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 ofdeveloping clinical obesity as well as several other non‐communicable diseases. Accordingto the World Health Organization (WHO) classification, obesity is defined based on theBody Mass Index (BMI), which is calculated as weight in kilograms divided by height inmeters squared. A BMI >29.9 kg/m² is classified as obesity, >34.9 kg/m² as grade Iobesity, >35 kg/m² as grade II obesity, and >40 kg/m² as morbid obesity. The newdefinition and classification of obesity proposed by Rubino F., Cummings D. et al. (2025)distinguishes preclinical obesity, characterized by excessive adiposity with preservedfunction of other tissues and organs, from clinical obesity, where chronic excessiveadiposity 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 adipocytehypertrophy, triggering sustained release of pro‐inflammatory cytokines (IL‐1β, IL‐6,TNF‐α), leading to a state of low‐grade chronic systemic inflammation. Cytokinesexpressed by adipose tissue reach the Central Nervous System (CNS) through thebloodstream, cross the blood‐brain barrier, and alter mechanisms that allow entry ofcytokines and fatty acids. This provokes glial cell reactivity, activating IL‐6 and TNF‐αexpression and leading to neuroinflammation, which may modify neuropeptide expressionthrough mechanisms such as reactive oxygen species (ROS) production, neuronal damage, andneuronal apoptosis. Hypothalamic alterations are directly associated with appetiteregulation, energy balance, and reward via AgRP/NPY neurons (hunger‐inducing) andPOMC/CART neurons (satiety‐promoting). Hypothalamic inflammation impairs these neurons,disrupting hunger‐satiety signaling and perpetuating obesity. Dysfunction of hypothalamiccircuits in structures such as the arcuate nucleus shows specific alterations in theAgRP/NPY and POMC pathways, with hyperactivation of AgRP/NPY increasing orexigenicneuropeptide Y and suppressing POMC/CART, reducing anorexigenic α‐MSH. These changes mayexplain the perpetuation of the hunger‐satiety cycle in people with obesity. Beyondgenetic factors, hypercaloric diets rich in saturated fats and simple carbohydrates,along with micronutrient deficiencies, can exacerbate obesity. In Mexico, overnutritioncoexists with nutritional deficiencies, increasing metabolic and cognitive risk. Currentstudies in obese patients have linked malnutrition to a higher risk of neurodegenerativediseases. Antioxidant vitamins such as ascorbic acid (vitamin C) and α‐tocopherol(vitamin E) are essential micronutrients that act as second messengers in cellularsignaling cascades. Evidence reports their protective role in the immune system and inpreventing neuronal cell death caused by oxidative stress.Vitamin C (ascorbic acid) reduces ROS, inhibits microglial activation, and decreasesinterleukin expression, protecting against cognitive decline. Studies (Xiao‐Ying Zhang etal., 2018) in murine models with cognitive impairment induced by lipopolysaccharidesshowed that vitamin C reduced oxidative stress and neuroinflammation, increasedsuperoxide dismutase (SOD) expression, decreased malondialdehyde (MDA), and inhibitedmicroglial activation.Vitamin E (α‐tocopherol) can attenuate oxidative stress in the hippocampus, improvememory, 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 Enormalized antioxidant mechanisms in the hippocampus and reduced neuroinflammationinduced 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 andTNF‐α expression and memory impairment (Takashi K. et al., 2019). Positive effects ofsupplementation include reduced ROS, increased SOD activity, and decreased TNF‐α/IL‐1βexpression 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 thecentral nervous system. Recent studies show that high‐fat diets induce neuronal apoptosisin the hypothalamus, affecting the regulation of key neuropeptides (POMC and AgRP/NPY)involved in appetite control.Although pharmacological treatments exist that modulate foodintake, they do not address the underlying neuroinflammation, limiting their long‐termeffectiveness. Therefore, it is a priority to investigate accessible, safe strategieswith sustainable effects on hypothalamic regulation of energy homeostasis.In thiscontext, ascorbic acid and α‐tocopherol emerge as promising alternatives due to theirantioxidant and anti‐inflammatory capacity, demonstrated in animal models. However,evidence in humans is required to understand their impact on hypothalamic neuroprotectionand their potential application as adjuvants in obesity management.This research seeks tocontribute to the development of evidence‐based nutritional interventions that improvenot only body weight but also neuronal function altered in obesity, offering acost‐effective and widely accessible strategy for the Mexican population.Intervention Design The protocol will be carried out in three phases, with a totalduration of 72 weeks (18 months).During the first 12 months, participants will be recruited, baseline measurements will betaken, and the intervention will begin.The intervention itself will last 12 weeks. The final 12 weeks will be dedicated tocompleting the intervention, conducting final measurements, and offering workshops and/ortalks to participants on the importance of proper nutrition and the health consequencesof obesity.Randomization and Sampling A simple probabilistic randomized sampling will be performedfor 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 isdistributed evenly among the study groups.Sample Size According to the sample size calculation for comparing differences betweengroups, 192 participants are required, with 48 per group.Body Mass Index Weight will be measured in kilograms using a Tanita scale, and heightwill be measured with a Seca stadiometer to calculate the BMI, which will be classifiedaccording to the WHO criteria. BMI will be assessed both before and aftersupplementation. For height measurement, participants will stand upright in theanatomical position, looking straight ahead in the Frankfurt plane.Determination of Total Body Fat Percentage The total body fat percentage will be measuredusing 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 garmentsas possible and to step barefoot onto the scale without carrying a cell phone orsmartwatch.Determination of Abdominal Circumference Abdominal circumference will be measured using aSeca measuring tape. The technique consists of placing the tape at the midpoint betweenthe last rib and the upper edge of the iliac crest (Figure 4). The tape will bepositioned snugly but without compressing the skin, and the measurement will be takenafter normal expiration.Evaluation of Dietary Habits A Food Frequency Questionnaire (FFQ) will be applied toevaluate dietary changes during the intervention and to control for confoundingvariables. These assessments will be conducted as part of patient follow‐up every twoweeks during the intervention. The questionnaire will be administered by trainedpersonnel.Physical Activity Evaluation The International Physical Activity Questionnaire (IPAQ) ‐Short Form will be applied. This instrument is standardized for use in population‐basedstudies and evaluates physical activity across several dimensions. The questionnaire canbe self‐administered or conducted through face‐to‐face interviews. It will be applied aspart of patient follow‐up every two weeks during the intervention to assess changes inphysical activity.Treatment Adherence The Morisky Medication Adherence Scale (MMAS‐8) will be used tomeasure medication adherence. This scale considers specific adherence‐related behaviorsassociated with medication intake. It has been validated in the Mexican population andconsists of seven questions plus one multiple‐choice item using a Likert‐type scale.Cognitive Impairment The Mini‐Mental State Examination (MMSE) screening tool will beapplied. The version adapted and validated by Reyes de Beaman S. and collaborators in theMexican population will be used. This test evaluates mild loss of cognitive abilitiesincluding thinking, memory, language, and spatial orientation. It is a brief and rapidtest 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, alongwith clinical data provided by the patient.Albumin‐Creatinine Index To evaluate renal disease prior to supplementation, participantswill provide a first‐morning urine sample collected in a sterile 60 ml container. Thealbumin‐creatinine index will be calculated using standardized reactive strips.Collection of Venous Blood Samples A 5 ml peripheral venous blood sample will be obtainedin EDTA vacutainer tubes. Samples will be stored at ‐20°C in a Thermo Scientificultrafreezer until RNA extraction. Blood collection will be performed by trainedpersonnel.Genomic RNA Isolation RNA isolation will be performed using the Trizol‐based technique,which contains guanidinium thiocyanate, a potent protein denaturant, to extract mRNA. RNApurity will be assessed by spectrophotometry. Subsequently, mRNA will bereverse‐transcribed into cDNA using reverse transcriptase and oligo dT primers.Expression of Neuroinflammation Markers Transcription of biomarkers included in theprotocol 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 neuronalinflammatory responses. For example, Glial Fibrillary Acidic Protein (GFAP) is abundantlyexpressed in astrocytes and is considered a specific marker of central nervous systemdiseases, related to harmful neuronal processes that compromise blood‐brain barrierintegrity. Additionally, the expression of neuropeptide Y (NPY) metabolites,agouti‐related peptide (AgRP), and proopiomelanocortin (POMC) will be measured.