Atherosclerosis (AS) is associated with a high incidence of cardiovascular events, yet the mechanisms underlying this association remain unclear. Our previous study found that Atherosclerotic endothelial injury is closely associated with ferroptosis in ApoE-/- mice. Ferroptosis is a novel mode of cell death induced by decreased antioxidant capacity of the organism and accumulation of reactive oxygen species. Nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy is an important regulator of sudden ferroptosis in cells. However, the role of NCOA4 in AS and the exact mechanism by which it regulates the ferritinophagy response remain unclear. Herein, we report that NCOA4 expression is elevated in ApoE-/- mice and endothelial cells and is significantly correlated with AS. NCOA4 expression promoted ferroptosis, and was positively correlated with ferritinophagy response. Mechanistically, our findings indicate that LOX-1 is a key upstream target that influences the function of NCOA4. The specific pathway is related to the activation of cGAS-STING signaling to upregulate NCOA4 expression. Moreover, our findings demonstrate the "Gualou-Xiebai" herb pair can regulate LOX-1 to inhibit ferroptosis. Collectively, our results provide evidence of a connection between NCOA4-mediated promotion of AS and suggest that targeting upstream molecules regulating NCOA4 could be a potential therapy for AS.

Ethnopharmacological relevance: Fufangmuniziqi formula (FFMN), a traditional Uyghur medicine used in China, is derived from an ancient Uyghur medical book and consists of 13 herbs. The herbs of FFMN, such as Peganum harmala L., Glycyrrhiza uralensis Fisch., and Nigella glandulifera, have been demonstrated to have acetylcholinesterase (AChE) inhibitory, anti-neuroinflammatory, or antioxidant effects. Therefore, FFMN may have a good anti-Alzheimer's disease (AD) effect, but its specific action and mechanism need to be further proven. Aim of the study: This study aims to investigate the anti-AD effects of FFMN and the role played by alkaloids, flavonoids, and saponins in anti-AD. Materials and methods: The alkaloids, flavonoids, and saponins fractions of FFMN were prepared by macroporous resin chromatography. The absorbed ingredients in the drug-containing serum were identified by UPLC ⁃Q ⁃TOF ⁃MS. An AD mouse model was established by intraperitoneal injection of scopolamine (SCO). The role of different fractions of FFMN in the anti-AD process was examined by Morris water maze (MWM), in-vitro cell, and AChE inhibition assay. Results: A total of 20 ingredients were identified in the serum samples collected after oral administration of FFMN, and seven compounds were selected as candidate active compounds. MWM experiments showed that different fractions of FFMN could significantly improve SCO-induced learning memory impairment in mice. The alkaloids fraction (ALK) regulated cholinergic function by inhibiting AChE activity, activating choline acetyltransferase activity, and protein expression. Flavonoids and saponins were more potent than the ALK in downregulating pro-inflammatory factors or inflammatory mediators, such as TNF-& alpha;, MPO, and nitric oxide. Western blot results further confirmed that flavonoids and saponins attenuated neuroinflammation by inhibiting the phosphorylation of I & kappa;B and NF-& kappa;B p65. This result was also verified by in-vitro cellular assays. FFMN enhanced antioxidant defense by increasing the activity of superoxide dismutase and reducing the production of MDA. Combined with cellular experiments, flavonoids and saponins were proven more protective against oxidative damage. Conclusion: FFMN improved cognitive and memory impairment in the SCO-induced AD mouse model. ALK mainly enhanced the function of the cholinergic system. Flavonoid and saponin fractions mainly attenuated neuroinflammation and oxidative stress by modulating the NF-& kappa;B pathway. All these findings strongly suggested that the combination of alkaloid, flavonoid, and saponin fractions derived from FFMN is a promising anti-AD agent that deserves further development.

Diabetic neuropathic pain (DNP), one of the most common complications of diabetes, is characterized by bilateral symmetrical distal limb pain and substantial morbidity. To compare the differences  is aimed at serum metabolite levels between 81 DNP and 73 T2DM patients without neuropathy and found that the levels of branched-chain amino acids (BCAA) are significantly lower in DNP patients than in T2DM patients. In high-fat diet/low-dose streptozotocin (HFD/STZ)-induced T2DM and leptin receptor-deficient diabetic (db/db) mouse models, it is verified that BCAA deficiency aggravated, whereas BCAA supplementation alleviated DNP symptoms. Mechanistically, using a combination of RNA sequencing of mouse dorsal root ganglion (DRG) tissues and label-free quantitative proteomic analysis of cultured cells, it is found that BCAA deficiency activated the expression of L-type amino acid transporter 1 (LAT1) through ATF4, which is reversed by BCAA supplementation. Abnormally upregulated LAT1 reduced Kv1.2 localization to the cell membrane, and inhibited Kv1.2 channels, thereby increasing neuronal excitability and causing neuropathy. Furthermore, intraperitoneal injection of the LAT1 inhibitor, BCH, alleviated DNP symptoms in mice, confirming that BCAA-deficiency-induced LAT1 activation contributes to the onset of DNP. These findings provide fresh insights into the metabolic differences between DNP and T2DM, and the development of approaches for the management of DNP.

ETHNOPHARMACOLOGICAL RELEVANCE:Atherosclerosis (AS) is a chronic vascular ailment characterized by inflammatory and lipid deposition in the arterial wall caused by endothelial injury. Ferroptosis is a novel type of cell death, and endothelial ferroptosis is a significant contributor to the progression of AS. Gualou-Xiebai (GLXB) is a renowned Chinese herb pair that serves a crucial function in treating AS. However, whether the underlying mechanism of GLXB plays a role in anti-atherosclerotic effects by inhibiting ferroptosis in endothelial cells has not been determined.AIM OF THE STUDY:To explore the influence of GLXB on endothelial ferroptosis and determine its underlying mechanism of action in AS.MATERIALS AND METHODS:In ApoE-/- mice, ultrasound was performed in mice fed a high-fat diet (HFD) for 12 weeks to assess the success of AS establishment. Then, ApoE-/- mice were treated with GLXB and Simvastatin (positive control) for 4 weeks. The effects of GLXB on AS pathology were assessed through aorta imaging and hematoxylin-eosin (HE) staining. To confirm the presence of ferroptosis, mitochondrial damage was observed using transmission electron microscope (TEM), along with analysis of free iron and lipid peroxidation levels. In vitro: ox-LDL-induced human vascular endothelial cells (HUVECs) injury and treated with GLXB, the ferroptosis inducer Erastin and an Nrf2 inhibitor ML385. Cell viability was evaluated using the CCK-8 assay in all groups. Flow cytometry was employed to detect lipid peroxidation and intracellular ferrous iron levels. Immunofluorescence staining microscopy verified Nrf2 nuclear translocation. Protein expression were measured by Western blot analysis.RESULTS:GLXB improved atherosclerotic aortic lesions and vascular plaques. GLXB inhibited endothelial injury in the aorta by decreasing the levels of inflammatory factors and adhesion factors, and by decreasing the shedding of endothelial cells. GLXB suppressed ferroptosis in ApoE-/- mice by attenuating mitochondrial damage in ECs, increasing the levels of glutathione (GSH) and superoxide dismutase (SOD) in aortic tissues and down-regulating the levels of levels of lipid peroxide (LPO) and malondialdehyde (MDA). Interestingly, Erastin was used to demonstrate in vitro that GLXB inhibition of ferroptosis attenuated ox-LDL-induced injuring effects on HUVECs that were reversed by Erastin. Mechanistically, GLXB activates the Nrf2 signaling pathway to inhibit ferroptosis by increasing downstream anti-ferroptosis target proteins and promoting the interaction between Nrf2 and SLC7A11. More convincingly, ML385 (Nrf2 inhibitor) reversed the anti-ferroptosis effect of GLXB.CONCLUSION:GLXB inhibits ferroptosis-mediated endothelial cell injury via activating the Nrf2 signaling pathway and further alleviates AS pathological damage.

ETHNOPHARMACOLOGICAL RELEVANCE:Diabetic retinopathy (DR) is a neurovascular disease that causes blindness in adults and is the most serious and common complication of diabetes mellitus. Retinal inflammation is an early stage of DR, and it is believed to play a crucial role in the development of DR. Panax notoginseng saponins (PNS) are the major active constituent in the main root of P. notoginseng, and they exhibit various biological activities, including anti-inflammatory, antioxidant, neuroprotective, and immunomodulatory functions. However, the protective effects and underlying mechanisms of PNS against DR remain unclear. AIM OF THE STUDY:This study aimed to investigate the alleviation effects of PNS on DR and the mechanisms involved. Furthermore, it intended to explore the major components that exert efficacy in vivo. MATERIALS AND METHODS:Streptozotocin (STZ) was administered intraperitoneally to Sprague Dawley rats, and PNS was administered orally for 1 month after 2 months of STZ injection. The morphological structure of the retina and retinal acellular capillaries were assessed via hematoxylin and eosin (H&E) staining assay. The disruption of the blood-retinal barrier (BRB) was detected through Evans blue dye leakage assay, and retinal leukocyte adhesion was achieved via fluorescein isothiocyanate-coupled concanavalin A lectin labeling assay. Immunofluorescence staining and Western blot assays were conducted to detect the expression of tight junction proteins, adhesion molecules, and the ionized calcium-binding adapter molecule-1 (Iba-1) in the retina. Enzyme-linked immunosorbent assay was performed to detect the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in serum. In addition, the protein expression levels of nuclear factor (NF)-κB p65, phosphorylated IκB kinase (p-IKK), phosphorylated NF-κB inhibitor (p-IκB), and phosphorylated NF-κB p65 (p-p65) were measured using Western blot assay. The ocular tissue distribution of PNS in normal and diabetic rats was determined through ultra-performance liquid chromatography-tandem mass spectrometry. The in vitro anti-inflammatory effects of PNS, notoginsenoside (NGR1), ginsenoside Rg1, Re, Rb1, and Rd (GRg1, GRe, GRb1, and GRd) were evaluated on human Müller (MIO-M1) cells. RESULTS:PNS increased the reduction in retinal inner nuclear layer thickness, reduced the increase in retinal acellular capillaries, and attenuated elevated BRB disruption by upregulating the decrease in protein expression of claudin-1 and occludin. Furthermore, PNS significantly abrogated microglial cell activation and reversed the increase in leukocyte adhesion by downregulating the increase in the protein expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Moreover, PNS reduced the elevated levels of TNF-α, IL-6, and IL-1β in serum and inhibited the increased protein expression of p-IKK, p-IκB, and p-p65, and the nuclear translocation of p65. The tissue distribution results revealed that NGR1, GRg1, GRe, GRb1, and GRd were detected in the ocular tissue, while GRg1 and GRb1 were found at the highest levels compared with the other components. The cellular results showed that PNS, NGR1, GRg1, GRe, GRb1, and GRd suppressed the development of cellular inflammatory responses by inhibiting the activation of the NF-κB signaling pathway in MIO-M1 cells and that their anti-inflammatory effects were comparable. CONCLUSION:PNS suppressed retinal inflammation by inhibiting the activation of the NF-κB signaling pathway, alleviating DR. GRg1 and GRb1 may be the primary components that exert anti-inflammatory effects in vivo.

Atherosclerosis (AS) is a metabolic disorder commonly correlated with a high-fat diet (HFD). There are many endogenous metabolic changes associated with AS development. Gualou-Xiebai (GLXB) is a traditional Chinese medicine herb pair that has been used to treat AS. However, the mechanism of GLXB herb pair on the process of AS is still essentially unknown. In this study, aortic histopathological examination and biochemical analyses were used to validate the anti-atherosclerotic effects of GLXB herb pair on ApoE-/- mice during the disease course of AS. The mechanism of GLXB herb pair were performed by metabolomics approach based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). As a result, GLXB herb pair has protective effects on AS lesion development and improves blood lipid levels in ApoE-/- mice. A total of 34, 39, and 49 metabolites were found to be profoundly altered in the 9-week, 14-week, and 19-week model groups compared with the corresponding control groups. Among them, 16, 18, and 18 metabolites showed a trend toward normal levels after pharmacological intervention. Metabolic pathway analysis found that GLXB herb pair mainly affects glycerophospholipid metabolism, pentose and glucuronate interconversions in 9 weeks; linoleic acid metabolism, cysteine and methionine metabolism, and arachidonic acid metabolism in 14 weeks; arachidonic acid metabolism and pentose and glucuronate interconversions in 19 weeks. The results demonstrated that GLXB herb pair mainly played a therapeutic role by regulating glycerophospholipid metabolism and pentose and glucuronate interconversions in the whole process of AS.