In this present study, the effect of Chlorella vulgaris supplementation on protein expression in connection with liver oxidative stress of rabbits was investigated. It is necessary to understand this because oxidative stress is a biochemical process causing the generation of reactive oxygen species, molecules, and or ions formed through incomplete electron reduction reaction of oxygen and this is strongly involved in pathways affecting normal cellular functions of the liver. Oxidative stress is a critical factor responsible for the progression of liver and non-liver dysfunctional conditions such as non-alcoholic fatty liver diseases (NAFLD), male infertility, pulmonary carcinogenesis, hypoxia, and vascular malfunctions [18,19,20,21,22]. However, despite the ubiquitous nature of oxidative stress, the supplementation of antioxidants have been widely reported as a way-out of its complications but the exact mechanisms underlying efficacies of some of the antioxidants in oxidative stress protection in-vivo is poorly understood [23].
Although, reports from recent investigations on antioxidant agent supplementation identified that gene expression and regulation modification for cellular protection against oxidative stress in rabbits and other models are mechanisms associated with intakes of antioxidant agents including microalgae [24, 25]. Therefore, in taking a step further, this present study confirmed some of the assertions by indicating that antioxidant agents could have some post-transcription modulation impacts against oxidative stress because the supplementation of Chlorella vulgaris in this study led to a higher antioxidant proteins expression in the liver of the supplemented rabbits. The outcome of this present study also showed that primary antioxidant enzymes such as superoxide dismutase, catalase, and glutathione could be active across the cytoplasmic and mitochondria phases of oxidative stress protections since observations recorded showed that the proteins are more in the treatment group versus the control. Meanwhile, this is in agreement with a previous report which stated that the increasing abundance of antioxidant proteins could be associated with the effectiveness of cytosolic and mitochondria oxidative stress protection in cells [26].
Furthermore, functional enrichment of the proteins upregulated in the supplemented rabbits indicated that in association with other proteins, they are involved in the scavenging of free radicals and their upregulation is required for reduced oxidative stress. This observation could be accepted as a confirmation that the microalga Chlorella vulgaris is a potential drug and nutraceutical candidate since the biological processes and pathways analysis of the proteome indicated that the supplementation of the microalga modulated cellular response of the liver to inhibit cellular oxidative stress damage basically via the hydrogen peroxide metabolism and detoxification of xenobiotics compounds. These cellular activities can be linked to prominent mechanisms involving catalytic removal of free radicals by superoxide dismutase, catalase, and peroxidase, and scavenging of reactive species by low molecular agents such as carotenoids present in the microalga Chlorella vulgaris [27].
The superoxide dismutase proteins were more abundant in the supplemented rabbits, and this could be regarded as the starting point of endogenous antioxidant protection mechanism whereby one superoxide radical is oxidized and the second one is reduced to eliminate continuous damage by radical oxygen. Although this mechanism usually leads to the production of hydrogen peroxide which is another reactive compound, the hydrogen peroxide produced is usually quickly acted upon by subsequent enzymatic actions. The catalase and glutathione peroxidases carry out further actions in the cytoplasm while in the mitochondria glutathione reductase carry out the inhibition of the free radical activities; these observations agreed with submissions of [27].
The inter-protein interactions and functional enrichment analyses in this present study indicated that the supplementation of Chlorella vulgaris biomass as an antioxidant in rabbit led to increased activities of the antioxidant enzymes activities and increased production of their respective proteins to prevent liver oxidative stress damages and progression in a mechanism similar to impacts of ascorbate which is a common conventional antioxidant agent. In agreement with this submission, Halliwell and Gutteridge [28] suggested that antioxidant agent supplements are expected to exhibit the capability of enhancing both enzymatic and non-enzymatic antioxidant activities. In upholding this submission, this present study demonstrated that the intake of the microalga increases the antioxidant enzymes activities and their respective proteins including the catalase (CAT), peroxiredoxin (PRDX6), macrophage migration inhibitory factor (MIF), and glutathione S-transferase Pi 1 (GSTP1) due to their higher abundance in the supplemented group compared with the control group.
The reduction of the lipid peroxidation biomarker malondialdehyde in the liver of the supplemented rabbits could be linked with the increased expression of the antioxidant proteins because of the microalga intakes and this can be described as a mechanism of oxidative stress protection in the liver promoted by the microalga. In specific, the higher expression of the catalase protein could be primarily linked with the protection of cells against the toxic effects of hydrogen peroxide as well as promoting the growth of immune cells including T cells and B cells; and hence, this could be described as double effects of oxidative stress protection and immune enhancement. This observation is in agreement with [29], which stated that antioxidant therapy focusing on reducing oxidative stress and enhancing activities of T cells and B cells could also lead to immune protection against oxidative stress-mediated diseases such as organ damage and comorbidities in systemic lupus erythematosus (SLE).
Similarly, increased expression of peroxiredoxin (PRDX6) in the supplemented rabbit affirm competency of the microalga supplemented to protect against oxidative stress. This is because as a protein-encoded gene and a member of the thiol-specific antioxidant protein family, it has a bifunctional enzyme with two distinct active sites involved in redox regulation of the cell [30]. The increased expression of peroxiredoxin in the supplement rabbits can reduce pro-oxidant activities of hydrogen peroxide and short-chain organic, fatty acid, and phospholipid hydroperoxides and it may also play a role in the regulation of phospholipid turnover as well as in protection against oxidative injury [7]. The microalga supplementation also increased MIF which indicates that it is a potent antioxidant agent since the gene MIF encodes a lymphokine involved in cell-mediated immunity, immunoregulation, and inflammation which plays roles in the regulation of macrophage function in host defense through the suppression of anti-inflammatory effects of glucocorticoids—a mechanism substantial for protection against environmental triggered stress at the cellular level [31].
In correlating the malondialdehyde concentration reduction with the increasing abundance of antioxidant proteins in the supplemented rabbits as observed in this study, it could be juxtaposed that supplementation of the microalga Chlorella vulgaris prevented attacks on lipid signaling and also led to the upregulation of its associated proteins as well as enhanced activities of the antioxidant enzymes. These submissions agreed with [32], which opined that under normal physiological conditions, there will be reduced malondialdehyde concentrations necessary for cellular stimulation, maintenance, and survival occurring through activities of antioxidant enzymes and antioxidant genes for promoting the constitutive antioxidants defense systems and protection of lipid signaling pathways as well as activation of genes expression required for upregulating antioxidants proteins needed for the formation of adequate protection and adaptation to the stress response. The underlying physiological importance of reduced malondialdehyde in association with the antioxidant protein abundance in the liver could therefore be described as critical for the promotion of healthy living. This is because if otherwise, the cells can induce inordinate apoptosis or necrosis resulting in cell deaths and facilitation of various pathological conditions [33].
The mechanism of oxidative stress promotion of liver diseases involves the activation of stellate cells by the free radicals leading to the synthesis of collagen and the extracellular matrix [34]. These are complications associated with liver diseases such as non-alcoholic fatty liver diseases, fibrosis, and encephalopathy. This present study suggests that Chlorella vulgaris could serve as an antioxidant supplement suitable for the alleviation of these liver diseases. This is in agreement with a report that the microalga improved liver health through the modulation of the fasting blood sugar and lipid profile in a double-blind randomized placebo-controlled clinical trial involving 60 NAFLD patients [35, 36]. Furthermore, the present study demonstrated a possible molecular basis for the effectiveness of the microalga as an antioxidant agent suitable for the management of liver damages because of the inter-protein interaction of the significantly upregulated antioxidant proteins in the supplemented group which revealed the involvement of the proteins in more than a hundred pathways associated with oxidative stress-protective activities (Supplementary Table 1).