柚皮素通过抑制NLRP3炎症小体激活改善糖尿病小鼠心肌重构

Naringenin ameliorates myocardial remodeling by inhibiting NLRP3 inflammasome activation in diabetic mice

摘要:

目的：研究柚皮素（naringenin，NAR）对糖尿病小鼠心肌重构的作用及对NLRP3炎症小体的影响。方法：将雄性C57BL/6小鼠35只随机分为正常（N）组（7只）和糖尿病模型组（28只），糖尿病模型组连续腹腔注射链脲佐菌素（streptozotocin，STZ）3 d，正常组腹腔注射相应溶剂，1周后断尾取血测随机血糖，以随机血糖大于16.7 mmol/L作为1型糖尿病小鼠成模标准。最终28只模型组小鼠均造模成功，并随机分为糖尿病（DM）组、柚皮素低剂量（DM+LN）组、柚皮素中剂量（DM+MN）组、柚皮素高剂量（DM+HN）组，每组7只。各组连续灌胃给药8周后处死小鼠，观察柚皮素对小鼠体质量及血糖的影响；HE及Masson染色观察心脏形态改变，并测量心肌胶原容积分数（collagen volume fraction，CVF）；TUNEL荧光染色观察心肌细胞凋亡；Western blot测定心肌组织NLRP3、ASC、Caspase-1、白介素（interleukin，IL）-1β、Ⅰ型胶原蛋白、纤连蛋白（fibronectin，FN）水平；PCR检测NLRP3、ASC、Caspase-1、IL-1β mRNA表达。结果：Masson染色结果分析显示，DM组[（37.68±1.14）%]的CVF较DM+LN组[（13.58±0.86）%]、DM+MN组[（9.50±0.59）%]、DM+HN组[（6.38±1.41）%]明显增加（P=0.000）；TUNEL荧光染色结果分析显示，DM组[（64.94±5.25）%]心肌细胞凋亡较DM+LN组[（48.80±6.11）%]、DM+MN组[（31.85±4.44）%]、DM+HN组[（13.64±1.79）%]明显增加（P=0.000）；Western blot结果显示，DM组（0.52±0.03）心肌NLRP3蛋白表达量较DM+LN组（0.33±0.07）、DM+MN组（0.23±0.06）、DM+HN组（0.15±0.04）增加（P=0.000）；同时DM组（0.69±0.09）心肌IL-1β蛋白表达也较DM+LN组（0.42±0.09）、DM+MN组（0.25±0.04）、DM+HN组（0.09±0.03）增加（P=0.000）。结论：NAR可以改善糖尿病小鼠心肌纤维化、减少心肌细胞凋亡，从而抑制心肌重构，其机制之一可能是抑制了NLRP3炎症小体激活，降低了炎症水平。

Abstract:

Objective：To study the effects of NAR on myocardial remodeling as well as its effects on NLRP3 inflammasome in dia－betic mice. Methods：Male C57BL/6 mice（n=35） were randomly divided into normal group（N group，n=7） and experimental group （n=28）. The mice in the experimental group were injected with STZ for 3 days to induce diabetes mellitus. Random blood glucose was measured after 7 days. Mice with random blood glucose over 16.7 mmol/L were regarded as diabetes mellitus. Finally，all the twenty-eight mice in the experimental group were successfully modeled. Twenty-eight diabetes mice were randomly divided into diabetes group（DM group），diabetes+low dose of NAR group（DM+LN group），diabetes+ middle dose of NAR group（DM+MN group），and dia－betes+high dose of NAR group（DM+HN group）. The mice in treatment group received different doses of NAR through intraperitoneal injection for 8 weeks. At the end of the study，the mice were sacrificed to observe the effects of NAR at different dose on the body weight and blood glucose. HE staining was used observe pathological changes of myocardial tissue. The myocardial CVF was calculated by Masson staining. Apoptosis was detected by TUNEL fluorescent staining. Western blot was applied to deter－mine the protein levels of NLRP3，ASC，Caspase-1，IL-1β，type Ⅰ collagen，and FN. PCR was used to analyze mRNA of NLRP3，ASC，Caspase-1，and IL-1β. Results：Masson staining showed that the CVF was significantly higher in DM group[（37.68±1.14）%] than in DM+LN group[（13.58±0.86）%]，DM+MN group[（9.50±0.59）%] and DM+HN group[（6.38±1.41）%]（P=0.000）. TUNEL fluorescent staining showed that cell apoptotic rate was significantly higher in DM group[（64.94±5.25）%] than in DM+LN group[（48.80±6.11）%]，DM+MN group[（31.85±4.44）%] and DM+HN group[（13.64±1.79）%]（P=0.000）. Western blot results demonstrated that the DM group（0.52±0.03） had significantly higher expression of NLRP3 compared with the DM+LN group（0.33±0.07），DM+MN group（0.23±0.06） and DM+HN group（0.15±0.04）（P=0.000）. Moreover，the DM group（0.69±0.09） had significantly higher expression of IL-1β than the DM+LN group（0.42±0.09），DM+MN group（0.25±0.04） and DM+HN group（0.09±0.03）（P=0.000）. Conclusion：NAR significantly attenuates the myocardial fibrosis and apoptosis in the diabetic mice，and its mechanism may be associated with inhibiting NLRP3 inflammasome activation，reducing the level of inflammation.