自噬对肾小管细胞毒性损伤后线粒体功能的影响及相关机制研究

目的研究自噬对肾小管细胞毒性损伤后线粒体功能的影响及相关机制。方法将人近曲肾小管上皮细胞(HK-2)分为杂化siRNA组(对照无关序列片段siRNA转染细胞)、杂化siRNA+顺铂组(对照无关序列片段siRNA转染细胞+10μmol/L顺铂溶液)、沉默Pink1+顺铂组(Pink1沉默转染细胞+10μmol/L顺铂溶液)和沉默Parkin+顺铂组(Parkin沉默转染细胞+10μmol/L顺铂溶液)。培养12 h后,细胞计数试剂盒8(CCK-8)法测定各组细胞存活率,JC-1法检测线粒体膜电位,荧光法检测各组细胞内三磷酸腺苷(ATP)的含量,Western blotting和免疫荧光探针检测自噬相关蛋白的相对表达。结果杂化siRNA+顺铂组的细胞存活率为(88.2±2.7)%,显著低于杂化siRNA组(101.3±3.1)%](P<0.05);沉默Pink1+顺铂组和沉默Parkin+顺铂组的存活率为(80.1±2.3)%、(79.4±3.0%),显著低于杂化siRNA+顺铂组(P<0.05)。杂化siRNA+顺铂组的线粒体膜电位和ATP含量为(0.90±0.01)、(0.82±0.01)nmol/mg,显著低于杂化siRNA组(1.01±0.02)、(1.00±0.04)nmol/mg](P<0.05);沉默Pink1+顺铂组和沉默Parkin+顺铂组的线粒体膜电位(0.79±0.02、0.77±0.02)和ATP(0.66±0.05)、(0.66±0.02)nmol/mg]含量显著低于杂化siRNA+顺铂组(P<0.05)。杂化siRNA+顺铂组的Pink1、Parkin、Beclin蛋白的相对表达量和LC3Ⅱ/LC3Ⅰ的比值(1.68±0.04、1.80±0.05、1.87±0.05、2.01±0.04)显著高于杂化siRNA组(1.00±0.04、1.00±0.05、1.01±0.01、1.04±0.02)(P<0.05);而沉默Pink1+顺铂组和沉默Parkin+顺铂组的Pink1、Parkin、Beclin蛋白的相对表达量和LC3Ⅱ/LC3Ⅰ的比值(1.17±0.05、0.69±0.05、1.37±0.05、1.43±0.02;1.22±0.04、0.57±0.06、1.39±0.03、1.38±0.10)显著低于杂化siRNA+顺铂组(P<0.05)。杂化siRNA+顺铂组的自噬阳性点数为(12.2±0.7)个,显著多于杂化siRNA组(2.4±0.3)个](P<0.05);而沉默Pink1+顺铂组和沉默Parkin+顺铂组的荧光点数(5.3±0.8)、(5.6±0.5)个]显著少于杂化siRNA+顺铂组(P<0.05)。结论顺铂处理可诱导HK-2细胞的线粒体自噬作用,从而改善顺铂引起的肾小管细胞毒性损伤和线粒体功能,其作用机制可能与Pink1/Parkin蛋白的表达情况有关。

Study of effect and mechanism of mitophagy on mitochondrial function after toxic injury

Objective To investigate the effect and mechanism of mitophagy on mitochondrial function after toxic injury. Methods Human renal proximal tubular(HK-2) cells were set as hybrid siRNA group(control irrelevant sequence fragment siRNA transfected cells),Hybrid siRNA + cisplatin group(control irrelevant sequence fragment siRNA transfected cells + 10 μmol/L cisplatin solution),silent Pink1 + cisplatin group(Pink1 silent transfected cells + 10 μmol/L cisplatin solution) and silent Parkin + cisplatin group(Parkin silent transfected cells + 10μmol/L cisplatin solution),after 12 hours of culture,the cell viability was detected by cell counting kit-8(CCK-8) method,the mitochondrial membrane potential was detected by JC-1 fluorescence method,the adenosine triphosphate(ATP) was determined with a ATP determination kit,the expressions of autophagy related protein was detected by western blotting and immunofluorescence probe. Results The cell survival rate of the hybrid siRNA + cisplatin group was(88. 2 ± 2. 7) %,which was significantly lower than that of the hybrid siRNA group (101. 3 ± 3. 1) %](P < 0. 05);The survival rate of the silent Pink1 + cisplatin group and Parkin + cisplatin group was(80. 1 ± 2. 3) %,(79. 4 ± 3. 0) %,which was significantly lower than that of the hybrid siRNA + cisplatin group(P < 0. 05). The mitochondrial membrane potential and ATP content of the hybrid siRNA + cisplatin group were 0. 90 ± 0. 01 and(0. 82 ± 0. 01) nmol/mg,which were significantly lower than the hybrid siRNA group (1. 01 ± 0. 02),(1. 00 ± 0. 04) nmol/mg](P < 0. 05);The mitochondrial membrane potential(0. 79 ± 0. 02,0. 77 ± 0. 02) and ATP(0. 66 ± 0. 05),(0. 66 ± 0. 02) nmol/mg]content of the silent Pink1 + cisplatin group and Parkin + cisplatin group were significantly lower than the hybridization siRNA + cisplatin group(P < 0. 05). The relative expression levels of Pink1,Parkin and Beclin proteins in the hybrid siRNA +cisplatin group and the ratio of LC3Ⅱ/LC3Ⅰ(1. 68 ± 0. 04,1. 80 ± 0. 05,1. 87 ± 0. 05,2. 01 ± 0. 04) were significantly higher than those in the hybrid siRNA group(1. 00 ± 0. 04,1. 00 ± 0. 05,1. 01 ± 0. 01,1. 04 ± 0. 02)(P < 0. 05);while the relative expression of Pink1,Parkin and Beclin protein in the silent Pink1 + cisplatin group and Parkin + cisplatin group and the ratio of LC3Ⅱ/LC3Ⅰ(1. 17 ± 0. 05,0. 69 ± 0. 05,1. 37 ± 0. 05,1. 43 ± 0. 02;1. 22 ± 0. 04,0. 57 ± 0. 06,1. 39 ± 0. 03,1. 38 ± 0. 10) were significantly lower than the hybrid siRNA + cisplatin group(P < 0. 05). The number of autophagy positive spots in the hybrid siRNA + cisplatin group(12. 2 ± 0. 7) was significantly more than that in the hybrid siRNA group(2. 4 ± 0. 3)(P < 0. 05);the number of fluorescent spots in the silent Pink1 + cisplatin group(5. 3 ± 0. 8) and Parkin + cisplatin group(5. 6 ± 0. 5) was significantly less than that of the hybrid siRNA + cisplatin group(P < 0. 05). Conclusion Cisplatin treatment can induce mitochondrial autophagy in HK-2 cells,thereby improving renal tubular cytotoxicity and mitochondrial function caused by cisplatin. Its mechanism may be related to the expression of Pink1/Parkin protein.