恩格列净对阿霉素诱导大鼠心脏损伤模型的改善作用及其机制

doi:10.13481/j.1671-587X.20260111

摘要/Abstract

摘要：

目的探讨恩格列净（EMPA）对阿霉素（DOX）诱导大鼠心脏损伤（HI）模型的改善作用，并阐明其作用机制。方法 24只6-7周龄雄性Wistar大鼠随机分为对照组（正常健康大鼠维持饲养）、HI组（建立DOX诱导的大鼠HI模型）和HI+EMPA组（在建立DOX诱导的大鼠HI模型6周后，每日给予大鼠灌胃10 mg·kg^-1 EMPA，连续灌胃14 d），每组8只。采用心脏超声心动图检测各组大鼠左室收缩末内径（LVIDs）、左室射血分数（LVEF）和左室短轴缩短率（LVFS），HE染色和Masson染色观察各组大鼠心肌组织病理形态表现和心肌组织中胶原纤维沉积情况，脱氧核糖核苷酸末端转移酶介导的dUTP缺口末端标记（TUNEL）法分析各组大鼠心肌细胞凋亡情况，酶联免疫吸附试验（ELISA）法检测各组大鼠血清中乳酸脱氢酶（LDH）和肌酸激酶（CK）水平。采用大鼠心肌细胞H9c2进行体外实验。体外建立DOX诱导的大鼠心肌细胞损伤模型（DOX组）。细胞实验分组-1分为对照组（H9c2细胞正常培养不做任何处理）、DOX组（H9c2细胞培养液中加入0.1 μmol·L^-1 DOX处理48 h，诱导心肌细胞损伤）、DOX+EMPA组（H9c2细胞培养液中加入0.1 μmol·L^-1 DOX和500 nmol·L^-1 EMPA，处理48 h）和DOX+EMPA+EX527［沉默信息调节因子相关酶1（SIRT1）抑制剂］组（H9c2细胞培养液中加入10 μmol·L^-1 EX527预处理1 h，然后加入0.1 μmol·L^-1 DOX和500 nmol·L^-1 EMPA，处理48 h）。细胞实验分组-2分为对照组（H9c2细胞正常培养不做任何处理）、DOX组（H9c2细胞培养液中加入0.1 μmol·L^-1 DOX处理48 h，诱导心肌细胞损伤）和DOX+动力学相关蛋白1（Drp-1）抑制剂组（H9c2细胞培养液中加入75 μmol·L^-1 Drp-1抑制剂Mdivi-1预处理2 h，然后0.1 μmol·L^-1 DOX处理48 h）。采用Western blotting法检测各组细胞中自噬相关蛋白微管相关蛋白1轻链3（LC3）、自噬关键分子酵母Atg6同系物BECLIN1、泛素结合蛋白P62、SIRT1、过氧化物酶体增殖物激活受体γ辅激活子1α（PGC-1α）、Drp-1、线粒体裂变1蛋白（Fis-1）和线粒体裂变因子（MFF）蛋白表达水平。结果对照组大鼠心脏心肌纤维排列整齐，间质未见明显炎症细胞浸润和胶原纤维沉积；与对照组比较，HI组大鼠心肌纤维排列紊乱，心肌间质扩大，可见炎症细胞浸润和胶原纤维沉积；与HI组比较，HI+EMPA组大鼠心肌纤维排列较为规整，间质未见明显炎症细胞浸润，可见少量胶原纤维沉积。与对照组比较，HI组大鼠心肌组织TUNEL阳性细胞数和LVIDs升高（P<0.05），LVEF和LVFS降低（P<0.05），血清中LDH和CK水平升高（P<0.05）。与HI组比较，HI+EMPA组大鼠心肌组织TUNEL阳性细胞数和LVIDs降低（P<0.05），LVEF和LVFS均升高（P<0.05），血清中LDH和CK水平降低（P<0.05）。与对照组比较，DOX组H9c2细胞中BECLIN1蛋白表达水平和LC3-Ⅰ/LC3-Ⅱ比值升高（P<0.05），P62、SIRT1和PGC-1α蛋白表达水平降低（P<0.05），Drp-1蛋白表达水平升高（P<0.05）。与DOX组比较，DOX+EMPA组H9c2细胞中BECLIN1蛋白表达水平和LC3-Ⅰ/LC3-Ⅱ比值降低（P<0.05），P62、SIRT1和PGC-1α蛋白表达水平升高（P<0.05），Drp-1蛋白表达水平降低（P<0.05）。与DOX+EMPA组比较，DOX+EMPA+EX527组H9c2细胞中BECLIN1蛋白表达水平和LC3-Ⅰ/LC3-Ⅱ比值升高（P<0.05），P62和Drp-1蛋白表达水平降低（P<0.05）。与对照组比较，DOX组H9c2细胞中Drp-1、Fis-1和MFF蛋白表达水平升高（P<0.05）；与DOX组比较，DOX+Drp-1抑制剂组H9c2细胞中Fis-1和MFF蛋白表达水平降低（P<0.05）。结论 EMPA可减轻DOX诱导的大鼠心肌病变和心脏功能异常，降低心肌细胞凋亡和自噬，其机制可能与EMPA上调SIRT1和PGC-1α蛋白表达、降低Drp1蛋白表达有关。

关键词: 阿霉素, 心脏毒性, 恩格列净, 线粒体裂变, 沉默信息调节因子相关酶1, 过氧化物酶体增殖物激活受体γ辅激活子1α, 动力学相关蛋白1

Abstract:

Objective To discuss the ameliorative effect of empagliflozin （EMPA） on the doxorubicin （DOX）-induced heart injury （HI） model in the rats， and to clarify its mechanism of action. Methods Twenty-four 6-7-week-old male Wistar rats were randomly divided into control group （normal healthy rats were maintained）， HI group （the DOX-induced rat HI model was established）， and HI+EMPA group （after 6 weeks of establishing the DOX-induced rat HI model， the rats were given 10 mg·kg?1 EMPA daily by gavage for 14 consecutive days）， with 8 rats in each group. Echocardiography was used to detect the left ventricular internal diameter at end-systole （LVIDs）， left ventricular ejection fraction （LVEF）， and left ventricular fractional shortening （LVFS） of the rats in various groups； HE staining and Masson staining were used to detect the pathomorphology of myocaridium tissue and collagen fibers in myocardium tissue of the rats in various groups； terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） method was used to analyze the apoptosis in the left ventricular myocardium cells of the rats in various groups； enzyme-linked immunosorbent assay （ELISA） was used to detect the serum levels of lactate dehydrogenase （LDH） and creatine kinase （CK） of the rats in various groups. Rat myocardial H9c2 cells were used for in vitro experiments. A DOX-induced rat cardiomyocyte injury model was established in vitro （DOX group）. Cell experiment grouping-1 was divided into control group （H9c2 cells were cultured normally without any treatment）， DOX group （0.1 μmol·L^-1 DOX was added to the H9c2 cell culture medium for 48 h to induce cardiomyocyte injury）， DOX+EMPA group （0.1 μmol·L^-1 DOX and 500 nmol·L^-1 EMPA were added to the H9c2 cell culture medium for 48 h）， and DOX+EMPA+sirtuin 1 （SIRT1） inhibitor （EX527） group （H9c2 cells were pretreated with 10 μmol·L?1 EX527 for 1 h， then 0.1 μmol·L^-1 DOX and 500 nmol·L^-1 EMPA were added for 48 h）. Cell experiment grouping-2 was divided into control group （H9c2 cells were cultured normally without any treatment）， DOX group （0.1 μmol·L^-1 DOX was added to the H9c2 cell culture medium for 48 h to induce cardiomyocyte injury）， and DOX+dynamin-related protein 1 （Drp-1） inhibitor group （H9c2 cells were pretreated with 75 μmol·L^-1 Drp-1 inhibitor Mdivi-1 for 2 h， then treated with 0.1 μmol·L^-1 DOX for 48 h）. Western blotting method was used to determine the expression levels of autophagy-related proteins microtubule-associated protein 1 light chain 3 （LC3）， autophagy key molecule yeast Atg6 homolog BECLIN1， and ubiquitin-binding protein P62， SIRT1， peroxisome proliferator-activated receptor gamma coactivator 1-alpha （PGC-1α）， and Drp-1； mitochondrial fission 1 protein （Fis-1）， and mitochondrial fission factor （MFF） proteins in the cells in various groups. Results In control group， the myocardial fibers of the rat hearts were arranged neatly， and no obvious inflammatory cell infiltration or collagen fiber deposition was seen in the interstitium； compared with control group， in HI group， the myocardial fibers of the rats were disordered， the myocardial interstitium was enlarged， and inflammatory cell infiltration and collagen fiber deposition were observed； compared with HI group， in HI+EMPA group， the myocardial fibers of the rats were more regular， no obvious inflammatory cell infiltration was seen in the interstitium， and a small amount of collagen fiber deposition was observed. The TUNEL staining results showed that compared with control group， the number of TUNEL-positive cells in the myocardium tissue of the rats in HI group was increased （P<0.05）. The echocardiography results showed that compared with control group， the LVIDs of the rats in HI group was increased （P<0.05）， and the LVEF and LVFS were decreased （P<0.05）； compared with HI group， the LVIDs of the rats in HI+EMPA group was decreased （P<0.05）， and the LVEF and LVFS were increased （P<0.05）. The ELISA results showed that compared with control group， the serum levels of LDH and CK of the rats in HI group were increased （P<0.05）； compared with HI group， the serum levels of LDH and CK of the rats in HI+EMPA group were decreased （P<0.05）. The Western blotting results showed that compared with control group， the BECLIN1 protein expression level and LC3-Ⅰ/LC3-Ⅱ ratio in the H9c2 cells in DOX group were increased （P<0.05）， the expression levels of P62， SIRT1， and PGC-1α proteins were decreased （P<0.05）， and the expression level of Drp-1 protein was increased （P<0.05）； compared with DOX group， the BECLIN1 protein expression level and LC3-Ⅰ/LC3-Ⅱ ratio in the H9c2 cells in DOX+EMPA group were decreased （P<0.05）， the expression levels of P62， SIRT1， and PGC-1α proteins were increased （P<0.05）， and the expression level of Drp-1 protein was decreased （P<0.05）； compared with DOX+EMPA group， the BECLIN1 protein expression level and LC3-Ⅰ/LC3-Ⅱ ratio in the H9c2 cells in DOX+EMPA+EX527 group were increased （P<0.05）， and the expression levels of P62 and Drp-1 proteins were decreased （P<0.05）. The Western blotting results showed that compared with control group， the expression levels of Drp-1， Fis-1， and MFF proteins in the H9c2 cells in DOX group were increased （P<0.05）； compared with DOX group， the expression levels of Fis-1 and MFF proteins in the H9c2 cells in DOX+Drp-1 inhibitor group were decreased （P<0.05）. Conclusion EEMPA can alleviate DOX-induced myocardial lesions and cardiac dysfunction in rats， and reduce cardiomyocyte apoptosis and autophagy， and its mechanism may be related to the up-regulation of SIRT1 and PGC-1α protein expressions and the down-regulation of Drp1 protein expression by EMPA.

Key words: Doxorubicin, Cardiotoxicity, Empagliflozin, Mitochondrial fission, Sirtuin 1, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha, Dynamin-related protein 1

中图分类号:

R541

李佳蔚,阿地力江null,吴莉,姜芸. 恩格列净对阿霉素诱导大鼠心脏损伤模型的改善作用及其机制[J]. 吉林大学学报(医学版), 2026, 52(1): 105-115.

Jiawei LI, Adilijiang,Li WU,Yun JIANG. Improvement effect of empagliflozin on ameliorating doxorubicin-induced myocardial injury rat model and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2026, 52(1): 105-115.

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Group	LVIDs	LVEF (η/%)	LVFS (η/%)
Control	4.06±1.11	82.4±1.3	53.2±2.6
HI	5.82±0.83^*	43.1±1.7^*	22.6±1.4^*
HI+EMPA	4.24±1.77^△	66.7±2.2^△	51.4±2.1^△
F	4.45	997.00	538.90
P	<0.05	<0.05	<0.05

Group	Serum LDH	Serum CK
Control	1 967±213	1 233±239
HI	3 056±261^*	4 136±312^*
HI+EMPA	1 745±188^△	875±178^△
F	79.39	412.40
P	<0.001	<0.001