Crosstalk between Oxidative Stress and Inflammation in Obesity

MIRELA ELENA EPINGEAC, MIHNEA ALEXANDRU GAMAN*, CAMELIA CRISTINA DIACONU, AMELIA MARIA GAMAN University of Medicine and Pharmacy of Craiova, Department of Pathophysiology, 2 Petru Rares Str., 200349, Craiova, Romania Carol Davila University of Medicine and Pharmacy, 8 Eroii Sanitari Blvd., 050474, Bucharest, Romania Filantropia City Hospital, Clinic of Hematology1 Filantropiei Str., 200143, Craiova, Romania

The aim of the study was to evaluate the levels of oxidative stress and inflammation markers in a group of patients stratified in different classes of obesity and to investigate the relationship between these variables.

Experimental part
We evaluated oxidative stress levels and inflammation markers in 85 obese patients vs. 30 non-obese controls. Patients were divided by sex, age and obesity class. The diagnosis of obesity was based on body mass index (BMI) values according to the WHO criteria: first class obesity -BMI of 30.0 to 34.9 kg/m 2 , second class obesity -BMI of 35.0 to 39.9 kg/m 2 , and third class obesity -BMI > 40kg/m 2 . To assess BMI, height and weight were measured with a standard height-weight scale, and BMI was calculated as weight (kg) divided by height (m) squared. The complete blood count was determined using an automated analyzer BM-800. Serum glucose levels, uric acid and serum iron were measured by spectrophotometry using a KONELAB601 analyzer. Oxidative stress levels were evaluated using a CR3000 analyzer. Reactive oxygen species levels were measured using the FORT (Free Oxygen Radicals Testing) test and the antioxidant capacity using the FORD (Free Oxygen Radical Defence). Inflammatory status was evaluated by acute phase proteins (fibrinogen, C-reactive protein, serum ferritin) and the neutrophil-to-lymphocyte ratio (NLR). Statistical analysis of data was performed using Microsoft Excel (Microsoft Office Professional Plus 2013) and GraphPad QuickCalcs (https://www.graphpad.com). Informed consent was obtained from all subjects involved. The study had the approval of the Ethics Committee of the University of Medicine and Pharmacy of Craiova (approval number: 40/27.03.2018).

Results and discussions
The  (Fig. 1).
Obesity can lead to oxidative stress via several biochemical mechanisms but other factors such as chronic inflammation, elevated lipid levels, postprandial ROS generation, low antioxidant defenses, hyperglycemia, mineral or vitamin deficiencies and endothelial dysfunction are main contributors nevertheless [10]. Furukawa et al. revealed that elevated levels of fatty acids from the adipose tissue of obese mice and from cultured adipocytes increased oxidative stress levels via NADPH oxidase activation which in turn enhanced local and systemic generation of pro-inflammatory adipocytokines, i.e. IL-6, TNF-α, IL-1β, monocyte chemotactic protein-1 (MCP-1), adiponectin and plasminogen activator inhibitor-1 (PAI-1) [19]. In obese subjects, the overexpression of pro-inflammatory cytokines is also generated by the activation of the c-Jun N-terminal kinase [9]. The most important cytokines in obesity are IL-6, the inflammasome-activated IL-1β and TNF-α, since these molecules are responsible for the chronic inflammation in obese subjects [1]. The inflammasome is an innate immune cell sensor activated by ROS, hyperglycemia, lipopolysaccharides, uric acid etc. which initiates the inflammatory response by activating a NOD-like receptor (NLRP3) which promotes, in the fat tissue, the activation of T-cells by mechanisms mediated by macrophages [9,20]. Nishimura et al. concluded that CD8+ T-cells are key actors in the initiation and propagation of inflammation via enlarged adipocytes found in the fat tissue. In turn, CD8+ T-cells that are activated by the enlarged adipocytes recruit and activate macrophages in the fat tissue [21]. On the other hand, the obesity-related inflammatory response is linked with a change in the macrophages' phenotype, particularly in the fat tissue located in the viscera. Under the influence of lipopolysaccharides and IFN-γ, these cells release pro-inflammatory cytokines (IL-6, TNFα) and generate ROS [17]. As previously mentioned, the increased levels of oxidative stress and pro-inflammatory cytokines in obese subjects might also be related to the hypoperfusion and subsequent hypoxia of the adipose tissue due to the progressive enlargement of adipocytes [15]. Thus, one important link in explaining the crosstalk between obesity, oxidative stress and chronic inflammation might be the overexpression of pro-inflammatory cytokines [9].
In obese subjects, the free fatty acid accumulation is responsible for the activation of several serine kinases with a proinflammatory activity (c-Jun N-terminal kinase, IκB kinase etc.). As an effect, fat tissue is stimulated to release IL-6 which, in turn, will enhance the production and secretion of CRP (a very sensitive inflammation marker) by liver cells [22]. Previous research has shown that there is a correlation between abdominal fat deposits, the elevated risk of cardiovascular events and CRP levels [9]. Some studies have shown that TNF-α is expressed and secreted by adipocytes and that the adipose body mass might mediate the relationship between chronic inflammation and obesity. Independently of the BMI, abdominal fat is linked to increased values of CRP; on the same hand, also independently of the BMI, increased values of high-sensitivity CRP are linked to visceral obesity [9,17,23].
Iron is a transition metal necessary for normal cell growth and proliferation. However, excessive amounts can catalyze the production of toxic ROS via the Fenton reaction, inducing oxidative tissue damage. Ferritin is an acute-phase protein whose expression can be up-regulated by inflammatory processes, infections, excessive production of ROS and uncontrolled proliferation of cells [24]. Serum ferritin levels are indicators of the total amount of iron stored in the body and increased serum ferritin concentrations are associated with various metabolic risk factors, including insulin resistance [25]. Uric acid is one of the most important endogenous antioxidants in the plasma, but its effects are controversial, since during generation of uric acid, ROS are produced. Xanthine oxidoreductase inhibition in obesity is associated with vascular alterations, cell differentiation, foam cell formation and insulin resistance [26][27][28].
Neutrophil-to-lymphocyte ratio (NLR) is an easy to measure laboratory parameter that can reflect the levels of inflammation of a certain subject. Studies have shown that it is a useful prognostic factor of morbidity and mortality in several types of cardiovascular disorders or malignancies, and as a marker of infection, inflammation or postoperative complications [29][30]. According to Forget et al., non-geriatric, healthy adults should have a NLR between 0.78 and 3.53 [31].
It is noteworthy to mention that, in susceptible individuals, including obese subjects, increased levels of oxidative stress and inflammation may contribute to an increased risk of developing solid and haematological cancers [32][33][34].

Conclusions
In our study, we found decreased levels of antioxidants and increased levels of reactive oxygen species and inflammation markers in obese subjects vs. healthy controls. The culprits and the pathophysiological events leading to obesity-induced chronic inflammation, as well as the relationship between cytokines or inflammation and obesity indices, are still not fully understood. Further studies are needed to investigate the crosstalk between obesity, oxidative stress and inflammation.