Effect of Tocopherol and Ascorbic Acid on Neuroinflammation Markers Associated With Neuropeptide Expression in the AgRP/NPY and POMC Pathways in Mexican Population With Obesity

Obesity is the accumulation of excessive adiposity that leads to a higher risk of developing clinical obesity as well as several other non-communicable diseases. According to the World Health Organization (WHO) classification, obesity is defined based on the Body Mass Index (BMI), which is calculated as weight in kilograms divided by height in meters squared. A BMI >29.9 kg/m² is classified as obesity, >34.9 kg/m² as grade I obesity, >35 kg/m² as grade II obesity, and >40 kg/m² as morbid obesity. The new definition and classification of obesity proposed by Rubino F., Cummings D. et al. (2025) distinguishes preclinical obesity, characterized by excessive adiposity with preserved function of other tissues and organs, from clinical obesity, where chronic excessive adiposity causes loss of organ and tissue function with symptoms.In Mexico, obesity (OB) remains a public health problem with a prevalence of 37.1% in adults aged ≥20 years (ENSANUT 2020-2023). Long-term accumulation of adipose tissue in obesity causes adipocyte hypertrophy, triggering sustained release of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), leading to a state of low-grade chronic systemic inflammation. Cytokines expressed by adipose tissue reach the Central Nervous System (CNS) through the bloodstream, cross the blood-brain barrier, and alter mechanisms that allow entry of cytokines and fatty acids. This provokes glial cell reactivity, activating IL-6 and TNF-α expression and leading to neuroinflammation, which may modify neuropeptide expression through mechanisms such as reactive oxygen species (ROS) production, neuronal damage, and neuronal apoptosis. Hypothalamic alterations are directly associated with appetite regulation, energy balance, and reward via AgRP/NPY neurons (hunger-inducing) and POMC/CART neurons (satiety-promoting). Hypothalamic inflammation impairs these neurons, disrupting hunger-satiety signaling and perpetuating obesity. Dysfunction of hypothalamic circuits in structures such as the arcuate nucleus shows specific alterations in the AgRP/NPY and POMC pathways, with hyperactivation of AgRP/NPY increasing orexigenic neuropeptide Y and suppressing POMC/CART, reducing anorexigenic α-MSH. These changes may explain the perpetuation of the hunger-satiety cycle in people with obesity. Beyond genetic factors, hypercaloric diets rich in saturated fats and simple carbohydrates, along with micronutrient deficiencies, can exacerbate obesity. In Mexico, overnutrition coexists with nutritional deficiencies, increasing metabolic and cognitive risk. Current studies in obese patients have linked malnutrition to a higher risk of neurodegenerative diseases. Antioxidant vitamins such as ascorbic acid (vitamin C) and α-tocopherol (vitamin E) are essential micronutrients that act as second messengers in cellular signaling cascades. Evidence reports their protective role in the immune system and in preventing neuronal cell death caused by oxidative stress.

Vitamin C (ascorbic acid) reduces ROS, inhibits microglial activation, and decreases interleukin expression, protecting against cognitive decline. Studies (Xiao-Ying Zhang et al., 2018) in murine models with cognitive impairment induced by lipopolysaccharides showed that vitamin C reduced oxidative stress and neuroinflammation, increased superoxide dismutase (SOD) expression, decreased malondialdehyde (MDA), and inhibited microglial activation.

Vitamin E (α-tocopherol) can attenuate oxidative stress in the hippocampus, improve memory, and counteract the effects of obesogenic diets, though more evidence is needed. It is a fat-soluble micronutrient with antioxidant and neuroprotective properties. Studies (Alzoubi K. et al., 2013; La Torre M. et al., 2021) reported that vitamin E normalized antioxidant mechanisms in the hippocampus and reduced neuroinflammation induced by microglial activity, but controversy remains regarding its efficacy.

Deficiency of vitamins C and E in the hippocampus has been linked to increased IL-6 and TNF-α expression and memory impairment (Takashi K. et al., 2019). Positive effects of supplementation include reduced ROS, increased SOD activity, and decreased TNF-α/IL-1β expression in the hippocampus.

Justification Obesity in Mexico represents one of the main public health challenges, being associated not only with metabolic diseases but also with alterations in the central nervous system. Recent studies show that high-fat diets induce neuronal apoptosis in the hypothalamus, affecting the regulation of key neuropeptides (POMC and AgRP/NPY) involved in appetite control.Although pharmacological treatments exist that modulate food intake, they do not address the underlying neuroinflammation, limiting their long-term effectiveness. Therefore, it is a priority to investigate accessible, safe strategies with sustainable effects on hypothalamic regulation of energy homeostasis.In this context, ascorbic acid and α-tocopherol emerge as promising alternatives due to their antioxidant and anti-inflammatory capacity, demonstrated in animal models. However, evidence in humans is required to understand their impact on hypothalamic neuroprotection and their potential application as adjuvants in obesity management.This research seeks to contribute to the development of evidence-based nutritional interventions that improve not only body weight but also neuronal function altered in obesity, offering a cost-effective and widely accessible strategy for the Mexican population.

Intervention Design The protocol will be carried out in three phases, with a total duration of 72 weeks (18 months).

During the first 12 months, participants will be recruited, baseline measurements will be taken, and the intervention will begin.

The intervention itself will last 12 weeks. The final 12 weeks will be dedicated to completing the intervention, conducting final measurements, and offering workshops and/or talks to participants on the importance of proper nutrition and the health consequences of obesity.

Randomization and Sampling A simple probabilistic randomized sampling will be performed for the assignment of participants to treatment groups using the statistical program R. Randomization is used in blinded trials to ensure that the number of participants is distributed evenly among the study groups.

Sample Size According to the sample size calculation for comparing differences between groups, 192 participants are required, with 48 per group.

Body Mass Index Weight will be measured in kilograms using a Tanita scale, and height will be measured with a Seca stadiometer to calculate the BMI, which will be classified according to the WHO criteria. BMI will be assessed both before and after supplementation. For height measurement, participants will stand upright in the anatomical position, looking straight ahead in the Frankfurt plane.

Determination of Total Body Fat Percentage The total body fat percentage will be measured using a Tanita bioimpedance scale, both at the beginning and at the end of the treatment. For the bioimpedance measurement, participants will be asked to remove as many garments as possible and to step barefoot onto the scale without carrying a cell phone or smartwatch.

Determination of Abdominal Circumference Abdominal circumference will be measured using a Seca measuring tape. The technique consists of placing the tape at the midpoint between the last rib and the upper edge of the iliac crest (Figure 4). The tape will be positioned snugly but without compressing the skin, and the measurement will be taken after normal expiration.

Evaluation of Dietary Habits A Food Frequency Questionnaire (FFQ) will be applied to evaluate dietary changes during the intervention and to control for confounding variables. These assessments will be conducted as part of patient follow-up every two weeks during the intervention. The questionnaire will be administered by trained personnel.

Physical Activity Evaluation The International Physical Activity Questionnaire (IPAQ) - Short Form will be applied. This instrument is standardized for use in population-based studies and evaluates physical activity across several dimensions. The questionnaire can be self-administered or conducted through face-to-face interviews. It will be applied as part of patient follow-up every two weeks during the intervention to assess changes in physical activity.

Treatment Adherence The Morisky Medication Adherence Scale (MMAS-8) will be used to measure medication adherence. This scale considers specific adherence-related behaviors associated with medication intake. It has been validated in the Mexican population and consists of seven questions plus one multiple-choice item using a Likert-type scale.

Cognitive Impairment The Mini-Mental State Examination (MMSE) screening tool will be applied. The version adapted and validated by Reyes de Beaman S. and collaborators in the Mexican population will be used. This test evaluates mild loss of cognitive abilities including thinking, memory, language, and spatial orientation. It is a brief and rapid test that detects cognitive impairment and dementia risk.

Determination of Triglycerides and Cholesterol Biochemical parameters including glucose, triglycerides, and cholesterol will be measured using standardized reactive strips, along with clinical data provided by the patient.

Albumin-Creatinine Index To evaluate renal disease prior to supplementation, participants will provide a first-morning urine sample collected in a sterile 60 ml container. The albumin-creatinine index will be calculated using standardized reactive strips.

Collection of Venous Blood Samples A 5 ml peripheral venous blood sample will be obtained in EDTA vacutainer tubes. Samples will be stored at -20°C in a Thermo Scientific ultrafreezer until RNA extraction. Blood collection will be performed by trained personnel.

Genomic RNA Isolation RNA isolation will be performed using the Trizol-based technique, which contains guanidinium thiocyanate, a potent protein denaturant, to extract mRNA. RNA purity will be assessed by spectrophotometry. Subsequently, mRNA will be reverse-transcribed into cDNA using reverse transcriptase and oligo dT primers.

Expression of Neuroinflammation Markers Transcription of biomarkers included in the protocol will be measured using qRT-PCR with SYBR Green. Expression of GFAP, IL-1β, IL-8, and S100B will be evaluated. Evidence has associated these markers with neuronal inflammatory responses. For example, Glial Fibrillary Acidic Protein (GFAP) is abundantly expressed in astrocytes and is considered a specific marker of central nervous system diseases, related to harmful neuronal processes that compromise blood-brain barrier integrity. Additionally, the expression of neuropeptide Y (NPY) metabolites, agouti-related peptide (AgRP), and proopiomelanocortin (POMC) will be measured.