线粒体靶向抗氧化肽SS-31对脓毒症大鼠血管通透性的保护作用
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摘要
目的探讨线粒体靶向抗氧化肽SS-31对脓毒症大鼠血管通透性的影响及其机制。方法40只12周龄SD大鼠(雌雄各半,体质量约220 g)分成5组(n=8):假手术组(只开腹,不结扎盲肠和穿孔)、脓毒症组(盲肠结扎穿孔术制作脓毒症模型)、常规治疗组(conventional treatment,CT组,脓毒症模型12 h后,股静脉输注LR)、SS-31早期治疗组(常规治疗基础上在盲肠结扎穿孔前30 min尾静脉给予SS-31 3 mg/kg)和SS-31晚期治疗组(常规治疗基础上在盲肠结扎穿孔12 h后从股静脉输注LR联合SS-31)。通过荧光蛋白透过率的方法测定大鼠肺脏和肾脏的血管通透性,伊文思蓝染色透过率检测大鼠肠道通透性;离体取原代肺静脉内皮细胞观察LPS刺激(LPS组)及SS-31预处理(SS-31预处理组)后对内皮细胞的闭锁小带蛋白1(zonula occludes-1,ZO-1)的表达和细胞跨膜电阻(trans electric resistance,TER),以及活性氧(reactive oxygen species,ROS)的影响,以无血清的基础培养基培养24 h的细胞作为正常对照组。结果 (1)与假手术组比较,脓毒症组大鼠肺脏、肾脏血管以及肠道组织的通透性明显增加,线粒体功能降低(P <0. 01);采用常规治疗(LR联合头孢呋辛钠和多巴胺同时输注)尽管可以降低脓毒症大鼠的通透性和减轻线粒体功能损害,但与脓毒症组比较,差异无统计学意义(P>0. 05); SS-31治疗可明显降低脓毒症大鼠肺脏、肾脏血管以及肠道组织的通透性和增加线粒体功能,与常规治疗组比较,差异有统计学意义(P <0. 01),其中SS-31早期治疗效果比晚期治疗效果更好。(2)与正常对照组比较,LPS组ZO-1表达明显降低,细胞结构排列紊乱,TER明显降低,ROS含量增加; SS-31预处理可使ZO-1表达增加,细胞间连接紧密,TER明显增加,ROS含量明显降低。结论 SS-31对脓毒症大鼠的血管通透性有保护作用,其机制可能与抑制线粒体氧化应激有关。
Objective To investigate the protective effect of mitochondria-targeted peptide SS-31 on vascular permeability in septic rats and explore its underlying mechanism. Methods Forty Sprague-Dawley rats( 12 weeks old,both genders,weighting about 220 g) were equally and randomly divided into 5 groups,sham operation group( sham group),sepsis group,sepsis + conventional treatment( CT) group,sepsis +early injection of SS-31 group and sepsis + later injection of SS-31 group. Cecal ligation and puncture( CLP)was performed to establish septic rats. Lactated Ringer 's solution( containing cefuroxime sodium and dopamine) or the solution containing 3 mg/kg SS-31 were injected through femoral vein to the rats of sepsis +CT group or sepsis + later injection group,and the agent was also given to the rats of the sepsis + early injection group in 30 min before CLP. Fluorescence albumin transmissivity in the tissue homogenate was measured for the vascular permeability of the lungs and kidneys. Intestinal permeability was measured by Evans blue staining. Primarily isolated and cultured pulmonary vein endothelial cells were treated with lipopolysaccharide( LPS group),and SS-31 followed by LPS addition( LPS + SS-31 group). Then the expression level of zonula occludes-1( ZO-1),intensity of transmembrane resistance( TER),and generation of reactive oxygen species( ROS) were detected in the endothelial cells. The cells cultured in serum-free medium for 24 h served as normal control cells. Results(1) The sepsis group had significantly more obvious vascular permeability of the lungs,kidneys and intestine,and damaged mitochondrial function when compared with the sham group( P < 0. 01). Though conventional treatment reduced the permeability and relieved mitochondrial dysfunction,the effects were not significant than the conditions in the sepsis group( P > 0. 05).Both early and later injection of SS-31 notably decreased the permeability and improved mitochondrial function than the conventional treatment( P < 0. 01),with the effects of early injection more significant.(2) Compared with the normal control cells,LPS significantly decreased the expression of ZO-1,leading to disordered cell structure and significantly reduced TER,but increased the generation of ROS. SS-31 pretreatment could enhance ZO-1 expression and TER,and decrease ROS content. Conclusion SS-31 exerts protective effect on vascular permeability of septic rats,which was probably related to the mitochondrial oxidative stress.
Objective To investigate the protective effect of mitochondria-targeted peptide SS-31 on vascular permeability in septic rats and explore its underlying mechanism. Methods Forty Sprague-Dawley rats( 12 weeks old,both genders,weighting about 220 g) were equally and randomly divided into 5 groups,sham operation group( sham group),sepsis group,sepsis + conventional treatment( CT) group,sepsis +early injection of SS-31 group and sepsis + later injection of SS-31 group. Cecal ligation and puncture( CLP)was performed to establish septic rats. Lactated Ringer 's solution( containing cefuroxime sodium and dopamine) or the solution containing 3 mg/kg SS-31 were injected through femoral vein to the rats of sepsis +CT group or sepsis + later injection group,and the agent was also given to the rats of the sepsis + early injection group in 30 min before CLP. Fluorescence albumin transmissivity in the tissue homogenate was measured for the vascular permeability of the lungs and kidneys. Intestinal permeability was measured by Evans blue staining. Primarily isolated and cultured pulmonary vein endothelial cells were treated with lipopolysaccharide( LPS group),and SS-31 followed by LPS addition( LPS + SS-31 group). Then the expression level of zonula occludes-1( ZO-1),intensity of transmembrane resistance( TER),and generation of reactive oxygen species( ROS) were detected in the endothelial cells. The cells cultured in serum-free medium for 24 h served as normal control cells. Results(1) The sepsis group had significantly more obvious vascular permeability of the lungs,kidneys and intestine,and damaged mitochondrial function when compared with the sham group( P < 0. 01). Though conventional treatment reduced the permeability and relieved mitochondrial dysfunction,the effects were not significant than the conditions in the sepsis group( P > 0. 05).Both early and later injection of SS-31 notably decreased the permeability and improved mitochondrial function than the conventional treatment( P < 0. 01),with the effects of early injection more significant.(2) Compared with the normal control cells,LPS significantly decreased the expression of ZO-1,leading to disordered cell structure and significantly reduced TER,but increased the generation of ROS. SS-31 pretreatment could enhance ZO-1 expression and TER,and decrease ROS content. Conclusion SS-31 exerts protective effect on vascular permeability of septic rats,which was probably related to the mitochondrial oxidative stress.
引文
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[10] HIRAKAWA A,TAKEYAMA N,NAKATANI T,et al.Mitochondrial permeability transition and cytochrome c,release in ischemia-reperfusion injury of the rat liver[J]. J Surg Res,2003,111(2):240-247. DOI:10. 1016/S0022-4804(03)00091-X.
[11] SZETO H H,LOVELACE J L,FRIDLAND G,et al. In vivo pharmacokinetics of selective mu-opioid peptide agonists[J]. J Pharmacol Exp Therapeut,2001,298(1):57-61.DOI:10. 1016/S1056-8719(01)00162-9.
[12] ZHAO K,LUO G,GIANNELLI S,et al. Mitochondriatargeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines[J]. Biochem Pharmacol,2005,70(12):1796-1806. DOI:10. 1016/j. bcp. 2005. 08. 022.
[13] THOMAS D A,STAUFFER C,ZHAO K,et al. Mitochondrial targeting with antioxidant peptide SS-31 prevents mitochondrial depolarization,reduces islet cell apoptosis,increases islet cell yield,and improves posttransplantation function[J]. J Am Soc Nephrol Jasn,2007,18(1):213-222.DOI:10. 1681/ASN. 2006080825.
[14] ZHAO K,ZHAO G M,WU D,et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury[J]. J Biol Chem,2004,279(33):34682-34690. DOI:10. 1074/jbc. M402999200.
[15] CAI J,JIANG Y,ZHANG M,et al. Protective effects of mitochondrion-targeted peptide SS-31 against hind limb ischemia-reperfusion injury[J]. J Physiol Biochem,2018,74(2):335-343. DOI:10. 1007/s13105-018-0617-1.
[16] IMAI T,MISHIRO K,TAKAGI T,et al. Protective effect of bendavia(SS-31)against oxygen/glucose-deprivation stress-induced mitochondrial damage in human brain microvascular endothelial cells[J]. Curr Neurovasc Res,2017,14(1):53-59. DOI:10. 2174/1567202614666161117110609.
[17] ZHANG W,TAM J, SHINOZAKI K, et al. Increased survival time with SS-31 after prolonged cardiac arrest in rats[J]. Heart Lung Circ,2018.[Epub ahead of print]. DOI:10. 1016/j. hlc. 2018. 01. 008.
[18]杨娇娇,邱丽丽,韩金凤,等.抗氧化剂SS-31肽对脓毒症小鼠海马神经元凋亡与炎症反应的影响[J].药学与临床研究,2014,22(5):394-397. DOI:10. 13664/j.cnki. pcr. 2014. 05. 034.YANG J J,QIU L L,HAN J F,et al. Effects of antioxidant SS-31 peptide on hippocampal apoptosis and inflammatory response in the mice with sepsis[J]. Pharm Clin Res,2014,22(5):394-397. DOI:10. 13664/j. cnki. pcr. 2014.05. 034.
[19]李国民,丁东亮,杨娇娇,等.线粒体靶向抗氧化剂SS-31肽对脓毒血症小鼠急性肺损伤的影响[J].临床麻醉学杂志,2016,32(5):476-479.LI G M,DING D L,YANG J J,et al. Effect of mitochondria-targeted antioxidant SS-31 on sepsis-induced acute lung injury in a mouse model[J]. J Clin Anesthesiol,2016,32(5):476-479.
[20]王月华,侯延娟,任韫卓,等.抗氧化肽SS-31对高糖诱导的小鼠肾小球系膜细胞凋亡的影响[J].中国药理学通报,2013,29(1):102-107. DOI:10. 3969/j. issn.1001-1978. 2013. 01. 022.WANG Y H,HOU Y J,REN W Z,et al. Influence of antioxidant peptide SS-31 on HG-induced mouse mesangial cells apoptosis[J]. Chin Pharm Bull,2013,29(1):102-107. DOI:10. 3969/j. issn. 1001-1978. 2013. 01. 022.
[21] LI J,CHEN X,XIAO W,et al. Mitochondria-targeted antioxidant peptide SS31 attenuates high glucose-induced injury on human retinal endothelial cells[J]. Biochem Biophy Res Commun,2011,404(1):349-356. DOI:10. 1016/j.bbrc. 2010. 11. 122.
[22] LI G,WU J,LI R,et al. Protective effects of antioxidant peptide SS-31 against multiple organ dysfunctions during endotoxemia[J]. Inflammation,2016,39(1):54-64.DOI:10. 1007/s10753-015-0222-1.
[2] ZHANG X S,ZHANG X,ZHOU M L,et al. Amelioration of oxidative stress and protection against early brain injury by astaxanthin after experimental subarachnoid hemorrhage[J].J Neurosurg,2014,121(1):42-54. DOI:10. 3171/2014.2. JNS13730.
[3] FORSTERMANN U,XIA N,LI H. Roles of vascular oxidative stress and nitric oxide in the pathogenesis of atherosclerosis.[J]. Circ Res,2017,120(4):713-735. DOI:10.1161/CIRCRESAHA. 116. 309326.
[4] WALTON E L. Oxidative stress and diabetes:Glucose response in the cROSsfire[J]. Biomed J,2017,40(5):241-244. DOI:10. 1016/j. bj. 2017. 10. 001.
[5] SZETO H H. Mitochondria-targeted cytoprotective peptides for ischemia-reperfusion injury[J]. Antioxid Redox Sign,2008,10(3):601-619. DOI:10. 1089/ars. 2007. 1892.
[6] ANDERSON E J,LUSTIG M E,BOYLE K E,et al. Mitochondrial H2O2emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans[J]. J Clin Invest,2009,119(3):573-581. DOI:10.1172/JCI37048.
[7] YANG L,Zhao K,CALINGASAN N Y,et al. Mitochondria targeted peptides protect against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Neurotoxicity[J]. Antioxid Red Sign,2009,11(9):2095-2104. DOI:10. 1089/ARS. 2009. 2445.
[8] ZHU Y,WU H L,WU Y. et al. Beneficial effect of intermedin1-53 in septic shock rats:contributions of Rho kinase and Bkca pathway-mediated improvement in cardiac function[J].Shock,2016,46(5):557-565. DOI:10. 1097/SHK.0000000000000639.
[9] ZHANG J,YANG G M,ZHU Y,et al. Role of connexin 43in vascular hyperpermeability and relationship to Rock1-MLC20 pathway in septic rats[J]. Am J Physiol Lung Cell Mol Physiol,2015,309(11):L1323-L1332. DOI:10.1152/ajplung. 00016. 2015.
[10] HIRAKAWA A,TAKEYAMA N,NAKATANI T,et al.Mitochondrial permeability transition and cytochrome c,release in ischemia-reperfusion injury of the rat liver[J]. J Surg Res,2003,111(2):240-247. DOI:10. 1016/S0022-4804(03)00091-X.
[11] SZETO H H,LOVELACE J L,FRIDLAND G,et al. In vivo pharmacokinetics of selective mu-opioid peptide agonists[J]. J Pharmacol Exp Therapeut,2001,298(1):57-61.DOI:10. 1016/S1056-8719(01)00162-9.
[12] ZHAO K,LUO G,GIANNELLI S,et al. Mitochondriatargeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines[J]. Biochem Pharmacol,2005,70(12):1796-1806. DOI:10. 1016/j. bcp. 2005. 08. 022.
[13] THOMAS D A,STAUFFER C,ZHAO K,et al. Mitochondrial targeting with antioxidant peptide SS-31 prevents mitochondrial depolarization,reduces islet cell apoptosis,increases islet cell yield,and improves posttransplantation function[J]. J Am Soc Nephrol Jasn,2007,18(1):213-222.DOI:10. 1681/ASN. 2006080825.
[14] ZHAO K,ZHAO G M,WU D,et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury[J]. J Biol Chem,2004,279(33):34682-34690. DOI:10. 1074/jbc. M402999200.
[15] CAI J,JIANG Y,ZHANG M,et al. Protective effects of mitochondrion-targeted peptide SS-31 against hind limb ischemia-reperfusion injury[J]. J Physiol Biochem,2018,74(2):335-343. DOI:10. 1007/s13105-018-0617-1.
[16] IMAI T,MISHIRO K,TAKAGI T,et al. Protective effect of bendavia(SS-31)against oxygen/glucose-deprivation stress-induced mitochondrial damage in human brain microvascular endothelial cells[J]. Curr Neurovasc Res,2017,14(1):53-59. DOI:10. 2174/1567202614666161117110609.
[17] ZHANG W,TAM J, SHINOZAKI K, et al. Increased survival time with SS-31 after prolonged cardiac arrest in rats[J]. Heart Lung Circ,2018.[Epub ahead of print]. DOI:10. 1016/j. hlc. 2018. 01. 008.
[18]杨娇娇,邱丽丽,韩金凤,等.抗氧化剂SS-31肽对脓毒症小鼠海马神经元凋亡与炎症反应的影响[J].药学与临床研究,2014,22(5):394-397. DOI:10. 13664/j.cnki. pcr. 2014. 05. 034.YANG J J,QIU L L,HAN J F,et al. Effects of antioxidant SS-31 peptide on hippocampal apoptosis and inflammatory response in the mice with sepsis[J]. Pharm Clin Res,2014,22(5):394-397. DOI:10. 13664/j. cnki. pcr. 2014.05. 034.
[19]李国民,丁东亮,杨娇娇,等.线粒体靶向抗氧化剂SS-31肽对脓毒血症小鼠急性肺损伤的影响[J].临床麻醉学杂志,2016,32(5):476-479.LI G M,DING D L,YANG J J,et al. Effect of mitochondria-targeted antioxidant SS-31 on sepsis-induced acute lung injury in a mouse model[J]. J Clin Anesthesiol,2016,32(5):476-479.
[20]王月华,侯延娟,任韫卓,等.抗氧化肽SS-31对高糖诱导的小鼠肾小球系膜细胞凋亡的影响[J].中国药理学通报,2013,29(1):102-107. DOI:10. 3969/j. issn.1001-1978. 2013. 01. 022.WANG Y H,HOU Y J,REN W Z,et al. Influence of antioxidant peptide SS-31 on HG-induced mouse mesangial cells apoptosis[J]. Chin Pharm Bull,2013,29(1):102-107. DOI:10. 3969/j. issn. 1001-1978. 2013. 01. 022.
[21] LI J,CHEN X,XIAO W,et al. Mitochondria-targeted antioxidant peptide SS31 attenuates high glucose-induced injury on human retinal endothelial cells[J]. Biochem Biophy Res Commun,2011,404(1):349-356. DOI:10. 1016/j.bbrc. 2010. 11. 122.
[22] LI G,WU J,LI R,et al. Protective effects of antioxidant peptide SS-31 against multiple organ dysfunctions during endotoxemia[J]. Inflammation,2016,39(1):54-64.DOI:10. 1007/s10753-015-0222-1.
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