冬凌草甲素通过LINC00707/miR-145-5p通路对MPP+诱导SK-N-SH细胞损伤的保护作用

Protective Effect of Oridonin on MPP^(+)-Induced SK-N-SH Cell Damage Via LPS00707/miR-145-5p Pathway

目的探讨冬凌草甲素(oridonin)对MPP+诱导SK-N-SH细胞损伤的作用及机制。方法实验分为对照组、MPP+组、冬凌草甲素低、中、高浓度组、pcDNA组、pcDNA-LINC00707组、si-NC组、si-LINC00707组、MPP++si-NC组、MPP++si-LINC00707组、MPP++冬凌草甲素+pcDNA组、MPP++冬凌草甲素+pcDNA-LINC00707组。流式细胞术检测细胞凋亡;蛋白质印迹(Western blot)法检测Bcl-2、Bax蛋白表达;乳酸脱氢酶(LDH)试剂盒检测细胞上清液中LDH释放量;丙二醛(MDA)试剂盒、谷胱甘肽(GSH)试剂盒分别检测细胞MDA、GSH含量;实时荧光定量PCR(RT-qPCR)检测LINC00707和miR-145-5p表达水平;双荧光素酶报告实验检测LINC00707和miR-145-5p的靶向关系。结果冬凌草甲素低、中、高浓度处理后,MPP+诱导的SK-N-SH细胞中细胞凋亡率显著降低,Bcl-2表达水平显著升高,Bax表达水平显著降低,LDH水平升高,MDA含量显著降低,GSH含量显著升高,LINC00707表达水平显著降低,miR-145-5p表达水平显著升高,且呈浓度依赖性(P<0.05)。LINC00707靶向调控miR-145-5p,抑制LINC00707表达抑制MPP+诱导SK-N-SH细胞凋亡和氧化应激;过表达LINC00707逆转了冬凌草甲素对MPP+诱导SK-N-SH细胞损伤的保护作用。结论冬凌草甲素可抑制MPP+诱导SK-N-SH细胞凋亡和氧化应激,其机制可能与LINC00707和miR-145-5p有关。

OBJECTIVE To investigate the effect and mechanism of oridonin on MPP^(+)-induced SK-N-SH cell damage.METHODS The experiment was divided into control group,MPP^(+)group,oridonin low,medium,and high concentration groups,pcDNA group,pcDNA-LINC00707 group,si-NC group,si-LINC00707 group,MPP^(+)+si-NC group,MPP^(+)+si-LINC00707 group,MPP^(+)+oridonin+pcDNA group,and MPP^(+)+oridonin+pcDNA-LINC00707 group.Flow cytometry was used to detect apoptosis;Western blot was used to detect Bcl-2 and Bax protein expression;lactate dehydrogenase(LDH)kit was used to detect LDH release in cell supernatant;malondialdehyde(MDA)kit and glutathione(GSH)kit were used to detect MDA and GSH content in cells;real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the expressions of LINC00707 and miR-145-5 p;dual luciferase reporter assay was used to detect the targeting relationship between LINC00707 and miR-145-5 p.RESULTS After treatment with oridonin at low,medium and high concentrations,the MDA content in SK-N-SH cells induced by MPP^(+)was significantly reduced,the apoptosis rate was significantly reduced,and the Bcl-2 expression was significantly increased,the expression of Bax was significantly reduced,the LDH level was increased,the MDA content was significantly reduced,and the GSH content was significantly increased,the expression of LINC00707 was significantly decreased,and the expression of miR-145-5 p was significantly and concentration-dependently increased(P<0.05).LINC00707 targets miR-145-5 p and inhibited the expression of LINC00707,MPP^(+)-induced apoptosis and oxidative stress in SK-N-SH cells;overexpression of LINC00707 reversed the protective effect of oridonin on MPP^(+)-induced SK-N-SH cell injury.CONCLUSION Oridonin can inhibit MPP^(+)-induced apoptosis and oxidative stress in SK-N-SH cells,and its mechanism may be related to LINC00707 and miR-145-5 p.