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siR-NA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...

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.