Humans are constantly facing multiple health challenges from both communicable and non-communicable diseases that significantly affect their health. Additionally, drug resistance or failure has made the situation even worse and poses serious challenges for researchers to develop new drugs. Hence, to address these problems, there is an urgent need to discover and develop timely and long-term-based therapeutic treatments from different sources. One such approach is harnessing the potential of plant secondary metabolites. Plants have been utilized for therapeutic purposes in addition to being used for nutritional benefits. In the last two decades, plant-based drug developments have been one of the effective means of treating human diseases owing to their multiple functions. More recently, anti-nutritional factors (ANFs) have emerged as one of the important targets for novel plant-based drug development due to their multifaceted and potential pharmacological properties. However, their anti-nutritional properties have been the major setback for their limited success in the pharmacological sector. In this review, we provide an overview of ANFs and their beneficial roles in preventing human diseases with multiple case studies. We also highlight the recent developments and applications of ANFs in the food industry, agriculture, and pharmaceutics with future perspectives. Furthermore, we evaluate meta-analyses on ANFs from the last 30 years in relation to their function in human health benefits. This review is an endeavor to reevaluate the merit of these natural compounds and explore their potential for both human and animal health.

Arterial medial calcification (AMC) is associated with crystallization of hydroxyapatite in the extracellular matrix and arterial smooth muscle cells (SMCs) leading to reduced arterial compliance. The study was performed to test whether lysosomal acid sphingomyelinase (murine gene code: Smpd1)‐derived ceramide contributes to the small extracellular vesicle (sEV) secretion from SMCs and consequently leads to AMC. In Smpd1^trg/SM^cre mice with SMC‐specific overexpression of Smpd1 gene, a high dose of Vit D (500 000 IU/kg/d) resulted in increased aortic and coronary AMC, associated with augmented expression of RUNX2 and osteopontin in the coronary and aortic media compared with their littermates (Smpd1^trg/SM^wt and WT/WT mice), indicating phenotypic switch. However, amitriptyline, an acid sphingomyelinase (ASM) inhibitor, reduced calcification and reversed phenotypic switch. Smpd1^trg/SM^cre mice showed increased CD63, AnX2 and ALP levels in the arterial wall, accompanied by reduced co‐localization of lysosome marker (Lamp‐1) with multivesicular body (MVB) marker (VPS16), a parameter for lysosome‐MVB interaction. All these changes related to lysosome fusion and sEV release were substantially attenuated by amitriptyline. Increased arterial stiffness and elastin disorganization were found in Smpd1^trg/SM^cre mice as compared to their littermates. In cultured coronary arterial SMCs (CASMCs) from Smpd1^trg/SM^cre mice, increased P_i concentrations led to markedly increased calcium deposition, phenotypic change and sEV secretion compared with WT CASMCs, accompanied by reduced lysosome‐MVB interaction. However, amitriptyline prevented these changes in P_i‐treated CASMCs. These data indicate that lysosomal ceramide plays a critical role in phenotype change and sEV release in SMCs, which may contribute to the arterial stiffness during the development of AMC.

Background: Pyroptosis, an inflammatory cell death, is involved in the progression of atherosclerosis. Pyroptosis in endothelial cells (ECs) and its underlying mechanisms in atherosclerosis are poorly understood. Here, we investigated the role of a caspase-4/5-NF-κB pathway in pyroptosis in palmitic acid (PA)-stimulated ECs and EVs as players in pyroptosis. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in an endothelial cell medium, treated with Ox-LDL, PA, caspase-4/5 inhibitor, NF-κB inhibitor, and sEV release inhibitor for 24 h, respectively. The cytotoxicity of PA was determined using an MTT assay, cell migration using a scratch-wound-healing assay, cell morphology using bright field microscopy, and lipid deposition using oil red O staining. The mRNA and protein expression of GSDM-D, CASP4, CASP5, NF-κB, NLRP3, IL-1β, and IL-18 were determined with RT-PCR and Western blot. Immunofluorescence was used to determine NLRP3 and ICAM-1 expressions. Extracellular vesicles (EVs) were isolated using an exosome isolation kit and were characterized by Western blot and scanning electron microscopy. Results: PA stimulation significantly changed the morphology of the HUVECs characterized by cell swelling, plasma membrane rupture, and increased LDH release, which are features of pyroptosis. PA significantly increased lipid accumulation and reduced cell migration. PA also triggered inflammation and endothelial dysfunction, as evidenced by NLRP3 activation, upregulation of ICAM-1 (endothelial activation marker), and pyroptotic markers (NLRP3, GSDM-D, IL-1β, IL-18). Inhibition of caspase-4/5 (Ac-FLTD-CMK) and NF-κB (trifluoroacetate salt (TFA)) resulted in a significant reduction in LDH release and expression of caspase-4/5, NF-κB, and gasdermin D (GSDM-D) in PA-treated HUVECs. Furthermore, GW4869, an exosome release inhibitor, markedly reduced LDH release in PA-stimulated HUVECs. EVs derived from PA-treated HUVECs exacerbated pyroptosis, as indicated by significantly increased LDH release and augmented expression of GSDM-D, NF-κB. Conclusions: The present study revealed that inflammatory, non-canonical caspase-4/5-NF-κB signaling may be one of the crucial mechanistic pathways associated with pyroptosis in ECs, and pyroptotic EVs facilitated pyroptosis in normal ECs during atherosclerosis.

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome is activated during atherogenesis, but how this occurs is unclear. Here, we explored the mechanisms activating and regulating NLRP3 inflammasomes via the acid sphingomyelinase (ASM)-ceramide signaling pathway. As a neointima formation model, partial left carotid ligations were performed on endothelial cell (EC)-specific ASM transgene mice (Smpd1^trg/EC^cre) and their control littermates (Smpd1^trg/WT and WT/WT) fed on the Western diet (WD). We found neointima formation remarkably increased in Smpd1^trg/EC^cre mice over their control littermates. Next, we observed enhanced colocalization of NLRP3 versus adaptor protein ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) or caspase-1 in the carotid ECs of WD-treated Smpd1^trg/EC^cre mice but not in their control littermates. In addition, we used membrane raft (MR) marker flotillin-1 and found more aggregation of ASM and ceramide in the intima of Smpd1^trg/EC^cre mice than their control littermates. Moreover, we demonstrated by in situ dihydroethidium staining, carotid intimal superoxide levels were much higher in WD-treated Smpd1^trg/EC^cre mice than in their control littermates. Using ECs from Smpd1^trg/EC^cre and WT/WT mice, we showed ASM overexpression markedly enhanced 7-ketocholesterol (7-Ket)-induced increases in NLRP3 inflammasome formation, accompanied by enhanced caspase-1 activity and elevated interleukin-1β levels. These 7-Ket-induced increases were significantly attenuated by ASM inhibitor amitriptyline. Furthermore, we determined that increased MR clustering with NADPH oxidase subunits to produce superoxide contributes to 7-Ket-induced NLRP3 inflammasome activation via a thioredoxin-interacting protein-mediated controlling mechanism. We conclude that ceramide from ASM plays a critical role in NLRP3 inflammasome activation during hypercholesterolemia via MR redox signaling platforms to produce superoxide, which leads to TXNIP dissociation.

Macrophages, key players in the myeloid series, are vital for innate immunity, inflammation, pathogen elimination, host defense, and tissue repair. Their polarization can produce M1 and M2 phenotypes. At locations of tissue damage, M1 macrophages are triggered by TLR agonists or IFN-γ, leading to the production of pro-inflammatory cytokines such as IL-1β, TNF-α, IFN-γ, and IL-12, as well as reactive oxygen species (ROS). In contrast, M2 macrophages that IL-4 and IL-13 have stimulated produce TGF-β, IL-6, and IL-10, which are anti-inflammatory cytokines. Additionally, these cells transform arginine into ornithine, which stimulates the production of collagen and cell division, which are essential for wound healing, neoangiogenesis, and tissue remodeling. The regulation of damage, inflammation, and tissue repair depends on macrophages’ capacity to dynamically alter their phenotype in response to external stimuli, including signals from pathogens, dying cells, and activated lymphocytes. M2 macrophages sustain tumor microenvironments, encourage endothelium development, and progress malignancy in neoplastic tissues. This chapter delves into the complex interplay of macrophage polarization, metabolism, and the advancement of illness. It emphasizes the molecular pathways, activation states, and potential therapeutic applications in conditions such as inflammation, cancer, cardiovascular diseases, and other afflictions.

Evogliptin, an anti-diabetic drug had positive impact on various cardiovascular events including inflammation and vascular calcification (VC), an active process driven by vascular smooth muscle cell (VSMC) phenotypic transition. Sphingolipids such as ceramide (CER) mediates inflammation and VC in the vascular tissue. We investigated whether evogliptin ameliorate phenotypic transition and pyroptosis in VSMCs as underlying cause of VC. In cultured VSMCs, isolated from the aorta of (C57/BL6) mouse, we observed more severe calcification with prior treatment of CER in P _i -treated VSMCs as detected by Alizarin Red Staining. Prior CER- stimulation led to a marked upregulation of osteogenic markers such as RUNX2, OPN, BMP2 and decreased contractile markers SM22-α and α- SMA in P _i -treated VSMCs as compared to control cells. In addition, increased expression of pyroptotic markers such as NLRP3, GSDM-D, IL-1 β , IL-18, and LDH release was observed with prior treatment of CER in P _i -treated VSMCs as compared to control cells. Furthermore, MCC950 (NLRP3 inhibitor), disulfiram (GSDM-D inhibitor) and evogliptin significantly downregulated osteogenic and pyroptotic markers including LDH release in both P _i ‐induced only and CER + P _i -treated VSMCs. Moreover, GW4869 (SMase inhibitor) and evogliptin significantly reduced SMase activity in sphingomyelin (SM)-induced VSMCs as compared to both P _i and SM only-treated groups. Also, the cleavage efficiency of GSDM-D was high in P _i and CER + P _i groups which was reduced with prior treatment of evogliptin. Hence, our data demonstrate that evogliptin alleviates VC by blocking phenotypic transition and associated pyroptosis via modulation of NLRP3/GSDM-D mediated pathway in CER-induced VSMCs.