高铜对肉鸡心肌细胞线粒体自噬的影响

Effects of high dietary copper on mitophagy in cardiomyocytes of broilers

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.