To investigate the effects of Yiqi Huoxue Formula (YQHX) on angiogenesis after myocardial infarction (MI) and to elucidate the underlying regulatory mechanisms involving mitochondria-associated endoplasmic reticulum membranes (MAMs).

Thirty male SD rats were randomly divided into sham operation group (n = 6) and modeling group (n = 24) using a random number table method. The MI model was established by ligating the left anterior descending (LAD) coronary artery. In the sham operation group, the LAD was threaded with a suture without ligation. Following successful modeling, the rats were randomly divided into four groups: model, YQHX low-dose (4.1 g/kg), YQHX high-dose (8.2 g/kg), and perindopril (4.0×10^-4 g/kg), with six rats per group. Following 28 d of consecutive oral gavage (once daily), all rats were sacrificed and the relevant indices were measured. Cardiac function parameters were evaluated using echocardiography. Serum lactate dehydrogenase (LDH) and endothelin-1 (ET-1) levels were measured using ELISA. Histopathological changes and collagen fiber proliferation in myocardial tissues were assessed via HE and Masson staining. Platelet endothelial cell adhesion molecule-1 (CD31) expression was detected using immunohistochemistry (IHC). Western blotting (WB) was performed to quantify the protein expression levels of CD31, vascular endothelial growth factor (VEGF), and MAM-associated proteins, including inositol 1,4,5-trisphosphate receptor type 2 (IP3R2), glucose-regulated protein 75 (GRP75), voltage-dependent anion channel 1 (VDAC1), and mitofusin-2 (MFN2).

Compared with the sham operation group, the cardiac function parameters of the model group exhibited significant deterioration, characterized by decreased left ventricular ejection fraction and left ventricular fractional shortening(P< 0.05), alongside increased left ventricular end-systolic volume and left ventricular end-diastolic volume (P < 0.05). These cardiac function parameters were improved after intervention with YQHX or perindopril (P<0.05). Serum LDH and ET-1 levels increased (P< 0.05) in the model group, while decreased in all drug treatment groups using ELISA (P < 0.05). Histopathological staining revealed disorganized myocardial arrangement, pronounced inflammatory infiltration, and extensive collagen fiber proliferation in the model group; these pathological alterations were alleviated in all drug treatment groups. IHC result showed that the staining intensity of CD31 in the myocardial tissue of the model group was higher than that of the sham operation group, and this positive expression was further enhanced in all drug treatment groups (P< 0.05). WB result showed that the protein expression levels of CD31 and VEGF in the model group were elevated compared to the sham operation group (P<0.05); these levels were further upregulated following drug treatments (P < 0.05). Concurrently, IP3R2, GRP75, and VDAC1 expressions were upregulated, whereas MFN2 was downregulated in the model group (P<0.05). All treatment groups reversed the aberrant expression levels of these proteins (P<0.05).

YQHX exerts a protective effect on cardiac function and myocardial structure following MI. This efficacy is likely attributable to the promotion of angiogenesis and the enhancement of endothelial cell function, which are mediated by the upregulation of CD31 and VEGF, as well as the restoration of the balance of IP3R2, GRP75, VDAC1, and MFN2.

To investigate the regulatory mechanism of Yiqi Huoxue Formula (YQHXF) on angiogenesis in myocardial infarction (MI) rats through transcriptome sequencing and network pharmacology.

An MI model was established via left anterior descending coronary artery ligation in 12 male SD rats. After successful modeling, the rats were divided into model and YQHXF groups using a random number table method, with six rats per group. The remaining six rats in the sham operation group were only threaded without undergoing ligation. On the second day after the operation, the YQHXF group received intragastric administration of YQHXF (8.2 g/kg), whereas the other two groups received an equal volume of distilled water, once a day for 28 days. Cardiac function was assessed using echocardiography to measure left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV). The appearance of the heart was observed and photographed. Inflammatory cell infiltration and collagen fiber proliferation in the MI border area were observed using hematoxylin-eosin (HE) staining and Masson staining. Immunofluorescence staining was used to detect the fluorescence expression of CD31 in the MI border area. Transcriptome sequencing was used to identify differentially expressed genes (DEGs) between the sham operation and model groups, and gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Network pharmacology was used to screen the targets of YQHXF. These DEGs were then intersected with targets of YQHXF, and GO and KEGG analyses were performed to predict core targets and pathways involved in YQHXF-mediated post-MI angiogenesis.

Hearts in the sham operation group appeared reddish and smooth with no significant necrosis, whereas those in the model group were pale, wrinkled, and exhibited apparent MI in the ligation area and surrounding regions. Hearts in the YQHXF group also showed whitish myocardial tissue but had a reduced MI size compared to the model group, along with significant cardiac enlargement. Compared to the sham operation group, the model group had significantly decreased LVEF and LVFS, and increased LVESV and LVEDV (P<0.05). Compared to the model group, the YQHXF group showed increased LVEF and LVFS, and decreased LVESV and LVEDV (P<0.05). HE staining showed neatly arranged and intact cardiomyocytes in the sham operation group. In contrast, the model group exhibited irregular cardiomyocyte morphology and arrangement with extensive inflammatory cell infiltration in the MI border area. The YQHXF group showed reduced inflammatory cell infiltration compared to the model group. Masson staining revealed red-stained, well-arranged, and clear myocardial fibers in the sham operation group. The model group had blurred myocardial cell boundaries and extensive blue collagen fiber proliferation. The YQHXF group showed reduced blue collagen fiber and a more orderly myocardial cell arrangement compared to the model group. Compared with the sham operation group, the CD31 fluorescence intensity in the model group was increased (P<0.05). Compared with the model group, the CD31 fluorescence intensity was increased in the YQHXF group (P<0.05). Transcriptome sequencing identified 691 DEGs between the model and sham operation groups. GO analysis involved cell biological processes, including extracellular matrix(ECM) and extracellular space structure, and KEGG enrichment was primarily in pathways such as ECM-receptor interaction and transforming growth factor-β signaling pathways. Network pharmacology retrieval identified 47 main components of YQHXF and 756 potential targets. Intersecting these with the DEGs of transcriptome sequencing, a total of 31 core targets for YQHXF in treating post-MI angiogenesis were obtained. GO and KEGG analyses predicted that YQHXF may regulate angiogenesis through calcium ion transport and the vascular endothelial growth factor(VEGF) pathway.

YQHXF may promote angiogenesis after MI through calcium ion transport and the VEGF pathway, thereby improving cardiac structure and function.

To investigate the cardioprotective effect of Yiqi Huoxue granule (YQHXG) in the regulation of autophagy in rats induced with myocardial infarction (MI).

An acute MI animal model was established by ligation of the left anterior descending branch of the coronary artery in Sprague-Dawley rats. Besides, 20 rats received sham operation were classified into a control group. The remaining 59 rats were randomly divided into MI model group (n = 19), YQHXG group (n = 20), and perindopril group (n = 20). Relevant indicators on days 7 and 28 were observed in each group. Left ventricular function was determined by echocardiography. The structure and morphology of mitochondria, and the number of autophagic vesicles, were observed by transmission electron microscopy. The mRNA and protein expression levels of LC3, FUNDC1, Beclin-1, and BNIP3 were examined in the tissue of the MI marginal area.

Compared with the MI model group, YQHXG showed obvious improvements in cardiac functions. Observing the microscopic morphology of the heart tissue, myocardial tissue damage attenuated, autophagic signs of autophagosomes and autolysosomes reduced, vacuolization in mitochondria mitigated, and mitochondria arranged in order. YQHXG could reduce the degree of tissue lesion after MI and regulate the expression of autophagy-related molecules at different stages. On Day 7, YQHXG significantly downregulated the expression of Fundc1, Becn1, Bnip3 mRNA and reduced the levels of FUNDC1, Beclin-1, BNIP3, and LC3 B proteins expression (all P < .001). On Day 28, YQHXG could upregulate the expression of Becn1, Fundc1 and Bnip3 mRNA and increased the levels of the corresponding proteins expression (all P < .001). Besides, it also increased LC3 B protein expression level (P = .0344).

YQHXG regulated the expression of mitochondrial autophagy-related factors in myocardial tissue and mitochondrial autophagic activity at different stages to protect the heart following MI.

In myocardial ischemia, hypoxia leads to destruction of the cytoskeleton, and especially the imbalance of microtubule polymerization-depolymerization, which seriously affects the structure and function of cardiomyocytes. We previously showed that a Yiqi Huoxue Decoction (YQHX) improves mitochondrial function and decreases anti-oxidative effects in hypoxia-induced H9c2 cell injury. Therefore, in this study we investigated whether YQHX protects against hypoxia-induced damage by decreasing damage to the cardiac cytoskeleton.

After reaching 70%–80% confluence, H9c2 cells were synchronized in serum-free Dulbecco's Modified Eagle Medium for 6 hours, then divided into control, model, and YQHX (100, 200, 400 μg/mL) groups, which were then grown in a hypoxic atmosphere for 12 hours. Cardiac cell viability was assessed using an xCELLigence system. The levels of lactate dehydrogenase, maleic dialdehyde, and superoxide dismutase in H9c2 cell supernatants were measured. Hoechst 33258 staining was employed to observe cardiac cell apoptosis. Confocal microscopy, immunofluorescence, and western blot analysis were performed to evaluate the protective effects of the YQHX against hypoxia-induced injury in the H9c2 cell line.

Cells that were pretreated with YQHX were more able to maintain their microtubule structure in the early stages of hypoxia and had better myocardial fitness in response to hypoxia compared with cells that were not pretreated. However, hypoxia-induced upregulation of α-tubulin and β-tubulin expression antagonized the protective effect of YQHX (100 μg/mL). In addition, YQHX (100 μg/mL) treatment significantly upregulated MAP4 protein expression (P = .003) and downregulated p-AMPKα protein expression (P < .001) compared with the model group.

The results indicate that YQHX plays a role in protecting against oxidative stress injury and apoptosis in H9c2 cells. Notably, our results suggested that the YQHX could mitigate the damage to the cardiac cytoskeleton and the dysregulation of AMPK-related protein signaling pathways that are induced by hypoxia.

Yiqi Huoxue Decoction (YQHX) has been widely used for clinical treatment of ischemic heart disease. While oxidative stress plays a key role in the pathogenesis of ischemic heart disease, the function and molecular mechanism underlying antioxidative protective effects of YQHX on H9c2 cardiomyocytes against ischemia/hypoxia (I/H) have yet to be well clarified.

H9c2 cells were subjected to 12 h of hypoxia with serum-free conditions and then treated with or without YQHX (100–400 μg/mL). Cell viability was examined using a CCK-8 assay. Maleic dialdehyde (MDA) and superoxide dismutase (SOD) activity were detected using commercial kits. Intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential were measured using fluorescence microscopy and confocal laser-scanning microscopy, respectively. Ultrastructural details of mitochondria in H9c2 cells were observed using transmission electron microscopy. The antioxidative protective pathway was assessed by measuring mRNA and protein expression of Nrf2 and HO-1, as well as AMPK activation.

I/H injury gradually induced oxidative stress. Treatment with YQHX significantly increased cell viability and reversed I/H-induced oxidative stress, including reducing the production of oxidative stress products (ROS and MDA), increasing SOD levels, improving mitochondrial morphology, and increasing mitochondrial membrane potential. YQHX was also observed to increase I/H-induced expression of Nrf2 and HO-1, and the activation effects of YQHX were blocked by an AMPK inhibitor. In addition, HPLC analysis showed that YQHX contained two active antioxidative constituents (calycosin and ferulic acid).

The results suggest that anti-oxidative effects exerted by YQHX in H9c2 cardiomyocytes may be linked to upregulation of the AMPK-mediated Nrf2/HO-1 pathway.

To investigate how Yiqi Huoxue (YQHX) prescription regulates mitochondrial biosynthesis and ATP synthesis via AMP-activated protein kinase (AMPK) and to reveal its molecular mechanism in preventing and treating post-MI myocardial remodeling.

The MI animal model of myocardial infarction were established by ligating Sprague Dawley (SD) rats' left anterior descending coronary arteries; the animals were randomly divided into MI group, YQHX prescription group and perindopril group, and a sham operation group was set at the same time. Related drug intervention was administered on the 2nd day after surgery, the YQHX group was given Astragalus, angelica, ginseng, Ligusticum wallichii and pseudoginseng, provided by the Dongzhimen Hospital of Beijing University of Chinese Medicine, once per day, at a dose of 21 g/kg body weight/day (the clinical equivalent dose based on a previous study), and the changes in relevant indicators were observed at 1 week and 4 weeks. Echocardiography (ECG) was used to observe the changes in rat cardiac structure and functions; the morphology-based technique was used to observe the changes in myocardial cells and mitochondria; the expression of AMPK signal pathway-related proteins and mRNA was detected using western blotting and real-time fluorescence quantification respectively, while fluorescence enzyme-labeled method was used for detecting ATP synthesis.

Cardiac structure and functions: Compared with the MI group at 1 week, the YQHX prescription group and the perindopril group exhibited increased LVEF and LVFS (P_all <.05); their LVEDs and LVEDd were reduced but suggested no statistically significant differences (F = 2.258, F = 0.3464, P_all >.05). At 4 weeks, both LVEF and LVFS were elevated in the YQHX prescription group (P =.008,.009) and the perindopril group (P =.279,.333), where differences in the later group indicated no statistical significance; the YQHX prescription group and the perindopril group were also featured by reduced LVEDs and LVEDd (P_all <.05). Morphology: Compared with the MI group at two time nodes, the YQHX prescription group and the perindopril group exhibited significant improvement in the pathological changes in myocardial cells and mitochondrial structure. Expression of AMPK signal pathway-related proteins and mRNA: The expression of both pLKB1 and pAMPK proteins followed a rising trend in the YQHX prescription group and the perindopril group at 1 week. The expression of LKB1mRNA and AMPKmRNA was elevated (P_all <.05). The increased expression of PGC-1α, NRF1 and mtTFA proteins demonstrated statistically significant differences (P_all <.05). The expression of mtDNA protein followed an increasing trend. At 4 weeks, the expression of both pLKB1 and pAMPK proteins was elevated (P_all <.05). Heightened expression was also reported in LKB1mRNA and AMPKmRNA (P_all <.05). The increased expression of PGC-1α, NRF1, mtTFA and mtDNA proteins demonstrated statistically significant differences (P_all <.05). ATP synthesis: ATP synthesis was increased in the YQHX prescription group and the perindopril group at both 1 week and 4 weeks (P_all <.001).

The possible mechanism of YQHX prescription for preventing and treating post-MI myocardial remodeling may function through strengthening the activation AMPK signal pathway by LKB1, thus further increasing the expression of downstream transcription factor proteins and initiating mitochondrial replication and transcription; as a result, the YQHX prescription can improve the post-MI damage in mitochondrial morphological structure in the tissue at heart marginal zone as well as enhance mitochondrial biosynthesis and ATP synthesis.

Atherosclerosis is the fundamental pathophysiologic component of cardiovascular disease, and Tiaozhi Tongmai Granules show great efficiency in the treatment of the disease. However, the mechanism of Tiaozhi Tongmai Granules is still unclear. In this study, we have combined experiments with network pharmacology to explore the anti-atherosclerosis mechanism of Tiaozhi Tongmai Granules.

120 male ApoE^−/− mice were randomly divided into three groups: the model group, Chinese herb group and Atorvastatin group. The model group, Atorvastatin group and Chinese herb group were fed with a high-fat diet, a high-fat diet plus atorvastatin (5.1 mg/kg/d) and a high-fat diet plus Tiaozhi Tongmai Granules (16.5 g/kg/d) for 16 weeks, respectively. Atherogenesis was identified by H&E staining. The colocalization of neutral lipid stain BODIPY and microtubule-associated protein 1 light chain 3 (LC3) and the colocalization of BODIPY and lysosomal-associated membrane protein 1 (LAMP1) within ApoE^−/− mice aortic plaques were tested using fluorescence confocal microscopy and the Pearson's coefficients were calculated. To further explore the anti-atherosclerosis mechanism of Tiaozhi Tongmai Granules, the network pharmacology was used to construct the herb-compound-target network.

The size of the aortic lipid plaque in the Chinese herb group and Atorvastatin group were smaller compared with the model group on the 16th week. Compared with the model group on the 16th week, the BODIPY and LC3 colocalization rate, the BODIPY and LAMP1 colocalization rate of the Chinese herb group and Atorvastatin group all presented significant increase in the aortic plaque (P <.001), showing that Tiaozhi Tongmai Granules could enhance autophagosome activity in the macrophage. In the herb-compound-target network, 17 active compounds and 27 targets were obtained through literature searching and using LHRI & DAVID Bioinformatics. It was found that 23 targets were correlated with the macrophage. Some of them participated in macrophage inflammatory response, and the other targets could promote/inhibit phagocytosis of the macrophage. It was hypothesized that the active compounds of Tiaozhi Tongmai Granules were acting on these targets and having y the biological effects.

In the progression stage of atherosclerosis, Tiaozhi Tongmai Granules can still make the macrophage have higher autophagosome activity, and play a role of anti-atherogenesis.

ATP-binding cassette transporter A1 (ABCA1) is an integral membrane protein that plays a key role in cellular lipid metabolism, preventing the accumulation of lipids that contribute to the initiation and progression of atherosclerosis. Tiaozhi Tongmai Granules are a Chinese herbal compound that is capable of treating atherosclerosis. This study was designed to explore the potential pharmacological mechanism by which Tiaozhi Tongmai Granules protect against atherosclerosis.

Forty-nine male New Zealand rabbits were randomly divided into seven groups: normal control group, normal diet; model groups 1 and 2: balloon injury and high-fat diet for 6 or 12 weeks; statin groups 1 and 2: balloon injury and high-fat diet plus atorvastatin for 6 or 12 weeks; and Chinese herb groups 1 and 2: balloon injury and high-fat diet plus Tiaozhi Tongmai Granules for 6 or12 weeks. The granules were administered at a dose of 1.14 g/kg/d, with atorvastatin (1.14 mg/kg/d) serving as positive control. Serum lipid profiles and liver function indices were measured. Atherogenesis was viewed after H&E staining and quantified by thickened intimal area percentage and maximal intimal thickness percentage. The ABCA1 protein expression in atherosclerotic plaque macrophages of the common carotid arteries (CCA), thoracic aortae (TA), and liver tissues were observed by immunohistochemical staining and evaluated using mean optical density (OD) value in macrophages and ABCA1-positive hepatocyte number.

Compared with model group 1 at week 6, Chinese herb group 1 and statin group 1 displayed significant reductions in total cholesterol (TC) (P = 0.027, 0.012) and low-density lipoprotein cholesterol (LDL-C) (P = 0.039, 0.028) levels, as well as marked increases in ABCA1-positive hepatocyte numbers (P all <0.001), and only statin group 1 displayed a markedly reduced maximal intimal thickness percentage in the CCA (P = 0.018). Compared with model group 2 at week 12, Chinese herb group 2 and statin group 2 all presented significant reductions in TC (P = 0.011, 0.003), LDL-C (P = 0.017, 0.010) and thickened intimal area percentage in the CCA (P = 0.001, 0.022), as well as prominent increases in the ABCA1 OD value of both the CCA (P = 0.001, 0.039) and TA (P = 0.001, 0.025) and positive hepatocyte number (P all <0.001). Chinese herb group 2 had a markedly reduced maximal intimal thickness percentage compared with model group 2 (P = 0.006) and a higher positive hepatocytes number than statin group 2 (P = 0.001).

Tiaozhi Tongmai Granules appear to have an anti-atherogenic effect that is most likely mediated by simultaneously upregulating the protein expression of ABCA1 in rabbit atherosclerotic plaque macrophages and in the liver.