复方健耳剂干预小鼠老年性耳蜗感音神经细胞凋亡径路
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摘要
目的探讨中药复方健耳剂干预老年性耳蜗感音神经细胞凋亡作用可能径路。方法选择刚断奶C57BL/6J小鼠15只随机分为2月龄对照组、7月龄对照组、7月龄中药组各5只,其中2月龄对照组、7月龄对照组每日饮用自来水直到出生后2、7个月止,7月龄中药组每日饮用中药复方健耳剂直到出生后7个月止。各组期满立刻取出耳蜗,利用实时聚合酶链反应技术,检测耳蜗Fas配体(L)、细胞色素(cyt) C基因、含半胱氨酸的天冬氨酸蛋白水解酶(caspase)-3表达量。结果 7月龄对照组耳蜗cyt C、Fas L、caspase-3表达量均显著高于7月龄中药组和2月龄对照组(P<0. 05)。结论 C57BL/6J小鼠老年性耳蜗感音神经细胞凋亡可能均通过线粒体或细胞膜途径介导caspase引起程序性细胞死亡。该中药防治老年性聋关键作用机制之一可能与保护线粒体,防止cyt C外泄或稳定细胞膜,抑制Fas L,以阻止其触发caspase引起细胞程序性死亡径路有关。
Objective To investigate possible pathway on effects of compound healthy ear agent( CHEA) from traditional Chinese medicine( TCM) on interfering in age-related cochlear sensorineural apoptosis. Methods 15 C57 BL/6 J mice at 4 weeks of age were randomly divided into three groups. Five animals drank tap water daily from ablactation till 2 months of age and terminated as 2-month old control group. Five animals drank tap water daily from ablactation till 7 months of age and terminated as 7-month old control group with presbycusis. Other five animals drank the CHEA TCM daily from ablactation till 7 months of age as CHEA treating group. The animal cochleae in each group at the termination of the experiment were immediately removed and then were used for detecting cyt C,Fas L and caspase-3 in the cochlear tissue by Real Time PCR technique. In the end,the data were statistically analyzed and compared. Results Cycs,Fas L and caspase-3 expression were higher in 7-month old control group than those in 2-month old control group or in CHEA treating group( P<0.05).Conclusions Age-related cochlear sensorineural apoptosis in C57 BL/6 J mouse is probably via caspase-inducing programmed cell death mediated by two pathways from mitochondrion and cell membrane. One of mechanisms of the key effects of the CHEA on preventing and treating presbycusis is perhaps related mainly to protecting mitochondria and preventing cytochrome C leakage,or stabilizing cell membrane and inhibiting Fas L and then preventing them triggering programmed cell death pathway.
Objective To investigate possible pathway on effects of compound healthy ear agent( CHEA) from traditional Chinese medicine( TCM) on interfering in age-related cochlear sensorineural apoptosis. Methods 15 C57 BL/6 J mice at 4 weeks of age were randomly divided into three groups. Five animals drank tap water daily from ablactation till 2 months of age and terminated as 2-month old control group. Five animals drank tap water daily from ablactation till 7 months of age and terminated as 7-month old control group with presbycusis. Other five animals drank the CHEA TCM daily from ablactation till 7 months of age as CHEA treating group. The animal cochleae in each group at the termination of the experiment were immediately removed and then were used for detecting cyt C,Fas L and caspase-3 in the cochlear tissue by Real Time PCR technique. In the end,the data were statistically analyzed and compared. Results Cycs,Fas L and caspase-3 expression were higher in 7-month old control group than those in 2-month old control group or in CHEA treating group( P<0.05).Conclusions Age-related cochlear sensorineural apoptosis in C57 BL/6 J mouse is probably via caspase-inducing programmed cell death mediated by two pathways from mitochondrion and cell membrane. One of mechanisms of the key effects of the CHEA on preventing and treating presbycusis is perhaps related mainly to protecting mitochondria and preventing cytochrome C leakage,or stabilizing cell membrane and inhibiting Fas L and then preventing them triggering programmed cell death pathway.
引文
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5宣伟军,丁大连.中药聪耳胶囊干预老年性耳蜗螺旋神经节和疆孔神经损害的实验研究[J].中华中医药杂志,2007;22(6):355-9.
6宣伟军,周仲昭.补肾健脾胶囊延缓老年性耳蜗毛细胞凋亡的实验研究[J].中华中医药杂志,2008;23(2):114-7.
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9 Boatriqht KM,Salvesen GS.Mechanisms of caspase activation[J].Curr Opin Cell Biol,2003;15(6):725-31.
10 Schultz DR,Harrington WJ Jr.Apoptosis:programmed cell death at a molecular level[J].Semin Arthritis Rheum,2003;32(6):345-69.
11 Choi C,Benveniste EN.Fas ligand/Fas system in the brain:regulator of immune and apoptotic responses[J].Brain Res Brain Res Rev,2004;44(1):65-81.
12 Caroppi P,Sinibaldi F,Fiorucci L,et al.Apoptosis and human diseases:mitochondrion damage and lethal role of released cytochrome Cas proapoptotic protein[J].Curr Med Chem,2009;16(31):4058-65.
13 Kulikov AV,Shilov ES,Mufazalov IA,et al.Cytochrome C:the Achilles'heel in apoptosis[J].Cell Mol Life Sci,2012;69(11):1787-97.
14 Li P,Nijhawan D,Budihardjo I,et al.Cytochrome C and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptoic protease cascade[J].Cell,1997;91(4):479-89.
15 Shakeri R,Kheirollahi A,Davoodi J.Apaf-1:regulation and function in cell death[J].Biochimie,2017;135(4):111-25.
16 Kam JH,Jeffery G.To unite or divide:mitochondrial dynamics in the murine outer retina that preceded age related photoreceptor loss[J].Oncotarget,2015;6(29):26690-701.
17 Depraetere V,Golstein P.Fas and other cell death signaling pathways[J].Semin Immunol,1997;9(2):93-107.
18 Waring P,Müllbacher A.Cell death induced by the Fas/Fas ligand pathway and its role in pathology[J].Immunol Cell Biol,1999;77(4):312-7.
19 Kavurma MM,Khachigian LM.Signaling and transcriptional control of Fas ligand gene expression[J].Cell Death Differ,2003;10(1):36-44.
20 Zhao H,Roychoudhury J,Doggett TA,et al.Age-dependent changes in Fas L(CD95L)modulate macrophage function in a model of agerelated macular degeneration[J].Invest Ophthalmol Vis Sci,2013;54(8):5321-31.
21 Sawhney M,Mathew M,Valarmathi MT,et al.Age related changes in Fas(CD95)and Fas ligand gene expression and cytokine profiles in healthy Indians[J].Asian Pac J Allergy Immunol,2006;24(1):47-56.
22宣伟军,褚汉启,宣毅.复方健耳剂对国产DBA/2J小鼠耳蜗组织BDNF表达影响的初步研究[J].中华中医药杂志,2013;28(9):2748-51.
23 Croll SD,Ip NY,Lindsay RM,et al.Expression of BDNF and trk B as a function of age and cognitive performance[J].Brain Res,1998;812(1-2):200-8.
24 Han BH,Holtzman DM.BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway[J].J Neurosci,2000;20(15):5775-81.
25 Jerónimo-Santos A,Fonseca-Gomes J,Guimar2es DA,et al.Brain-derived neurotrophic factor mediates neuroprotection against Aβ-induced toxicity through a mechanism independent on adenosine 2Areceptor activation[J].Growth Factors,2015;33(4):298-308.
26 Han BH,D'Costa A,Back SA,et al.BDNF blocks caspase-3 activation in neonatal hypoxia-ischemia[J].Neurobiol Dis,2000;7(1):38-53.
2宣伟军,丁大连.复方健耳剂对抗小鼠老年性耳蜗螺旋神经节神经元凋亡效应及机制研究[J].中华老年医学杂志,2016;35(12):1329-33.
3宣伟军,唐俊波,陈壮,等.中药复方健耳剂对C57BL/6J小鼠老年性聋的防护效应[J].中国中西医结合杂志,2016;36(10):1261-6.
4宣伟军,黄正团,丁大连.中药健耳Ⅱ号胶囊对抗C57BL/6J小鼠老年性耳蜗损害的实验研究[J].听力学及言语疾病杂志,2007;15(1):47-50.
5宣伟军,丁大连.中药聪耳胶囊干预老年性耳蜗螺旋神经节和疆孔神经损害的实验研究[J].中华中医药杂志,2007;22(6):355-9.
6宣伟军,周仲昭.补肾健脾胶囊延缓老年性耳蜗毛细胞凋亡的实验研究[J].中华中医药杂志,2008;23(2):114-7.
7陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993:1103-5.
8 Savitskaya MA,Onishchenko GE.Mechanisms of apoptosis[J].Biochemistry(Mosc),2015;80(11):1393-405.
9 Boatriqht KM,Salvesen GS.Mechanisms of caspase activation[J].Curr Opin Cell Biol,2003;15(6):725-31.
10 Schultz DR,Harrington WJ Jr.Apoptosis:programmed cell death at a molecular level[J].Semin Arthritis Rheum,2003;32(6):345-69.
11 Choi C,Benveniste EN.Fas ligand/Fas system in the brain:regulator of immune and apoptotic responses[J].Brain Res Brain Res Rev,2004;44(1):65-81.
12 Caroppi P,Sinibaldi F,Fiorucci L,et al.Apoptosis and human diseases:mitochondrion damage and lethal role of released cytochrome Cas proapoptotic protein[J].Curr Med Chem,2009;16(31):4058-65.
13 Kulikov AV,Shilov ES,Mufazalov IA,et al.Cytochrome C:the Achilles'heel in apoptosis[J].Cell Mol Life Sci,2012;69(11):1787-97.
14 Li P,Nijhawan D,Budihardjo I,et al.Cytochrome C and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptoic protease cascade[J].Cell,1997;91(4):479-89.
15 Shakeri R,Kheirollahi A,Davoodi J.Apaf-1:regulation and function in cell death[J].Biochimie,2017;135(4):111-25.
16 Kam JH,Jeffery G.To unite or divide:mitochondrial dynamics in the murine outer retina that preceded age related photoreceptor loss[J].Oncotarget,2015;6(29):26690-701.
17 Depraetere V,Golstein P.Fas and other cell death signaling pathways[J].Semin Immunol,1997;9(2):93-107.
18 Waring P,Müllbacher A.Cell death induced by the Fas/Fas ligand pathway and its role in pathology[J].Immunol Cell Biol,1999;77(4):312-7.
19 Kavurma MM,Khachigian LM.Signaling and transcriptional control of Fas ligand gene expression[J].Cell Death Differ,2003;10(1):36-44.
20 Zhao H,Roychoudhury J,Doggett TA,et al.Age-dependent changes in Fas L(CD95L)modulate macrophage function in a model of agerelated macular degeneration[J].Invest Ophthalmol Vis Sci,2013;54(8):5321-31.
21 Sawhney M,Mathew M,Valarmathi MT,et al.Age related changes in Fas(CD95)and Fas ligand gene expression and cytokine profiles in healthy Indians[J].Asian Pac J Allergy Immunol,2006;24(1):47-56.
22宣伟军,褚汉启,宣毅.复方健耳剂对国产DBA/2J小鼠耳蜗组织BDNF表达影响的初步研究[J].中华中医药杂志,2013;28(9):2748-51.
23 Croll SD,Ip NY,Lindsay RM,et al.Expression of BDNF and trk B as a function of age and cognitive performance[J].Brain Res,1998;812(1-2):200-8.
24 Han BH,Holtzman DM.BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway[J].J Neurosci,2000;20(15):5775-81.
25 Jerónimo-Santos A,Fonseca-Gomes J,Guimar2es DA,et al.Brain-derived neurotrophic factor mediates neuroprotection against Aβ-induced toxicity through a mechanism independent on adenosine 2Areceptor activation[J].Growth Factors,2015;33(4):298-308.
26 Han BH,D'Costa A,Back SA,et al.BDNF blocks caspase-3 activation in neonatal hypoxia-ischemia[J].Neurobiol Dis,2000;7(1):38-53.