强心方对慢性心力衰竭大鼠心肌线粒体能量代谢与自噬的影响

Effect of Qiangxin Formula on Myocardial Mitochondrial Energy Metabolism and Autophagy in Rats with Chronic Heart Failure

【目的】探讨强心方(由温阳活血利水方药组成)对慢性心力衰竭大鼠的治疗机制。【方法】从72只大鼠中选取60只大鼠建立慢性心力衰竭模型,感染死亡5只。将55只成功造模的大鼠按体质量分为模型组,卡托普利组,强心方低、中、高剂量组,每组11只。另取11只大鼠设为假手术组,仅切口穿线不结扎。建模第1天,强心方低、中、高剂量组大鼠分别灌胃3.96、7.92、15.84(生药)g·kg^(-1)·d^(-1)的强心方混悬液,卡托普利组大鼠灌胃10 g/mL的卡托普利。假手术组和模型组分别灌胃等体积生理盐水溶液。每日1次,连续4周。采用酶联免疫吸附法(ELISA)检测血清中单磷酸腺苷(AMP)、二磷酸腺苷(ADP)、三磷酸腺苷(ATP)含量,考马斯亮蓝法检测心肌组织Na+-K+ATP酶、Ca2+-Mg2+ATP酶活性,苏木素-伊红(HE)染色法观察心肌组织病理形态,免疫荧光法检测心肌组织自噬蛋白微管相关蛋白轻链3Ⅱ(LC3-Ⅱ)阳性表达情况,蛋白免疫印迹(Western Blot)法检测心肌组织中p38丝裂原活化蛋白激酶(p38MAPK)、磷酸化p38MAPK(p-p38MAPK)及过氧化物酶体增殖物激活受体(PPAR)-γ蛋白表达。【结果】(1)与假手术组比较,模型组血清ATP含量和心肌组织Na+-K+ATP酶活性、Ca2+-Mg2+ATP酶活性降低,血清ADP、AMP含量升高(P<0.05);与模型组比较,强心方各剂量组和卡托普利组血清ATP含量和心肌组织Na+-K+ATP酶活性、Ca2+-Mg2+ATP酶活性升高,血清ADP、AMP含量降低(P<0.05)。(2)HE染色结果:模型组大鼠心肌组织纤维排列紊乱或断裂,强心方各剂量组与卡托普利组大鼠心肌组织损伤改善。(3)与假手术组比较,模型组心肌组织LC3-Ⅱ阳性表达率升高(P<0.05);与模型组比较,强心方低、中、高剂量组和卡托普利组LC3-Ⅱ阳性表达率降低(P<0.05)。(4)与假手术组比较,模型组心肌组织p38MAPK、p-p38MAPK蛋白表达水平升高,PPAR-γ蛋白表达水平降低(P<0.05);与模型组比较,强心方各剂量组和卡托普利组心肌组织p38MAPK、p-p38MAPK蛋白表达水平降低,PPAR-γ蛋白表达水平升高(P<0.05)。此外,强心方高剂量组上述各指标与卡托普利组比较,差异均无统计学意义(P>0.05)。【结论】强心方可能通过抑制p38MAPK活化及促进PPAR-γ蛋白表达进而增强心肌线粒体能量代谢,减少心肌细胞自噬,从而起抗慢性心力衰竭的作用。

Objective To explore the therapeutic mechanism of Qiangxin Formula(involved in yang-warming,blood-activating and diuresis-inducing Chinese herbal medicines)in rats with chronic heart failure.Methods Sixty rats were selected from 72 rats to establish a chronic heart failure model.Five rats died of infection and 55 successfully modeled rats were classified by body mass into the model group,the Captopril group,and the Qiangxin Formula low-,medium-and high-dose groups,11 rats in each group.The remaining 11 rats were set up as a sham-operation group,with only the incision threaded without ligature.On day 1,rats in the Qiangxin Formula low-,medium-and high-dose groups were gavaged with 3.96,7.92 and 15.84(raw drug)g·kg^(-1)·d^(-1)of Qiangxin Formula suspension,respectively,and rats in the Captopril group were gavaged with 10 g/mL of Captopril.The sham-operation group and model group were gavaged with equal volume of normal saline solution.The rats were treated once daily for 4 weeks.Adenosine monophosphate(AMP),adenosine diphosphate(ADP)and adenosine triphosphate(ATP)levels in serum were measured by enzyme-linked immunosorbent assay(ELISA),Na+-K+ATP enzyme and Ca2+-Mg2+ATP enzyme activities in myocardial tissues were quantified by Kormas Brilliant Blue method,myocardial histopathological morphology was observed by hematoxylin-eosin(HE)staining,and the positive expression of autophagy microtubule-associated protein light chain 3Ⅱ(LC3-Ⅱ)was detected by immunofluorescence assay.The protein expression of p38 mitogen-activated protein kinase(p38MAPK),phosphorylated p38MAPK(p-p38MAPK)and peroxisome proliferator activated receptor(PPAR)-γin myocardium were detected by Western Blot.Results(1)Compared with the sham-operation group,serum ATP content and myocardial tissue Na+-K+ATP enzyme activity and Ca2+-Mg2+ATP enzyme activity were decreased and serum ADP and AMP content were increased in the model group(P<0.05);compared with the model group,serum ATP content and myocardial tissue Na+-K+ATP enzyme activity and Ca2+-Mg2+ATP enzyme activity were increased and serum ADP and AMP content were decreased in the Qiangxin Formula dose groups and the Captopril group(P<0.05).(2)HE staining results:the myocardial tissue fibers in the model group were disorganized and broken,and the myocardial tissue damage in the rats of each dose group of Qiangxin Formula and Captopril group was improved.(3)Compared with the sham-operation group,the positive expression rate of LC3-Ⅱin the myocardial tissue of the model group was increased(P<0.05);compared with the model group,the positive expression rate of LC3-Ⅱin the low-,medium-and high-dose groups of Qiangxin Formula and the Captopril group was decreased(P<0.05).(4)Compared with the sham-operation group,the protein expression levels of myocardial tissue p38MAPK and p-p38MAPK were increased and protein expression level of PPAR-γwas decreased in the model group(P<0.05);compared with the model group,protein expression levels of myocardial tissue p38MAPK and p-p38MAPK were decreased and protein expression level of PPAR-γwas increased(P<0.05).In addition,there was no statistically significant difference in the above-mentioned indexes between the Qiangxin Formula high-dose group and the Captopril Group(P>0.05).Conclusion The Qiangxin Formula may act against chronic heart failure by enhancing myocardial mitochondrial energy metabolism and reducing cardiomyocyte autophagy through inhibiting p38MAPK activation and promoting PPAR-γexpression.