A simplified herbal decoction attenuates myocardial infarction by regulating macrophage metabolic reprogramming and phenotypic differentiation via modulation of the HIF-1α/PDK1 axis.

BACKGROUND: Myocardial infarction (MI) poses a global public health challenge, often associated with elevated mortality rates and a grim prognosis. A crucial aspect of the inflammatory injury and healing process post-MI involves the dynamic differentiation of macrophages. A promising strategy to alleviate myocardial damage after MI is by modulating the inflammatory response and orchestrating the shift from pro-inflammatory (M1) to anti-inflammatory (M2) macrophages, aiming to achieve a reduced M1/M2 ratio. Nuanxinkang (NXK), a simplified herbal decoction, has demonstrated noteworthy cardioprotective, inflammation-regulating, and myocardial energy metabolism-regulating properties. METHODS: In this study, we constructed an MI model by ligating coronary arteries to investigate the efficacy of NXK in improving ventricular remodeling and cardiac function. Mice were administered NXK (1.65 g/kg/d) or an equivalent volume of regular saline via gavage for 28 consecutive days, commencing the day after surgery. Then, we conducted echocardiography to assess the cardiac function, Masson staining to illustrate the extent of myocardial fibrosis, TUNEL staining to reveal myocardial apoptosis, and flow cytometry to analyze the polarization of M1 and M2 macrophages in the hearts. Besides, a lipopolysaccharide (LPS)-induced pro-inflammatory macrophage (M1) polarization model was implemented in RAW264.7 cells to elucidate the underlying mechanism of NXK in regulating macrophage polarization. RAW264.7 cells were pre-treated with or without NXK-containing serum. Oxidative stress was detected by MitoSox staining, followed by Seahorse energy metabolism assay to evaluate alterations in mitochondrial metabolic patterns and ATP production. Both In vivo and in vitro, HIF-1α and PDK1 were detected by fluorescent quantitative PCR and Western blotting. RESULTS: In vivo, MI mice exhibited a decline in cardiac function, adverse ventricular remodeling, and an increase in glycolysis, coupled with M1-dominant polarization mediated by the HIF-1α/PDK1 axis. Notably, robust responses were evident with high-dose NXK treatment (1.65 g/kg/day), leading to a significant enhancement in cardiac function, inhibition of cardiac remodeling, and partial suppression of macrophage glycolysis and the inflammatory phenotype in MI mice. This effect was achieved through the modulation of the HIF-1α/PDK1 axis. In vitro, elevated levels of mitochondrial ROS production and glycolysis were observed in LPS-induced macrophages. Conversely, treatment with NXK notably reduced the oxidative stress damage induced by LPS and enhanced oxidative phosphorylation (OXPHOS). Furthermore, NXK demonstrated the ability to modify the energy metabolism and inflammatory characteristics of macrophages by modulating the HIF-1α/PDK1 axis. The influence of NXK on this axis was partially counteracted by the HIF-1α agonist DMOG. And NXK downregulated PDK1 expression, curtailed glycolysis, and reversed LPS-induced M1 polarization in macrophages, similar to the PDK1 inhibitor DCA. CONCLUSION: In conclusion, NXK protects against MI-induced cardiac remodeling by inducing metabolic reprogramming and phenotypic differentiation of macrophages, achieved through the modulation of the HIF-1α/PDK1 axis. This provides a novel and promising strategy for the treatment of MI.

Analysis and validation of hub genes in neutrophil extracellular traps for the long-term prognosis of myocardial infarction.

INTRODUCTION: The study focuses on the long-term prognosis of myocardial infarction (MI) influenced by neutrophil extracellular traps (NETs). It also aims to analyze and validate relative hub genes in this process, in order to further explore new therapeutic targets that can improve the prognosis of MI. MATERIALS AND METHODS: We established a MI model in mice by ligating the left anterior descending branch (LAD) and conducted an 8-week continuous observation to study the dynamic changes in the structure and function of the heart in these mice. Meanwhile, we administered Apocynin, an inhibitor of NADPH Oxidase, which has also been shown to inhibit the formation of NETs, to mice undergoing MI surgery in order to compare. This study employed hematoxylin-eosin (HE) staining, echocardiography, immunofluorescence, and real-time quantitative PCR (RT-qPCR) to examine the impact of NETs on the long-term prognosis of MI. Next, datasets related to MI and NETs were downloaded from the GEO database, respectively. The Limma package of R software was used to identify differentially expressed genes (DEGs). After analyzing the "Robust Rank Aggregation (RRA)" package, we conducted a screening for robust differentially expressed genes (DEGs) and performed pathway enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to determine the functional roles of these robust DEGs. The protein-protein interaction (PPI) network was visualized and hub genes were filtered using Cytoscape. RESULTS: Immunofluorescence and qPCR results showed an increase in the expression of Myeloperoxidase (MPO) at week 1 and week 8 in the hearts of mice after MI. HE staining reveals a series of pathological manifestations in the heart of the MI group during 8 weeks, including enlarged size, disordered arrangement of cardiomyocytes, infiltration of inflammatory cells, and excessive deposition of collagen fibers, among others. The utilization of Apocynin could significantly improve these poor performances. The echocardiography displayed the cardiac function of the heart in mice. The MI group has a reduced range of heart movement and decreased ejection ability. Moreover, the ventricular systolic movement was found to be abnormal, and its wall thickening rate decreased over time, indicating a progressive worsening of myocardial ischemia. The Apocynin group, on the contrary, showed fewer abnormal changes in the aforementioned aspects. A total of 81 DEGs and 4 hub genes (FOS, EGR1, PTGS2, and HIST1H4H) were obtained. The results of RT-qPCR demonstrated abnormal expression of these four genes in the MI group, which could be reversed by treatment of Apocynin. CONCLUSION: The NETs formation could be highly related to MI and the long-term prognosis of MI can be significantly influenced by the NETs formation. Four hub genes, namely FOS, EGR1, PTGS2, and HIST1H4H, have the potential to be key genes related to this process. They could also serve as biomarkers for predicting MI prognosis and as targets for gene therapy.

Atractylodinol prevents pulmonary fibrosis through inhibiting TGF-ß receptor 1 recycling by stabilizing vimentin.

Pirfenidone and nintedanib are only anti-pulmonary fibrosis (PF) drugs approved by the FDA. However, they are not target specific, and unable to modify the disease status. Therefore, it is still desirable to discover more effective agents against PF. Vimentin (VIM) plays key roles in tissue regeneration and wound healing, but its molecular mechanism remains unknown. In this work, we demonstrated that atractylodinol (ATD) significantly inhibits TGF-ß1-induced epithelial-mesenchymal transition and fibroblast-to-myofibroblast transition in vitro. ATD also reduces bleomycin-induced lung injury and fibrosis in mice models. Mechanistically, ATD inhibited TGF-ß receptor I recycling by binding to VIM (KD = 454 nM) and inducing the formation of filamentous aggregates. In conclusion, we proved that ATD (derived from Atractylodes lancea) modified PF by targeting VIM and inhibiting the TGF-ß/Smad signaling pathway. Therefore, VIM is a druggable target and ATD is a proper drug candidate against PF. We prove a novel VIM function that TGF-ß receptor I recycling. These findings paved the way to develop new targeted therapeutics against PF.

Nuanxinkang prevents the development of myocardial infarction-induced chronic heart failure by promoting PINK1/Parkin-mediated mitophagy.

BACKGROUND: Mitochondrial dysfunction is an important pathological feature of chronic heart failure (CHF). Regulation of mitophagy can effectively maintain mitochondrial homeostasis and energy metabolism, thereby inhibiting the development of CHF. Nuanxinkang (NXK), a Chinese herbal compound preparation, has significant cardioprotective effects on CHF; however, its underlying mechanism on mitophagy has not been completely clarified. This research intended to investigate the mechanism of NXK in treating myocardial infarction (MI)-induced CHF. METHODS: The left anterior descending coronary artery (LAD) ligation surgery was performed to establish an MI-induced CHF model in male C57BL/6 mice. From 1 day after surgery, mice were given NXK (0.41, 0.82 or 1.65 g/kg/d), Perindopril (PDPL, 0.607 mg/kg/d), or an equivalent amount of sterile water by gavage for 28 continuous days. Then, mice were examined for cardiac function, myocardial fibrosis, cardiomyocyte apoptosis, mitochondrial structure and mitophagy levels of cardiomyocytes, etc. In addition, a hypoxic injury model was created using HL-1 cardiomyocytes from wild-type (WT) mice. HL-1 cells were pretreated with or without NXK-containing serum. Mitochondrial function and mitophagy levels were examined in HL-1 cells. RESULTS: In MI-induced CHF mice, cardiac dysfunction, severe cardiac remodeling, elevated levels of oxidative stress, reduced ATP levels, and inhibition of PINK1/Parkin-mediated mitophagy were observed. High-dose NXK treatment (1.65 g/kg/d) significantly improved myocardial energy metabolism, inhibited cardiac remodeling, improved cardiac function, and restored cardiac PINK1/Parkin-mediated mitophagy levels to some extent in MI mice. In vitro, elevated levels of mitochondrial reactive oxygen species (ROS) with impaired mitochondrial membrane potential (ΔΨm) were observed in hypoxic HL-1 cells. While NXK treatment significantly protected cardiomyocytes from hypoxia-induced mitochondrial dysfunction, which is consistent with the in vivo results. Further studies showed that NXK could increase PINK1/Parkin-mediated mitophagy levels in cardiomyocytes, which could be blocked by the mitophagy inhibitor Mdivi-1. CONCLUSION: In conclusion, NXK could prevent cardiac mitochondrial dysfunction and improve cardiac function against MI-induced CHF by promoting Pink1/Parkin-mediated mitophagy, which represents a very prospective strategy for the treatment of CHF.

Nuanxinkang protects against ischemia/reperfusion-induced heart failure through regulating IKKß/IκBα/NF-κB-mediated macrophage polarization.

BACKGROUND: Heart failure (HF) is a leading cause of death worldwide. Nuanxinkang (NXK) is an effective Chinese herbal formula used in treating HF, but its underlying potential mechanisms have not been fully elucidated. PURPOSE: To explore the protective activities of NXK in ischemia/reperfusion (IR)-induced HF through modulating the ratio of proinflammatory (M1) and anti-inflammatory (M2) macrophage populations and leading to the alleviation of inflammation. MATERIALS AND METHODS: In vivo, mice were subjected to myocardial IR to generate HF mouse models. Mice in the NXK group were treated with NXK for 28 days. Cardiac function was detected by echocardiography. Major lesions on mouse hearts were determined by hematoxylin-eosin (HE) staining, Masson staining, and TUNEL staining. Inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and qPCR examination. Flow cytometric analyses and qPCR examination were utilized for monitoring the temporal dynamics of macrophage infiltration following IR. In vitro, two polarized models were established by stimulating RAW264.7 cells with 200 ng/ml lipopolysaccharide (LPS) or 20 ng/ml interleukin-4 (IL-4). The RAW264.7 cells with nuclear factor-κB (NF-κB) overexpression was generated by transient transfection of NF-κB plasmids, and NXK intervention was conducted on this cell model to further clarify the involvement of NF-κB signaling in the NXK-mediated HF process. RESULTS: In the present study, NXK was found to significantly contribute the cardiac function and ameliorate cardiac fibrosis and apoptosis after myocardial IR injury in vivo, which may be partially due to a decrease in inflammation. We therefore hypothesized that NXK reduced inflammatory damage by modulating subtypes of macrophages. And the results demonstrated that the percentage of proinflammatory macrophages infiltrated in the post-IR period was reduced with NXK treatment, and thereby blunting the wave of proinflammatory response and shifting the peak of the anti-inflammatory macrophage-mediated wound healing process towards an earlier time point. The further investigation showed that macrophage polarization was mediated by NXK through inhibiting the phosphorylation and the nuclear translocation of NF-κB. Besides, the phosphorylated IKKß and IκBα, upstream mediators of the NF-κB pathway, also decreased by NXK. Moreover, the overexpression of NF-κB partially reversed the NXK-induced favorable activities; and successfully compensated the suppressive effect on inflammation and the phosphorylation of NF-κB. CONCLUSION: In conclude, our results demonstrated that NXK induced the cardioprotective effects against IR injury through a regulatory axis of IKKß/IκBα/NF-κB-mediated macrophage polarization. The information gained from this study provide a possible natural strategy for anti-inflammatory treatment of HF.

Huang-Qi San ameliorates hyperlipidemia with obesity rats via activating brown adipocytes and converting white adipocytes into brown-like adipocytes.

BACKGROUND: Brown adipose tissue (BAT) activation is a promising therapeutic target to treat hyperlipidemia with obesity. Huang-Qi San (HQS), an traditional Chinese medicine, can ameliorate hyperlipidemia with obesity, but its mechanism of action (MOA) is not understood. PURPOSE: To articulate the MOA for HQS with animal models. METHODS: The main chemical constituents of HQS were identified by high-performance liquid chromatography (HPLC) based assay. Hyperlipidemia with obesity rat models induced by high-fat diet were employed in the study. The levels of the fasting plasma glucose (FPG), triglyceride (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) were measured to evaluate the ability of HQS to ameliorate hyperlipidemia with obesity. Pathological analyses of organs were conducted with Oil Red O staining, hematoxylin-eosin (H&E) staining and transmission electron microscopy. The expression of mRNAs related to thermogenic genes, fatty acid oxidation-related genes and mitochondria biogenic genes were examined by quantitative real-time PCR. The protein expressions of uncoupling protein 1 (UCP1) were investigated by immunohistochemistry and western blot. Simultaneously, the protein expression of PR domain containing 16 (PRDM16), ATP synthase F1 subunit alpha (ATP5A) was detected by western blot. RESULTS: HQS ameliorates metabolic disorder, lipid ectopic deposition, obesity and maintained glucose homeostasis in hyperlipidemia with obesity rats. HQS can significantly increase the number of mitochondria and reduced the size of the intracellular lipid droplets in BAT, and increase the expression of BAT activation-related genes (UCP1, PGC1α, PGC1ß, Prdm16, CD137, TBX1, CPT1a, PPARα, Tfam, NRF1 and NRF2) in vivo. Furthermore, UCP1, PRDM16 and ATP5A proteins of BAT were increased. CONCLUSION: HQS can activate BAT and browning of S-WAT (subcutaneous white adipose tissue) through activating the PRDM16/PGC1α/UCP1 pathway, augmenting mitochondrial biogenesis and fatty acid oxidation to increase thermogenesis and energy expenditure, resulting in a significant amelioration of hyperlipidemia with obesity. Therefore, HQS is an effective therapeutic medicine for the treatment of hyperlipidemia with obesity.

The design and synthesis of a novel compound of berberine and baicalein that inhibits the efficacy of lipid accumulation in 3T3-L1 adipocytes.

The combination of berberine and baicalein may have a better therapeutic effect against disease. To explore the combined effect of baicalein and berberine in the treatment of obesity, we designed and synthesized a hybrid compound, and its biological activities were evaluated in 3T3-L1 adipocytes. The structures of the berberine-baicalein (BBS) compounds were confirmed by 1H NMR, 13C NMR, ultraviolet spectroscopy and high resolution mass spectrometry (HRMS). The present study showed that the IC50 values of the inhibitory effects of baicalein, berberine and BBS against 3T3-L1 cells were 29.81±0.90, 21.84±1.67 and 9.42±0.60µM, respectively. The expression of mRNAs related to lipolysis and lipogenesis were examined by quantitative real-time PCR. The results showed that BBS could up-regulate the expression of the Atgl gene and down-regulate the mRNA expression of Srebp-1c, Fasn, Scd1, and Acc in 3T3-L1 adipocytes. These results indicate that BBS may have a stronger effect than baicalein and berberine on the viability of 3T3-L1 preadipocytes. In addition, BBS may have therapeutic effects and pharmacological activities against obesity. This "medicine couple" may be beneficial for studies of traditional Chinese medicine.