To the best of our knowledge, the present study demonstrates for the first time that EOFAZ (the composition was significantly different from previous reports and from the essential oil extract from the fruit of A. zerumbet) protects against endothelial cell injury induced by ox-LDL via ameliorating oxidative stress, which may be the main underlying mechanism.
Human umbilical vein endothelial cells (HUVECs) have played a major role as a model system for studying the regulation of endothelial cell function and the role of the endothelium in the response of the blood vessel wall to stretch, shear forces, and the development of atherosclerotic plaques and angiogenesis . It is well known that the atherosclerotic lesion is characterized by an accumulation of lipids carried by lipoproteins, such as low-density lipoprotein (LDL). LDL becomes susceptible to (non)enzymatic oxidative modifications when retained in the artery wall . These modifications make LDL a potent effector of cellular functions. Multiple lines of evidence suggest that oxidative stress, characterized by an elevated generation of ROS, is involved in the pathogenesis of AS, which implies that oxidized-LDL may promote the development of AS through oxidative stress, and is one of the most important risk factors for AS and cardiovascular morbidity . Endothelial dysfunction elicited by ox-LDL has been demonstrated as the key step in the initiation of AS. It is widely accepted that ox-LDL-induced endothelial dysfunction is associated with an alteration of the cell redox status, and ameliorating the redox status has been a key therapeutic strategy against AS in the clinic .
The cell injury was evaluated by the MTT assay, TBES, and LDH release, which are widely accepted methods for cell injury evaluation. The MTT assay is a quantitative colorimetric method to determine cell proliferation or injury. It utilizes the yellow MTT, which is metabolized by the mitochondrial succinate dehydrogenase to yield a purple formazan reaction product. The TBES method is widely used to determine the number of viable cells and is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, eosin and propidium, whereas dead cells do not. Lactate dehydrogenase (LDH) is a stable enzyme in the cytosol, present in all cell types, that is rapidly released into the medium upon damage of the plasma membrane; hence it is a biomarker for cell membrane damage. The cell injury induced by incubation with ox-LDL for 24 h was confirmed by the MTT assay (OD570 decrease), trypan blue staining ratio increase, and the LDH activity increase. Pretreatment with EOFAZ or PRA ameliorated the cell injury.
Membranes of bioplasm are very rich in polyunsaturated fatty acids, which are especially sensitive to free radical-induced lipid peroxidation. In oxidative stress, superoxide anion and hydrogen peroxide are formed and cannot be readily scavenged because of the low activities of CAT, SOD and GSH-Px in the endothelial cell . Augmentation of endogenous antioxidants (SOD, CAT and GSH-Px) has been recognized as an important pharmacological property present in natural as well as many synthetic compounds . This constitutes a major mechanism of protection against oxidative stress [30, 31]. The most abundant ROS generated in living systems is the superoxide radical, which is acted upon by SOD to produce hydrogen peroxide, which in turn is broken down by catalase and/or GSH-Px into water and oxygen. Thus, increase in both SOD and catalase along with GSH-Px activity is considered to be more beneficial in the event of oxidative stress.
Exposure to 100 mg/L ox-LDL for 24 h, increased the MDA contents, decreased the GSH contents, and inhibited the enzymes’ antioxidant activity, indicating that the redox status was exacerbated. In the present study, a 30 min preincubation with EOFAZ significantly elevated the activities of SOD, CAT and GSH-Px, whereas PRA only enhanced the activity of SOD and CAT. Obviously, EOFAZ scavenged hydrogen peroxide and superoxide anion, further decreasing the formation of hydroxyl radicals and attenuating lipid peroxidative damage after the HUVECs were exposed to ox-LDL. However, it is not clear whether EOFAZ induces the expression of the endogenous antioxidant enzymes or whether it has direct protective effects of endogenous antioxidants. PRA, a hydroxymethylglutaryl-CoA reductase inhibitor, is a member of the statins drug class and was used here as a positive control. PRA, which is used for lowering cholesterol and preventing cardiovascular disease, and is widely used to prevent AS, has anti-oxidative effects . The present results confirmed previous research.
Oxidative metabolites are involved in the functional inactivation of endothelial cells by increasing permeability and as potent inducers of endothelial cell death. The level of MDA reflects the extent of cell damage by oxidative stress . GSH is an intracellular reductant that plays major roles in catalysis, metabolism and transport, and protects cells against free radicals, peroxides, and other toxic compounds, as it is a critical factor involved in the glutathione system to scavenge hydrogen peroxide and organic hydroperoxides . It is well known that a deficiency in GSH within living organisms can lead to tissue disorders and injury. The present study demonstrated that EOFAZ alleviated the increase in MDA and the decrease in GSH contents caused by the ox-LDL-induced endothelial cell damage, suggesting it has the potential to protect the membranes of HUVECs from lipid peroxidative damage.
Endothelial dysfunction is an early feature of both atherosclerosis and vascular diseases, which may lead to an improvement of prognosis in patients with cardiovascular risk factors preventing the development of atherosclerosis and consequently promoting a reduction in cardiovascular events . Chronic inflammation and oxidative stress play crucial roles in endothelial dysfunction and atherosclerosis . Controlling a variety of risk factors causing inflammation and oxidative stress with combination therapy may simultaneously address multiple mechanisms underlying the pathogenesis of atherosclerosis. EOFAZ has been shown to be an anti-inflammatory agent in previous research  and the current results confirmed it is also an antioxidant, which may provide a novel therapy for atherosclerosis in the clinic.
In summary, the present research indicates that increase in HUVECs’ lipid peroxidation and depletion of endogenous antioxidants support the occurrence of oxidative stress after exposure to ox-LDL. Furthermore, it was accompanied by cell viability decrease, which was confirmed by the MTT assay, trypan blue exclusion, and LDH release. EOFAZ protected the cells against oxidative stress, as evidenced by inhibition of the increased MDA contents and decreased GSH contents, alleviation of CAT, SOD and GSH-Px activities, and finally, enhancement of cell viability. The mechanism of this protection can be attributed to the augmented endogenous antioxidant reserve in HUVECs and/or a direct antioxidant effect. This study shows that EOFAZ from natural products may be an excellent protecting agent against endothelial cell injury in the clinic; the mechanism will be explored in detail and a clinical evaluation conducted in future research.