Myocardial infarction and following reperfusion therapy-induced myocardial ischemia/reperfusion (I/R) injury have been recognized as an important subject of cardiovascular disease with high mortality. As the antiarrhythmic agent, Wenxin Granule (WXG) is widely used to arrhythmia and heart failure. In our pilot study, we found the antioxidative potential of WXG in the treatment of myocardial I/R. This study is aimed at investigating whether WXG could treat cardiomyocyte hypoxia/reoxygenation (H/R) injury by inhibiting oxidative stress in mitochondria. The H9c2 cardiomyocyte cell line was subject to H/R stimuli to mimic I/R injury in vitro. WXG was added to the culture medium 24 h before H/R exposing as pretreatment. Protein kinase C-δ (PKC-δ) inhibitor rottlerin or PKC-δ lentivirus vectors were conducted on H9c2 cells to downregulate or overexpress PKC-δ protein. Then, the cell viability, oxidative stress levels, intracellular and mitochondrial ROS levels, mitochondrial function, and apoptosis index were analyzed. In addition, PKC-δ protein expression in each group was verified by western blot analysis. Compared with the control group, the PKC-δ protein level was significantly increased in the H/R group, which was remarkably improved by WXG or rottlerin. PKC-δ lentivirus vector-mediated PKC-δ overexpression was not reduced by WXG. WXG significantly improved H/R-induced cell injury, lower levels of SOD and GSH/GSSG ratio, higher levels of MDA, intracellular and mitochondrial ROS content, mitochondrial membrane potential and ATP loss, mitochondrial permeability transition pore opening, NOX2 activation, cytochrome C release, Bax/Bcl-2 ratio and cleaved caspase-3 increasing, and cell apoptosis. Similar findings were obtained from rottlerin treatment. However, the protective effects of WXG were abolished by PKC-δ overexpression, indicating that PKC-δ was a potential target of WXG treatment. Our findings demonstrated a novel mechanism by which WXG attenuated oxidative stress and mitochondrial dysfunction of H9c2 cells induced by H/R stimulation via inhibitory regulation of PKC-δ/NOX2/ROS signaling.

BACKGROUND Saffron (Crocus sativus L.) has been traditionally used as food, spice, and medicine. Crocetin (CRT), as main bioactive component of saffron, has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion (I/R) injury. However, the mechanisms are poorly explored. This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation (H/R) and elucidated the possible underlying mechanism. METHODS H/R attack was performed on H9c2 cells. Cell counting kit-8 was used to detect the cell viability. Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and cellular adenosine triphosphate (ATP) content. Various fluorescent probes were used to detect cell apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) content, mitochondrial morphology, mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (mPTP) opening. Proteins were evaluated via Western Blot. RESULTS H/R exposure severely reduced cell viability and increased LDH leakage. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) suppression and dynamin-related protein 1 (Drp1) activation were coincided with excessive mitochondrial fission, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse in H9c2 cells treated with H/R. Mitochondria fragmentation under H/R injury induced ROS over-production, oxidative stress, and cell apoptosis. Notably, CRT treatment significantly prevented mitochondrial fission, mPTP opening, MMP loss, and cell apoptosis. Moreover, CRT sufficiently activated PGC-1α and inactivated Drp1. Interestingly, mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction, oxidative stress and cell apoptosis. However, silencing PGC-1α with small interfering RNA (siRNA) abolished the beneficial effects of CRT on H9c2 cells under H/R injury, accompanied with increased Drp1 and p-Drp1^ser616 levels. Furthermore, over-expression of PGC-1α with adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells. CONCLUSIONS Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission. We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury. Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack, and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.

Diabetic cardiomyopathy (DCM), a major cause of diabetic mortality, lacks effective therapies. This study investigated the cardioprotective role of Heterophyllin B (HET-B), a natural compound and its underlying mechanisms in DCM. Using streptozotocin-induced DCM mice and high glucose (HG)-treated H9C2/neonatal cardiomyocytes, we assessed cardiac function, mitochondrial homeostasis, and apoptosis. HET-B significantly improved cardiac function, indicated by increased ejection fraction (EF) and fractional shortening (FS). It also reduced cardiomyocyte apoptosis (in vivo/vitro), and ameliorated HG-induced mitochondrial damage, characterized by dysfunction, fragmentation, and excessive reactive oxygen species (ROS) production. HET-B enhanced mitochondrial fusion protein OPA1 expression and reduced Bax/Bcl2, cytochrome C (Cyt C) release and caspase-3 cleavage. Molecular docking and cellular thermal shift assays identified mitochondrial antiviral-signaling protein (MAVS) as a potential target of HET-B. HET-B reversed MAVS downregulation induced by HG/DCM in vitro and in vivo. Importantly, MAVS knockdown via siRNA abolished HET-B's protection against HG-induced apoptosis and mitochondrial damage. Furthermore, HET-B restored HG-impaired autophagic flux, reducing autolysosome accumulation and normalizing LC3-II. Collectively, the natural compound HET-B alleviates diabetic myocardial injury by targeting MAVS to enhance autophagy, maintain mitochondrial homeostasis, and inhibit cardiomyocyte apoptosis, positioning it as a promising therapeutic candidate for DCM.

Peripheral arterial disease (PAD) is a common complication associated with diabetes, which can lead to foot ischemia. The condition is often accompanied by infection and necrosis, ultimately leading to diabetic foot ulcers and the risk of amputation. Brown adipose tissue (BAT) and its secreted cytokines play an essential role in the regulation of glucose homeostasis, the modulation of inflammatory responses, and vascular endothelial cell proliferation. The transplantation of BAT into ischemic regions may offer therapeutic benefits in alleviating the symptoms associated with PAD. A diabetic mouse model was established via intraperitoneal administration of streptozocin. Subsequently, a diabetic lower limb ulcer model was constructed by transection of the femoral artery and ligation of the femoral vein. BAT harvested from the subscapular region of the mouse was employed as an adipose graft. The research utilized Laser Doppler monitoring, Western blot analysis, hematoxylin-eosin (HE) staining, immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA) to evaluate blood flow recovery in ischemic regions, histopathological changes, angiogenesis and tissue remodeling, inflammatory responses, and M1/M2 macrophage polarization. BAT transplantation significantly enhanced blood flow recovery in ischemic regions of diabetic lower limb ulcer mice while concurrently reducing necrotic tissue. Pathological analyses demonstrate that BAT transplantation mitigates ischemic tissue damage, stimulates angiogenesis, and supports tissue remodeling. Furthermore, the Western blotting, immunofluorescence, and ELISA results revealed that BAT transplantation significantly reduces inflammatory levels in ischemic tissues, increases the expression of angiogenic factors, and promotes the polarization of macrophages from the M1 to the M2 phenotype. The research has demonstrated that BAT transplantation can mitigate ischemic injury in diabetic lower limb ulcer mice, attenuate inflammatory responses, and facilitate the restoration of blood flow. These effects may be linked to alterations in macrophage polarization.

Crocin (CRO), the main active component of Crocus sativus (saffron), exhibits significant therapeutic potential for the treatment of myocardial infarction (MI). However, its clinical application is hindered by poor oral administration due to the intestinal barrier. To overcome this limitation, a CRO-incorporated biodegradable microneedle (CRO-DMN) patch was developed to transdermally deliver CRO into the bloodstream, enhancing its therapeutic effect against MI. The microneedle patch, fabricated from sodium hyaluronate (HA), demonstrated excellent mechanical strength, effectively penetrating the stratum corneum and facilitating CRO infiltration to a depth exceeding 300 μm successfully. Studies revealed that CRO was fully released within 30 min postapplication, and the skin self-healed within 90 min after patch removal. Importantly, administration of CRO-DMNs to MI mice resulted in significant improvements in cardiac function and reductions in infarct size compared with CRO treatment alone. Additionally, CRO-DMNs also provided superior protective effects on the myocardium and mitochondria and significantly increased angiogenesis in the infarction border area. In conclusion, CRO-DMNs represent a promising transdermal drug delivery system that effectively improves the therapeutic efficacy of CRO in the treatment of MI.