CAJ | 학술논문

目的观察对氧磷酶1(PON1)基因过表达对急性敌敌畏中毒所致小鼠膈肌细胞损伤的影响.方法常规培养小鼠膈肌细胞,转染过表达慢病毒载体,将细胞分为正常对照组,敌敌畏(DDVP)组,LV-GFP+ DDVP组,LV-PON1+ DDVP组,CCK-8法检测细胞存活率、流式细胞术检测细胞的凋亡情况、RT-PCR及Western印迹法检测小鼠膈肌细胞PON1和Nrf2mRNA及蛋白的表达情况、ELISA法检测细胞乙酰胆碱酯酶(AchE)、血红素氧合酶1(HO-1)、醌氧化还原酶1(NQO-1)水平,化学比色法检测细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活力及丙二醛(MDA)水平.结果 0、80、160、320、640 μmol/L DDVP诱导小鼠膈肌细胞24 h后,细胞的生存率分别为(100±3.82)％、(82.13 ±2.60)％、(53.57±5.05)％、(30.77±3.30)％、(14.20 ±2.19)％,呈浓度依赖性;以160μmol／L DDVP分别诱导小鼠膈肌细胞0、6、12、24 h后,细胞的存活率分别为(100.17±2.74)％、(76.13±6.01)％、(66.53±3.55)％、(53.57±5.05)％,呈时间依赖性(P＜0.05).LV-PON1组细胞PON1蛋白表达高于空白对照组(0.370±0.015与0.232 ±0.004,0.197±0.015与0.037±0.003,P＜0.05).DDVP诱导后,不同时间点LV-PON1组细胞存活率高于DDVP组(P＜0.05).诱导24 h,LV-PON1组凋亡率及MDA含量低于DDVP组(P＜0.05).LV-PON1组AchE水平、PON1和Nrf2蛋白表达水平、SOD和CAT、HO-1和NQO-1水平均高于DDVP组(P＜0.05).结论 PON1基因过表达可以有效减轻DDVP对小鼠膈肌细胞AchE的抑制作用,可以诱导Nrf2的表达,发挥抗氧化作用,从而保护小鼠膈肌细胞.
목적 관찰대양린매1(PON1)기인과표체대급성활활외중독소치소서격기세포손상적영향.방법 상규배양소서격기세포,전염과표체만병독재체,장세포분위정상대조조,활활외(DDVP)조,LV-GFP+ DDVP조,LV-PON1+ DDVP조,CCK-8법검측세포존활솔、류식세포술검측세포적조망정황、RT-PCR급Western인적법검측소서격기세포PON1화Nrf2mRNA급단백적표체정황、ELISA법검측세포을선담감지매(AchE)、혈홍소양합매1(HO-1)、곤양화환원매1(NQO-1)수평,화학비색법검측세포내초양화물기화매(SOD)、과양화경매(CAT)활력급병이철(MDA)수평.결과 0、80、160、320、640 μmol/L DDVP유도소서격기세포24 h후,세포적생존솔분별위(100±3.82)％、(82.13 ±2.60)％、(53.57±5.05)％、(30.77±3.30)％、(14.20 ±2.19)％,정농도의뢰성;이160μmol／L DDVP분별유도소서격기세포0、6、12、24 h후,세포적존활솔분별위(100.17±2.74)％、(76.13±6.01)％、(66.53±3.55)％、(53.57±5.05)％,정시간의뢰성(P＜0.05).LV-PON1조세포PON1단백표체고우공백대조조(0.370±0.015여0.232 ±0.004,0.197±0.015여0.037±0.003,P＜0.05).DDVP유도후,불동시간점LV-PON1조세포존활솔고우DDVP조(P＜0.05).유도24 h,LV-PON1조조망솔급MDA함량저우DDVP조(P＜0.05).LV-PON1조AchE수평、PON1화Nrf2단백표체수평、SOD화CAT、HO-1화NQO-1수평균고우DDVP조(P＜0.05).결론 PON1기인과표체가이유효감경DDVP대소서격기세포AchE적억제작용,가이유도Nrf2적표체,발휘항양화작용,종이보호소서격기세포.
Objective To investigate the effect of paraoxonase1 (PON1) overexpression on mouse diaphragmatic muscle cells injury caused by acute dichlorvos poisoning.Methods Mouse diaphragmatic muscle cells were cultured routinely and infected with overexpression lentivirus.Cells were divided into normal control group,DDVP group,LV-GFP + DDVP group,LV-PON1 + DDVP group.Cell viability was determined by CCK-8 assay.Flow cytometry was used to detect cell apoptosis.The mRNA and protein expression of PON1 and Nrf2 in mouse diaphragmatic muscle cells was measured by RT-PCR and Western blot.Enzyme-linked immunosorbent assay was used to determine levels of acetyl cholinesterase (AchE),heme oxygenase 1 (HO-1) and quinone oxidoreductase-1 (NQO-1) in mouse diaphragmatic muscle cells.The activity of superoxide dismutase (SOD) and catalase (CAT) as well as malondialdehyde (MDA) content in cells was measured by chemical colorimetry.Results After induced by 0,80,160,320,640μmol/L DDVP for 24 hours,the viability of mouse diaphragmatic muscle cells was (100 ± 3.82) ％,(82.13 ±2.60)％,(53.57 ±5.05)％,(30.77 ±3.30)％,(14.20 ± 2.19)％ respectively,changing in a concentration-dependent manner(P ＜ 0.05).After induced by 160 μmol/L DDVP for 0,6,12,24 hours,the viability of mouse diaphragmatic muscle cells was (100.17 ± 2.74) ％,(76.13 ± 6.01) ％,(66.53 ±3.55) ％,(53.57 ± 5.05) ％,changing in a time-dependent manner(P ＜ 0.05).The PON1 protein level in LV-PON1 group was higher than that of blank control group (0.370 ± 0.015 vs 0.232 ± 0.004,0.197 ± 0.015 vs 0.037 ±0.003,P ＜0.05).The cell viability of LV-PON1 group is higher than that of DDVP group at different time point after induction of DDVP (P ＜ 0.05).After induced by DDVP for 24 hours,the cell apoptosis rate and MDA content in LV-PON1 group were lower than those of DDVP group(P ＜ 0.05).While levels of AchE,PON1 and Nrf2 protein expression,SOD and CAT,HO-1 and NQO-1 were higher than those of DDVP group (P ＜ 0.05).Conclusions The overexpression of PON1 could effectively alleviate AchE inhibition by DDVP and induce Nrf2 expression to exert antioxidant effect,thus protected the mouse diaphragmatic muscle cells.