Xinyang tablet alleviated cardiac dysfunction in a cardiac pressure overload model by regulating the receptor-interacting serum/three-protein kinase 3/FUN14 domain containing 1-mediated mitochondrial unfolded protein response and mitophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Xinyang tablet (XYT) has been used for heart failure (HF) for over twenty years in clinical practice, but the underlying molecular mechanism remains poorly understood. AIMS OF THE STUDY: In the present study, we aimed to explore the protective effects of XYT in HF in vivo and in vitro. MATERIALS AND METHODS: Transverse aortic constriction was performed in vivo to establish a mouse model of cardiac pressure overload. Echocardiography, tissue staining, and real-time quantitative PCR (qPCR) were examined to evaluate the protective effects of XYT on cardiac function and structure. Adenosine 5'-triphosphate production, reactive oxygen species staining, and measurement of malondialdehyde and superoxide dismutase was used to detect mitochondrial damage. Mitochondrial ultrastructure was observed by transmission electron microscope. Immunofluorescence staining, qPCR, and Western blotting were performed to evaluate the effect of XYT on the mitochondrial unfolded protein response and mitophagy, and to identify its potential pharmacological mechanism. In vitro, HL-1 cells and neonatal mouse cardiomyocytes were stimulated with Angiotensin II to establish the cell model. Western blotting, qPCR, immunofluorescence staining, and flow cytometry were utilized to determine the effects of XYT on cardiomyocytes. HL-1 cells overexpressing receptor-interacting serum/three-protein kinase 3 (RIPK3) were generated by transfection of RIPK3-overexpressing lentiviral vectors. Cells were then co-treated with XYT to determine the molecular mechanisms. RESULTS: In the present study, XYT was found to exerta protective effect on cardiac function and structure in the pressure overload mice. And it was also found XYT reduced mitochondrial damage by enhancing mitochondrial unfolded protein response and restoring mitophagy. Further studies showed that XYT achieved its cardioprotective role through regulating the RIPK3/FUN14 domain containing 1 (FUNDC1) signaling. Moreover, the overexpression of RIPK3 successfully reversed the XYT-induced protective effects and significantly attenuated the positive effects on the mitochondrial unfolded protein response and mitophagy. CONCLUSIONS: Our findings indicated that XYT prevented pressure overload-induced HF through regulating the RIPK3/FUNDC1-mediated mitochondrial unfolded protein response and mitophagy. The information gained from this study provides a potential strategy for attenuating mitochondrial damage in the context of pressure overload-induced heart failure using XYT.

Emodin alleviates CRS4-induced mitochondrial damage via activation of the PGC1α signaling.

Cardiorenal syndrome type 4 (CRS4), a progressive deterioration of cardiac function secondary to chronic kidney disease (CKD), is a leading cause of death in patients with CKD. In this study, we aimed to investigate the cardioprotective effect of emodin on CRS4. C57BL/6 mice with 5/6 nephrectomy and HL-1 cells stimulated with 5% CKD mouse serum were used for in vivo and in vitro experiments. To assess the cardioprotective potential of emodin, we employed a comprehensive array of methodologies, including echocardiography, tissue staining, immunofluorescence staining, biochemical detection, flow cytometry, real-time quantitative PCR, and western blot analysis. Our results showed that emodin exerted protective effects on the function and structure of the residual kidney. Emodin also reduced pathologic changes in the cardiac morphology and function of these mice. These effects may have been related to emodin-mediated suppression of reactive oxygen species production, reduction of mitochondrial oxidative damage, and increase of oxidative metabolism via restoration of PGC1α expression and that of its target genes. In contrast, inhibition of PGC1α expression significantly reversed emodin-mediated cardioprotection in vivo. In conclusion, emodin protects the heart from 5/6 nephrectomy-induced mitochondrial damage via activation of the PGC1α signaling. The findings obtained in our study can be used to develop effective therapeutic strategies for patients with CRS4.

Baicalin induces ferroptosis in osteosarcomas through a novel Nrf2/xCT/GPX4 regulatory axis.

BACKGROUND: Osteosarcomas (OS) is a kind of malignant bone tumor which occurs primarily in children and adolescents, and the clinical therapeutics remain disappointing. As a new programmed cell death, ferroptosis is characterized by iron dependent and intracellular oxidative accumulation, which provides a potential alternative intervene for the OS treatment. Baicalin, a major bioactive flavone derived from traditional Chinese medicine Scutellaria baicalensis, has been proved to have anti-tumor properties in OS. Whether ferroptosis participated in the baicalin mediated anti-OS activity is an interesting project. PURPOSE: To explore the pro-ferroptosis effect and mechanisms of baicalin in OS. METHODS/STUDY DESIGN: Pro-ferroptosis effect of baicalin on cell death, cell proliferation, iron accumulation, lipid peroxidation production was determined in MG63 and 143B cells. The levels of glutathione (GSH), oxidized (GSSG) glutathione and malondialdehyde (MDA) were determined by enzyme linked immunosorbent assay (ELISA). The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), Glutathione peroxidase 4 (GPX4) and xCT were detected by western blot in baicalin-mediated ferroptosis regulation. In vivo, a xenograft mice model was adopted to explore the anticancer effect of baicalin. RESULTS: In the present study, it was found that baicalin significantly suppress tumor cell growth in vitro and in vivo. By promoting the Fe accumulation, ROS formation, MDA production and suppressing the ratio of GSH/GSSG, baicalin was found to trigger ferroptosis in OS and ferroptosis inhibitor ferrostatin-1 (Fer-1) successfully reversed these suppressive effects, indicating that ferroptosis participated in the baicalin mediated anti-OS activity. Mechanistically, baicalin physically interacted with Nrf2, a critical regulator of ferroptosis, and influenced its stability via inducing ubiquitin degradation, which suppressed the Nrf2 downstream targets GPX4 and xCT expression, and led to stimulating ferroptosis. CONCLUSIONS: Our findings for the first time indicated that baicalin exerted anti-OS activity through a novel Nrf2/xCT/GPX4-dependent ferroptosis regulatory axis, which hopefully provides a promising candidate for OS treatment.

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.