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  • Title: [Effects of microRNA-34a on regulating silent information regulator 1 and influence of the factor on myocardial damage of rats with severe burns at early stage].
    Author: Bai XZ, He T, Zhang JL, Liu Y, Cao MY, Zhang JN, Cai WX, Jia YH, Shi JH, Su LL, Hu DH.
    Journal: Zhonghua Shao Shang Za Zhi; 2018 Jan 20; 34(1):21-28. PubMed ID: 29374923.
    Abstract:
    Objective: To explore the effects of microRNA-34a on regulating silent information regulator 1 (SIRT1) and influence of SIRT1 on myocardial damage of rats with severe burns at early stage. Methods: (1) Twenty-four Sprague-Dawley (SD) rats were divided into sham injury (SI) group, simple burns (SB) group and SIRT1 agonist (SA) group according to the random number table (the same grouping method below), with 8 rats in each group. Rats in groups SB and SA were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burns) on the back, and rats in group SI were sham injuried on the back. Immediately after injury, rats in groups SI and SB were intraperitoneally injected with normal saline of 50 mL/kg, and rats in group SA were intraperitoneally injected with normal saline of 50 mL/kg and 1 mg/mL resveratrol of 50 mg/kg. At 6 h post injury, abdominal aortic blood was collected to make serum and myocardial tissue of rats was collected. (2) Myocardial cells of twelve neonatal SD rats were collected and divided into microRNA-34a mimic control (MMC) group, microRNA-34a mimic (MM) group, microRNA-34a inhibitor control (MIC) group, and microRNA-34a inhibitor (MI) group, which were respectively transfected with gene sequences of mimic control, mimic, inhibitor control, and inhibitor of microRNA-34a. The microRNA-34a expression level and protein expression level of SIRT1 in myocardial cells were respectively detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Another batch of myocardial cells were divided into microRNA-34a inhibitor control+ burn serum (MCB) group, microRNA-34a inhibitor+ burn serum (MB) group, and microRNA-34a inhibitor+ burn serum + EX527 (MBE) group. Myocardial cells in group MCB were transfected with gene sequence of inhibitor control, and myocardial cells in the later groups were transfected with gene sequence of inhibitor of microRNA-34a. After transfection of 48 h, myocardial cells in group MBE were cultured in Dulbecco's modified Eagle's medium (DMEM) solution for 6 hours, with serum in group SB of volume fraction of 10% and final amount-of-substance concentration of 1 mol/L, and myocardial cells in the other 2 groups were cultured in DMEM solution with serum from rats of group SB of volume fraction of 10%. The protein expression levels of myocardial cells of SIRT1, cleaved-caspase-3, and Bax were detected by Western blotting. (3) Myocardial tissue from (1) was collected to detect expression levels of microRNA-34a and mRNA of SIRT1 in groups SI and SB by real-time fluorescence quantitative RT-PCR. Morphology of myocardial tissue of rats in groups SI, SB, and SA was observed with biological image navigator. The mRNA expression levels of interleukin 1β (IL-1β) and tumor necrosis factor (TNF-α) of rats in groups SI, SB, and SA were detected by real-time fluorescence quantitative RT-PCR. The expression levels of cleaved-caspase-3, and Bax of myocardial tissue of rats in groups SI, SB, and SA were detected by Western blotting. Data were processed with one-way analysis of variance and least-significant difference test. Results: (1) After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MM was 4.67±0.92, significantly higher than 1.03±0.04 in group MMC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MM was 0.35±0.06, significantly lower than 1.12±0.11 in group MMC (P<0.01). After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MI was 0.26±0.07, significantly lower than 1.33±0.07 in group MIC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MIC was 1.12±0.16, significantly lower than 1.74±0.34 in group MI (P<0.01). At 6 h after culture, compared with those in group MCB, the SIRT1 protein expression level of myocardial cells in group MB was significantly increased (P<0.05), while cleaved-caspase-3 and Bax protein expression levels of myocardial cells in group MB were significantly decreased (P<0.05). Compared with those in group MB, the SIRT1 protein expression level of myocardial cells in group MBE was with no significantly statistical difference (P>0.05), and cleaved-caspase-3 and Bax protein expression levels were significantly increased (P<0.05). (2) At 6 h post injury, compared with that in group SI, the microRNA-34a expression level of myocardial tissue in group SB was significantly increased (P<0.01), and the mRNA expression level of SIRT1 of myocardial tissue in group SB was significantly decreased (P<0.01). At 6 h post injury, myocardial cells in group SI arranged neatly with normal nucleus and no inflammatory cells infiltration; myocardial cells in group SB arranged disorderly, with no abnormal nucleus, and obvious inflammatory cells infiltration; myocardial cells in group SA arranged neatly, with normal nucleus and little inflammatory cells infiltration. At 6 h post injury, compared with those in group SB, the mRNA expression levels of IL-1β and TNF-α, and the protein expression levels of cleaved-caspase-3 and Bax of myocardial tissue in groups SI and SA were significantly decreased (P<0.01). Conclusions: The microRNA-34a expression level of myocardial tissue of rats with severe burns at early stage increases, which decreases the expression level of SIRT1, and increases the expression levels of IL-1β, TNF-α, cleaved-caspase-3 and Bax, leading to obvious myocardial damage. Activation of SIRT1 can alleviate myocardial damage of rats with severe burns at early stage through decreasing expression levels of IL-1β, TNF-α, cleaved-caspase-3, and Bax. 目的： 探讨微小RNA-34a对沉默信息调节因子1(SIRT1)的调节作用及SIRT1对严重烧伤大鼠早期心肌损伤的影响。 方法： (1)将24只SD大鼠按照随机数字表法(分组方法下同)分为假伤组、单纯烧伤组和SIRT1激动剂组，每组8只。单纯烧伤组、SIRT1激动剂组大鼠背部造成30%体表总面积Ⅲ度烫伤(以下称烧伤)，假伤组大鼠背部致假伤。伤后即刻，假伤组、单纯烧伤组大鼠腹腔注射生理盐水50 mL/kg，SIRT1激动剂组大鼠腹腔注射生理盐水50 mL/kg+1 mg/mL白藜芦醇(50 mg/kg)。伤后6 h，抽取腹主动脉血制备血清，取心肌组织。(2)取12只SD大鼠乳鼠心肌细胞，分为微小RNA-34a模拟物对照组、微小RNA-34a模拟物组、微小RNA-34a抑制物对照组及微小RNA-34a抑制物组，分别用微小RNA-34a的模拟物对照、模拟物、抑制物对照、抑制物序列进行转染。实时荧光定量反转录-聚合酶链反应(RT-PCR)法检测心肌细胞微小RNA-34a表达，蛋白质印迹法检测心肌细胞SIRT1的蛋白表达。另取心肌细胞，分为微小RNA-34a抑制物对照+烧伤血清组、微小RNA-34a抑制物+烧伤血清组及微小RNA-34a抑制物+烧伤血清+EX527组。微小RNA-34a抑制物对照+烧伤血清组心肌细胞转染微小RNA-34a抑制物的对照序列，后2组心肌细胞转染微小RNA-34a抑制物序列。转染48 h，微小RNA-34a抑制物+烧伤血清+EX527组心肌细胞用含体积分数10%的单纯烧伤组大鼠血清+终物质的量浓度为1 mol/L EX527的DMEM培养液培养6 h，其余2组心肌细胞用含体积分数10%的单纯烧伤组大鼠血清的DMEM培养液培养。蛋白质印迹法检测心肌细胞SIRT1及剪切型半胱氨酸天冬氨酸蛋白酶3、Bax的蛋白表达。(3)取来自(1)的心肌组织，实时荧光定量RT-PCR法检测假伤组和单纯烧伤组大鼠心肌组织中微小RNA-34a及SIRT1的mRNA表达；生物图像导航仪下观察假伤组、单纯烧伤组和SIRT1激动剂组大鼠心肌组织形态；实时荧光定量RT-PCR法检测假伤组、单纯烧伤组和SIRT1激动剂组大鼠心肌组织中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)的mRNA表达；蛋白质印迹法检测假伤组、单纯烧伤组和SIRT1激动剂组大鼠心肌组织剪切型半胱氨酸天冬氨酸蛋白酶3和Bax的蛋白表达。对数据行单因素方差分析及LSD检验。 结果： (1)转染48 h后，微小RNA-34a模拟物组心肌细胞微小RNA-34a的表达量为4.67±0.92，显著高于微小RNA-34a模拟物对照组的1.03±0.04(P<0.01)；微小RNA-34a模拟物组心肌细胞SIRT1的蛋白表达量为0.35±0.06，显著低于微小RNA-34a模拟物对照组的1.12±0.11(P<0.01)。转染48 h后，微小RNA-34a抑制物组心肌细胞微小RNA-34a表达量为0.26±0.07，低于微小RNA-34a抑制物对照组的1.33±0.07(P<0.01)；微小RNA-34a抑制物对照组心肌细胞SIRT1的蛋白表达量为1.12±0.16，显著低于微小RNA-34a抑制物组的1.74±0.34(P<0.01)。培养6 h后，与微小RNA-34a抑制物对照+烧伤血清组比较，微小RNA-34a抑制物+烧伤血清组心肌细胞SIRT1蛋白表达水平显著升高(P<0.05)，剪切型半胱氨酸天冬氨酸蛋白酶3、Bax蛋白表达水平显著降低(P<0.05)。与微小RNA-34a抑制物+烧伤血清组比较，微小RNA-34a抑制物+烧伤血清+EX527组心肌细胞SIRT1的蛋白表达水平差异无统计学意义(P>0.05)；剪切型半胱氨酸天冬氨酸蛋白酶3、Bax蛋白表达水平显著升高(P<0.05)。(2)伤后6 h，与假伤组比较，单纯烧伤组大鼠心肌组织微小RNA-34a表达显著升高(P<0.01)；SIRT1的mRNA表达量明显降低(P<0.01)。伤后6 h，假伤组大鼠心肌细胞排列整齐，细胞核形态正常，未见炎性细胞浸润；单纯烧伤组大鼠心肌细胞排列紊乱，细胞核消失或形态异常，炎性细胞浸润明显；SIRT1激动剂组大鼠心肌细胞排列整齐，细胞核形态正常，少量炎性细胞浸润。伤后6 h，与单纯烧伤组比较，假伤组、SIRT1激动剂组大鼠心肌组织IL-1β、TNF-α的mRNA表达量及剪切型半胱氨酸天冬氨酸蛋白酶3和Bax蛋白水平显著降低(P<0.01)。 结论： 严重烧伤大鼠早期心肌组织微小RNA-34a表达升高，导致SIRT1表达降低，IL-1β、TNF-α及剪切型半胱氨酸天冬氨酸蛋白酶3、Bax表达升高，心肌损伤明显。SIRT1活化后，可通过降低IL-1β、TNF-α及剪切型半胱氨酸天冬氨酸蛋白酶3、Bax的表达，减轻大鼠严重烧伤早期诱发的心肌损伤。.
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