非抗凝肝素衍生物ROH对脓毒症小鼠脑损伤的保护作用及机制研究
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摘要
目的研究非抗凝肝素衍生物——ROH对脂多糖(LPS)诱导的脓毒症小鼠脑损伤的保护作用及机制。方法按数表法将Balb/c小鼠随机分为4组:正常组、模型组、对照组和实验组。小鼠腹腔注射LPS 10 mg·kg~(-1)建立脓毒症急性脑损伤模型。对照组和实验组在造模前15 min,分别尾静脉注射肝素2. 5mg·kg~(-1)、ROH 7. 5 mg·kg~(-1)进行干预,正常组注射等体积0. 9%Na Cl。以免疫组化法检测脑组织乙酰肝素酶(HPA)和金属基质蛋白酶-9(MMP-9)表达水平;伊文思蓝染色法检测血脑屏障通透性; TBA法测定脑组织中丙二醛(MDA)的含量。结果正常组、模型组、对照组和实验组的HPA表达水平(光密度值)分别为(8. 0±8. 0)×10~(-5),(3. 0±1. 8)×10~(-3),(2. 3±1. 1)×10~(-4)和(5. 4±3. 3)×10~(-4);上述这4组的MMP-9表达水平(光密度值)分别为(8. 0±8. 0)×10~(-5),(3. 6±1. 8)×10~(-3),(3. 9±1. 4)×10~(-4)和(4. 9±3. 3)×10~(-4);上述这4组的脑内伊文思蓝的含量分别为(4. 36±1. 58),(78. 60±14. 75),(36. 28±9. 16)和(44. 09±8. 95)μg·g~(-1);上述这4组的MDA含量分别为(6. 91±1. 73),(10. 88±2. 08),(6. 68±1. 58)和(5. 41±1. 50)nmol·mL~(-1)。模型组与正常组相比,上述指标的差异均有统计学意义(P <0. 05,P <0. 01);对照组和实验组与模型组相比,上述指标的差异均有统计学意义(P <0. 05,P <0. 01)。结论 ROH与肝素一样,通过抑制脓毒症时LPS所致的HPA和MMP-9高表达,保护内皮糖萼的完整性,从而发挥脑保护作用,并提高脓毒症小鼠的生存率。
Objective To study the protective effect and mechanism of non-anticoagulated heparin derivatives reduced oxyheparin( ROH) on brain damage induced by lipopolysaccharide( LPS) in septic mice.Methods The Balb/c mice were randomly divided into 4 groups: normal group,model group,control group,and experimental group. LPS( 10 mg · kg~(-1)) was injected by intraperitoneal injection to establish acute brain injury septic mice model. Heparin( 2. 5 mg · kg~(-1)) and ROH( 7. 5 mg·kg~(-1)) were respectively injected by intravenous injention in control group and experimental group at the time of 15 min before modeling,and normal group was injected with the same volume of normal saline. Immunohistochemistry was used to detect the expression of heparanase( HPA) and matrix metalloproteinase-9( MMP-9) in brain tissue. Evans blue staining was used to detect blood-brain barrier permeability. TBA method was used to measure the content of dialdehyde( MDA) in brain tissue. Results The expression level( optical density,OD) of HPA in normal group,model group,control group and experimental group were( 8. 0 ± 8. 0) × 10~(-5),( 3. 0 ± 1. 8) × 10~(-3),( 2. 3 ± 1. 1) × 10~(-4),and( 5. 4 ± 3. 3) × 10~(-4); the expression level( OD) of MMP-9 in these four groups were( 8. 0 ± 8. 0) × 10~(-5),( 3. 6 ± 1. 8) × 10~(-3),( 3. 9 ± 1. 4) × 10~(-4),and( 4. 9 ± 3. 3) × 10~(-4); the contents of evans blue in these four groups were( 4. 36 ± 1. 58),( 78. 60 ± 14. 75),( 36. 28 ± 9. 16),and( 44. 09 ± 8. 95) μg·g~(-1); the concentrations of MDA in these four groups were( 6. 91 ± 1. 73),( 10. 88 ± 2. 08),( 6. 68 ± 1. 58),( 5. 41 ± 1. 50)nmol·m L~(-1). Comparison between model group and normal group,the difference of the factors were significantly( P < 0. 05,P < 0. 01); comparison between experimental group and control group with model group,the difference of the factors were significantly( P < 0. 05,P < 0. 01). Conclusion ROH,like heparin,protects the integrity of endothelial glycosides by inhibiting the high expression of HPA and MMP-9 induced by LPS in sepsis,thereby exerting brain protection and improving the survival rate of septic mice.
Objective To study the protective effect and mechanism of non-anticoagulated heparin derivatives reduced oxyheparin( ROH) on brain damage induced by lipopolysaccharide( LPS) in septic mice.Methods The Balb/c mice were randomly divided into 4 groups: normal group,model group,control group,and experimental group. LPS( 10 mg · kg~(-1)) was injected by intraperitoneal injection to establish acute brain injury septic mice model. Heparin( 2. 5 mg · kg~(-1)) and ROH( 7. 5 mg·kg~(-1)) were respectively injected by intravenous injention in control group and experimental group at the time of 15 min before modeling,and normal group was injected with the same volume of normal saline. Immunohistochemistry was used to detect the expression of heparanase( HPA) and matrix metalloproteinase-9( MMP-9) in brain tissue. Evans blue staining was used to detect blood-brain barrier permeability. TBA method was used to measure the content of dialdehyde( MDA) in brain tissue. Results The expression level( optical density,OD) of HPA in normal group,model group,control group and experimental group were( 8. 0 ± 8. 0) × 10~(-5),( 3. 0 ± 1. 8) × 10~(-3),( 2. 3 ± 1. 1) × 10~(-4),and( 5. 4 ± 3. 3) × 10~(-4); the expression level( OD) of MMP-9 in these four groups were( 8. 0 ± 8. 0) × 10~(-5),( 3. 6 ± 1. 8) × 10~(-3),( 3. 9 ± 1. 4) × 10~(-4),and( 4. 9 ± 3. 3) × 10~(-4); the contents of evans blue in these four groups were( 4. 36 ± 1. 58),( 78. 60 ± 14. 75),( 36. 28 ± 9. 16),and( 44. 09 ± 8. 95) μg·g~(-1); the concentrations of MDA in these four groups were( 6. 91 ± 1. 73),( 10. 88 ± 2. 08),( 6. 68 ± 1. 58),( 5. 41 ± 1. 50)nmol·m L~(-1). Comparison between model group and normal group,the difference of the factors were significantly( P < 0. 05,P < 0. 01); comparison between experimental group and control group with model group,the difference of the factors were significantly( P < 0. 05,P < 0. 01). Conclusion ROH,like heparin,protects the integrity of endothelial glycosides by inhibiting the high expression of HPA and MMP-9 induced by LPS in sepsis,thereby exerting brain protection and improving the survival rate of septic mice.
引文
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[3]CASU B,GUERRINI M,GUGLIERI S,et al.Undersulfated and glycol-split heparins endowed with antiangiogenic activity[J].JMed Chem,2004,47(4):838-848.
[4]ZHOU W,WANG J,LI Z,et al.MicroRNA-205-5b inhibits HMGB1 expression in LPS-induced sepsis[J].Int J Mol Med,2016,38(1):312-318.
[5]王长安.乙酰肝素酶和基质金属蛋白酶-9在非小细胞肺癌中的表达及临床意义[D].四川泸州:泸州医学院,2012.
[6]GARSEN M,ROPS M,RABELINK T J,et al.The role of heparanase and the endothelial glycocalyx in the development of proteinuria[J].Nephrol Dialysis Transplantat,2014,29(1):49-55.
[7]梁薇,谭年花,潘子怡,等.益智仁-乌药醚提取物对糖尿病肾病氧化应激Mn-SOD、MDA影响实验研究[J].山西中医学院学报,2016,17(3):20-22.
[8]郭阿茹,马莉,李杜鹏,等.大黄对脓毒症相关大鼠脑功能障碍的作用机制研究[J].中国急救医学,2017,37(10):878-883.
[9]MCKENZIE E.Heparanase:a target for drug discovery in cancer and inflammation[J].Br J Pharmacol,2010,151(1):1-14.
[10]谢志超,廖雪莲,康焰.脓毒症相关性脑病的诊疗与研究新进展[J].中国感染与化疗杂志,2015,15(6):609-613.
[11]RAMNATH R,FOSTER R R,QIU Y,et al.Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor:a contributor to endothelial cell glycocalyx dysfunction[J].FASEB J,2014,28(11):4686-4699.