杭白菊总黄酮通过下调miR-384-5p抑制MPP^(+)诱导的SK-N-SH凋亡及氧化应激

The total flavonoids of Chrysanthemum morifolium inhibited MPP^(+)-induced SK-N-SH apoptosis and oxidative stress by down-regulating miR-384-5p

目的探讨杭白菊总黄酮对1-甲基-4-苯基吡啶离子(MPP^(+))诱导的人神经母细胞瘤细胞(SK-N-SH细胞)凋亡和氧化应激的影响及可能机制。方法利用2.5 mmol/L MPP^(+)诱导SK-N-SH细胞建立细胞损伤模型,MPP^(+)诱导SK-N-SH细胞的同时加入0.1、0.2、0.4 mg/mL杭白菊总黄酮或(和)转染miR-384-5p模拟物(mimics),将加入杭白菊总黄酮的SK-N-SH细胞分为对照组(正常培养)、MPP^(+)组、MPP^(+)+杭白菊总黄酮低剂量(0.1 mg/mL)组、MPP^(+)+杭白菊总黄酮中剂量(0.2 mg/mL)组和MPP^(+)+杭白菊总黄酮高剂量(0.4 mg/mL)组,将转染miR-384-5p mimics的SK-N-SH细胞分为MPP^(+)+miR-384-5p组、MPP^(+)+杭白菊总黄酮组和MPP^(+)+杭白菊总黄酮+miR-384-5p组。细胞计数试剂盒-8(CCK-8)法检测细胞增殖,流式细胞仪检测细胞凋亡,蛋白质印迹法检测细胞中活化的半胱天冬酶-3(Cleavedcaspase3)蛋白表达,相应试剂盒检测细胞培养上清液中乳酸脱氢酶(LDH)水平、细胞中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,RT-qPCR检测miR-384-5p表达。结果与对照组比较,MPP^(+)组SK-N-SH细胞光密度(OD)值和SOD活性降低,细胞凋亡率、Cleaved-caspase3蛋白表达、LDH漏出量、细胞中MDA和miR-384-5p表达升高,差异均有统计学意义(P均<0.05);与MPP^(+)组比较,MPP^(+)+杭白菊总黄酮低、中、高剂量组SK-N-SH细胞OD值和SOD活性依次升高,细胞凋亡率、Cleaved-caspase3蛋白表达、LDH漏出量和细胞中MDA和miR-384-5p表达依次降低,差异均有统计学意义(P均<0.05),且杭白菊总黄酮呈剂量依赖性。与MPP^(+)组比较,MPP^(+)+miR-384-5p组SK-NSH细胞OD值、SOD活性降低,细胞凋亡率、Cleaved-caspase3蛋白表达、LDH漏出量和MDA含量升高,差异均有统计学意义(P均<0.05);与MPP^(+)+杭白菊总黄酮组比较,MPP^(+)+杭白菊总黄酮+miR-384-5p组SK-N-SH细胞OD值、SOD活性降低,细胞凋亡率、Cleaved-caspase3蛋白表达、LDH漏出量、MDA含量升高,差异均有统计学意义(P均<0.05)。结论杭白菊总黄酮可靶向下调miR-384-5p来抑制MPP^(+)诱导的SK-N-SH细胞凋亡和氧化应激。

Objective To explore the effect of total flavonoids of Chrysanthemum morifolium on SK-N-SH cells apoptosis and oxidative stress induced by 1-methyl-4-phenylpyridine ion(MPP^(+))and its possible mechanism.Methods SK-N-SH cells were induced by 2.5 mmol/L MPP^(+)to establish cell injury model;MPP^(+)induced SK-N-SH cells while adding 0.1,0.2,0.4 mg/mL total flavonoids of Chrysanthemum morifolium or(and)transfected miR-384-5p mimics.SK-N-SH cells induced by MPP^(+)were divided into control group(normal culture),MPP^(+)group,MPP^(+)+low dose of total flavonoids of Chrysanthemum morifolium group,MPP^(+)+medium dose total flavonoids of Chrysanthemum morifolium group and MPP^(+)+high dose total flavonoids of Chrysanthemum morifolium group.SK-N-SH cells transfected with miR-384-5p mimics were divided into MPP^(+)+miR-384-5p group,MPP^(+)+total flavonoids of Chrysanthemum morifolium group and MPP^(+)+total flavonoids of Chrysanthemum morifolium+miR-384-5p group.Cell counting kit-8(CCK-8)was used to detect cell proliferation,flow cytometry was used to detect cell apoptosis,Western blot was used to detect the expression of activated caspase-3(Cleaved-caspase3)protein in cells,and the kit was used to detect the level of lactate dehydrogenase(LDH),the content of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)in cell culture supernatant,the expression of miR-384-5p was detected by RT-qPCR.Results Compared with the control group,the optical density(OD)value and SOD activity of SK-N-SH cells in MPP^(+)group decreased,and the rate of apoptosis,the expression of Cleaved-caspase3 protein,the amount of LDH leakage,the expression of MDA and miR-384-5p in cells increased significantly(P all<0.05);compared with MPP^(+)group,the OD value and SOD activity of SK-N-SH cells in MPP^(+)+low,medium and high dose total flavonoids of Chrysanthemum morifolium groups increased in turn,and the rate of apoptosis,the expression of Cleaved-caspase3 protein,the amount of LDH leakage and the expression of MDA and miR-384-5p in cells decreased in turn,with statistical significance(P all<0.05),and total flavonoids of Chrysanthemum morifolium could be dose-dependent.Compared with MPP^(+)group,the OD value and SOD activity of SK-N-SH cells in MPP^(+)+miR-384-5p group decreased,and the rate of apoptosis,the expression of Cleaved-caspase3 protein,the amount of LDH leakage and the content of MDA increased,with significant differences(P all<0.05);compared with MPP^(+)+total flavonoids of Chrysanthemum morifolium group,the OD value and SOD activity of SK-NSH cells in MPP^(+)+total flavonoids of Chrysanthemum morifolium+miR-384-5p group decreased,and the rate of apoptosis,the expression of Cleaved-caspase3 protein,the amount of LDH leakage,and the content of MDA increased,with significant differences(P all<0.05).Conclusion The total flavonoids of Chrysanthemum morifolium might inhibit MPP^(+)-induced apoptosis and oxidative stress of SK-N-SH cells by down-regulating the expression of miR-384-5p.