人源性长寿保障基因2对脂多糖诱导的PC12细胞损伤的影响

Effects of Homo sapiens longevity assurance homologue 2 on lipopolysaccharide-induced PC12 cell injury

目的探讨沉默人源性长寿保障基因2(LASS2)基因对脂多糖(LPS)诱导的PC12细胞活力、凋亡及核转录因子kappaB(NF-κB)信号通路的影响。方法将大鼠肾上腺嗜铬细胞瘤(PC12)细胞分为4组:空白组(无任何处理)、LPS组(200μg·mL^-1 LPS处理PC12细胞)、LPS+NC组(转染阴性对照siRNA质粒后用200μg·mL^-1LPS处理)、LPS+si-LASS2组(转染LASS2的特异性siRNA质粒后用200μg·mL^-1LPS处理)。以蛋白质印迹法检测LASS2、磷酸化NF-κB(p-NF-κB)、活化的半胱氨酸天冬氨酸蛋白酶-3(Cleaved caspase-3)和环氧化酶-2(COX-2)蛋白表达。细胞检测试剂盒-8和流式细胞术分别检测细胞活力(OD值)与凋亡率。活性氧(ROS)试剂盒检测ROS含量(相对荧光强度)。结果空白组、LPS组、LPS+NC组、LPS+si-LASS2组的LASS2蛋白相对表达量分别为0.11±0.01,0.80±0.07,0.79±0.06和0.16±0.02;这4组的细胞活力分别为0.81±0.06,0.40±0.04,0.41±0.04和0.68±0.06;这4组的细胞凋亡率分别为(4.51±0.46)%,(31.33±1.75)%,(30.06±1.61)%和(16.74±1.01)%;这4组的ROS水平分别为248.20±15.30,521.80±23.60,518.90±22.30,396.60±18.70;这4组的COX-2蛋白相对表达量分别为0.07±0.01,0.66±0.06,0.67±0.06和0.17±0.02;这4组的p-NF-κB蛋白相对表达量分别为0.10±0.01,0.51±0.05,0.53±0.05和0.18±0.02;这4组的Cleaved-caspase3蛋白相对表达量分别为0.02±0.01,0.29±0.03,0.28±0.03和0.06±0.01。上述指标:LPS组和LPS+NC组与空白组相比,差异均有统计学意义(均P<0.05);LPS+si-LASS2组与LPS组相比,差异均有统计学意义(均P<0.05)。结论沉默LASS2表达可增强PC12细胞活力,抑制细胞凋亡,从而对神经损伤有保护作用,其机制可能与抑制Cleaved-caspase3、p-NF-κB、COX-2表达和ROS水平有关。

Objective To investigate the effect and mechanism of silencing Homo sapiens longevity assurance homologue2(LASS2)gene on lipopolysaccharide(LPS)-induced PC12 cell viability,apoptosis and nuclear factor kappaB(NF-κB)signaling pathway.Methods Rat adrenal pheochromocytoma(PC12)cells were divided into four groups:blank group(PC12 cells without any treatment),LPS group(PC12 cells treated with 200μg·mL^-1 LPS),LPS+NC group(treated with 200μg·mL^-1 LPS after transfected negative control siRNA plasmid),LPS+si-LASS2 group(treated with 200μg·m L^-1 LPS after transfected LASS2 specific siRNA plasmid).Expressions of LASS2,phosphorylated NF-κB(p-NF-κB),Cleaved cysteine-containing aspartate-specific proteases-3(Cleaved caspase-3)and loop Oxidase-2(COX-2)protein were detected by Western blotting.Cell viability(OD value)and apoptosis rate were detected by cell detection kit-8 and flow cytometry,respectively.The ROS content was measured by a reactive oxygen species(ROS:relative fluorescence intensity)kit.Results The relative expressions of LASS2 protein in blank group,LPS group,LPS+NC group and LPS+si-LASS2 group were0.11±0.01,0.80±0.07,0.79±0.06,0.16±0.02,respectively;the level of cell viability in the four groups were0.81±0.06,0.40±0.04,0.41±0.04,and 0.68±0.06,respectively;the apoptosis rates in the four groups were(4.51±0.46)%,(31.33±1.75)%,(30.06±1.61)%,(16.74±1.01)%,respectively;ROS levels in the four groups were 248.20±15.30,521.80±23.60,518.90±22.30,396.60±18.70,respectively;the relative expressions of COX-2 protein were 0.07±0.01,0.66±0.06,0.67±0.06,0.17±0.02,respectively;the relative expression of p-NF-κB protein were 0.10±0.01,0.51±0.05,0.53±0.05,0.18±0.02,respectively;the relative expression of Cleaved-caspase3 protein were 0.02±0.01,0.29±0.03,0.28±0.03,0.06±0.01,respectively.Comparison between LPS group,LPS+NC group and blank group,the difference of the factors were significantly(all P<0.05);comparison between LPS+si-LASS2 group and LPS group,the difference of the factors were significantly(all P<0.05).Conclusion The silencing of LASS2 expression can enhance the viability of PC12 cells,inhibit apoptosis and protect the nerve from injury.The mechanism may be related to the inhibition of Cleaved-caspase 3,p-NF-κB and COX-2 expression and ROS level.