基于HMGB_1/TLR4/NF-κB信号通路考察木瓜提取物对脊髓损伤大鼠的保护作用
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摘要
目的:探讨木瓜醇提取物对脊髓损伤(spinal cord injury,SCI)大鼠神经功能的保护作用,并初步探讨其可能机制。方法:制备SCI大鼠模型,根据处理方式分为假手术组(Sham组),模型组(SCI组),木瓜提取物治疗组(Pap组);重组高迁移率族蛋白B1(high mobility group histone B1,HMGB_1)处理组(rHMGB_1组);重组HMGB_1+木瓜提取物处理组(rHMGB_1+Pap组)。应用basso beattie bresnahan(BBB)评分评估大鼠后肢运动功能;检测脊髓组织含水量评估脊髓水肿情况;应用苏木素-伊红(HE)染色进行组织学评估;运用酶联免疫吸附检测白细胞介素-1β(interleuki-1β,IL-1β),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α),白细胞介素-6(interleuki-6,IL-6)水平;原位末端转移酶标记法(TUNEL)检测脊髓组织细胞凋亡情况;应用免疫组织化学和蛋白免疫印迹法(Western blot)检测组织中HMGB_1,Toll样受体4(Toll-like receptors 4,TLR4),核转录因子κB(nuclear factor-κB,NF-κB)p65蛋白表达情况;Western blot检测半胱氨酸蛋白酶(Caspase)-3蛋白;应用实时荧光定量PCR(Real-time PCR)检测HMGB_1,TLR4,NF-κB p65 mRNA表达情况。结果:与Sham组比较,SCI组大鼠BBB评分明显降低(P<0.05)。SCI组和rHMGB_1组脊髓组织松散,呈现嗜中性粒细胞浸润的组织学损伤;而Pap组和rHMGB_1+Pap组中组织学损伤情况得到明显改善。与Sham组比较,SCI组大鼠脊髓组织中含水量,HMGB_1,TLR4,NF-κB p65 mRNA和蛋白表达水平,IL-1β,IL-6,TNF-α水平,凋亡阳性细胞数,Caspase-3蛋白表达水平均明显增加(P<0.05)。Pap组和rHMGB_1+Pap组含水量,HMGB_1,TLR4,NF-κB p65 mRNA和蛋白表达水平,IL-1β,IL-6,TNF-α水平,凋亡阳性细胞数,Caspase-3蛋白表达水平均明显低于SCI组(P<0.05)。rHMGB_1+Pap组含水量,HMGB_1,TLR4,NF-κB p65 mRNA和蛋白表达水平,IL-1β,IL-6,TNF-α水平,凋亡阳性细胞数,Caspase-3蛋白表达水平明显低于rHMGB_1组,差异均有统计学意义(P<0.05)。结论:木瓜提取物能够通过抑制HMGB_1/TLR4/NF-κB p65信号通路活化,减轻SCI后的炎症反应、减少脊髓组织细胞凋亡,从而诱导脊髓组织修复和运动功能恢复。
Objective: To investigate the protective effect of Chaenomelis Fructus extracts on neurological function in rats with spinal cord injury(SCI),and explore its possible mechanism. Method: SCI rat model was prepared and was divided into sham operation group(Sham group),model group(SCI group),Chaenomelis Fructus alcohol extracts treatment group(Pap group),recombinant high mobility group histone B1(HMGB_1)treatment group(rHMGB_1 group),and recombinant HMGB_1 + Chaenomelis Fructus extracts treatment group(rHMGB_1+ Pap group) according to the treatment methods. Rat hindlimb motor function was evaluated by Basso Beattie Bresnahan(BBB) score; water content in spinal cord tissue was detected to assess the spinal cord edema;Histological evaluation was performed by hematoxylin-eosin(HE) staining. The levels of interleuki-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interleuki-6(IL-6) were detected by enzyme-linked immunosorbent assay(ELISA). Terminal dexynucleotidyl transferase(Td T)-mediated d UTP nick end labeling(TUNEL) was used to detect the apoptosis of spinal cord cells. The protein expression levels of HMGB_1,Toll-like receptors 4(TLR4)and nuclear factor-κB(NF-κB) p65 were detected by immunohistochemistry and Western blot,and their mRNA expression levels were detected by real time fluorescence quantitative polymerase chain reaction(Real-time PCR).Cysteine aspartate-specific protease-3(Caspase-3) was detected by Western blot. Result: As compared with the Sham group,the BBB score of the SCI group was significantly lower(P < 0. 05); spinal cord tissue was loose in SCI group and rHMGB_1 group,showing histological damage of neutrophil infiltration; and histological damage was significantly improved in Pap group and rHMGB_1+ Pap group. As compared with Sham group,the water content in spinal cord tissue,mRNA and protein expression levels of HMGB_1,TLR4,NF-κB p65 mRNA and protein,and IL-1β, IL-6, TNF-α levels,count of apoptosis-positive cells,and Caspase-3 protein expression level were significantly increased in SCI group(P < 0. 05). The water content in spinal cord tissue,mRNA and protein expression levels of HMGB_1,TLR4,NF-κB p65 mRNA and protein,and IL-1β,IL-6,TNF-α levels,count of apoptosis-positive cells,and Caspase-3 protein expression level in Pap and rHMGB_1+ Pap group were significantly lower than the SCI group(P < 0. 05). The water content in spinal cord tissue,mRNA and protein expression levels of HMGB_1,TLR4,NF-κB p65 mRNA and protein,and IL-1β,IL-6,TNF-α levels,count of apoptotic-positive cells and Caspase-3 protein expression level in rHMGB_1+ Pap group were significantly lower than those in rHMGB_1 group,with statistically significant differences(P < 0. 05). Conclusion: Chaenomelis Fructus extracts can induce spinal cord tissue repair and motor function recovery by inhibiting the activation of HMGB_1,TLR4,NF-κB p65 signaling pathway,reducing the inflammatory response after SCI and reducing the apoptosis of spinal cord tissue.
Objective: To investigate the protective effect of Chaenomelis Fructus extracts on neurological function in rats with spinal cord injury(SCI),and explore its possible mechanism. Method: SCI rat model was prepared and was divided into sham operation group(Sham group),model group(SCI group),Chaenomelis Fructus alcohol extracts treatment group(Pap group),recombinant high mobility group histone B1(HMGB_1)treatment group(rHMGB_1 group),and recombinant HMGB_1 + Chaenomelis Fructus extracts treatment group(rHMGB_1+ Pap group) according to the treatment methods. Rat hindlimb motor function was evaluated by Basso Beattie Bresnahan(BBB) score; water content in spinal cord tissue was detected to assess the spinal cord edema;Histological evaluation was performed by hematoxylin-eosin(HE) staining. The levels of interleuki-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interleuki-6(IL-6) were detected by enzyme-linked immunosorbent assay(ELISA). Terminal dexynucleotidyl transferase(Td T)-mediated d UTP nick end labeling(TUNEL) was used to detect the apoptosis of spinal cord cells. The protein expression levels of HMGB_1,Toll-like receptors 4(TLR4)and nuclear factor-κB(NF-κB) p65 were detected by immunohistochemistry and Western blot,and their mRNA expression levels were detected by real time fluorescence quantitative polymerase chain reaction(Real-time PCR).Cysteine aspartate-specific protease-3(Caspase-3) was detected by Western blot. Result: As compared with the Sham group,the BBB score of the SCI group was significantly lower(P < 0. 05); spinal cord tissue was loose in SCI group and rHMGB_1 group,showing histological damage of neutrophil infiltration; and histological damage was significantly improved in Pap group and rHMGB_1+ Pap group. As compared with Sham group,the water content in spinal cord tissue,mRNA and protein expression levels of HMGB_1,TLR4,NF-κB p65 mRNA and protein,and IL-1β, IL-6, TNF-α levels,count of apoptosis-positive cells,and Caspase-3 protein expression level were significantly increased in SCI group(P < 0. 05). The water content in spinal cord tissue,mRNA and protein expression levels of HMGB_1,TLR4,NF-κB p65 mRNA and protein,and IL-1β,IL-6,TNF-α levels,count of apoptosis-positive cells,and Caspase-3 protein expression level in Pap and rHMGB_1+ Pap group were significantly lower than the SCI group(P < 0. 05). The water content in spinal cord tissue,mRNA and protein expression levels of HMGB_1,TLR4,NF-κB p65 mRNA and protein,and IL-1β,IL-6,TNF-α levels,count of apoptotic-positive cells and Caspase-3 protein expression level in rHMGB_1+ Pap group were significantly lower than those in rHMGB_1 group,with statistically significant differences(P < 0. 05). Conclusion: Chaenomelis Fructus extracts can induce spinal cord tissue repair and motor function recovery by inhibiting the activation of HMGB_1,TLR4,NF-κB p65 signaling pathway,reducing the inflammatory response after SCI and reducing the apoptosis of spinal cord tissue.
引文
[1]吕威.脉冲电针对脊髓损伤大鼠受损局部Mash1,Neuro D2表达的影响[D].北京:北京中医药大学,2017.
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[4]戴晨.弱激光对骨髓来源巨噬细胞极化水平的调控研究及对大鼠脊髓损伤的影响[D].西安:第四军医大学,2016.
[5]尹继庭,姜力,姜永新,等.白花木瓜营养成分分析[J].营养学报,2013,35(2):201-202.
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[9]况作品,周亚菁,谢晓梅,等.高效液相色谱法测定木瓜水溶性有机酸含量的样品处理方法研究[J].安徽医药,2016,20(1):54-57.
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[11]高梦丹,林敬铨,童亮,等.姜黄素抑制NF-κB信号通路对脊髓损伤修复作用的研究进展[J].中国药理学与毒理学杂志,2016,30(3):272-277.
[12]张芬.MicroRNA-124a对SCI继发性损伤的影响及相关机制研究[J].郑州大学学报:理学版,2016,48(1):73-80.
[13]WANG T L,GU B,LI H N,et al.Inflammatory response and anti-inflammatory therapy after acute spinal cord injury[J].Chin Pharmacol Bull,2015,31(4):452-457.
[14]XU J,E X Q,LIU H Y,et al.Angelica sinensis attenuates inflammatory reaction in experimental rat models having spinal cord injury[J].Int J Clin Exp Pathol,2015,8(6):6779-6785.
[15]LI L,YAN L,MIN H,et al.Heparin inhibits the inflammatory response induced by LPS and HMGB1 by blocking the binding of HMGB1 to the surface of macrophages[J].Cytokine,2015,72(1):36-42.
[16]WANGY S,LI Y Y,WANG L H,et al.Tanshinone IIA attenuates chronic pancreatitis-induced pain in rats via downregulation of HMGB1 and TRL4 expression in the spinal cord[J].Pain Physician,2015,18(4):E615.
[17]MA B,Hottiger M O.Crosstalk between Wnt/β-catenin and NF-κB signaling pathway during inflammation[J].Front Immunol,2016,7(3):378-388.
[18]WANG Y,SUN L,SONG Z,et al.Maspin inhibits macrophage phagocytosis and enhances inflammatory cytokine production via activation of NF-κB signaling[J].Mol Immunol,2017,82(12):94-103.
[19]Maeda T,Ozaki M,Kobayashi Y,et al.HMGB1 as a potential therapeutic target for neuropathic pain[J].J Pharmacol Sci,2013,123(4):301-305.
[20]KANG N,HAI Y,YANG J,et al.Hyperbaric oxygen intervention reduces secondary spinal cord injury in rats via regulation of HMGB1/TLR4/NF-κB signaling pathway[J].Int J Clin Exp Pathol,2015,8(2):1141-1153.
[21]田慧群,崔倩倩,任东明,等.木瓜提取物的抗炎活性研究[J].华西药学杂志,2015,30(3):287-288.
[22]吕静波,陈强.少突胶质细胞凋亡与脑白质脱髓鞘病变发病机制关系的研究进展[J].浙江医学,2014,36(23):1965-1968.
[23]韩晓三,梁高林.自组装纳米荧光探针用于Caspase-3的检测[J].中国科学技术大学学报,2015,45(8):643-648.
[24]SUN L,CHEN S,GAO H,et al.Visfatin induces the apoptosis of endothelial progenitor cells via the induction of pro-inflammatory mediators through the NF-κB pathway[J].Int J Mol Med,2017,40(3):637-646.
[2]李顺国,王猛.继发性脊髓损伤的药物治疗进展[J].现代医药卫生,2015,31(3):368-371.
[3]ZHAO H,CHENG L,DU X,et al.Transplantation of cerebral dopamine neurotrophic factor transducted BMSCs in contusion spinal cord injury of rats:promotion of nerve regeneration by alleviating neuroinflammation[J].Mol Neurobiol,2016,53(1):187-199.
[4]戴晨.弱激光对骨髓来源巨噬细胞极化水平的调控研究及对大鼠脊髓损伤的影响[D].西安:第四军医大学,2016.
[5]尹继庭,姜力,姜永新,等.白花木瓜营养成分分析[J].营养学报,2013,35(2):201-202.
[6]周丽娜,叶文博.野木瓜注射液及其提取物对脊髓神经元的氧化保护和生长促进作用[J].上海师范大学学报:自然科学版,2011,40(5):540-545.
[7]LIU D,HUANG Y,JIA C,et al.Administration of antagomir-223 inhibits apoptosis,promotes angiogenesis and functional recovery in rats with spinal cord injury[J].Cell Mol Neurobiol,2015,35(4):1-9.
[8]向婷婷,霍元秀,刘朝奇,等.木瓜提取物对脂肪性肝纤维化的预防保护作用及机制研究[J].中药新药与临床药理,2015,26(4):489-494.
[9]况作品,周亚菁,谢晓梅,等.高效液相色谱法测定木瓜水溶性有机酸含量的样品处理方法研究[J].安徽医药,2016,20(1):54-57.
[10]李娜,金敬红,姜洪芳,等.宣木瓜总有机酸的纯化及镇痛抗炎作用[J].中国实验方剂学杂志,2011,17(1):113-116.
[11]高梦丹,林敬铨,童亮,等.姜黄素抑制NF-κB信号通路对脊髓损伤修复作用的研究进展[J].中国药理学与毒理学杂志,2016,30(3):272-277.
[12]张芬.MicroRNA-124a对SCI继发性损伤的影响及相关机制研究[J].郑州大学学报:理学版,2016,48(1):73-80.
[13]WANG T L,GU B,LI H N,et al.Inflammatory response and anti-inflammatory therapy after acute spinal cord injury[J].Chin Pharmacol Bull,2015,31(4):452-457.
[14]XU J,E X Q,LIU H Y,et al.Angelica sinensis attenuates inflammatory reaction in experimental rat models having spinal cord injury[J].Int J Clin Exp Pathol,2015,8(6):6779-6785.
[15]LI L,YAN L,MIN H,et al.Heparin inhibits the inflammatory response induced by LPS and HMGB1 by blocking the binding of HMGB1 to the surface of macrophages[J].Cytokine,2015,72(1):36-42.
[16]WANGY S,LI Y Y,WANG L H,et al.Tanshinone IIA attenuates chronic pancreatitis-induced pain in rats via downregulation of HMGB1 and TRL4 expression in the spinal cord[J].Pain Physician,2015,18(4):E615.
[17]MA B,Hottiger M O.Crosstalk between Wnt/β-catenin and NF-κB signaling pathway during inflammation[J].Front Immunol,2016,7(3):378-388.
[18]WANG Y,SUN L,SONG Z,et al.Maspin inhibits macrophage phagocytosis and enhances inflammatory cytokine production via activation of NF-κB signaling[J].Mol Immunol,2017,82(12):94-103.
[19]Maeda T,Ozaki M,Kobayashi Y,et al.HMGB1 as a potential therapeutic target for neuropathic pain[J].J Pharmacol Sci,2013,123(4):301-305.
[20]KANG N,HAI Y,YANG J,et al.Hyperbaric oxygen intervention reduces secondary spinal cord injury in rats via regulation of HMGB1/TLR4/NF-κB signaling pathway[J].Int J Clin Exp Pathol,2015,8(2):1141-1153.
[21]田慧群,崔倩倩,任东明,等.木瓜提取物的抗炎活性研究[J].华西药学杂志,2015,30(3):287-288.
[22]吕静波,陈强.少突胶质细胞凋亡与脑白质脱髓鞘病变发病机制关系的研究进展[J].浙江医学,2014,36(23):1965-1968.
[23]韩晓三,梁高林.自组装纳米荧光探针用于Caspase-3的检测[J].中国科学技术大学学报,2015,45(8):643-648.
[24]SUN L,CHEN S,GAO H,et al.Visfatin induces the apoptosis of endothelial progenitor cells via the induction of pro-inflammatory mediators through the NF-κB pathway[J].Int J Mol Med,2017,40(3):637-646.