达格列净通过激活AMPK/mTOR自噬途径保护脓毒症小鼠心肌

Protection effect of dapagliflozin on the myocardium of septic mice by activating the AMPK/mTOR autophagy pathway

目的探索达格列净对脓毒症诱导的小鼠心肌损伤的作用及机制。方法将30只雄性C57BL/6J小鼠随机分为对照组(Control组)、脂多糖处理组(LPS组)、达格列净处理组(LPS+Dapa组)、腺苷酸激活蛋白激酶抑制剂预处理组(LPS+Dapa+Compound C组)和腺苷酸激活蛋白激酶自噬抑制剂预处理组(LPS+Dapa+3-MA组)。LPS+Dapa组、LPS+Dapa+Compound C组和LPS+Dapa+3-甲基腺嘌呤(3-MA)组接受达格列净预处理1周,Compound C和3-MA分别用于抑制腺苷酸激活蛋白激酶(AMPK)的激活和自噬。第7天,使用LPS建立脓毒症心肌功能障碍小鼠模型。每组小鼠检查心脏超声,进行苏木精-伊红染色、微管相关蛋白轻链3(LC3)免疫荧光染色,检测血清肌酸激酶同工酶(CK-MB)、肌钙蛋白T(cTnT)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)水平、心肌AMPK、动物雷帕霉素靶蛋白(mTOR)、精氨酸酶1(Arg-1)、诱导型一氧化氮合酶(iNOS)、LC3Ⅱ/LC3Ⅰ、螯合体1(p62)蛋白水平及Arg-1和iNOS基因表达情况。结果与对照组比较,LPS组小鼠心功能显著受损,心肌细胞紊乱、稀疏、肥大,心肌损伤标志物、炎症因子水平升高,而达格列净可以改善小鼠心功能、心肌病理变化,并降低心肌损伤标志物及炎症因子水平。免疫印记结果显示,LPS组较对照组Arg-1、LC3Ⅱ/LC3Ⅰ比值略微升高,iNOS、p-mTOR明显升高,p62、p-AMPK表达下降(均P<0.05)。与LPS组比较,LPS+Dapa组Arg-1、p-AMPK表达增加,LC3Ⅱ/LC3Ⅰ进一步增加,iNOS、p62、p-mTOR表达降低(均P<0.05)。免疫荧光证实了达格列净能促进LC3Ⅱ表达。Arg-1和iNOS的基因表达结果与蛋白表达情况一致(P<0.05)。在上述结果中,在LPS+Dapa处理后添加3-MA或Compound C均能逆转达格列净的作用。结论达格列净能减轻脓毒症小鼠心肌损伤,其机制可能与促进AMPK/mTOR自噬途径,减轻炎症反应有关,巨噬细胞表型可能也参与其中。

Objective To explore the effect and mechanism of dapagliflozin(Dapa)on sepsis-induced myocardial damage.Methods Random allocation was performed to assign male C57BL/6J mice into various groups including control group,LPS group,LPS+Dapa group,LPS+Dapa+Compound C(adenylate activated protein kinase inhibitor)group,and LPS+Dapa+3-MA(3-methyladenine)group,with 6 mice in each group.LPS+Dapa group,LPS+Dapa+Compound C group and LPS+Dapa+3-MA group were pretreated with dapagliflozin(10 mg/kg)once a day by gavage for one week.Compounds C and 3-MA were used to inhibit the activation and autophagy of adenosine monophosphate-activated protein kinase(AMPK),respectively.On the 7th day,septic cardiomyopathy was induced by administering lipopolysaccharide(LPS)in mice.Cardiac function was assessed through echocardiography,hematoxylin-eosin staining and LC3 immunofluorescence staining.The phenotype of autophagy-related proteins were evaluated by using immunofluorescence and Western blot.The phenotype of macrophages proteins were evaluated by using enzyme-linked immunosorbent assay(ELISA),Western blot and RT-qPCR.The expression of AMPK and mTOR were evaluated by Western blot.Results Compared to the control group,the mice in LPS group exhibited significant differences in cardiac function and myocardium,such as disorganized,hypertrophic and ruptured myocardial cells,the increase of myocardial injury markers and inflammatory factors.The dapagliflozin demonstrated a significant amelioration in cardiac function and myocardial pathological changes in mice,concomitant with a reduction in the levels of myocardial injury markers and inflammatory factors.The Western blot results showed that the LPS group had a slight increase in Arg-1and LC3Ⅱ/LC3Ⅰratio compared to the control group,a significant increase in iNOS and p-mTOR,and the decrease of p62 and p-AMPK expression were observed(P<0.05).Compared to the group treated with LPS alone,the LPS+Dapa group showed a significant increase in the expression of Arg-1 and p-AMPK.Additionally,there was a further enhancement in LC3Ⅱ/LC3Ⅰlevels,while iNOS,p-mTOR and p62 expression exhibited a decrease(P<0.05).Immunofluorescence analysis provided the evidences for supporting the notion that dapagliflozin can promote the expression of LC3Ⅱ.The results of Arg-1 and iNOS gene were consistent with the protein expression(P<0.05).In the above results,adding 3-MA or Compound C to the LPS+Dapa group can reverse the effect of dapagliflozin.Conclusions In septic mice,dapagliflozin has the potential to mitigate myocardial damage by potentially enhancing the AMPK/mTOR autophagy pathway and diminishing inflammatory response,and macrophage phenotype may also be involved.