柚皮素通过调控AMPK/Nrf2/HO-1信号通路减轻糖尿病小鼠心肌损伤

目的:探讨柚皮素(naringenin,NAR)对糖尿病小鼠心肌的保护作用及对腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)/核因子E2相关因子2(nuclear factor-E2-related factor 2,Nrf2)/血红素加氧酶1(heme oxygenase 1,HO-1)信号通路的影响。方法:将50只C57BL/6小鼠随机分为正常组(N组)和模型组。模型组采用连续腹腔注射链脲佐菌素的方法建立1型糖尿病小鼠模型,成模后将其分为糖尿病组(D组)、低剂量NAR干预组(D+L-NAR组)、中等剂量NAR干预组(D+M-NAR组)和高剂量NAR干预组(D+H-NAR组),对干预组小鼠分别给予低、中、高剂量NAR灌胃,N组和D组给予与D+M-NAR组等体积的生理盐水灌胃。6周后处死小鼠,观察NAR对小鼠体重及血糖的影响;HE和Masson染色观察心脏形态学改变,测量心肌胶原容积分数(collagen volume fraction,CVF);免疫组化观察心脏组织中白细胞介素6(interleukin-6,IL-6)和IL-10的蛋白水平;TUNEL染色检测心肌组织凋亡情况;荧光探针DHE测定心肌细胞中活性氧簇(reactive oxygen species, ROS)的生成;超氧化物歧化酶(superoxide dismutase, SOD)及丙二醛(malondialdehyde, MDA)试剂盒测定心肌细胞中SOD活性及MDA的含量;Western blot测定心脏组织p-AMPKα、AMPKα、Nrf2、HO-1、NAD(P)H:醌氧化还原酶1NAD(P)H:quinone oxidoreductase 1, NQO1]和cleaved caspase-3的蛋白水平。结果:与N组相比,D组成模后血糖明显升高,体重明显下降;与D组相比较,NAR干预组的血糖有所下降,体重有所增高。与N组相比,D组CVF明显增大,凋亡率明显增加,IL-6和cleaved caspase-3蛋白水平升高,IL-10、p-AMPKα、Nrf2、HO-1和NQO1蛋白水平降低,ROS生成率和MDA含量升高,SOD活性降低(P<0.05);与D组相比较,NAR干预组CVF明显降低,IL-6和cleaved caspase-3蛋白水平降低,IL-10、p-AMPKα、Nrf2、HO-1和NQO1蛋白水平升高(P<0.05);与D组比较,D+L-NAR组ROS成率、MDA含量及SOD活性的差异无统计学显著性,D+M-NAR组及D+H-NAR组的ROS生成率降低、MDA含量降低,SOD活性升高。结论:NAR能减轻糖尿病小鼠心肌损伤,其机制可能与激活AMPK/Nrf2/HO-1信号通路,增强抗氧化反应,减轻心肌纤维化、凋亡及炎症反应有关。

Naringenin attenuates myocardial injury by regulating AMPK/Nrf2/HO-1 signaling pathways in diabetic mice

AIM: To investigate the effects of naringenin(NAR) on the myocardium as well as its effects on adenosine monophosphate-activated protein kinase(AMPK)/nuclear factor(NF)-E2-related factor 2(Nrf2)/heme oxygenase 1(HO-1) signaling pathways in diabetic mice. METHODS: C57 BL/6 mice(n=50) were randomly divided into normal group(N group) and model group. The mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin(STZ), then the mice were divided into diabetes group(D group), diabetes+low dose of NAR intervention group(D+L-NAR group), diabetes+middle dose of NAR intervention group(D+M-NAR group) and diabetes+high dose of NAR intervention group(D+H-NAR group). The mice in intervention groups were received NAR at low, middle and high doses respectively by gavage, and the mice in N group and D group were received equal volume of normal saline. After 6 weeks, the mice were sacrificed to observe the effects of NAR at different doses on the body weight and blood glucose. The histopathological changes of the cardiac tissues were observed with HE staining. The myocardial collagen volume fraction(CVF) was calculated by Masson staining. Immumohistochemical staining was used to test the protein levels of interleukin-6(IL-6) and IL-10, and the TUNEL was used to observe the apoptosis of myocardial tissues. The production of reactive oxygen species(ROS) in the myocardial cells was analyzed by fluorescence probe of DHE, and superoxide dismutase(SOD) activity and malondiodehyde(MDA) content in the myocardial cells were measured by SOD and MDA kits. Western blot was applied to determine the protein levels of p-AMPKα, AMPKα, Nrf2, HO-1, NAD(P)H:quinone oxidoreductase 1(NQO1) and cleaved caspase-3 in the myocardial tissues. RESULTS: Compared with N group, the blood glucose of the mice in D groups was increased and the body weight was decreased significantly. Compared with D group, the blood glucose of the mice in NAR intervention groups was decreased and the body weight was increased. Compared with N group, the CVF, apoptotic rate and the protein levels of IL-6, cleaved caspase-3 were increased, while the protein levels of IL-10, p-AMPKα, Nrf2, HO-1, NQO1 and SOD activity were decreased, the ROS production rate and MDA content was increased significantly in D group(P<0.05). Compared with D group, the CVF, apoptotic rate and the protein levels of IL-6, cleaved caspase-3 were relatively decreased, conversely the protein levels of IL-10, p-AMPKα, Nrf2, HO-1, NQO1 were increased in NAR intervention groups(P<0.05). No significantly difference of the ROS production rate, SOD activity and MDA content between D group and D+L-NAR group was observed. However, the ROS production rate and MDA content was decreased,SOD activity were increased in D+M-NAR group and D+H-NAR group as compared with D group. CONCLUSIONS: NAR attenuates myocardial injury in diabetic mice, and its mechanism may be related to regulation of AMPK/Nrf2/HO-1 signaling pathway, enhancement of the antioxidant reaction, reduction of myocardial fibrosis, apoptosis and inflammation.