Total extracts from Abelmoschus manihot (L.) alleviate radiation-induced cardiomyocyte ferroptosis via regulating redox imbalances mediated by the NOX4/xCT/GPX4 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Radiation-induced heart disease (RIHD) is one of the most serious complications in patients receiving chest radiotherapy, partially offsetting its benefits. At present, there is a lack of effective treatments for RIHD. Ferroptosis is a newly discovered type of cell death that results from iron-dependent lipid peroxide accumulation. It was recently shown that irradiation generates severe ferroptosis, providing new insights for the treatment of RIHD. Abelmoschus manihot (L.) possesses excellent pharmacological properties and is widely used in treating various ischemic heart and brain diseases; however, its efficacy and mechanism in treating RIHD are unknown. AIM: This study aimed to investigate the efficacy and mechanism of A. manihot (L.) total extracts (TEA) in treating RIHD. MATERIALS AND METHODS: C57BL/6 mice and H9C2 cells were exposed to irradiation to induce RIHD in vivo and in vitro, respectively. In vivo, we evaluated the protective effects of TEA (150 and 300 mg/kg) on RIHD. Body and heart weight changes of mice were calculated in each group, and malondialdehyde (MDA) level, glutathione /oxidized glutathione (GSH/GSSH) and nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) ratios, western blot, heart histology, and immunohistochemistry were used to evaluate TEA effectiveness. We identified the potential mechanism of radiation-induced cardiomyocyte injury in H9C2 cells treated with small interfering RNA. We determined the effective dose of TEA (0.6 mg/mL) using a Cell Counting Kit-8 assay. Intracellular Fe2+ and lipid peroxidation levels were detected by Phen Green™ SK diacetate probe, BODIPY 581/591 C11 staining, and MDA, GSH, and NADPH kits, and the level of target protein was evaluated by immunofluorescence and western blot. RESULTS: Radiation inhibited system Xc-cystine (xCT)/glutathione peroxidase 4 (GPX4) expression and activity in cardiomyocytes in a time and dose-dependent manner. After silencing xCT/GPX4, MDA significantly increased and GSH/GSSH and NADPH/NADP+ ratios were reduced. xCT/GPX4 inhibition drove ferroptosis in radiation-induced H9C2 injury. Oxidative stress in H9C2 was significantly enhanced by irradiation, which also significantly increased NADPH oxidase (NOX4) expression and inhibited nuclear factor E2-related factor 2 (Nrf2) expression in vivo and in vitro. Inhibition of xCT/GPX4 drove ferroptosis in radiation-induced H9C2 injury, which was aggravated by inactivation of Nrf2 and alleviated by inhibition of NOX4. Compared with the ionizing radiation-only group, TEA improved body weight loss, MDA levels, and histological changes induced by irradiation in mice hearts, and increased the ratio of GSH/GSSH and NADPH/NADP+in vivo; it also reduced lipid peroxidation and intracellular Fe2+ accumulation, restored MDA levels, and elevated the ratios of GSH/GSSH and NADPH/NADP+ in irradiation-injured H9C2 cells. TEA up-regulated Nrf2, xCT, and GPX4 expression and inhibited NOX4 expression in vivo and in vitro. CONCLUSIONS: Ferroptosis induced by redox imbalance mediated through the NOX4/xCT/GPX4 axis is a potential mechanism behind radiation-induced cardiomyocyte injury, and can be prevented by TEA.

Ziyuglycoside II, a triterpene glycoside compound in Sanguisorbae officinalis l. extract, suppresses metastasis in osteosarcoma via CBX4-mediated Wnt/ß-catenin signal pathway.

BACKGROUND: Osteosarcoma (OS), the most prevalent primary bone malignancy, exhibits rapid growth and a high tendency for lung metastasis, posing significant treatment challenges. Ziyuglycoside II (ZGS II), a main active compound derived from Sanguisorba officinalis l., has shown potential in cancer treatment. However, the effects of ZGS II and its potential mechanism in OS remain elusive. PURPOSE: This study aims to explore the anti-metastatic potential of ZGS II in OS, offering a novel therapeutic strategy for improved patient outcomes. METHODS: Cell viability and proliferation was detected by cell counting kit-8 (CCK-8) and clone formation assay, respectively. Transwell and wound-healing assay were applied to evaluate the potential metastatic abilities of OS cells in vitro. More critically, the chromobox protein homolog 4 (CBX4) and Wnt/ß-catenin signaling pathway was investigated utilizing Western blotting, immunohistochemistry, shRNA knockdown and immunofluorescence. An orthotopic metastasis mouse model was utilized to evaluate the efficacy of ZGS II in suppressing OS metastasis in vivo, with molecular docking studies conducted to elucidate the interaction between ZGS II and the CBX4 protein. RESULTS: Our study demonstrated the potent inhibitory effects of ZGS II on OS cell proliferation and induced apoptosis in vitro, as evidenced by decreased cell viability, enhanced caspase-3 activation, and mitochondrial dysfunction. Furthermore, using an orthotopic metastasis mouse model, we illustrated that ZGS II effectively suppressed tumor growth and lung metastasis in vivo. Notably, our investigation revealed that the antitumor action of ZGS II is dependent on the reduction of CBX4 levels, leading to the attenuation of the Wnt/ß-catenin signaling pathway activation. Molecular docking analyses supported this pathway's suppression, showing that ZGS II has the capability to directly bind and disrupt CBX4 function. To further confirm this mechanism, we utilized shRNA to silence CBX4 in OS cells, which significantly enhanced the inhibitory impact of ZGS II on cell migration. CONCLUSION: Our study findings reveal that ZGS II efficiently suppresses both metastasis and tumor growth in OS by a novel mechanism that entails the inhibition of the CBX4-regulated Wnt/ß-catenin pathway. These outcomes highlight the promising potential of ZGS II as a therapeutic agent for managing metastatic OS, thus justifying the need for additional clinical investigations.

Aucubin supplementation alleviate diabetes induced-disruption of blood-testis barrier and testicular damage via stabilizing cell junction integrity.

Disruption of blood-testis barrier (BTB) was a crucial pathological feature of diabetes induced-testicular injury at early phase. Aucubin (AU), a main active component in Eucommiae Cortex, has drawn attention for its benefits against male reproductive system disease. The current study was aimed at investigating the protective role of AU and exploring the underlying mechanism in diabetic model. A murine model of type 2 diabetes mellitus (T2DM) was induced by high-fat diet (HFD) combined with streptozocin (STZ). Testicular weight index and morphology, sperm quality, integrity of BTB and protein levels were analyzed. The underlying mechanism of the protective effect of AU was further explored in Sertoli cells (SCs) cultured with high glucose (HG). Our results showed AU inhibited testicular structural destruction, restored disruption of BTB and improved abnormal spermatogenic function in diabetic mice. Consistent with in vivo results, HG induced decreased transcellular resistance and increased permeability in SCs monolayers, while AU exposure reverses this trend. Meanwhile, reduced expression of Zonula occludin-1(ZO-1) and Connexin43(Cx43) in testicular tissue diabetic mice and HG-induced SCs was prominently reversed via AU treatment. Mechanistic studies suggested a high affinity interaction between AU and c-Src protein was identified based on molecular docking, and the activation of c-Src was significantly inhibited in AU treatment. Furthermore, AU significantly increased the expression of Cx43 and ZO-1 proteins HG-induced SCs, which can be further enhanced in gene-silenced c-Src cells to some extent. Our results suggested that AU ameliorated disruption of BTB and spermatogenesis dysfunction in diabetic mice via inactivating c-Src to stabilize cell junction integrity.

Aucubin ameliorates liver fibrosis and hepatic stellate cells activation in diabetic mice via inhibiting ER stress-mediated IRE1α/TXNIP/NLRP3 inflammasome through NOX4/ROS pathway.

Type 2 diabetes (T2DM) is closely associated with hepatic injury, which could promote/exacerbate hepatic inflammation, steatosis, and accelerate liver fibrosis progression. Aucubin (AU), as an active ingredient isolated from Eucommia ulmoides, exists a nutritional value in hepatoprotective effect and diabetic complications. However, whether it possesses more outstanding features on improving liver injury in diabetic conditions and the underlying mechanism is unclear. Our research investigated the treatment of AU on liver fibrosis and potential mechanisms on high-fat diet/streptozotocin-induced diabetic mice and high glucose (HG)&TGF-ß1-induced LX-2 cells. Results showed that AU restored hepatic function without affecting blood sugar levels in diabetic mice. Meanwhile, the enhanced levels of total cholesterol, triglycerides, and LDL-c were reversed in hepatic tissue after AU treatment. Histomorphology assays including H&E, Masson, PAS, Oil red and Sirius red staining showed that AU treatment reduced liver swelling, steatosis and fibrosis. Mechanistic studies showed that AU alleviated NLRP3 inflammasome activation and inflammatory responses via inhibiting ER stress-mediated IRE1α/TXNIP signaling pathway, which could postpone the development of T2DM induced hepatic fibrosis. In addition, the ROS generation and the up-regulated expression of NADHP oxidase 4 (NOX4) in the liver tissue were suppressed by AU treatment. Moreover, in vitro model, NOX4 activation was prominently enhanced and AU treatment blocked HG&TGF-ß1-induced NOX4 derived superoxide generation and thereby ameliorating hepatic stellate cell activation, which can be abrogated in the overexpression of NOX4 LX-2 cells. In addition, inhibition effects on ER stress-mediated IRE1α/TXNIP/NLRP3 inflammasome by AU treatment also were abolished in the overexpression of NOX4 LX-2 cells. Meanwhile, molecular docking results indicated that AU and NOX4 protein have a higher affinity. Taken together, AU might be a potential nutraceutical or therapeutic drug to ameliorate hepatic impairment and fibrosis in T2DM.