CAJ | 학술논문

目的：研究 microRNA-99a(miR-99a)对过氧化氢所诱导神经细胞 neuro-2a 氧化损伤的影响。方法常规培养 neuro-2a细胞并分为3组：正常对照组,过氧化氢100μmol/ L 刺激组,miR-99a 预处理组(转染 miRNA-99a mimics+过氧化氢100μmol/ L刺激),用 CCK-8试剂盒检测细胞存活率,生化试剂盒检测还原型烟酰胺腺嘌呤二核苷酸( nicotinamide adenine dinucleotide, NADH)含量和总超氧化物歧化酶(total superoxide dismutase,T-SOD)、锰超氧化物歧化酶(manganese superoxide dismutase,Mn-SOD)活性,Western blotting 检测突触小体相关蛋白(synaptosoma associated protein of molecular mass 25000,SNAP25)及 Mn-SOD、细胞外超氧化物歧化酶(extracellular SOD,EC-SOD)的蛋白表达水平。结果与正常对照组相比,过氧化氢刺激的2组细胞存活率明显下降,分别为85%和89%(P<0.05),但 miR-99a 预处理组的细胞存活率下降程度较小仅为11%(P<0.05)。进一步研究发现,过氧化氢刺激引起细胞 T-SOD、Mn-SOD 活性降低(P<0.05),转染 miR-99a mimics 不但能增强 T-SOD 和 Mn-SOD 的活性,还能促进 Mn-SOD 和 EC-SOD 的表达(P<0.05)。过氧化氢导致细胞中 NADH 含量降低(P<0.05),miR-99a 能够增加 NADH 含量甚至高于正常细胞水平(P<0.05)。 miR-99a 还有增加 neuro-2a 细胞表达 SNAP25的趋势。结论 miR-99a 对过氧化氢诱导neuro-2a 细胞引起的氧化损伤具有保护作用。
목적：연구 microRNA-99a(miR-99a)대과양화경소유도신경세포 neuro-2a 양화손상적영향。방법상규배양 neuro-2a세포병분위3조：정상대조조,과양화경100μmol/ L 자격조,miR-99a 예처리조(전염 miRNA-99a mimics+과양화경100μmol/ L자격),용 CCK-8시제합검측세포존활솔,생화시제합검측환원형연선알선표령이핵감산( nicotinamide adenine dinucleotide, NADH)함량화총초양화물기화매(total superoxide dismutase,T-SOD)、맹초양화물기화매(manganese superoxide dismutase,Mn-SOD)활성,Western blotting 검측돌촉소체상관단백(synaptosoma associated protein of molecular mass 25000,SNAP25)급 Mn-SOD、세포외초양화물기화매(extracellular SOD,EC-SOD)적단백표체수평。결과여정상대조조상비,과양화경자격적2조세포존활솔명현하강,분별위85%화89%(P<0.05),단 miR-99a 예처리조적세포존활솔하강정도교소부위11%(P<0.05)。진일보연구발현,과양화경자격인기세포 T-SOD、Mn-SOD 활성강저(P<0.05),전염 miR-99a mimics 불단능증강 T-SOD 화 Mn-SOD 적활성,환능촉진 Mn-SOD 화 EC-SOD 적표체(P<0.05)。과양화경도치세포중 NADH 함량강저(P<0.05),miR-99a 능구증가 NADH 함량심지고우정상세포수평(P<0.05)。 miR-99a 환유증가 neuro-2a 세포표체 SNAP25적추세。결론 miR-99a 대과양화경유도neuro-2a 세포인기적양화손상구유보호작용。
Objective To investigate the effects of microRNA-99a(miR-99a) on neuro-2a cells against oxidative injury induced by hydrogen peroxide(H2 O2 ). Methods Neuro-2a cells were divided into 3 groups by different treatments: control group, control+H2 O2 (100 μmol/ L) group and miR-99a mimics+H2 O2 group. Cell viability was measured by cell counting kit-8. Total superoxide dismutase (T-SOD) activity, manganese superoxide dismutase ( Mn-SOD) activity, and content of reduced nicotinamide adenine dinucleotide (NADH) were assayed by a variety of biochemical kits according to the instructions respectively. Protein expression levels of synaptosoma associated protein of molecular mass 25 000(SNAP25), Mn-SOD and extracellular SOD(EC-SOD) were assessed by Western blotting. Results Compared with control group, cell viability of neuro-2a in the control + H2 O2 group and miR-99a + H2 O2 group, decreased significantly to 85% and 89% respectively upon hydrogen peroxide stimulation(P<0. 05). However, miR-99a+H2 O2 group appeared to have less reduction of 11% than control+H2 O2 group(P<0. 05). Further studies showed that enzyme activities of T-SOD and Mn-SOD were depressed in control+H2 O2 group in comparison with control group( P<0. 05). Whereas, with pretreatment of miR-99a mimics transfection, T-SOD and Mn-SOD activities were enhanced to a significantly higher level, relative to the normal level of control group(P<0. 05), as well as greatly increased protein expression of Mn-SOD and EC-SOD, in contrast to control group and control+H2 O2 group(P<0. 05). Besides, NADH content of neuro-2a in control+H2 O2 group was reduced compared with control group(P<0. 05), while in miR-99a+H2 O2 group, NADH content exceeded the normal level of control group( P<0. 05). It was also found that protein expression of SNAP25 increased in the two groups of hydrogen peroxide stimulation. And miR-99a+H2 O2 group had a tendency of more increase in protein expression of SNAP25, compared to control + H2 O2 group. Conclusion miR-99a can effectively protect neuro-2a cells from oxidative injury induced by hydrogen peroxide.