Yang-xin-xue keli exerts therapeutic effects via regulating mitochondrial homeostasis and function in doxorubicin-induced rat heart failure

Figures

• 

  FIGURE 1. The fingerprint of the YXX keli by HPLC. (A). The reference standard. (B). The 20 characteristic peaks of the YXX keli fingerprint. The No. 1 peak (Gallic acid), No. 5 peak (Chlorogenic acid), No. 8 peak (Paeoniflorin), No. 9 peak (Ferulic Acid), No. 12 peak (Spinosin), No. 17 peak (Ginsenoside Rb1), and No. 20 peak (Catalpol) are determined compared with the reference standard.

• 

  FIGURE 2. Model building and transthoracic echocardiography to evaluate the cardiac function of rats. (A) building of heart failure of a rat model. UC + PBS: untreated group fed with the same volume of PBS; HF + PBS: heart failure group fed with the same volume of PBS; HF + LD: heart failure group fed with a low dose of YXX keli; HF + MD: heart failure group fed with a middle dose of YXX keli; HF + HD: heart failure group fed with a high dose of YXX keli; HF + Benazepril: heart failure group fed with Benazepril. (B). Transthoracic echocardiography was performed after all experiments. (C). In different groups, cardiac function was performed. *p < 0.05, vs. Model group. (D). ANP and BNP in different groups were measured. *p < 0.05, vs. Model group.

• 

  FIGURE 3. Detection of heart injury and metabolic function after the YXX keli treatment. To evaluate the myocardial injury, H&E (A) and Masson’s Trichrome (B) stained sections was performed. After being sacrificed, the left ventricle was freshly prepared and detected for ATP synthesis (C) and mitochondrial DNA mass (D). *p < 0.05, vs. model group; **p < 0.01, vs. model group.

• 

  FIGURE 4. YXX keli protects RCM against DOX-induced cell injury. (A). After 24-h incubation under a range concentration of DOX, the cell viability of H9c2 was measured by performing a CCK-8 assay. After the addition of medicated serum for co-incubation with 1 μM of DOX for 24 h, cell viability was measured by performing CCK-8 assay (B), cell cycle distribution was measured by performing PI staining followed by flow cytometry (C) and proliferating cells were stained using EdU assay (D). *p < 0.05, vs. 1 μM DOX + PBS-medicated serum group. (E) After the addition of medicated serum for co-incubation with 1.54 μM of DOX for 24 h, cell apoptosis was measured by performing Annexin V-FITC/PI double staining followed by flow cytometry assay. *p < 0.05, mock group; # p < 0.05, DOX + PBS-medicated serum.

• 

  FIGURE 5. YXX keli protects cardiomyocytes against DOX-induced cell injury. (A). RCMs were isolated and imaged after 1.5 μmol/L DOX treatment. (B). To evaluate the effect of medicated serum on DOX-induced cell cycle arrest, H9c2 cells were exposed to 1 μmol/L DOX for 24 h, and cell cycle distribution was measured after PI staining. *p < 0.05, mock group; # p < 0.05, DOX + PBS-medicated serum. (C). After addition of medicated serum for co-incubation with 1.54 μM of DOX for 24 h, apoptotic cell death of H9c2 was measured by performing Annexin V-FITC/PI double staining followed by flow cytometry assay. *p < 0.05, mock group; # p < 0.05, DOX + PBS-medicated serum.

• 

  FIGURE 6. YXX keli decreased ROS accumulation induced by DOX in RCM. (A). After DOX treatment, ROS accumulation was detected. *p < 0.05, vs. mock group; # p < 0.05, vs. DOX/PBS-medicated serum. (B). Mitochondrial membrane potential was measured by performing JC-1 staining. (C). Mitotracker staining was performed to detect mitochondria.

• 

  FIGURE 7. YXX keli protected mitochondrial function potentially via decreasing accumulated ROS induced by DOX in RCM. (A). ATP synthesis was measured. *p < 0.05, mock group; # p < 0.05, DOX + PBS-medicated serum. (B). Mitochondrial DNA mass was measured by quantitative PCR. *p < 0.05, mock group; # p < 0.05, DOX + PBS-medicated serum. (C). Mitochondrial transcriptional activity was measured by performing RT-qPCR to detect the expression level of mitochondrial coded genes.

• 

  FIGURE 8. YXX keli inhibited autophagy/mitophagy in H9c2 cells. (A). Formation of autophagosomes was measured by introducing GFP-RFP-LC3. (B). Autophagic and mitophagic vacuoles were observed by performing electron microscopy (EM). (C). Western blot was performed to detect PINK1, Parkin and cleavage of LC3 I and II. *p < 0.05, mock group; # p < 0.05, DOX + PBS-medicated serum. (D). Western blot was further performed to detect Bax and Bcl-2 protein. *p < 0.05, mock group; # p < 0.05, DOX + PBS-medicated serum.