Gualou-Xiebai herb pair and its active ingredients act against atherosclerosis by suppressing VSMC-derived foam cell formation via regulating P2RY12-mediated lipophagy.

BACKGROUND: Atherosclerosis (AS) is a chronic disease characterized by lipid accumulation in the aortic wall and the formation of foam cells overloaded with large lipids inclusions. Currently, Western medicine is primarily used to improve lipid metabolism disorders and reduce inflammatory reactions to delay AS progression, but these medicines come with serious side effects and drug resistance. Gualou-Xiebai (GLXB) is a renowned herb pair that has been proven effective against AS. However, the potential molecular mechanism through which GLXB exerts the anti-atherosclerotic effects of increasing lipophagy in vascular smooth muscle cells (VSMCs) remains unknown. PURPOSE: This study aims to explore the role of lipophagy and the therapeutic mechanism of GLXB in AS. METHODS: UPLC-Q-TOF-MS for the determination of the main components of GLXB-containing serum. An AS mouse model was established by feeding a high-fat diet (HFD) to ApoE-/- mice for 12 weeks. Ultrasonography monitoring was used to confirm the successful establishment of the AS model. Plaque areas and lipid deposition were evaluated using HE staining and aorta imagingafter GLXB treatment. Immunofluorescence staining and Western blotting were utilized to observe the P2RY12 and lipophagy levels in AS mice. VSMCs were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The degree of lipophagy and the related molecular mechanisms were assessed after treating the VSMCs with GLXB-containing serum or si-P2RY12 transfection. The active components of GLXB-containing serum that act on P2RY12 were screened and verified by molecular docking and dual-luciferase reporter assays. RESULTS: Seventeen components of GLXB were identified in rat serum by UPLC-Q-TOF-MS. GLXB significantly reduced lipid deposition in HFD-fed ApoE-/- mice and ox-LDL-induced VSMCs. GLXB strikingly increased lipophagy levels by downregulating P2RY12, p62, and plin2, upregulating LC3Ⅱ protein expression, and increasing the number of autophagosomes. Notably, the lipophagy inhibitor CQ and the P2RY12 receptor agonist ADPß abolished the GLXB-induced increase in lipophagy. Last, we confirmed that albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin from GLXB significantly inhibited P2RY12. CONCLUSION: GLXB activates lipophagy and inhibits lipid accumulation-associated VSMC-derived foam cell formation through suppressing P2RY12 activation, resulting in anti-atherosclerotic effects. The GLXB components albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin are the potential active effectors against P2RY12.

Gualou-Xiebai herb pair ameliorate atherosclerosis in HFD-induced ApoE-/- mice and inhibit the ox-LDL-induced injury of HUVECs by regulating the Nrf2-mediated ferroptosis.

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.

["Trichosanthis Fructus-Allii Macrostemonis Bulbus" combination inhibits NLRP3 inflammasome activation and reduces inflammatory response in RAW264.7 macrophage by inducing autophagy].

This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1ß, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1ß in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1ß, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1ß. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.