| Background:Because the pathogenesis of heart failure is intertwined and complex,the current drug development for heart failure treatment has entered a plateau.Myocardial mitochondria are the main site of cardiometabolism,the foundation of cardiac exercise,and the key target for the treatment of heart failure.Traditional Chinese medicine has shown unique advantages in the treatment of heart failure due to its multi-site synergistic effect.Many Chinese medicines have also been shown to affect mitochondria.Therefore,research on the mechanism of traditional Chinese medicine for the treatment of heart failure with mitochondria as the target may provide more theoretical basis for the treatment of heart failure.Objective:To explore the effects of Linggui Zhugan Decoction on cardiac function,myocardial mitochondria and related key signaling molecules in the SIRT1-AMPKPGC1α signaling pathway in chronic heart failure rats after myocardial infarction.Methods: 1.A rat model of chronic heart failure after myocardial infarction was established by ligation of the left anterior descending coronary artery.Rats with successful modeling were divided into model group（n=11）,Lingguizhugan decoction group（n=12）and captopril group（n=11）,while sham operation group（n=12）served as control groups.All groups were given intragastric intervention for 6 weeks.The Linggui Zhugan Decoction group was given Linggui Zhugan Decoction 5.4 g/（kg d）,the captopril group was given captopril 6.75 mg/（kg d）,and the sham group and the model group were given the same amount of distilled water,and comparing the differences between the groups;2.Observe the general condition of the rats,measure the exercise tolerance of the rats in each group by the exhaustive swimming method,and measure the cardiac function of the rats by small animal color Doppler ultrasound,including the determination of LVEF,LVFS,LVIDd,and LVIDs;3.ELISA method was used to measure the levels of s ST2 and NTpro BNP in rat plasma,SOD activity was measured by WST-1 method,and MDA content was measured by TBA method;4.HE staining and Masson staining were used to observe rat myocardial tissue morphology and collagen deposition;5.The ultrastructure of rat myocardium was observed by TEM,and the level of myocardial mitochondrial membrane potential was detected by JC-1 staining;6.RT-PCR was used to detect the expression levels of SIRT1,AMPK,PGC-1α,NRF1,TFAM,m RNA and mt DNA in rat myocardial tissue;7.Western blot was used to detect the protein expression levels of SIRT1,AMPK,p-AMPK and PGC-1α in rat myocardial tissue.Results: 1.The cardiac structure and the cardiac function: The rats that were successfully modeled were given drug intervention of Lingguizhugan Decoction and captopril for 6 weeks,was found that they are had better general conditions than the model group,the time of exhaustive swimming was significantly prolonged（P<0.05）,and the whole heart mass index（HW/BW）decreased（P<0.05）.Ultrasound detection showed that the LVEF and LVFS of the two groups of drug intervention rats were increased compared with the model group,and the LVIDd and LVIDs were decreased,and the difference was statistically significant（P<0.05）.However,compared with the captopril group,the Lingguizhugan decoction group had a significant decrease in LVIDs（P<0.05）,and there was no significant difference in other ultrasound indicators.2.The markers of heart failure: Compared with the sham group,the plasma NTpro BNP and s ST2 levels in the model group were significantly increased（P<0.05 or P<0.001）.The level of the Lingguizhugan decoction group and the captopril group were lower than those in the model group（P<0.05 or P<0.01）,and there was no difference in NT-pro BNP and ST-2 between the Lingguizhugan decoction group and the captopril group（P>0.05）.3.The oxidative stress injury: Compared with the sham group,the model group significantly decreased the plasma SOD activity（P<0.01）,and the MDA content was significantly increased（P<0.001）;compared with the model group,about the Lingguizhugan decoction group,the activity of SOD in the plasma of rats was significantly increased（P<0.05）,and the content of MDA was significantly decreased（P<0.01）.Compared with the model group,although there was no significant change in the activity of plasma SOD in the captopril group（P>0.05）,the content of MDA decreased significantly.（P<0.01）.4.The pathological structure of myocardial tissue: HE staining showed that the myocardial cells in the sham-operated group were well-structured,uniform in size,neatly arranged,and the muscle fibers were rarely damaged.The cardiomyocytes in the model group were of different sizes,various shapes,irregular arrangement,disordered muscle fibers,and even some of them were broken and necrotic,with inflammatory cell infiltration.Compared with the model group,the degree of myocardial structural damage in the Lingguizhugan decoction group and the captopril group was improved,the infiltration of inflammatory cells was reduced,and the muscle fibers were looser.Masson staining showed that there was very little or no collagen fibrous tissue deposition in the sham-operated group,while large fibrous tissue could be seen in the model group.Some sheet-like fibrous tissues were seen in the Lingguizhugan decoction group and captopril group,which were less than those in the model group under microscope.Using the collagen volume fraction for quantitative comparison,the CVF of the model group was significantly higher than that of the sham-operated group（P<0.001）.Compared with the model group,the degree of myocardial fibrosis in the captopril group and Linggui Zhugan decoction group was significantly reduced（P<0.01）.5.The myocardial ultrastructure and mitochondrial membrane potential: The sham operation group showed that the myocardial cell membrane was intact,the myocardial fiber striated was clear,the sarcomere was complete,the mitochondria between the muscle fibers were arranged regularly,the number was large,and the morphological structure Normal,the inner mitochondrial cristae are clear and tightly arranged.The myocardial structure of the rats in the model group was severely damaged,the myocardial sarcomere was destroyed,the transverse striations were blurred,the mitochondria were degenerated and swollen,the outer membrane was incomplete,the fusion was blurred,the internal cristae were disordered and partially destroyed.Compared with the model group,the pathological state of myocardial ultrastructure in Lingguizhugan decoction and captopril groups was improved,with regular sarcomeres,slightly blurred horizontal stripes,decreased mitochondrial cristae expansion,decreased cristae disorder,and increased membrane clarity.Fluorescence microscope observation of JC-1-stained mitochondria showed that the myocardial MMP of the model group was significantly lower than that of the sham group（P<0.001）;In the captopril group,MMP increased,but the difference was not statistically significant（P>0.05）.6.The key signaling molecules in SIRT1-AMPK-PGC1α signaling pathway: RTPCR results showed that the m RNA levels of SIRT1 and PGC-1α in the model group were significantly lower than those in the sham group（P<0.01）;The level of PGC-1α m RNA was higher than the model group（P<0.05）.Although the captopril group was higher than the model group,the difference was not statistically significant（P>0.05）.There was no significant difference in AMPK among the groups.In addition,compared with the model group,the m RNA levels of NRF1 and TFAM in the Linggui Zhugan decoction group were increased（P<0.05）,but there was no significant change in the captopril group;in terms of mitochondrial DNA content in myocardial tissue,the Linggui Zhugan decoction group and the cardtopril group had no significant changes.Both groups were significantly increased（P<0.01）.Western blot results showed that the expression of SIRTI and PGC-1α proteins in the model group was relatively attenuated compared with the sham operation group（P<0.01）.After the intervention of Lingguizhugan decoction,the relative expressions of SIRTI and PGC-1α proteins were significantly increased compared with those in the model group（P<0.01）,the relative expression of p-AMPK protein increased,and there was no significant change in AMPK protein,and the level of p-AMPK/AMPK increased.High（P<0.05）.Compared with the model group,the SIRTI and pAMPK/AMPK protein levels of the captopril group were almost unchanged,but the PGC-1α protein level increased（P<0.05）.Conclusion: 1.Linggui Zhugan Decoction can delay left ventricular remodeling,enhance cardiac pumping function,improve symptoms,and increase exercise tolerance in chronic heart failure rats.2.Linggui Zhugan Decoction can exert antioxidant effect and improve myocardial pathological structure and myocardial fibrosis in chronic heart failure rats.3.Lingguizhugan decoction can reduce the damage of mitochondrial structure and stabilize mitochondrial membrane potential in chronic heart failure rats.4.Linggui Zhugan Decoction exerts the protective effect on mitochondria by stimulating the SIRT1-AMPK-PGC1α pathway in chronic heart failure rats. |
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