西咪替丁对低剂量率照射比格犬肝细胞凋亡的影响及其机制研究
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摘要
目的:研究西咪替丁对低剂量率照射比格犬肝细胞凋亡的影响及其机制。方法:将健康雄性比格犬随机分为正常对照组、模型对照组、阳性药物组(香菇多糖,21.33 mg/kg)和西咪替丁低、中、高剂量组(5.33、10.67、21.33 mg/kg),每组4只。除正常对照组外,其余各组比格犬均予~(60)Co-γ射线(剂量率:0.040 8 mGy/min)累积照射23 d,各给药组于每日照射前口服相应药物1次。停止照射24 h后,采用TUNEL法检测各组比格犬肝细胞凋亡情况,并计算凋亡细胞百分比;采用免疫组化法检测其肝组织中凋亡相关蛋白[Bax、Bcl-2、胱天蛋白酶3(Caspase-3)、p53]的表达水平。结果:与正常对照组比较,模型对照组比格犬肝组织中凋亡细胞以及Bax、Caspase-3、p53阳性细胞均明显增加,凋亡细胞百分比以及Bax、Caspase-3、p53蛋白表达水平均显著升高;Bcl-2阳性细胞明显减少,其蛋白表达水平均显著降低(P<0.05或P<0.01)。与模型对照组比较,各给药组比格犬肝组织中上述阳性细胞均有不同程度的变化,其中各给药组凋亡细胞百分比和p53蛋白表达水平,阳性药物组和西咪替丁低、高剂量组Bax蛋白表达水平以及西咪替丁各剂量组Caspase-3蛋白表达水平均显著降低;西咪替丁各剂量组Bcl-2蛋白表达水平均显著升高;且西咪替丁中、高剂量组凋亡细胞百分比以及西咪替丁各剂量组Caspase-3蛋白表达水平均显著低于阳性对照组,西咪替丁低剂量组p53蛋白表达水平显著高于阳性药物组(P<0.05或P<0.01)。结论:西咪替丁可抑制低剂量率照射所致的比格犬肝细胞凋亡,对其具有一定的辐射保护作用。这种作用可能与上调Bcl-2蛋白的表达,下调Bax、Caspase-3、p53蛋白的表达有关。
OBJECTIVE:To study the effects of cimetidine on low dose rate irradiation-induced liver cell apoptosis in Beagle dogs. METHODS:Healthy male Beagle dogs were randomly divided into normal control group,model control group,positive drug group(lentinan,21.33 mg/kg)and cimetidine low-dose,medium-dose and high-dose groups(5.33,10.67,21.33 mg/kg),with 4 Beagle dogs each. Except for normal control group,other groups were given ~(60)Co-γ accumulative irradiation(dosage rate: 0.040 8 mGy/min)for 23 d;the medication groups were given relevant medicine orally before irradiation,once a day. Twenty-four hours after stopping irradiation,TUNEL method was used to detect the apoptosis of liver cells in Beagle dogs. The percentage of apoptotic cells was calculated. The expression level of apoptosis-related proteins(Bax,Bcl-2,Caspase-3,p53)in liver tissue was detected by immunohistochemistry. RESULTS:Compared with normal control group,apoptotic cells and Bax,Caspase-3,p53 positive cells were increased significantly in liver tissue of Beagle dogs in model control group;the percentage of apoptotic cells,protein expression levels of Bax,Caspase-3 and p53 were increased significantly;Bcl-2 positive cells were decreased significantly,and its protein expression level was decreased significantly(P<0.05 or P<0.01). Compared with model control group,above positive cells of liver tissue in Beagle dogs were changed to different extents in medication groups;the percentage of apoptotic cells and protein expression levels of p53 in medication groups,protein expression levels of Bax in positive drug group,cimetidine low-dose and high-dose groups as well as protein expression levels of Caspase-3 in cimetidine groups were decreased significantly;protein expression levels of Bcl-2 were increased significantly in cimetidine groups. The percentage of apoptotic cells in cimetidine medium-dose and high-dose groups as well as protein expression levels of Caspase-3 in cimetidine groups were all lower than positive control group. Protein expression level of p53 in cimetidine low-dose group was significantly higher than positive drug group(P<0.05 or P<0.01). CONCLUSIONS:Cimetidine can inhibit the low dose rate irradiation-induced apoptosis of liver cells in Beagle dogs,and certainly protect liver cells against irradiation. The mechanism of it may be associated with up-regulating the protein expression of Bcl-2 and down-regulating the protein expression of Bax,Caspase-3 and p53 in liver cells.
OBJECTIVE:To study the effects of cimetidine on low dose rate irradiation-induced liver cell apoptosis in Beagle dogs. METHODS:Healthy male Beagle dogs were randomly divided into normal control group,model control group,positive drug group(lentinan,21.33 mg/kg)and cimetidine low-dose,medium-dose and high-dose groups(5.33,10.67,21.33 mg/kg),with 4 Beagle dogs each. Except for normal control group,other groups were given ~(60)Co-γ accumulative irradiation(dosage rate: 0.040 8 mGy/min)for 23 d;the medication groups were given relevant medicine orally before irradiation,once a day. Twenty-four hours after stopping irradiation,TUNEL method was used to detect the apoptosis of liver cells in Beagle dogs. The percentage of apoptotic cells was calculated. The expression level of apoptosis-related proteins(Bax,Bcl-2,Caspase-3,p53)in liver tissue was detected by immunohistochemistry. RESULTS:Compared with normal control group,apoptotic cells and Bax,Caspase-3,p53 positive cells were increased significantly in liver tissue of Beagle dogs in model control group;the percentage of apoptotic cells,protein expression levels of Bax,Caspase-3 and p53 were increased significantly;Bcl-2 positive cells were decreased significantly,and its protein expression level was decreased significantly(P<0.05 or P<0.01). Compared with model control group,above positive cells of liver tissue in Beagle dogs were changed to different extents in medication groups;the percentage of apoptotic cells and protein expression levels of p53 in medication groups,protein expression levels of Bax in positive drug group,cimetidine low-dose and high-dose groups as well as protein expression levels of Caspase-3 in cimetidine groups were decreased significantly;protein expression levels of Bcl-2 were increased significantly in cimetidine groups. The percentage of apoptotic cells in cimetidine medium-dose and high-dose groups as well as protein expression levels of Caspase-3 in cimetidine groups were all lower than positive control group. Protein expression level of p53 in cimetidine low-dose group was significantly higher than positive drug group(P<0.05 or P<0.01). CONCLUSIONS:Cimetidine can inhibit the low dose rate irradiation-induced apoptosis of liver cells in Beagle dogs,and certainly protect liver cells against irradiation. The mechanism of it may be associated with up-regulating the protein expression of Bcl-2 and down-regulating the protein expression of Bax,Caspase-3 and p53 in liver cells.
引文
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[3]GORI T,MUNZEL T.Biological effects of low-dose radiation:of harm and hormesis[J].Eur Heart J,2012,33(3):292-295.
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[7]张俊玲,何颖,王庆蓉,等.西咪替丁对0.7 Gy照射小鼠嗜多染红细胞微核的保护作用[J].解放军医学杂志,2015,40(12):950-954.
[8]赵忆宁,何颖,沈先荣,等.西咪替丁对低剂量电离辐射致大鼠氧化应激的保护作用[J].解放军医学杂志,2017,42(2):128-133.
[9]何颖,赵忆宁,沈先荣,等.西咪替丁对低剂量辐射比格犬的保护作用[J].解放军药学学报,2018,34(2):124-126.
[10]闵锐.低剂量/低剂量率电离辐射与辐射生物效应[J].辐射研究与辐射工艺学报,2014,32(6):3-11.
[11]郝光荣.实验动物学[M].上海:第二军医大学出版社,1999:232.
[12]MOZDARANI H.Can cimetidine be used as a radioprotector?[J].J Iran Univ Med Sci,1996,3(1/2):106-117.
[13]王庆蓉,张俊玲,何颖,等.西咪替丁对急性照射小鼠存活率及造血系统改变的影响[J].解放军医学杂志,2017,42(1):61-65.
[14]XING F,ZHAN Q,HE Y,et al.1 800 mHz microwave induces p53 and p53-mediated caspase-3 activation leading to cell apoptosis in vitro[J].PLo S One,2016.DOI:10.1371/journal.pone.0163935.
[15]LIU Q,SI T,XU X,et al.Electromagnetic radiation at900 mHz induces sperm apoptosis through Bcl-2,Bax and caspase-3 signaling pathways in rats[J].Reprod Health,2015.DOI:10.1186/s12978-015-0062-3.
[16]USHAKOVA T,MELKONYAN H,KONOVA L,et al.Modification of gene expression by dietary antioxidants in radiation-induced apoptosis of mice splenocytes[J].Free Radic Biol Med,1999,26(7/8):887-891.
[17]KIRKOVA M,ATANASSOVA M,RUSSANOV E.Effects of cimetidine and its metal complexes on nitroblue tetrazolium and ferricytochrome C reduction by superoxide radicals[J].Gen Pharmacol,1999,33(3):271-276.
[18]MIURA Y,ANZAI K,UEDA J,et al.Novel approach to in vivo screening for radioprotective activity in whole mice:in vivo electron spin resonance study probing the redox reaction of nitroxyl[J].J Radiat Res,2000,41(2):103-111.
[19]MOZDARANI H,KHOSHBIN-KHOSHNAZAR AR.In vivo protection by cimetidine against fast neutron-induced micronuclei in mouse bone marrow cells[J].Cancer Letters,1998,124(1):65-71.
[20]JIANG DW,WANG QR,SHEN XR,et al.Radioprotective effects of cimetidine on rats irradiated by long-term,low-dose-rate neutrons and60Coγ-rays[J].Mil Med Res,2017.DOI:10.1186/s40779-017-0116-7.
[21]柳迎华,李明春.香菇多糖注射液对慢性低剂量辐射损伤小鼠的防护作用[J].解放军药学学报,2012,28(3):200-204.
[22]高东伟.原发性肝癌正常肝组织放射生物效应的早期实验研究[D].济南:山东省医学科学院,2007.
[23]崔玉芳,丁彦青,杜雪梅.细胞凋亡与辐射免疫损伤研究进展[J].国外医学放射医学核医学分册,2002,26(6):271-274.
[24]姜成钢,李嘉斌,徐惠绵,等.西咪替丁对胃癌细胞SGC-7901增殖及凋亡的影响[J].世界华人消化杂志,2007,15(2):118-122.
[25]JIANG CG,LIU FR,YU M,et al.Cimetidine induces apoptosis in gastric cancer cells in vitro and inhibits tumor growth in vivo[J].Oncol Rep,2010,23(3):693-700.
[26]郑溢声,武宁,宁允叶,等.西咪替丁对人肺腺癌A549细胞增殖和凋亡的影响[J].肿瘤防治研究,2011,38(10):1097-1100.
[2]MARTIN LM,MARPLES B,LYNCH TH,et al.Exposure to low dose ionizing radiation:Molecular and clinical consequences[J].Cancer Lett,2014,349(1):98-106.
[3]GORI T,MUNZEL T.Biological effects of low-dose radiation:of harm and hormesis[J].Eur Heart J,2012,33(3):292-295.
[4]FARHAN F,KAZEMIAN A,ALAGHEBAND H.in vitro radioprotective effects of histamine H2receptor antagonists against gamma-rays induced chromosomal aberrations in human lymphocytes[J].Iran J Radiat Res,2003,1(2):99-104.
[5]KOJIMA Y,KONDO T,ZHAO QL.Protective effects of cimetidine on radiation-induced micronuclei and apoptosis in human peripheral blood lymphocytes[J].Free Radic Res,2002,36(3):255-563.
[6]MOZDARANI H,SALIMI M,FROUGHIZADEH M.Effect of cimetidine and famotidine on survival of lethally gamma irradiated mice[J].Iran J Radiat Res,2008,5(4):187-194.
[7]张俊玲,何颖,王庆蓉,等.西咪替丁对0.7 Gy照射小鼠嗜多染红细胞微核的保护作用[J].解放军医学杂志,2015,40(12):950-954.
[8]赵忆宁,何颖,沈先荣,等.西咪替丁对低剂量电离辐射致大鼠氧化应激的保护作用[J].解放军医学杂志,2017,42(2):128-133.
[9]何颖,赵忆宁,沈先荣,等.西咪替丁对低剂量辐射比格犬的保护作用[J].解放军药学学报,2018,34(2):124-126.
[10]闵锐.低剂量/低剂量率电离辐射与辐射生物效应[J].辐射研究与辐射工艺学报,2014,32(6):3-11.
[11]郝光荣.实验动物学[M].上海:第二军医大学出版社,1999:232.
[12]MOZDARANI H.Can cimetidine be used as a radioprotector?[J].J Iran Univ Med Sci,1996,3(1/2):106-117.
[13]王庆蓉,张俊玲,何颖,等.西咪替丁对急性照射小鼠存活率及造血系统改变的影响[J].解放军医学杂志,2017,42(1):61-65.
[14]XING F,ZHAN Q,HE Y,et al.1 800 mHz microwave induces p53 and p53-mediated caspase-3 activation leading to cell apoptosis in vitro[J].PLo S One,2016.DOI:10.1371/journal.pone.0163935.
[15]LIU Q,SI T,XU X,et al.Electromagnetic radiation at900 mHz induces sperm apoptosis through Bcl-2,Bax and caspase-3 signaling pathways in rats[J].Reprod Health,2015.DOI:10.1186/s12978-015-0062-3.
[16]USHAKOVA T,MELKONYAN H,KONOVA L,et al.Modification of gene expression by dietary antioxidants in radiation-induced apoptosis of mice splenocytes[J].Free Radic Biol Med,1999,26(7/8):887-891.
[17]KIRKOVA M,ATANASSOVA M,RUSSANOV E.Effects of cimetidine and its metal complexes on nitroblue tetrazolium and ferricytochrome C reduction by superoxide radicals[J].Gen Pharmacol,1999,33(3):271-276.
[18]MIURA Y,ANZAI K,UEDA J,et al.Novel approach to in vivo screening for radioprotective activity in whole mice:in vivo electron spin resonance study probing the redox reaction of nitroxyl[J].J Radiat Res,2000,41(2):103-111.
[19]MOZDARANI H,KHOSHBIN-KHOSHNAZAR AR.In vivo protection by cimetidine against fast neutron-induced micronuclei in mouse bone marrow cells[J].Cancer Letters,1998,124(1):65-71.
[20]JIANG DW,WANG QR,SHEN XR,et al.Radioprotective effects of cimetidine on rats irradiated by long-term,low-dose-rate neutrons and60Coγ-rays[J].Mil Med Res,2017.DOI:10.1186/s40779-017-0116-7.
[21]柳迎华,李明春.香菇多糖注射液对慢性低剂量辐射损伤小鼠的防护作用[J].解放军药学学报,2012,28(3):200-204.
[22]高东伟.原发性肝癌正常肝组织放射生物效应的早期实验研究[D].济南:山东省医学科学院,2007.
[23]崔玉芳,丁彦青,杜雪梅.细胞凋亡与辐射免疫损伤研究进展[J].国外医学放射医学核医学分册,2002,26(6):271-274.
[24]姜成钢,李嘉斌,徐惠绵,等.西咪替丁对胃癌细胞SGC-7901增殖及凋亡的影响[J].世界华人消化杂志,2007,15(2):118-122.
[25]JIANG CG,LIU FR,YU M,et al.Cimetidine induces apoptosis in gastric cancer cells in vitro and inhibits tumor growth in vivo[J].Oncol Rep,2010,23(3):693-700.
[26]郑溢声,武宁,宁允叶,等.西咪替丁对人肺腺癌A549细胞增殖和凋亡的影响[J].肿瘤防治研究,2011,38(10):1097-1100.
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