Huoxue Huatan Decoction Ameliorates Myocardial Ischemia/Reperfusion Injury in Hyperlipidemic Rats via PGC-1α-PPARα and PGC-1α-NRF1-mtTFA Pathways

Abstract

Objective: The aim of this study was to eluc\idate the preventive and therapeutic effects and the underlying mechanisms of Huoxue Huatan Decoction (HXHT) on myocardial ischemia/reperfusion (I/R) injury in hyperlipidemic rats.

Methods: An I/R model was established in hyperlipidemic Wistar rats. After 4-8 weeks of HXHT treatment, the physical signs of rats were observed. Lipid metabolism, myocardial enzyme spectrum, cardiac function, myocardial histomorphology, and mitochondrial biosynthesis were investigated by a biochemical method, ultrasonography, electron microscopy, pathological examination, real-time PCR, and Western blot.

Results: HXHT can affect lipid metabolism at different time points and significantly reduce the levels of cholesterol (CHO), triglyceride (TG), high-density lipid-cholesterol (HDL-C), and low-density lipid-cholesterol (LDL-C) in hyperlipidemic rats (P < 0.05 or P < 0.01); it can significantly reduce the levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), reduce the myocardial infarct size and myocardial ischemic area, and improve cardiac function. The results of myocardial histomorphology showed that HXHT could protect myocardial cells, relieve swelling, reduce the number of cardiac lipid droplets, and improve myocardial mitochondrial function. HXHT could significantly increase the levels of total superoxide dismutase (T-SOD) and succinate dehydrogenase (SDH) (P < 0.05 or P < 0.01), increase CuZn-superoxide dismutase (CuZn-SOD) and glutathione-peroxidase (GSH-Px) levels, and decrease the levels of malondialdehyde (MDA) (P < 0.05); it could increase the mRNA and protein expression levels of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor alpha (PPARα), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (mtTFA) (P < 0.05 or P < 0.01), and increase the synthesis of mitochondrial DNA (mtDNA) (P < 0.01).

Conclusion: HXHT can reduce myocardial I/R injury in hyperlipidemic rats. The protective mechanisms may involve a reduction in blood lipids, enhancement of PGC-1α-PPARα pathway activity, and, subsequently, an increase in fatty acid β-oxidation, which may provide the required input for mitochondrial energy metabolism. HXHT can additionally enhance PGC-1α-NRF1-mtTFA pathway activity and, subsequently, increase the antioxidant capacity, promote mtDNA synthesis, and reduce mitochondrial damage. The two pathways use PGC-1α as the intersection point to protect mitochondrial structure and function, reduce I/R-induced injury, and improve cardiac function.

Keywords: Huoxue Huatan Decoction; PGC-1α; hyperlipidemia; ischemia/reperfusion (I/R); mitochondria.

Figure 1

Effects of Huoxue Huatan Decoction (HXHT) on cholesterol (CHO) serum levels in hyperlipidemic rats $(\overline{x}\pm s)$. The num ber of animals per group at 4 weeks is n = 24; the number of animals per group at 8 weeks is n = 16. ^b P < 0.01 compared with the Normal group; ^c P < 0.05, ^d P < 0.01 compared with the Model group. HXHT, Huoxue Huatan Decoction; CHO, cholesterol.

Figure 2

Effects of Huoxue Huatan Decoction (HXHT) on triglyceride (TG) serum levels in hyperlipidemic rats $(\overline{x}\pm s)$. The number of animals per group at 4 weeks is n = 24; the number of animals per group at 8 weeks is n = 16. ^b P < 0.01 compared with the Normal group; ^c P < 0.05, ^d P < 0.01 compared with the Model group. HXHT, Huoxue Huatan Decoction; TG, triglyceride.

Figure 3

Effects of Huoxue Huatan Decoction (HXHT) on high-density lipid-cholesterol (HDL-C) serum levels in hyperlipidemic rats $(\overline{x}\pm s)$. The number of animals per group at 4 weeks is n = 24; the number of animals per group at 8 weeks is n = 16. ^b P < 0.01 compared with the Normal group; ^c P < 0.05 compared with the Model group. HXHT, Huoxue Huatan Decoction; HDL-C, high-density lipid-cholesterol.

Figure 4

Effects of Huoxue Huatan Decoction (HXHT) on low-density lipid-cholesterol (LDL-C) serum levels in hyperlipidemic rats $(\overline{x}\pm s)$. The number of animals per group at 4 weeks is n = 24; the number of animals per group at 8 weeks is n = 16. ^a P < 0.05, ^b P < 0.01 compared with the Normal group; ^c P < 0.05, ^d P < 0.01 compared with the Model group. HXHT, Huoxue Huatan Decoction; LDL-C, low-density lipid-cholesterol.

Figure 5

Effects of Huoxue Huatan Decoction (HXHT) on creatine kinase-MB (CK-MB) serum levels in hyperlipidemic ischemia/reperfusion (I/R) rats $(\overline{x}\pm s)$ The number of animals per group at 4 weeks is n = 24; the number of animals per group at 8 weeks is n = 16. ^a P < 0.05, ^b P < 0.01 compared with the Normal group; ^c P < 0.05, ^d P < 0.01 compared with the Model group.

Figure 6

Effects of Huoxue Huatan Decoction (HXHT) on lactate dehydrogenase (LDH) serum levels in hyperlipidemic ischemia/reperfusion (I/R) rats $(\overline{x}\pm s)$. The number of animals at 4 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 7; n _{Control B} = 8; n _HXHT-H = 7; n _HXHT-M = 8; n _HXHT-L = 8. The number of animals at 8 weeks is n _Normal = 16; n _Model = 15; n _{Control A} = 16; n _{Control B} = 15; n _HXHT-H = 16; n _HXHT-M = 16; n _HXHT-L = 15. ^b P < 0.01 compared with the Normal group; ^c P < 0.05, ^d P < 0.01 compared with the Model group. HXHT, Huoxue Huatan Decoction; CK-MB, creatine kinase-MB; LDH, lactate dehydrogenase; I/R, ischemia/reperfusion.

Figure 7

B-ultrasound of hyperlipidemic ischemia/reperfusion (I/R) rats.

Figure 8

Effects of Huoxue Huatan Decoction (HXHT) on left ventricular ejection fraction (LVEF) in hyperlipidemic ischemia/reperfusion (I/R) rats $(\overline{x}\pm s)$. TThe number of animals at 4 weeks is n _Normal = 8; n _Model = 7; n _Control A = 7; N _Control B = 8; n _HXHT-H = 7; n _HXHT-M = 8; n _HXHT-L = 8. The number of animals at 8 weeks is n _Normal = 16; n _Model = 15; n _Control A = 16; n _Control B = 15; n _HXHT-H = 16; n _HXHT-M = 16; n _HXHT-L = 15. ^a P < 0.05 compared with the Normal group; ^c P < 0.05 compared with the Model group.

Figure 9

Effects of Huoxue Huatan Decoction (HXHT) on left ventricular end-diastolic inner diameter (LVIDd) in hyperlipidemic ischemia/reperfusion (I/R) rats $(\overline{x}\pm s)$. The number of animals at 4 weeks is n _Normal = 8; n _Model = 7; n _Control A = 7; N _Control B = 8; n _HXHT-H = 7; n _HXHT-M = 8; n _HXHT-L = 8. The number of animals at 8 weeks is n _Normal = 16; n _Model = 15; n _Control A = 16; n _Control B = 15; n _HXHT-H = 16; n _HXHT-M = 16; n _HXHT-L = 15. ^a P < 0.05 compared with the Normal group; ^c P < 0.05 compared with the Model group.

Figure 10

Effects of Huoxue Huatan Decoction (HXHT) on left ventricular end-systolic inner diameters (LVIDs) in hyperlipidemic ischemia/reperfusion (I/R) rats $(\overline{x}\pm s)$. The number of animals at 4 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 7; n _{Control B} = 8; n _HXHT-H = 7; n _HXHT-M = 8; n _HXHT-L = 8. The number of animals at 8 weeks is n _Normal = 16; n _Model = 15; n _{Control A} = 16; n _{Control B} = 15; n _HXHT-H = 16; n _HXHT-M = 16; n _HXHT-L = 15. ^a P < 0.05 compared with the Normal group; ^c P < 0.05, ^d P < 0.01 compared with the Model group.

Figure 11

Effects of Huoxue Huatan Decoction (HXHT) on myocardial infarct size and ischemic area in hyperlipidemic ischemia/reperfusion (I/R) rats $(\overline{x}\pm s)$. The number of animals at 4 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 7; n _{Control B} = 8; n _HXHT-H = 7; n _HXHT-M = 8; n _HXHT-L = 8. The number of animals at 8 weeks is n _Normal = 8; n _Model = 8; n _{Control A} = 8; n _{Control B} = 7; n _HXHT-H = 8; n _HXHT-M = 8; n _HXHT-L = 7. ^a P < 0.05 compared with the Normal group; ^c P < 0.05, ^d P < 0.01 compared with the Model group. HXHT, Huoxue Huatan Decoction; I/R, ischemia/reperfusion.

Figure 12

Effects of Huoxue Huatan Decoction (HXHT) on heart histopathology after myocardial ischemia/reperfusion (I/R) injury in hyperlipidemic rats (HE staining, 200×). (A) Normal control; (B) Model control; (C) Control A; (D) HXHT-M. The number of animals at 8 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 8; n _HXHT-M = 8. HXHT, Huoxue Huatan Decoction; I/R, ischemia/reperfusion.

Figure 13

Effects of Huoxue Huatan Decoction (HXHT) on heart histopathology after myocardial ischemia/reperfusion (I/R) injury in hyperlipidemic rats (Oil Red O staining, 100×). (A) Normal control; (B) Model control; (C) Control A; (D) HXHT-M. The number of animals at 8 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 8; n _HXHT-M = 8. HXHT, Huoxue Huatan Decoction; I/R, ischemia/reperfusion.

Figure 14

Effects of Huoxue Huatan Decoction (HXHT) on myocardial mitochondria after myocardial ischemia/reperfusion (I/R) injury in hyperlipidemic rats (transmission electron microscopy, 40,000×). (A) Normal control; (B) Model control; (C) Control A; (D) HXHT-M. The number of animals at 8 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 8; n _HXHT-M = 8. HXHT, Huoxue Huatan Decoction; I/R, ischemia/reperfusion.

Figure 15

Effects of Huoxue Huatan Decoction (HXHT) on oxidative stress indicators in hyperlipidemic ischemia/reperfusion (I/R) rats. ^a P < 0.05, ^b P < 0.01 compared with the Normal group; ^c P < 0.05, ^d P < 0.01 compared with the Model group. The number of animals at 8 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 8; n _HXHT-M = 8. HXHT, Huoxue Huatan Decoction; I/R, ischemia/reperfusion.

Figure 16

Effects of Huoxue Huatan Decoction (HXHT) on mRNA levels in hyperlipidemic ischemia/reperfusion (I/R) rats $(\overline{x}\pm s)$. The results were calculated using the 2^−ΔΔCt method, where 2^−ΔΔCt = (Ct_{target gene} − Ct_{housekeeper gene}) experimental group − (Ct_{target gene} − Ct_{housekeeper gene}) control group. The amplification factors are presented for comparison reasons. ^b P < 0.01 compared with the Normal group; ^c P < 0.05 compared with the Model group. The number of animals at 8 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 8; n _HXHT-M = 8. HXHT, Huoxue Huatan Decoction; I/R, ischemia/reperfusion.

Figure 17

Effects of Huoxue Huatan Decoction (HXHT) on proteins in hyperlipidemic ischemia/reperfusion (I/R) rats $(\overline{x}\pm s)$. The images were scanned and analyzed with ImageJ software. The gray-scale value was calculated for each band. The relative content of each target protein was calculated by dividing the gray-scale value of the target protein by the gray-scale value of β-actin. ^b P < 0.01 compared with the Normal group; ^c P < 0.05, ^d P < 0.01 compared with the Model group. The number of animals at 8 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 8; n _HXHT-M = 8. HXHT. (A) Normal control; (B) Model control; (C) Control A; (D) HXHT-M. HXHT, Huoxue Huatan Decoction; I/R, ischemia/reperfusion.

Figure 18

Effects of Huoxue Huatan Decoction (HXHT) on mitochondrial DNA (mtDNA) copy number in hyperlipidemic ischemia/reperfusion (I/R) rats $(\overline{x}\pm s)$. ^b P < 0.01 compared with the Normal group; ^d P < 0.01 compared with the Model group. The number of animals at 8 weeks is n _Normal = 8; n _Model = 7; n _{Control A} = 8; n _HXHT-M = 8. HXHT, Huoxue Huatan Decoction; I/R, ischemia/reperfusion; mtDNA, mitochondrial DNA.