CAJ | 학술논문

目的观察Ras相关的C3肉毒素底物1(Rac1)在草酸所致的肾小管上皮细胞氧化损伤中的作用.方法体外培养犬肾小管上皮细胞(MDCK细胞)制成爬片后,随机分为空白对照组(A组)、Rac1抑制组(B组)、草酸组(C组)、Rac1抑制+草酸组(D组).B组和D组分别暴露在含Rac1抑制剂NSC23766(100 μmol/L)培养液中,30 min后C组及D组分别更换为含草酸(5 mmol/L)的培养液,余对照更换为磷酸盐缓冲液(PBS).4h后分别测定各组培养液中过氧化氢(H2 O2)浓度、乳酸脱氢酶(LDH)活性和细胞还原型烟酰胺腺嘌呤二核苷酸(NADPH)氧化酶活性,免疫细胞化学染色观察细胞Rac1表达,向培养液中加入草酸钙(0.75 mmol/L)15 min后在倒置显微镜下观察细胞对草酸钙结晶的黏附.结果 A组H2O2浓度、LDH活性及细胞NADPH氧化酶活性分别为(24.23±2.44) mmol/L、(0.62 ±0.06) U/ml、(1 042.83±61.00) RLU/(min· mg);B组分别为(24.46±3.39) mmol/L、(0.56±0.06) U/ml、(1 096.67±66.12) RLU/(min· mg);C组分别为(67.84±4.25) mmol/L、(1.71 ±0.08) U/ml、(2 852.50±105.71) RLU/(min· mg);D组分别为(34.79±3.07) mmol/L、(0.96±0.07) U/ml、(1 118.83±57.56) RLU/(min· mg).与A组比较,C组细胞Rac1表达增强(P＜0.05),NADPH氧化酶活性、H2O2浓度、LDH活性明显增强(P＜0.05),细胞对草酸钙结晶的黏附数量增多;而与C组比较,D组细胞Rac1表达降低(P＜0.05),NADPH氧化酶活性、H2O2浓度、LDH活性均显著降低(P＜0.05),黏附在细胞表面的草酸钙结晶数量减少.结论草酸通过Rac1调节的NADPH氧化酶致肾小管上皮细胞氧化损伤,抑制Rac1可显著减少活性氧物质的产生及细胞损伤.
목적 관찰Ras상관적C3육독소저물1(Rac1)재초산소치적신소관상피세포양화손상중적작용.방법 체외배양견신소관상피세포(MDCK세포)제성파편후,수궤분위공백대조조(A조)、Rac1억제조(B조)、초산조(C조)、Rac1억제+초산조(D조).B조화D조분별폭로재함Rac1억제제NSC23766(100 μmol/L)배양액중,30 min후C조급D조분별경환위함초산(5 mmol/L)적배양액,여대조경환위린산염완충액(PBS).4h후분별측정각조배양액중과양화경(H2 O2)농도、유산탈경매(LDH)활성화세포환원형연선알선표령이핵감산(NADPH)양화매활성,면역세포화학염색관찰세포Rac1표체,향배양액중가입초산개(0.75 mmol/L)15 min후재도치현미경하관찰세포대초산개결정적점부.결과 A조H2O2농도、LDH활성급세포NADPH양화매활성분별위(24.23±2.44) mmol/L、(0.62 ±0.06) U/ml、(1 042.83±61.00) RLU/(min· mg);B조분별위(24.46±3.39) mmol/L、(0.56±0.06) U/ml、(1 096.67±66.12) RLU/(min· mg);C조분별위(67.84±4.25) mmol/L、(1.71 ±0.08) U/ml、(2 852.50±105.71) RLU/(min· mg);D조분별위(34.79±3.07) mmol/L、(0.96±0.07) U/ml、(1 118.83±57.56) RLU/(min· mg).여A조비교,C조세포Rac1표체증강(P＜0.05),NADPH양화매활성、H2O2농도、LDH활성명현증강(P＜0.05),세포대초산개결정적점부수량증다;이여C조비교,D조세포Rac1표체강저(P＜0.05),NADPH양화매활성、H2O2농도、LDH활성균현저강저(P＜0.05),점부재세포표면적초산개결정수량감소.결론 초산통과Rac1조절적NADPH양화매치신소관상피세포양화손상,억제Rac1가현저감소활성양물질적산생급세포손상.
Objective To explore the potential role of ras-related C3 botulinum toxin substrate (Rac1) in renal tubular epithelial cell oxidative injury induced by oxalate.Methods The Madin-Darby canine kidney (MDCK) cells were cultured in vitro and divided randomly into group A (blank control group),B (Rae1 inhibitor group),C (oxalate group) and D (Rae1 inhibitor and oxalate group).Group B and D were exposed in medium with Rac1 inhibitor NSC23766 (100 μmol/L) thirty minutes,and then 5 mmol/L oxalate were added to group C and D.In other control procedures,cells were treated with PBS.Four hours later,hydrogen peroxide (H2O2) and lactate dehydrogenase (LDH) were measured in each medium and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity were measured in cells,Rac1 expression were determined using immunocytochemical staining,and calcium oxalate crystallization adhered to cells were observed microscopically after adding 0.75 mmol/L calcium oxalate to the medium.Results H2 O2,LDH and NADPH oxidase activity in cells of group A were (24.23 ± 2.44) mmol/L,(0.62 ± 0.06) U/ml,(1 042.83 ±61.00) RLU/ (min·mg),group B were (24.46 ±3.39) mmol/L,(0.56 ± 0.06) U/ml,(1 096.67 ± 66.12) RLU/ (min· mg),group C were (67.84 ± 4.25) mmol/L,(1.71 ± 0.08) U/ml,(2 852.50 ± 105.71) RLU/ (min· mg),group D were (34.79 ± 3.07) mmol/L,(0.96 ± 0.07) U/ml,(1118.83 ±57.56) RLU/ (min·mg).Compared with group A,the level of Rac1 expression,NADPH oxidase activity,H2 O2 generation and LDH release in cells of group C increased significantly (all P ＜ 0.05),and the number of calcium oxalate adhered to cells in group C also increased,while compared with group C,the above indicators observed in group D decreased significantly (all P ＜ 0.05).Conclusion Oxalate cause renal tubular epithelial cell oxidative injury via Rac1 regulated-NADPH oxidase.Inhibition of Rac1 results in decreased reactive oxygen species (ROS) production and a reduction in cell injury.