藁本内酯通过调控miR-133b-5p表达对脂多糖所致心肌损伤的保护作用

Protective effect of ligustilide on myocardial injury induced by LPS by regulating miR-133b-5p expression

目的:探讨藁本内酯(LIG)对脂多糖(LPS)所致心肌损伤的影响及分子机制。方法:用质量浓度为10 mg·L-1的LPS处理心肌细胞,记为LPS组,正常培养的细胞为对照(Con)组;用浓度分别为10,20,40μmol·L-1的藁本内酯处理心肌细胞后再用10 mg·L-1的LPS处理,作为藁本内酯低、中、高浓度组;将miR-NC、miR-133b-5p转染至心肌细胞中再用10 mg·L-1的LPS处理,记为LPS+miR-NC组、LPS+miR-133b-5p组;将anti-miR-NC、anti-miR-133b-5p转染至心肌细胞中再用40μmol·L-1的藁本内酯、10 mg·L-1的LPS处理,记为LPS+LIG+anti-miR-NC组、LPS+LIG+anti-miR-133b-5p组。酶联免疫吸附法(ELISA)法检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平;流式细胞术检测细胞凋亡;蛋白质印迹(Western blot)法检测B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达;实时荧光定量PCR(RT-qPCR)检测miR-133b-5p表达水平。结果:藁本内酯低、中、高浓度处理后,LPS诱导的心肌细胞中TNF-α、IL-6水平显著降低,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,miR-133b-5p表达水平显著升高,且呈浓度依赖性(P<0.05)。miR-133b-5p过表达可抑制LPS诱导的心肌细胞中TNF-α、IL-6水平和细胞凋亡。抑制miR-133b-5p表达逆转了藁本内酯对LPS所致的心肌细胞损伤的保护作用。结论:藁本内酯通过上调miR-133b-5p表达可抑制LPS诱导的心肌细胞凋亡和炎症反应,对LPS诱导的心肌损伤具有保护作用。

OBJECTIVE To investigate the effect and molecular mechanism of ligustilide(LIG)on cardiomyocytes injury induced by lipopolysaccharide(LPS).METHODS Cardiomyocytes were treated with LPS at a concentration of 10 mg·L-1,recorded as the LPS group,and cells in normal culture as the control(Con)group;cardiomyocytes were treated with ligustilide at concentrations of 10,20,and 40μmol·L-1,respectively,and then treated with 10 mg·L-1 LPS as low,medium,and high concentration groups of the ligustilide;miR-NC,miR-133 b-5 p were transfected into cardiomyocytes and then treated with 10 mg·L-1 LPS;recorded as LPS+miR-NC group and LPS+miR-133 b-5 p group;anti-miR-NC and anti-miR-133 b-5 p were transfected into cardiomyocytes,and then treated with 40μmol·L-1 ligustilide and 10 mg·L-1 LPS and recorded as LPS+LIG+anti-miR-NC group and LPS+LIG+anti-miR-133 b-5 p group.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6);flow cytometry was used to detect apoptosis;Western blot method was used to detect the protein expressions of B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2 related X protein(Bax);real-time fluorescent quantitative PCR(RT-qPCR)was used to detect miR-133 b-5 p expression.Results After treatment with low,medium and high concentrations of ligustilide,the levels of TNF-αand IL-6 in cardiomyocytes induced by LPS were significantly reduced,the apoptosis rate was significantly reduced,the expression of Bcl-2 was significantly increased,and the expression of Bax was significantly decreased,the expression of miR-133 b-5 p was significantly increased,and it was concentration-dependent(P<0.05).Overexpression of miR-133 b-5 p can reduce the levels of TNF-αand IL-6 in cardiomyocytes induced by LPS and inhibit apoptosis..Inhibition of miR-133 b-5 p expression reversed the protective effect of ligustilide on cardiomyocyte injury induced by LPS.Conclusions Ligustilide can inhibit the cardiomyocytes apoptosis and inflammatory response induced by LPS through up-regulating the expression of miR-133 b-5 p,and has protective effect on myocardial injury induced by LPS.