化合物BAM15减轻大鼠原代肝细胞冷保存损伤的实验研究
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摘要
目的探讨化合物BAM15对大鼠离体肝脏原代细胞冷保存损伤的影响。方法采用胶原酶灌注法提取大鼠肝脏原代细胞,根据细胞培养条件的不同,将细胞分为4组:A组(含250 nmol/L BAM15的Hibernate细胞培养液);B组(含500 nmol/L BAM15的Hibernate细胞培养液);C组(含1 000 nmol/L BAM15的Hibernate细胞培养液);对照组(Hibernate细胞培养液)。各组细胞均冷保存12 h。荧光显微镜下观察肝脏原代细胞的纯度。同时测定各组细胞的增殖能力、凋亡情况以及线粒体活性氧(ROS)变化情况。结果 B组和C组的细胞增殖能力均强于对照组(均为P<0.05)。A、B、C组的细胞凋亡率分别为(33.7±2.2)%、(19.7±1.1)%、(28.7±1.2)%,均明显低于对照组[(82.7±4.2)%,均为P<0.05]。A、B、C组的细胞内ROS阳性率分别为(11.8±4.0)%、(7.6±1.3)%、(8.9±1.6)%,均明显低于对照组[(27.4±4.5)%,均为P<0.05]。结论化合物BAM15能有效减轻大鼠肝脏原代细胞的冷损伤,其保护机制可能与BAM15减少冷缺血期间ROS生成有关。
Objective To investigate the effect of compound BAM15 on the primary hepatocyte injury induced by cold storage in rats. Methods The primary rat hepatocytes were extracted by collagenase perfusion method. According to different cell culture conditions, the cells were divided into 4 groups: group A(Hibernate cell culture solution containing250 nmol/L BAM15), group B(Hibernate cell culture solution containing 500 nmol/L BAM15), group C(Hibernate cell culture solution containing 1 000 nmol/L BAM15), control group(Hibernate cell culture solution). The cells of each group were cryopreserved for 12 h. The purity of primary hepatocytes was observed under fluorescence microscope.The changes in the cell proliferation ability, cell apoptosis rate and mitochondrial reactive oxygen species(ROS) were measured in each group. Results The cell proliferation ability in groups B and C was significantly higher than that in the control group(both P<0.05). The apoptosis rates in groups A, B and C were(33.7±2.2)%,(19.7±1.1)% and(28.7±1.2)%,which were significantly lower than(82.7±4.2)% in the control group(all P<0.05). The positive rates of intracellular ROS in groups A, B and C were(11.8±4.0)%,(7.6±1.3)% and(8.9±1.6)%, remarkably lower than(27.4±4.5)% in the control group(all P<0.05). Conclusions Compound BAM15 can effectively mitigate the primary hepatocyte injury in rats induced by cryopreservation. The underlying mechanism is probably associated with the role of BAM15 in reducing ROS generation during cold ischemia.
Objective To investigate the effect of compound BAM15 on the primary hepatocyte injury induced by cold storage in rats. Methods The primary rat hepatocytes were extracted by collagenase perfusion method. According to different cell culture conditions, the cells were divided into 4 groups: group A(Hibernate cell culture solution containing250 nmol/L BAM15), group B(Hibernate cell culture solution containing 500 nmol/L BAM15), group C(Hibernate cell culture solution containing 1 000 nmol/L BAM15), control group(Hibernate cell culture solution). The cells of each group were cryopreserved for 12 h. The purity of primary hepatocytes was observed under fluorescence microscope.The changes in the cell proliferation ability, cell apoptosis rate and mitochondrial reactive oxygen species(ROS) were measured in each group. Results The cell proliferation ability in groups B and C was significantly higher than that in the control group(both P<0.05). The apoptosis rates in groups A, B and C were(33.7±2.2)%,(19.7±1.1)% and(28.7±1.2)%,which were significantly lower than(82.7±4.2)% in the control group(all P<0.05). The positive rates of intracellular ROS in groups A, B and C were(11.8±4.0)%,(7.6±1.3)% and(8.9±1.6)%, remarkably lower than(27.4±4.5)% in the control group(all P<0.05). Conclusions Compound BAM15 can effectively mitigate the primary hepatocyte injury in rats induced by cryopreservation. The underlying mechanism is probably associated with the role of BAM15 in reducing ROS generation during cold ischemia.
引文
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[3]GILBO N,MONBALIU D.Temperature and oxygenation during organ preservation:friends or foes?[J].Curr Opin Organ Transplant,2017,22(3):290-299.DOI:10.1097/MOT.0000000000000416.
[4]JING L,YAO L,ZHAO M,et al.Organ preservation:from the past to the future[J].Acta Pharmacol Sin,2018,39(5):845-857.DOI:10.1038/aps.2017.182.
[5]NIGMET Y,HATA K,TAMAKI I,et al.Human atrial natriuretic peptide in cold storage of donation after circulatory death rat livers:an old but new agent for protecting vascular endothelia?[J].Transplantation,2019,103(3):512-521.DOI:10.1097/TP.0000000000002552.
[6]OU J,BALL JM,LUAN Y,et al.iPSCs from a hibernator provide a platform for studying cold adaptation and its potential medical applications[J].Cell,2018,173(4):851-863.e16.DOI:10.1016/j.cell.2018.03.010.
[7]YANG Z,CHEN C,ZHAO J,et al.Hypoglycemic mechanism of a novel proteoglycan,extracted from Ganoderma lucidum,in hepatocytes[J].Eur J Pharmacol2018,820:77-85.DOI:10.1016/j.ejphar.2017.12.020.
[8]LATCHANA N,PECK JR,WHITSON BA,et al Preservation solutions used during abdominal transplantation:current status and outcomes[J].World J Transplant2015,5(4):154-164.DOI:10.5500/wjt.v5.i4.154.
[9]CAMERON AM,BARANDIARAN CORNEJO JF.Organ preservation review:history of organ preservation[J]Curr Opin Organ Transplant,2015,20(2):146-151.DOI:10.1097/MOT.0000000000000175.
[10]王诚,李选鹏,郭霜,等.流体剪切力在移植肾机械灌注中的机制及研究进展[J].器官移植,2018,9(2):159-161,168.DOI:10.3969/j.issn.1674-7445.2018.02.013.WANG C,LI XP,GUO S,et al.The mechanism and research progress of fluid shear force in mechanical perfusion of transplanted kidney[J].Organ Transplant,2018,9(2):159-161,168.DOI:10.3969/j.issn.1674-7445.2018.02.013.
[11]AKATEH C,BEAL EW,KIM JL,et al.Intrahepatic delivery of pegylated catalase is protective in a rat ischemia/reperfusion injury model[J].J Surg Res,2019238:152-163.DOI:10.1016/j.jss.2019.01.028.
[12]BO H,JIANG N,MA G,et al.Regulation of mitochondrial uncoupling respiration during exercise in rat heart:role of reactive oxygen species(ROS)and uncoupling protein 2[J].Free Radic Biol Med,2008,44(7):1373-1381.DOI:10.1016/j.freeradbiomed.2007.12.033.
[13]HRYCAY EG,BANDIERA SM.Involvement of cytochrome P450 in reactive oxygen species formation and cancer[J].Adv Pharmacol,2015,74:35-84.DOI:10.1016/bs.apha.2015.03.003.
[14]HE L,HE T,FARRAR S,et al.Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species[J].Cell Physiol Biochem,2017,44(2):532-553DOI:10.1159/000485089.
[15]HINDER LM,SAS KM,O'BRIEN PD,et al.Mitochondrial uncoupling has no effect on microvascular complications in type 2 diabetes[J].Sci Rep,2019,9(1):881.DOI:10.1038/s41598-018-37376-y.
[16]JARMUSZKIEWICZ W,SZEWCZYK A.Energydissipating hub in muscle mitochondria:potassium channels and uncoupling proteins[J].Arch Biochem Biophys,2019664:102-109.DOI:10.1016/j.abb.2019.01.036.
[17]CHENG WC,TSUI YC,RAGUSA S,et al.Uncoupling protein 2 reprograms the tumor microenvironment to support the anti-tumor immune cycle[J].Nat Immunol2019,20(2):206-217.DOI:10.1038/s41590-018-0290-0.
[18]SCHIFFER TA,CHRISTENSEN M,GUSTAFSSONH,et al.The effect of inactin on kidney mitochondrial function and production of reactive oxygen species[J]PLoS One,2018,13(11):e0207728.DOI:10.1371/journal.pone.0207728.
[19]PITT MA.Overexpression of uncoupling protein-2 in cancer:metabolic and heat changes,inhibition and effects on drug resistance[J].Inflammopharmacology,201523(6):365-369.DOI:10.1007/s10787-015-0250-3.
[20]LIU Y,YANG L,TAO K,et al.Protective effects of hydrogen enriched saline on liver ischemia reperfusion injury by reducing oxidative stress and HMGB1release[J].BMC Gastroenterol,2014,14:12.DOI:10.1186/1471-230X-14-12.