan University, approval No. NR001803.RESULTS AND CONCLUSION:(1) Compared with the sham group, the Basso, Beattie Bresnahan scores and the maximal title angle in tilt board experiment in the spinal cord injury group were significantly decreased at different time points after administration(P < 0.01). Compared with the spinal cord injury group, the Basso, Beattie Bresnahan scores and the maximal title angle in tilt board experiment in the moderateand high-dose extract of piper auritum groups were significantly at 7, 14 and 28 days after administration(P < 0.05, P < 0.01).(2) Compared with the sham group, the spinal cord injury was more serious in the spinal cord injury group, the apoptosis rate, the levels of Bax, Caspase-3,malondialdehyde, interleukin-1β, interleukin-6 and tumor necrosis factor-α, p-IκBα, NF-κB p65, brain-derived neurotrophic factor, and neuronal/glial cell antigen were significantly increased(P < 0.05, P < 0.01), and the levels of Bcl-2, IκBα, superoxide dismutase, and glutathione peroxidase were significantly decreased(P < 0.05, P < 0.01). Compared with the spinal cord injury group, the degree of spinal cord injury in each extract of piper auritum group was alleviated, the apoptosis rate, the levels of Bax, Caspase-3, malondialdehyde,interleukin-1β, interleukin-6 and tumor necrosis factor-α, p-IκBα, NF-κB p65, brain-derived neurotrophic factor, and neuronal/glial cell antigen were significantly decreased(P < 0.05, P < 0.01), and the levels of Bcl-2, IκBα, superoxide dismutase, and glutathione peroxidase were significantly increased(P < 0.05, P < 0.01).(3) These findings imply that extract of piper auritum can attenuate acute spinal cord injury by inhibiting inflammation and oxidative stress and also plays a neuroprotective role in spinal cord injury, and the mechanism may be related to inhibit the activation of NF-κB signaling pathway.
BACKGROUND: Extract of piper auritum plays a protective role in streptozotocin-induced diabetic rats, and the mechanism is associated with anti-oxidative damage. However, whether it can alleviate acute spinal cord injury by anti-oxidative stress is unknown.OBJECTIVE: To investigate the effects of extract of piper auritum on oxidative stress, inflammation and nerve injury of acute spinal cord injury rats.METHODS: One hundred rats(provided by Chengdu Dashuo Experimental Animal Company) were randomly divided into sham group, spinal cord injury group, and low-, moderate-, and high-dose extract of piper auritum groups. Except for sham group, the rats in the other groups were used to establish acute spinal cord injury model by Allen's method. Afterwards, the rats in the extract of piper auritum groups were given100, 200 and 400 mg/(kg·d) extract of piper auritum via gavage, respectively, for 28 consecutive days. Basso, Beattie Bresnahan scores and tilt board experiment were carried out at 3, 7, 14 and 28 days after administration. The concentrations of superoxide dismutase, glutathione peroxidase, malondialdehyde, interleukin-1β, interleukin-6 and tumor necrosis factor-α in serum were measured by ELISA. The protein levels of Bcl-2, Bax, Caspase-3, brain-derived neurotrophic factor, neuronal/glial cell antigen, IκBα, p-IκBα and NF-κB p65 were detected by western blot assay. Spinal cord tissues underwent hematoxylin-eosin staining and TUNEL staining. The study was approved by the Laboratory Animal Ethics Committee of Institute of Tropical Agriculture and Forestry, Hainan University, approval No. NR001803.RESULTS AND CONCLUSION:(1) Compared with the sham group, the Basso, Beattie Bresnahan scores and the maximal title angle in tilt board experiment in the spinal cord injury group were significantly decreased at different time points after administration(P < 0.01). Compared with the spinal cord injury group, the Basso, Beattie Bresnahan scores and the maximal title angle in tilt board experiment in the moderateand high-dose extract of piper auritum groups were significantly at 7, 14 and 28 days after administration(P < 0.05, P < 0.01).(2) Compared with the sham group, the spinal cord injury was more serious in the spinal cord injury group, the apoptosis rate, the levels of Bax, Caspase-3,malondialdehyde, interleukin-1β, interleukin-6 and tumor necrosis factor-α, p-IκBα, NF-κB p65, brain-derived neurotrophic factor, and neuronal/glial cell antigen were significantly increased(P < 0.05, P < 0.01), and the levels of Bcl-2, IκBα, superoxide dismutase, and glutathione peroxidase were significantly decreased(P < 0.05, P < 0.01). Compared with the spinal cord injury group, the degree of spinal cord injury in each extract of piper auritum group was alleviated, the apoptosis rate, the levels of Bax, Caspase-3, malondialdehyde,interleukin-1β, interleukin-6 and tumor necrosis factor-α, p-IκBα, NF-κB p65, brain-derived neurotrophic factor, and neuronal/glial cell antigen were significantly decreased(P < 0.05, P < 0.01), and the levels of Bcl-2, IκBα, superoxide dismutase, and glutathione peroxidase were significantly increased(P < 0.05, P < 0.01).(3) These findings imply that extract of piper auritum can attenuate acute spinal cord injury by inhibiting inflammation and oxidative stress and also plays a neuroprotective role in spinal cord injury, and the mechanism may be related to inhibit the activation of NF-κB signaling pathway.
引文
[1]Zeng HH,Huang YR,Li ZJ,et al.Effects of emodin on oxidative stress and inflammatory response in rats with acute spinal cord injury.Zhongguo Zhong Yao Za Zhi.2018;43(9):1886-1893.
[2]Moon YJ,Lee JY,Oh MS,et al.Inhibition of inflammation and oxidative stress by Angelica dahuricae radix extract decreases apoptotic cell death and improves functional recovery after spinal cord injury.J Neurosci Res.2012;90(1):243-256.
[3]Paterniti I,Impallizzeri D,Di Paola R,et al.Docosahexaenoic acid attenuates the early inflammatory response following spinal cord injury in mice:in-vivo and in-vitro studies.JNeuroinflammation.2014;11:6.
[4]Dietz V,Fouad K.Restoration of sensorimotor functions after spinal cord injury.Brain.2014;137:654-667.
[5]Coll-MiróM,Francos-Quijorna I,Santos-Nogueira E,et al.Beneficial effects of IL-37 after spinal cord injury in mice.Proc Natl Acad Sci U S A.2016;113(5):1411-1416.
[6]Zheng H,Jia L,Liu CC,et al.TREM2 Promotes Microglial Survival by Activating Wnt/β-Catenin Pathway.J Neurosci.2017;37(7):1772-1784.
[7]Sun Y,Gong F,Yin J,et al.Therapeutic effect of apocynin through antioxidant activity and suppression of apoptosis and inflammation after spinal cord injury.Exp Ther Med.2017;13(3):952-960.
[8]Gutierrez RMP.Effect of the hexane extract of Piper auritum on insulin release fromβ-cell and oxidative stress in streptozotocin-induced diabetic rat.Pharmacogn Mag.2012;8(32):308-313.
[9]Gonzalez AM,Gutierrez RM,Cotera LB.Antidiabetic activity of Piper auritum leaves in streptozotocin-induced diabetic rat,beneficial effect on advanced glycation endproduct.Chin JIntegr Med.2014.[Epub ahead of print]
[10]郑利强,伍亚民,石永江,等.黄芪多糖对大鼠脊髓损伤后运动功能和脊髓病理的效果[J].中国康复理论与实践,2016,22(11):1269-1275.
[11]Zhao H,hen S,Gao K,et al.Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway.Neuroscience.2017;348:241-251.
[12]Ahuja CS,Nori S,Tetreault L,et al.Traumatic Spinal Cord Injury-Repair and Regeneration.Neurosurgery.2017;80(3S):S9-S22.
[13]Sun X,Jones ZB,Chen XM,et al.Multiple organ dysfunction and systemic inflammation after spinal cord injury:a complex relationship.J Neuroinflammation.2016;13(1):260.
[14]Hazekawa M,Hideshima Y,Ono K,et al.Anti-inflammatory effects of water extract from bell pepper(Capsicum annuum L.var.grossum)leaves in vitro.Exp Ther Med.2017;14(5):4349-4355.
[15]Agbor GA,Vinson JA,Oben JE,et al.In vitro antioxidant activity of three Piper species.J Herb Pharmacother.2007;7(2):49-64.
[16]Parker D,McClelland TJ.Neuromodulator interactions and spinal cord injury in lamprey.Neural Regen Res.2018;13(4):643-644.
[17]Seo JY,Kim YH,Kim JW,et al.Effects of Therapeutic Hypothermia on Apoptosis and Autophagy following Spinal Cord Injury in Rats.Spine.2015;40(12):883-890.
[18]Bahney J,Von CB.The Cellular Composition and Glia-Neuron Ratio in the Spinal Cord of a Human and a Non-Human Primate:Comparison with other Species and Brain Regions.Anat Rec.2018;301(4):697-710.
[19]Li H,Jia Z,Li G,et al.Neuroprotective effects of exendin-4 in rat model of spinal cord injury via inhibiting mitochondrial apoptotic pathway.Int J Clin Exp Pathol.2015;8(5):4837-4843.
[20]Xia P,Gao X,Duan L,et al.Mulberrin(Mul)reduces spinal cord injury(SCI)-induced apoptosis,inflammation and oxidative stress in rats via miroRNA-337 by targeting Nrf-2.Biomed Pharmacother.2018;107:1480-1487.
[21]Liu JT,Zhang S,Gu B,et al.Methotrexate combined with methylprednisolone for the recovery of motor function and differential gene expression in rats with spinal cord injury.Neural Regen Res.2017;12(9):1507-1518.
[22]Tanaka T,Kai S,Matsuyama T,et al.General anesthetics inhibit LPS-induced IL-1βexpression in glial cells.PLoS One.2013;8(12):e82930.
[23]Zhang W,Cheng L,Hou Y,et al.Plumbagin Protects Against Spinal Cord Injury-induced Oxidative Stress and Inflammation in Wistar Rats through Nrf-2 Upregulation.Drug Res.2015;65(09):495-499.
[24]Hirata N,Naruto S,Inaba K,et al.Histamine release inhibitory activity of Piper nigrum leaf.Biol Pharm Bull.2008;31(10):1973-1976.
[25]Tan Y,Yu L,Zhang C,et al.miRNA-146a attenuates inflammation in an in vitro spinal cord injury model via inhibition of TLR4 signaling.Exp Ther Med.2018;16(4):3703-3709.
[2]Moon YJ,Lee JY,Oh MS,et al.Inhibition of inflammation and oxidative stress by Angelica dahuricae radix extract decreases apoptotic cell death and improves functional recovery after spinal cord injury.J Neurosci Res.2012;90(1):243-256.
[3]Paterniti I,Impallizzeri D,Di Paola R,et al.Docosahexaenoic acid attenuates the early inflammatory response following spinal cord injury in mice:in-vivo and in-vitro studies.JNeuroinflammation.2014;11:6.
[4]Dietz V,Fouad K.Restoration of sensorimotor functions after spinal cord injury.Brain.2014;137:654-667.
[5]Coll-MiróM,Francos-Quijorna I,Santos-Nogueira E,et al.Beneficial effects of IL-37 after spinal cord injury in mice.Proc Natl Acad Sci U S A.2016;113(5):1411-1416.
[6]Zheng H,Jia L,Liu CC,et al.TREM2 Promotes Microglial Survival by Activating Wnt/β-Catenin Pathway.J Neurosci.2017;37(7):1772-1784.
[7]Sun Y,Gong F,Yin J,et al.Therapeutic effect of apocynin through antioxidant activity and suppression of apoptosis and inflammation after spinal cord injury.Exp Ther Med.2017;13(3):952-960.
[8]Gutierrez RMP.Effect of the hexane extract of Piper auritum on insulin release fromβ-cell and oxidative stress in streptozotocin-induced diabetic rat.Pharmacogn Mag.2012;8(32):308-313.
[9]Gonzalez AM,Gutierrez RM,Cotera LB.Antidiabetic activity of Piper auritum leaves in streptozotocin-induced diabetic rat,beneficial effect on advanced glycation endproduct.Chin JIntegr Med.2014.[Epub ahead of print]
[10]郑利强,伍亚民,石永江,等.黄芪多糖对大鼠脊髓损伤后运动功能和脊髓病理的效果[J].中国康复理论与实践,2016,22(11):1269-1275.
[11]Zhao H,hen S,Gao K,et al.Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway.Neuroscience.2017;348:241-251.
[12]Ahuja CS,Nori S,Tetreault L,et al.Traumatic Spinal Cord Injury-Repair and Regeneration.Neurosurgery.2017;80(3S):S9-S22.
[13]Sun X,Jones ZB,Chen XM,et al.Multiple organ dysfunction and systemic inflammation after spinal cord injury:a complex relationship.J Neuroinflammation.2016;13(1):260.
[14]Hazekawa M,Hideshima Y,Ono K,et al.Anti-inflammatory effects of water extract from bell pepper(Capsicum annuum L.var.grossum)leaves in vitro.Exp Ther Med.2017;14(5):4349-4355.
[15]Agbor GA,Vinson JA,Oben JE,et al.In vitro antioxidant activity of three Piper species.J Herb Pharmacother.2007;7(2):49-64.
[16]Parker D,McClelland TJ.Neuromodulator interactions and spinal cord injury in lamprey.Neural Regen Res.2018;13(4):643-644.
[17]Seo JY,Kim YH,Kim JW,et al.Effects of Therapeutic Hypothermia on Apoptosis and Autophagy following Spinal Cord Injury in Rats.Spine.2015;40(12):883-890.
[18]Bahney J,Von CB.The Cellular Composition and Glia-Neuron Ratio in the Spinal Cord of a Human and a Non-Human Primate:Comparison with other Species and Brain Regions.Anat Rec.2018;301(4):697-710.
[19]Li H,Jia Z,Li G,et al.Neuroprotective effects of exendin-4 in rat model of spinal cord injury via inhibiting mitochondrial apoptotic pathway.Int J Clin Exp Pathol.2015;8(5):4837-4843.
[20]Xia P,Gao X,Duan L,et al.Mulberrin(Mul)reduces spinal cord injury(SCI)-induced apoptosis,inflammation and oxidative stress in rats via miroRNA-337 by targeting Nrf-2.Biomed Pharmacother.2018;107:1480-1487.
[21]Liu JT,Zhang S,Gu B,et al.Methotrexate combined with methylprednisolone for the recovery of motor function and differential gene expression in rats with spinal cord injury.Neural Regen Res.2017;12(9):1507-1518.
[22]Tanaka T,Kai S,Matsuyama T,et al.General anesthetics inhibit LPS-induced IL-1βexpression in glial cells.PLoS One.2013;8(12):e82930.
[23]Zhang W,Cheng L,Hou Y,et al.Plumbagin Protects Against Spinal Cord Injury-induced Oxidative Stress and Inflammation in Wistar Rats through Nrf-2 Upregulation.Drug Res.2015;65(09):495-499.
[24]Hirata N,Naruto S,Inaba K,et al.Histamine release inhibitory activity of Piper nigrum leaf.Biol Pharm Bull.2008;31(10):1973-1976.
[25]Tan Y,Yu L,Zhang C,et al.miRNA-146a attenuates inflammation in an in vitro spinal cord injury model via inhibition of TLR4 signaling.Exp Ther Med.2018;16(4):3703-3709.