高铜对肉鸡心肌细胞线粒体自噬的影响
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摘要
为了探究高铜(copper,Cu)是否能诱导肉鸡心肌细胞线粒体发生自噬,本研究选用48只1日龄健康白羽肉鸡,随机将其分为4组,每组12只,分别喂以对照日粮(11 mg/kg Cu,对照组)和高铜日粮(110mg/kg Cu,高铜I组;220 mg/kg Cu,高铜Ⅱ组;330 mg/kg Cu,高铜Ⅲ组),并于49日龄进行心脏采集,实时荧光定量PCR检测肉鸡心肌细胞中线粒体自噬相关基因PIN K 1、Parkin、LC3-I、LC3-Ⅱ、p62和NIX的mRNA转录水平,免疫印迹和免疫组织化学技术分别检测蛋白LC3-Ⅱ/LC3-I的表达与LC3-Ⅱ的定位。结果显示,高铜可显著上调线粒体自噬相关基因PINK1、Parkin、LC3-I、LC3-Ⅱ和NIX的mRNA转录水平(P<0.05),但p62的mRNA转录水平变化不显著(P>0.05),蛋白LC3-Ⅱ/LC3-I也显著升高(P<0.05),且由免疫组织化学结果可看出肉鸡心肌细胞中LC3-Ⅱ的棕色阳性颗粒增加(P<0.05)。上述结果表明,高铜可诱导肉鸡心肌细胞线粒体发生自噬。
To investigate whether high-copper can induce mitophagy in cardiomyocytes of broilers,48 one-day-old healthy white feather broilers were randomly divided into 4 groups with 12 each in this study.The broilers were fed as follows:control diet(11 mg/kg Cu,control group),and high-copper diets(110 mg/kg Cu,high-copper group Ⅰ;220 mg/kg Cu,high-copper group Ⅱ;330 mg/kg Cu,high-copper groupⅢ).The heart tissues were collected on day-old 49.The m RNA expression levels of mitophagy-related genes(PINK1,Parkin,LC3-Ⅰ,LC3-Ⅱ,p62 and NIX) were detected by real-time quantitative PCR.The protein expressions of LC3-II/LC3-Ⅰ and the distribution of LC3-Ⅱ protein were determined by Western-blot and immunohistochemistry,respectively.In result,the m RNA expression levels of PINK1,Parkin, LC3-Ⅰ,LC3-Ⅱand NIX were significantly upregulated compared to the control group(P<0.05).However,there was no significant difference in p62 m RNA expression between the control group and the Cu-treated groups(P>0.05).The protein level of LC3-Ⅱ/LC3-Ⅰwas also remarkably increased(P<0.05).Moreover,Immunohistochemical results showed the brown positive particles of LC3-Ⅱwere increased.The above-mentioned results showed that high-copper can induce mitophagy in cardiomyocytes of broiler.
To investigate whether high-copper can induce mitophagy in cardiomyocytes of broilers,48 one-day-old healthy white feather broilers were randomly divided into 4 groups with 12 each in this study.The broilers were fed as follows:control diet(11 mg/kg Cu,control group),and high-copper diets(110 mg/kg Cu,high-copper group Ⅰ;220 mg/kg Cu,high-copper group Ⅱ;330 mg/kg Cu,high-copper groupⅢ).The heart tissues were collected on day-old 49.The m RNA expression levels of mitophagy-related genes(PINK1,Parkin,LC3-Ⅰ,LC3-Ⅱ,p62 and NIX) were detected by real-time quantitative PCR.The protein expressions of LC3-II/LC3-Ⅰ and the distribution of LC3-Ⅱ protein were determined by Western-blot and immunohistochemistry,respectively.In result,the m RNA expression levels of PINK1,Parkin, LC3-Ⅰ,LC3-Ⅱand NIX were significantly upregulated compared to the control group(P<0.05).However,there was no significant difference in p62 m RNA expression between the control group and the Cu-treated groups(P>0.05).The protein level of LC3-Ⅱ/LC3-Ⅰwas also remarkably increased(P<0.05).Moreover,Immunohistochemical results showed the brown positive particles of LC3-Ⅱwere increased.The above-mentioned results showed that high-copper can induce mitophagy in cardiomyocytes of broiler.
引文
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[18]KATSURAGI Y,ICHIMURA Y,KOMATSU M.p62/SQSTM1 functions as a signaling hub and an autophagy adaptor[J].FEBS Journal,2015,282(24):4672-4678.
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[21]PIQUEREAU J,GODIN R,DESCHNES S,et al.Protective role of PARK2/Parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction[J].Autophagy,2013,9(11):1837-51.
[22]SU H,LI F,RANEK M J,et al.COP9 signalosome regulates autophagosome maturation[J].Circulation,2011,124(19):2117-2128.
[23]SANDOVAL H,THIAGARAJAN P,DASGUPTA S K,et al.Essential role for Nix in autophagic maturation of erythroid cells[J].Nature,2008,454(7201):232-235.
[24]GAO F,CHEN D,SI J M,et al.The mitochondrial protein BNIP3L is the substrate of PARK2 and mediates mitophagy in PINK1/PARK2 pathway[J].Human Molecular Genetics,2015,24(9):2528-2538.
[2]GEISLER S,HOLMSTRM K M,SKUJAT D,et al.PINK1/Parkin-mediated mitophagy is dependent on VDAC1and p62/SQSTM1[J].Nature Cell Biology,2010,12(2):119-131.
[3]NOVAK I,KIRKIN V,Mc Ewan D G,et al.Nix is a selective autophagy receptor for mitochondrial clearance[J].EMBO Reports,2010,11(1):45-51.
[4]XIE R,NGUYEN S,MCKEEHAN W L,et al.Acetylated microtubules are required for fusion of autophagosomes with lysosomes[J].BMC Cell Biology,2010,11(1):89.
[5]HOSSEINI M J,SHAKI F,GHAZI-KHANSARI M,et al.Toxicity of copper on isolated liver mitochondria:impairment at complexes I,II,and IV leads to increased ROS production[J].Cell Biochemistry and.Biophysics,2014,70:367-381.
[6]崔恒敏,陈怀涛.铜中毒对雏鸭某些血液指标的影响[J].中国兽医学报,2005,3(25):311-313.CUI Heng-min,CHEN Huai-tao.Effect of copper toxicity on some blood parameters in ducklings[J].Chinese Journal of Veterinary Science,2005,3(25):311-313.(in Chinese)
[7]曹华斌,苏荣胜,李和平,等.铜对肉鸡肝细胞线粒体膜通透性及呼吸的影响[J].中国兽医科学,2007,37(04):342-345.CAO Hua-bin,SU Rong-sheng,LI H P,et al.Effects of copper on membrane permeability and respiration of mitochondria from broiler hepatocyte[J].Chinese Veterinary Science,2007,37(04):342-345.(in Chinese)
[8]YANG F,LIAO J Z,PEI R N,et al.Autophagy attenuates copper-induced mitochondrial dysfunction by regulating oxidative stress in chicken hepatocytes[J].Chemosphere,2018,204:36-43.
[9]DENOYER D,MASALDAN S,LA F S,et al.Targeting copper in cancer therapy:'Copper That Cancer'[J].Metallomics,2015,7(11):1459-1476.
[10]苏荣胜,贾雪霞,刘好朋,等.高铜日粮对肉鸡肝脏Bax、Bcl-2蛋白表达的影响[J].中国兽医学报,2014,34(8):1377-1379.SU Rong-sheng,JIA Xue-xia,LIU Hao-peng,et al.Effect of high-level copper diet on expression of Bax,Bcl-2 protein in broilers liver[J].Chinese Journal of Veterinary Science,2014,34(8):1377-1379.(in Chinese)
[11]吴柳燕,康振龙,乔娜,等.铜离子在诱导鸡原代肝细胞凋亡方面及其对Drp1基因表达水平的影响[J].中国兽医科学,2018,48(07):918-923.WU Liu-yan,KANG Zhen-long,QIAO Na,et al.Effects in apoptosis induced by copper ions and expression level of dynamin-related protein 1 gene in primary cultured chicken hepatocytes[J].Chinese Veterinary Science,2018,48(07:918-923.(in Chinese)
[12]PEREIRA T C,CAMPOS M M,BOGO M R.Copper toxicology,oxidative stress and inflammation using zebrafish as experimental model[J].Journal of Applied Toxicology,2016,36(7):876-885.
[13]LI S,ZHAO H,WANG Y,et al.Regulation of autophagy factors by oxidative stress and cardiac enzymes imbalance during arsenic or/and copper induced cardiotoxicity in Gallus gallus[J].Ecotoxicology and Environmental Safety,2018,148:125-134.
[14]GLADKOVA C,MASLEN S L,SKEHEL J M,et al.Mechanism of parkin activation by PINK1[J].Nature,2018,559(7714):410-414.
[15]CORNELISSEN T,VERSTREKEN P,VANDENBERGHE W.Imaging mitophagy in the fruit fly[J].Autophagy,2018,14(9):1656-1657.
[16]WHITWORTH A J,PALLANCK L J.PINK1/Parkin mitophagy and neurodegeneration-what do we really know in vivo[J].Current Opinion in Genetics&Development,2017,44:47-53.
[17]HAN J Y,KANG M J,KIM K H,et al.Nitric oxide induction of Parkin translocation in PTEN-induced putative kinase 1(PINK1)deficiency:functional role of neuronal nitric oxide synthase during mitophagy[J].Journal of Biological Chemistry,290(16):10325-35.
[18]KATSURAGI Y,ICHIMURA Y,KOMATSU M.p62/SQSTM1 functions as a signaling hub and an autophagy adaptor[J].FEBS Journal,2015,282(24):4672-4678.
[19]SCHLFLI AM,BEREZOWSKA S,ADAMS O,et al.Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry[J].European Journal of Histochemistry,2015:59(2):2481.
[20]PEREZ S E,HE B,NADEEM M,et al.Hippocampal endosomal,lysosomal,and autophagic dysregulation in mild cognitive impairment:correlation with aβand tau pathology[J].Journal Neuropathology and Experimental Neurology,2015,74(4):345-358.
[21]PIQUEREAU J,GODIN R,DESCHNES S,et al.Protective role of PARK2/Parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction[J].Autophagy,2013,9(11):1837-51.
[22]SU H,LI F,RANEK M J,et al.COP9 signalosome regulates autophagosome maturation[J].Circulation,2011,124(19):2117-2128.
[23]SANDOVAL H,THIAGARAJAN P,DASGUPTA S K,et al.Essential role for Nix in autophagic maturation of erythroid cells[J].Nature,2008,454(7201):232-235.
[24]GAO F,CHEN D,SI J M,et al.The mitochondrial protein BNIP3L is the substrate of PARK2 and mediates mitophagy in PINK1/PARK2 pathway[J].Human Molecular Genetics,2015,24(9):2528-2538.