CAJ | 학술논문

目的:研究亚硝酸钠(NaNO_2)预适应对过氧化氢(H_2O_2)损伤鼠肾上腺嗜铬细胞瘤(PC12)细胞的保护作用.方法:分别以系列浓度的NaNO_2作用PC12细胞24 h,细胞的增殖活性采用四甲基偶氮唑蓝(MTT)法和活细胞计数法,用Hoechst33258染色计数凝集和碎核细胞占细胞总数的百分比为细胞凋亡率.以3 mmol/L浓度的NaNO_2预适应细胞24 h后,加或不加一氧化氮清除剂亚铁血红蛋白(hemo),再用1.1 mmol/L H_2O_2暴露6 h,细胞存活率检测用MTT、凋亡检测用流式细胞术和Hoechst 33258/PI染色.比色法测定丙二醛(MDA)含量和酶活性.结果:NaNO_2对PC12细胞增殖作用呈典型的β型曲线,最大低促效应出现在第24 h,最大低促效应剂量为1.4 mmol/L,最大促增殖效应为对照组的156%,未观察到不良效应水平(NOAEL)为6 mmol/L,细胞增殖半数抑制率(IC_(50))值为45 mmol/L.用3 mmol/L NaNO_2预适应24 h,然后用1.1 mmol/L H_2O_2暴露6 h,细胞增殖活性比单纯H_2O_2组明显升高(P<0.05).用1.1 mmol/L H_2O_2暴露6 h,非预适应组细胞凋亡率为44.9%;3 mmol/L NaNO_2预适应组细胞凋亡率为19.1%,差异显著(P<0.05),这种现象可以被一氧化氮清除剂亚铁血红蛋白(hemo)所抑制.3 mmol/L NaNO_2预适应组细胞超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和谷胱甘肽过氧化物酶(GSH-Px)水平升高,MDA含量降低(P<0.05).结论:低于6 mmol/L NaNO_2暴露能够引起PC12细胞的低促效应.低浓度NaNO_2预适应,通过降低细胞脂质过氧化,提高抗氧化酶活性,保护过氧化氢毒害的PC12细胞.亚硝酸盐还原为一氧化氮在细胞保护过程中起关键作用.
목적:연구아초산납(NaNO_2)예괄응대과양화경(H_2O_2)손상서신상선기락세포류(PC12)세포적보호작용.방법:분별이계렬농도적NaNO_2작용PC12세포24 h,세포적증식활성채용사갑기우담서람(MTT)법화활세포계수법,용Hoechst33258염색계수응집화쇄핵세포점세포총수적백분비위세포조망솔.이3 mmol/L농도적NaNO_2예괄응세포24 h후,가혹불가일양화담청제제아철혈홍단백(hemo),재용1.1 mmol/L H_2O_2폭로6 h,세포존활솔검측용MTT、조망검측용류식세포술화Hoechst 33258/PI염색.비색법측정병이철(MDA)함량화매활성.결과:NaNO_2대PC12세포증식작용정전형적β형곡선,최대저촉효응출현재제24 h,최대저촉효응제량위1.4 mmol/L,최대촉증식효응위대조조적156%,미관찰도불량효응수평(NOAEL)위6 mmol/L,세포증식반수억제솔(IC_(50))치위45 mmol/L.용3 mmol/L NaNO_2예괄응24 h,연후용1.1 mmol/L H_2O_2폭로6 h,세포증식활성비단순H_2O_2조명현승고(P<0.05).용1.1 mmol/L H_2O_2폭로6 h,비예괄응조세포조망솔위44.9%;3 mmol/L NaNO_2예괄응조세포조망솔위19.1%,차이현저(P<0.05),저충현상가이피일양화담청제제아철혈홍단백(hemo)소억제.3 mmol/L NaNO_2예괄응조세포초양화물기화매(SOD)、과양화경매(CAT)활성화곡광감태과양화물매(GSH-Px)수평승고,MDA함량강저(P<0.05).결론:저우6 mmol/L NaNO_2폭로능구인기PC12세포적저촉효응.저농도NaNO_2예괄응,통과강저세포지질과양화,제고항양화매활성,보호과양화경독해적PC12세포.아초산염환원위일양화담재세포보호과정중기관건작용.
AIM: To study the cytoprotective role of NaNO_2 preconditioning against H_2O_2 induced damage in PC12 cells. METHODS: PC12 cells were treated with different concentrations of NaNO_2 for 6 h, 12 h, 24 h and 48 h, respectively. The viability of the cells was measured by MTT method and cell counting. The apoptotic rate of PC12 was determined by Hoechst 33258 staining to calculate the ratio of the cells between concentrated and broken nucleus in the total cell count. PC12 cells were pretreated with NaNO_2 at concentration of 3 mmol/L for 24 h. The cytoprotective role to the toxicity of H_2O_2 at concentration of 1.1 mmol/L for 6 h was observed by MTT. The cell apoptosis was measured by flow cytometry and staining with Hoechst 33258 and PI. The activities of catalase (CAT), superoxide dismutase (SOD), and the changes of glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured by the method of colorimetry. RESULTS: The dose-response results showed that the effect of NaNO_2 on PC12 cell proliferation was a typical β-shape curve. The maximal stimulatory response was at 24 h, and the concentration of the maximum stimulatory response was 1.4 mmol/L. The maximal stimulation of the concentration-responses was 156% above the control. No observable adverse effect level (NOAEL) was 6 mmol/L. IC_(50) was 45 mmol/L. When the cells were pretreated by NaNO_2 at concentration of 3 mmol/L for 24 h, and then exposed to H_2O_2 at concentration of 1.1 mmol/L for 6 h, the proliferation rate was increased as compared to the cells treated with H_2O_2 alone. Under the conditions of treating the cells with NaNO_2 at concentration of 3 mmol/L to induce the adaptive response, then exposing the cells to H_2O_2 at concentration of 1.1 mmol/L, the apoptosis rate in non-preconditioning group was 44.9%, the apoptosis rate in preconditioning group was 19.1%, the difference was significant (P<0.05). The cytoprotective effect of NaNO_2 was inhibited by nitric oxide (NO) scavenger ferrohemoglobin. The activities of SOD, CAT and the level of GSH-Px were markedly increased, the content of MDA decreased in preconditioning group. CONCLUSION: Exposure of NaNO_2 at concentration of <6 mmol/L induces hormesis on PC12 cells. Low dose of nitrite plays an important role in cytoprotection by reducing nitrite to NO, indicating that decrease in lipid peroxidation and increase in endogenous antioxidants may play a key role in cytoprotection induced by preconditioning with low dose of NaNO_2 in PC12 cells.