| 于兆海,于悦.粗糠柴毒素上调微小 RNA-204-5p对脂多糖所致心肌损伤的保护机制研究[J].安徽医药,2022,26(12):2368-2374. |
| 粗糠柴毒素上调微小 RNA-204-5p对脂多糖所致心肌损伤的保护机制研究 |
| Study on the protective mechanism of rottlerin up-regulates miR-204-5p on cardiomyocytes damage induced by LPS |
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| DOI：10.3969/j.issn.1009-6469.2022.12.008 |
| 中文关键词: 野桐属植物 高迁移率族蛋白 B1 粗糠柴毒素 微小 RNA-204-5p 脂多糖 心肌损伤 |
| 英文关键词: Mallotus plant HMGB1 Rottlerin MiR-204-5p LPS Cardiomyocyte damage |
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| 中文摘要: |
| 目的探讨粗糠柴毒素对脂多糖( LPS)所致心肌损伤的影响及其保护机制。方法该研究自 2018年 9月至 2019年 4月，用终浓度为 10 mg/L的 LPS刺激 HCM、AC16心肌细胞 6h，分别加入终浓度为 0.5 μmol/L、1.0 μmol/L、1.5 μmol/L、2.0 μmol/L粗糠柴毒素( Rottlerin)再处理 12 h后分别记为 LPS+0.5 μmol/L Rottlerin组、 LPS+1.0 μmol/L Rottlerin组、 LPS+1.5 μmol/L Rottler. in组、 LPS+2.0 μmol/L Rottlerin组，以单纯 10 mg/L LPS处理的为 LPS组，以未经粗糠柴毒素和 LPS处理的为对照组。将模拟物阴性对照( miR-NC)、微小 RNA-204-5p模拟物( miR-204-5p)、抑制剂阴性对照( anti-miR-NC)、 miR-204-5p抑制剂( anti-miR-204-5p)分别转染至未经任何处理的 HCM细胞中，记为 miR-NC组、 miR-204-5p组、 anti-miR-NC组、 anti-miR-204-5p组。将 miR-NC， miR-204-5p转染至单纯 10 mg/L LPS处理的 HCM细胞中，记为 LPS+miR-NC组， LPS+miR-204-5p组。将 anti-miR-NC、anti-miR-204-5p、过表达空载体( pcDNA3.1)、高迁移率族蛋白 B1(HMGB1)过表达载体( pcDNA3.1-HMGB1)转染至 2.0 μmol/L粗糠柴毒素和 10 mg/L LPS处理 HCM细胞中，记为 LPS+Rottlerin+anti-miR-NC组、 LPS+Rottlerin+anti-miR-204-5p组、 LPS+Rottlerin+pcD. NA3.1组、 LPS+Rottlerin+pcDNA3.1-HMGB1组。 MTT法检测各组细胞增殖；流式细胞术检测细胞凋亡；实时荧光定量逆转录聚合酶链反应( qRT-PCR)检测 miR-204-5p和 HMGB1 mRNA的表达水平；蛋白质印迹法检测蛋白表达；双荧光素酶报告基因检测实验检测荧光活性。结果与对照组相比， LPS组心肌细胞 HCM、AC16抑制率显著升高， HCM细胞中 miR-204-5p(0.30±0.03比 1.99±0.08)和 B淋巴细胞瘤 -2(Bcl-2)表达显著下降， Bax、HMGB1(1.04±0.10比 0.26±0.02)表达和凋亡率［( 27.79±2.23)%比(5.61±0.05)%］显著升高；相较于 LPS组， LPS+0.5、1.0、1.5、2.0 μmol/L Rottlerin组 HCM、AC16细胞抑制率显著降低， HCM细胞中 miR-204-5p(0.46±0.04、0.54±0.05、0.76±0.04、0.88±0.07比 0.30±0.03)和 Bcl-2表达显著升高， Bax、HMGB1(0.93±0.09、0.80±0.08、0.62±0.06、0.53±0.05比 1.04±0.10)表达和凋亡率［( 22.61±2.14)%、(20.06±1.94)%、(11.71±1.01)%、(9.16±1.00)%比(27.79±2.23)%］显著降低，呈浓度依赖型。与 LPS+miR-NC组相比， LPS+miR-204-5p组 HCM细胞中 miR-204-5p(0.86±0.07比 0.42±0.03)和 Bcl-2表达升高， Bax表达及细胞抑制率和凋亡率［( 15.76±1.32)%比( 27.26±2.25)%］降低。 miR-204-5p可靶向调控 HMGB1；抑制 miR-204-5p表达、过表达 HMGB1均能逆转 Rottlerin对 LPS作用的心肌细胞 HCM的保护作用。结论粗糠柴毒素对 LPS所致的心肌细胞损伤有保护作用，其机制主要是通过上调 miR-204-5p表达，进而抑制 HMGB1的表达发挥作用。 |
| 英文摘要: |
| Objective To explore the effects of Rottlerin on cardiomyocytes damage induced by LPS and its protective mechanism. Methods The study was conducted from September 2018 to April 2019. HCM, AC16 cardiomyocytes were stimulated with LPS at a fi.nal concentration of 10 mg/L for 6. before a final concentration of 0.5 μmol/L, 1.0 μmol/L, 1.5 μmol/L and 2.0 μmol/L Rottlerin wasadded and retreated for 12 h, respectively, which were denoted as LPS+0.5 μmol/L Rottlerin group and LPS+1.0 μmol/L Rottleringroup, LPS+1.5 μmol/L Rottlerin group, LPS+2.0 μmol/L Rottlerin group. Cells treated with 10 mg/L LPS were designated as the LPSgroup, while those treated without crude bran firewood toxin and LPS were used as control groups .The mimic negative control (miRNC),microrNA-204-5p mimic (miR-204-5p), inhibitor negative control (anti-miR-NC), and miR-204-5p inhibitor (anti-miR-204-5p) were transfected into HCM cells without any treatment, respectively. They were denoted as miR-NC group, miR-204-5p group, anti-miR-NC group, anti-miR-204-5p group. MiR-NC and miR-204-5p were transfected into HCM cells treated with 10 mg/L LPS, which were denot. ed as LPS+miR-NC group, LPS+miR-204-5p group. Anti-miR-NC, anti-miR-204-5p, overexpression vector (pcDNA3.1) and high mo. bility group protein B1 overexpression vector (pcDNA3.1-HMGB1) were transfected into HCM cells treated with 2.0 μmol/L Rottlerinand 10 mg/L LPS. They were denoted as LPS+Rottlerin+anti-miR-NC group, LPS+Rottlerin+anti-miR-204-5p group, LPS+Rottlerin+ pcDNA3.1 group, LPS+Rottlerin+ pcDNA3.1-HMGB1 group. The cell proliferation was detected by MTT; apoptosis was detected by flow cytometry; expression of miR-204-5p and HMGB1 mRNA was detected by qRT-PCR; protein expression was detected by Western blotting; fluorescence activity was detected by double luciferase reporter gene assay.Results Compared with the Con group, the inhibi. tion rate of HCM and AC16 in LPS group was significantly increased, the expression of miR-204-5p (0.30±0.03 vs. 1.99±0.08) and Bcl-2 in HCM cells was significantly decreased, and the expression of Bax and HMGB1 (1.04±0.10 vs. 0.26±0.02) and apoptosis rate [(27.79±2.23)% vs. (5.61±0.05)%] were significantly increased. Compared with the LPS group, the inhibition rate of HCM and AC16cells in LPS+0.5, 1.0, 1.5, 2.0 μmol/L Rottlerin group was significantly decreased, the expression of miR-204-5p (0.46±0.04, 0.54± 0.05, 0.76±0.04, 0.88±0.07 vs. 0.30±0.03) and Bcl-2 in HCM cells was significantly increased, the expression of Bax and HMGB1 (0.93±0.09, 0.80±0.08, 0.62±0.06, 0.53±0.05 vs. 1.04±0.10) and the apoptosis rate [(22.61±2.14)%, (20.06±1.94)%, (11.71±1.01)%, (9.16±1.00)% vs. (27.79±2.23)%] were significantly decreased, showing a concentration dependent pattern. Compared with LPS+miR-NC group, the expression of miR-204-5p (0.86±0.07 vs. 0.42±0.03) and Bcl-2 in HCM cells in LPS+miR-204-5p group was increased, and the expression of Bax, cell inhibition rate and apoptosis rate [(15.76±1.32)% vs. (27.26±2.25)%] were decreased. miR-204-5p could target HMGB1; Inhibition of miR-204-5p expression and overexpression of HMGB1 reversed the protective effect of Rottlerin on HMC treated with LPS.Conclusion Rottlerin has a protective effect against cardiomyocyte damage induced by LPS. The mechanism is mainly attributed to up-regulating the expression of miR-204-5p and inhibiting the expression of HMGB1. |
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