Qingxin Jieyu Granule Regulates Mitophagy To Improve Ventricular Remodeling After Myocardial Infarction

Ventricular Remodeling(VR)after myocardial infarction(MI)is the main pathological basis for the occurrence and development of heart failure(HF)after MI,and improving VR after MI is an important way to prevent HF after MI.Studies have shown that the imbalance of myocardial energy metabolism can promote myocardial remodeling and the progression of HF,mitochondria play an important role in it,and its dysfunction can cause cardiomyocyte remodeling,which is an important target for the treatment of HF.Mitochondria can remove dysfunctional mitochondria through autophagy,control the quality of the mitochondria,and ensure the function of the mitochondria,which is of great significance for improving VR and preventing HF.In this study,aiming at the key pathological link of mitophagy weakening in the process of VR,the mechanism of VR after MI was studied by Qingxin Jieyu Granule(QXJYG)to improve the mechanism of VR after MI,under the guidance of the theory of ’blood stasis and toxin’ proposed by KJ Chen et al.,it is of great significance for improving MI prognosis and preventing and treating HF.Part 1 Effect of Qingxin Jieyu Granule on ventricular remodeling after myocardial infarction in C57BL/6 miceObjective:To observe the effect of QXJYG on VR after MI in mice.Methods:The model of VR after MI was established by ligation of the left anterior descending coronary artery of C57BL/6 mice.The mice were randomly divided into Sham group,MI group,QXJYG-L group(1300 mg/kg/d),QXJYG-M group(2600 mg/kg/d),QXJYG-H group(5200 mg/kg/d)and Sacubitril Valsartan Sodium Tablets group(SVST,30 mg/kg/d)by computer generated random numbers,with 6 mice in each group.Mice were intragastric for 4 weeks after MI.Ultrasound system for small animals Vevo 2100 was used to detect LVPWs,LVPWd,LVIDs,LVIDd,LV Vols,LV Vold,LVFS and LVEF to observe the cardiac function of mice.Compensatory hypertrophy of the myocardium after MI was observed by heart mass index(HMI)and heart-tibial ratio(HW/TL).The levels of BNP,CK-MB,Ang Ⅱ and LDH were detected by ELISA to observe the changes of serological indexes in mice after MI.HE and Masson staining were used to observe the myocardial fibrosis of mice after MI.The expressions of Vascular Endothelial Growth Factor(VEGF)and Basic Fibroblast Growth Factor(bFGF)in MI mice were observed by immunohistochemistry.Results:(1)Compared with Sham group,LVPWs,LVPWd and LVIDs were decreased in MI group,SVST group,QXJYG-L group,QXJYG-M group and QXJYG-H group(P<0.05),LVIDd was increased in MI group and QXJYG-M group(P<0.05),LV Vols was increased in MI group,QXJYG-M group and QXJYG-H group(P<0.05),LVFS and LVEF were decreased in MI group,SVST group,QXJYG-L group,QXJYG-M group and QXJYG-H group(P<0.05).Compared with MI group,LVIDs,LV Vols and LV Vold were decreased,and LVFS,LVEF were increased in SVST group,QXJYG-L group,QXJYG-M group and QXJYG-H group(P<0.05),LVPWs and LVPWd were increased in QXJYG-L and QXJYG-H groups(P<0.05),LVIDd was decreased in SVST group,QXJYG-L group and QXJYG-H group(P<0.05).Compared with SVST group,LVPWs and LVPWd were increased in QXJYG-L group(P<0.05).Compared with QXJYG-L group,LVPWs and LVPWd were decreased in QXJYG-M group(P<0.05),LVIDd was increased in QXJYG-M group(P<0.05).(2)Compared with Sham group,HMI was increased in MI group,QXJYG-M group and QXJYG-H group,and HW/TL was increased in MI group and QXJYG-H group(P<0.05).(3)Compared with Sham group,serum BNP levels were significantly increased in MI group(P<0.05),and serum BNP levels were significantly decreased after SVST and QXJYG intervention(P<0.05).Compared with MI group,CK-MB levels decreased in the GXJYG-L,GXJYG-M,GXJYG-H groups(P<0.05).Compared with the Sham group,the serum LDH levels in the MI group were significantly increased(P<0.05),and the serum LDH levels decreased after SVST and GXJYG-M intervention(P<0.05).Compared with the MI group,the SVST group and the GXJYG-L group had a decrease in Ang Ⅱ(P<0.05).(4)Compared with the Sham group,pathological changes such as myocardial fiber thickening,disordered arrangement,loose gaps,and accompanied by vacuoles and interstitial visible inflammatory cell infiltration were improved after SVST and QXJYG intervention.Compared with the Sham group,the MI group had a large amount of collagen fiber deposition,cardiomyocyte alignment disorder,and collagen fiber reduction after SVST and QXJYG intervention.Compared with the Sham group,the positive regional AOD increased in the MI group,the QXJYG-L group and the QXJYG-H group(P<0.05),and the positive regional AOD decreased in the SVST group and the QXJYG-M group compared with the MI group(P<0.05).(5)Compared with the MI group,VEGF and bFGF expression were seen in the myocardial tissue of mice after MI after SVST and QXJYG intervention.Conclusion:QXJYG improved cardiac dysfunction after MI in mice.Part 2 Transcriptomics study of Qingxin Jieyu Granule improving ventricular remodeling after myocardial infarctionObjective:To provide a possible molecular mechanism for QXJYG to improve VR after MI.Methods:Total RNA was extracted from mouse myocardium tissues of Sham group,MI group and QXJYG group,respectively,and then total RNA was detected to establish transcriptome library,which was sequenced by Illumina HiSeqTM 2500 sequencer.Key genes and signaling pathways of QXJYG to improve VR after MI were screened by GO and KEGG enrichment analysis.Results:(1)There were 167 differentially expressed genes in QXJYG group and MI group,85 up-regulated genes and 82 down-regulated genes.There were 1155 differentially expressed genes in Sham group and MI group,including 389 up-regulated genes and 766 down-regulated genes.There were 4 identical up-regulated genes in QXJYG group and Sham group,and 3 identical down-regulated genes in QXJYG group and Sham group.(2)There were 1141 signaling pathways involved in differentially expressed genes in Sham group and MI group.The differentially expressed genes were mainly concentrated in ECM-receptor interaction,Protein digestion and absorption,and PI3K-Akt signaling pathway,Phagosome,Cytokine-cytokine receptor interaction.There were 221 signaling pathways involved in differentially expressed genes in QXJYG group and MI group.Differentially expressed genes are mainly enriched in Ubiquinone and other terpenoid-quinone biosynthesis,Phenylalanine metabolism,Tyrosine metabolism,AMPK signaling pathway.Conclusion:QXJYG may regulate mitophagy through AMPK signaling pathway to improve VR after MI.Part 3 Qingxin Jieyu Granule regulates mitophagy through AMPK/ULK1 signaling pathway to improve ventricular remodeling after myocardial infarction in C57BL/6 miceObjective:To observe the effect of QXJYG on AMPK/ULK1 signaling pathway in mouse myocardium.Methods:Mitochondrial morphology of mouse myocardial tissue after MI was observed by 3%uranium-dioxy acetate and lead citrate double staining.Mitochondrial Membrane Potential(MMP)changes in mouse myocardial tissue after MI were detected by JC-1 kit.Western-Blot was used to detect AMPK/ULK1 signaling pathway-related protein expression levels(AMPK,p-AMPK,mTOR、p-mTOR、ULK1、p-ULK1).Results:(1)Compared with Sham group,mitophagy inactivation in mouse myocardial tissue resulted in mitochondrial damage,mitochondrial swelling,crest shattered and myocardial fiber distortion in MI group.After QXJYG treatment,mitochondrial swelling was reduced,and autophagosomes and lysosomes appeared.(2)Compared with Sham group,MMP decreased in MI group,SVST group,QXJYG-L group,QXJYG-M group and QXJYG-H group,MMP decreased most obviously in MI group,and MMP increased after QXJYG and SVST treatment.Compared with Sham group,red AOD were increased in MI group,SVST group and QXJYG group(P<0.05).Compared with MI group,red AOD were decreased in SVST and QXJYG group(P<0.05).There was no significant difference in green AOD among all groups.(3)Compared with Sham group,the expressions of AMPK,p-AMPK,ULK1 and p-ULK1 in MI group were down-regulated(P<0.05),the expression of mTOR and p-mTOR was up-regulated(P<0.05),the expression of ULK1 and p-ULK1 was down-regulated in QXJYG-L group(P<0.05).Compared with MI group,the expressions of AMPK and p-AMPK were up-regulated in QXJYG-M and SVST groups(P<0.05),the expression of mTOR and p-mTOR was down-regulated in QXJYG-M group,QXJYG-H group and SVST group(P<0.05),ULK1 expression was up-regulated in QXJYG-M group(P<0.05),the expression of p-ULK1 was up-regulated in QXJYG-H group(P<0.05).Conclusion:QXJYG repaired damaged mitochondria and improved their function,and QXJYG may regulate mitophagy through AMPK/ULK1 signaling pathway to improve VR after MI.Part 4 Qingxin Jieyu Granule regulates mitophagy by activating AMPK/ULK1 signaling pathway to improve mitochondrial injury in H/R cardiomyocytesObjective:To observe the effect of QXJYG on AMPK/ULK1 signaling pathway in H/R cardiomyocytes.Methods:H9C2 cardiomyocyte H/R model was established,and the optimal drug concentration of QXJYG was determined by CCK8 method.H9C2 cardiomyocytes at logarithmic growth stage were divided into Control group,H/R group,QXJYG group(6.25 μg/mL),QXJYG+Compound C group(Q+Compound C).Mitochondrial ROS(mtROS)levels were measured by MitoSOX Red fluorescent probe.MMP of myocardial cells was measured by JC-1.Mitophagy-related proteins(AMPK,p-AMPK,mTOR,p-mTOR,ULK1,p-ULK1)in cardiomyocytes were detected by Western-Blot.Results:(1)Compared with the Control group,the production of mtROS was increased in H/R group(P<0.05);Compared with H/R group,the production of mtROS was decreased in QXJYG group(P<0.05).(2)Compared with the Control group,the intensity of red fluorescence decreased in the H/R group,indicating that the formation of JC-1 polymer in the H/R group was less,and MMP decreased;Compared with H/R group,the red fluorescence intensity was increased in QXJYG group,indicating that QXJYG treatment can maintain the stability of the MMP of H/R cardiomyocytes.(3)Compared with the Control group,AMPK,ULK1 and p-ULK1 expressions were down-regulated in H/R group and Q+Compound C group(P<0.05),p-AMPK expression was down-regulated in H/R group(P<0.05),mTOR and p-mTOR expression was down-regulated in QXJYG group and Q+Compound C group(P<0.05).Compared with H/R group,the expressions of AMPK,p-AMPK and ULK1 were up-regulated in QXJYG group(P<0.05),mTOR and p-ULK1 expression were down-regulated in Q+Compound C group(P<0.05).Compared with QXJYG group,AMPK,ULK1 and p-ULK1 expressions were down-regulated in Q+Compound C group(P<0.05).Conclusion:QXJYG reduced the level of mtROS in H/R cardiomyocytes,maintained the stability of MMP in H/R cardiomyocytes,and improved mitophagy injury in H/R cardiomyocytes by activating AMPK/ULK1 signaling pathway and regulating mitophagy.