The regulation of inflammatory markers is of great interest in hyperlipidemia management, as chronic inflammation is closely linked to the development and progression of the disease. In this study, the effect of novel anti-hyperlipidemic compounds on specific inflammatory markers was explored in Triton WR-1339 induced acute hyperlipidemic rat model to determine these compounds’ effects on the gene expression of these markers. Triton WR-1339 has been commonly used for the induction of hyperlipidemia in animal models. It is known to inhibit extrahepatic tissues from absorbing circulating lipoproteins, elevating blood lipoprotein levels thus increasing the plasma lipid profile [15, 16]. In this model, Triton WR-1339 elevated TG levels. Interestingly, all carboxamide compounds significantly reduced TG levels. Moreover, all compounds led to reduced levels of LDL while NF3BP also significantly lowered TC levels. These compounds have shown significant changes in the gene expression of key enzymes related to triglycerides metabolism and transport to the liver, they mainly caused the over expression of adipose and endothelial tissues lipoprotein lipase [17], along with the activation of PPAR-α hepatic metabolic pathway [17, 18].

Changes in gene expression levels of several genes encoding inflammatory markers were observed to be induced by Triton WR-1339 and novel compounds administration in different tissues. IL-1β, a pro-inflammatory cytokine, can be associated to the emergence of CVDs by promoting formation of atherosclerotic plaques through triggering inflammation in blood vessel walls [19]. IL-1β enhances the emergence of AS by boosting the accumulation of inflammatory cells within blood vessels and promoting their invasion into the local intima of blood vessels. Additionally, an inflammatory response is triggered IL-1β by in endothelial cells as it raised the expression of chemokines and adhesion factors [20]. Hence, when its levels decrease, the risk of AS decreases. Moreover, uncontrolled hyperlipidemia is suggested to stimulate NOD-like receptor protein 3 (NLRP3) inflammasome activation in macrophages, thus leading to increased expression of IL-1β [21]. Significant down regulation of IL-1β by the compounds in endothelial tissues and its central role in the protein-protein interaction network showed the importance of these compounds in modulating the role of IL-1β in the inflammation associated with hyperlipidemia. IL-1β can enhance the expression of IL-6, thus increasing the inflammatory response. Similarly, IL-1β works in conjugation with TNF-α, to increase inflammation and tissue damage. IL-1β can also induce the expression of both ICAM-1 and VCAM-1 on endothelial tissues, enhancing immune cells migration to inflamed tissues. In addition, IL-1β stimulates the liver to produce CRP, Overall, IL-1β acts as a central mediator in coordinating the inflammatory cascade. On the other hand, IL-38 is known to competitively bind to the IL-1R receptor preventing the signal transduction of IL-1β and blocking the activation of NF-κB, ERK and several signaling pathways [9].

CRP is an acute-phase protein, a type of protein synthesized mostly by the liver and also by adipocytes and vascular smooth muscle cells in response to inflammation [22]. Increasing levels of lipids in the bloodstream leads to an increase in the synthesis of proinflammatory cytokines including IL-6 and TNF-α, which can consequently promote the liver to express CRP [8]. Compound NF4AP significantly downregulated CRP gene expression in liver tissue. CRP can be considered a predictive marker of CVDs since elevated CRP levels in the blood is a sign of systemic inflammation [19, 23]. Thus, decreasing in CRP expression by NF4AP may minimize the risk of CVDs.

TNF-α, a pro-inflammatory cytokine, is a protein which has a major role in the progression of inflammation and immune response [24]. It has a predictive property for CVDs and has an important role in hyperlipidemia [19]. Studies have shown that elevated amounts of blood lipids are correlated with increased concentrations of TNF-α in which certain lipids, such as oxLDL, can stimulate inflammatory cytokines expression, including TNF-α [25]. In liver tissue, NF3BP led to a significant downregulation in TNF-α gene expression. The emergence of AS may correlate with TNF- α as it can promote expression of different adhesion molecules (VCAM-1, ICAM-1) and pro-inflammatory cytokines from immune cells, enhancing the adhesion of immune cells to atrial wall, and ultimately resulting in the accumulation of immune cells and fatty plaques formation [26]. Therefore, reducing TNF- α expression could have therapeutic benefits for the management of AS.

Also, all carboxamide derivatives and fenofibrate caused a significant downregulation of the expression of CXCL-16 in endothelial tissue. CXCL-16 belongs to the CXC chemokine group expressed due to inflammation [27]. It has been proven that the expression of CXCL-16 is increased in AS, and the plasma concentrations of CXCL-16 have been considered a marker for CVDs [8].

IL-6 is an interleukin synthesized by various types of cells, such as monocytes, macrophages, endothelial cells, and adipose tissue cells. Its production increases in response to inflammation [28]. IL-6 possesses multiple properties that contribute to the emergence of CVDs including stimulation of endothelial cells, enhancing of smooth muscle proliferation and macrophage lipid buildup as well as pro-thrombotic impacts on platelets [29]. Moreover, elevation in IL-6 expression is suggested to elevate adhesion molecules as well as enhancing the expression of acute phase markers such as TNF-α and CRP, which in turn increases the risk of AS and hyperlipidemia [30]. Notably, treatment with NF4AP showed a significant downregulating effect on the expression level of IL-6 in endothelial cells, suggesting it potential role in inflammation and hyperlipidemia management.

VCAM-1 and ICAM-1 are endothelial adhesion molecules produced in response to different stimuli such as oxidative stress and inflammation. They are major contributors to the induction and adhesion of immune cells to the endothelium and might have a role in atherogenesis by allowing monocyte aggregation in the arterial intima [31]. In addition, studies have shown that increased LDL levels coupled with its oxidation by ROS, can induce VCAM-1 and ICAM-1 expression from endothelial cells, leading to endothelial dysfunction and immune cells recruitment to the arterial wall, which is considered a critical stage in the emergence of AS [8]. A reduction of VCAM-1 and ICAM-1 levels has been suggested as a protective measure against the risk of AS. ICAM-1 expression in endothelial tissue was significantly downregulated in this study by both NF3BP and NF4AP indicating a promising effect of these compounds in reducing inflammation.

IL-38 has anti-inflammatory effects through interacting with IL-36 and IL-1R receptors leading to stop the signaling pathways [32]. It has a structure related to those of the IL-36 receptor antagonist (IL-36Ra). Therefore, it hinders IL36α, IL36β, and IL36γ from binding to IL36R receptors, which prevents pro-inflammatory cytokine expression. In addition, IL-38 may suppress the CD4 T lymphocytes, thus reducing the inflammatory response [33]. Therefore, IL-38 has anti-atherosclerotic properties that minimize the risk of CVDs [9]. In this study, fenofibrate and NF4BP caused a significant overexpression of IL-38 in cardiac tissue. The observed overexpression of IL-38 following the treatment with NF4BP highlights its potential in alleviating the inflammation associated with hyperlipidemia.

The need to produce new compounds to control hyperlipidemia is necessary to decrease cardiovascular complications. The primary strength of this study lies in its use of novel carboxamide derivatives in an acute animal model of Triton WR-1339-induced hyperlipidemia and the assessment of their effect on lipid profile and inflammatory mediators’ gene expression using single dose. Thus, giving a fast and reliable testing of their potential effects. Additionally, measuring specific inflammatory markers provided valuable information about the systemic inflammatory response to these compounds in targeted organs. Even though these findings highlight the important role of these potential compounds in modulating the inflammatory responses in hyperlipidemia, this study has its limitations since the molecular changes of acute hyperlipidemic models might not be the same as in chronic models. Also, significant changes in gene expression levels might not be as significant at protein levels. Future experiments would focus on overcoming these limitations and targeted to determine which derivative of the three has the most potential to be tested clinically. Overall, the reporting of the anti-inflammatory activity of the novel antihyperlipidemic compounds opens new avenues for the development of targeted therapies to inflammation-associated diseases.