Protective role of irisin against diabetic cardiomyopathy via Nrf2/Sirt3 signaling pathway
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摘要:
目的 明确鸢尾素（Irisin）在糖尿病心肌损伤中的作用及机制。 方法 将小鼠以随机数字表法分为4组：NS组、Irisin组、糖尿病心肌病（DCM）组、DCM-Irisin干预组；体外实验分组：高糖高脂组（HG/HF）、HG/HF-Irisin干预组、HG/HF-Irisin干预-Nrf2抑制剂组、HG/HF-Nrf2抑制剂组、HG/HF-Nrf2激动剂组、HG/HF-Nrf2激动剂-Sirt3抑制剂组、HG/HF- Irisin干预-Sirt3抑制剂组。采用Western blot、RT-PCR检测自噬及凋亡相关分子表达、CCK-8检测细胞活力、MMP和ATP检测评估线粒体功能、TUNEL染色检测细胞凋亡。 结果 糖尿病小鼠心肌组织及高糖高脂诱导的H9c2细胞中Irisin的蛋白及mRNA表达量显著降低（P<0.05），而LC3II/I和Cleaved caspase 3的表达量显著增加（P<0.01），同时可见Nrf2及P62的蛋白表达量显著降低（P<0.01）。Irisin处理后，可部分缓解DCM导致的心肌细胞损伤。在给予Nrf2抑制剂ML385后，Irisin的保护作用被部分抵消，同时观察到Sirt3蛋白表达被显著抑制（P<0.01）。而在高糖高脂条件下，抑制Sirt3活性则可消除因上调Nrf2对H9c2细胞的有益作用。 结论 Irisin通过激活Nrf2/Sirt3信号通路减轻糖尿病心肌病诱导的心肌细胞损伤。 Abstract:AIM To clarify the role and mechanism of irisin in diabetic cardiomyopathy. METHODS Mice were randomly divided into 4 groups: NS group, Irisin group, DCM group, and DCM-Irisin group. For in vitro experiments, mice were divided into HG/HF group, HG/HF-Irisin group, HG/HF-Irisin-Nrf2 inhibitor group, HG/HF-Nrf2 inhibitor group, HG/HF-Nrf2 agonist group, HG/HF-Nrf2 agonist-Sirt3 inhibitor group, and HG/HF-Irisin-Sirt3 inhibitor group. The expressions of autophagy and apoptosis-related proteins were detected by Western blot and RT-PCR, cell viability was detected by CCK-8, mitochondrial function was detected by MMP and ATP, and cell apoptotic rate was detected by TUNEL staining. RESULTS The expressions of irisin protein and mRNA levels were decreased significantly in myocardial tissue of diabetic mice and HG/HF-induced H9c2 cells (P<0.05), while the expressions of LC3II/I and Cleaved caspase3 were increased significantly (P<0.01), and the protein expression of Nrf2 and P62 were decreased significantly (P<0.01). Irisin treatment partially alleviated the cardiomyocytes injury caused by DCM. The administration of Nrf2 inhibitor ML385 partially offset the protective effect of irisin, and significant inhibition of Sirt3 protein expression was observed (P<0.01). Under the condition of HG/HF, inhibiting Sirt3 activity eliminated the beneficial effect of Nrf2 up-regulation on H9c2 cells. CONCLUSION Irisin mitigates the cardiomyocyte injury induced by DCM via activating Nrf2/Sirt3 signaling pathway. -
Key words:
- irisin /
- diabetic cardiomyopathy /
- Nrf2 /
- Sirt3 /
- myocardial injury
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图 1 糖尿病小鼠和HG/HF诱导细胞中Irisin及Insulin的变化
n=3。与NS组比较，aP<0.05，bP<0.01；与PBS组比较，cP<0.05，dP<0.01
图 2 Irisin对糖尿病小鼠中自噬性细胞死亡相关蛋白的影响
1：NS组；2：Irisin组；3：DCM组；4：DCM-Irisin组。n=3。与NS组比较，aP<0.05，bP<0.01；与DCM组比较，cP<0.05
图 3 Irisin对高糖高脂处理的H9c2细胞中自噬性细胞死亡相关蛋白的影响
1：PBS组；2：Irisin组；3：HG/HF组；4：HG/HF-Irisin组。n=3。与PBS组比较，bP<0.01；与HG/HF组比较，cP<0.05
图 4 Irisin和ML385对HG/HF诱导细胞中自噬性细胞死亡相关蛋白、细胞活力以及血清LDH的影响
1：HG/HF组；2：HG/HF-Irisin组；3：HG/HF-Irisin-ML385组；4：HG/HF-ML385组。n=3。与HG/HF组比较，aP<0.05，bP<0.01；与HG/HF-Irisin组比较，cP<0.05，dP<0.01 ；与PBS组比较，fP<0.01
图 5 Irisin和ML385对HG/HF诱导细胞中MMP及ATP含量的影响
1：HG/HF组；2：HG/HF-Irisin组；3：HG/HF-Irisin-ML385组；4：HG/HF-ML385组。n=3。与HG/HF组比较，aP<0.05，bP<0.01；与HG/HF-Irisin组比较，cP<0.05
图 6 Oltipraz和3-TYP对HG/HF诱导的细胞中自噬性细胞死亡相关蛋白及ATP含量的影响
1：HG/HF组；2：HG/HF-Oltipraz组；3：HG/HF-Oltipraz-3-TYP组。n=3。与HG/HF组比较，aP<0.05，bP<0.01；与HG/HF-Oltipraz组比较，cP<0.05，dP<0.01
图 7 Oltipraz和3-TYP对HG/HF诱导细胞凋亡及血清LDH的影响
1：HG/HF组；2：HG/HF-Oltipraz组；3：HG/HF-Oltipraz-3-TYP组。n=3。与HG/HF组比较，aP<0.05，bP<0.01；与HG/HF-Oltipraz组比较，cP<0.05
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