Ethical statement

This study was conducted in accordance with the principles outlined in the Declaration of Helsinki and was approved by the Institutional Review Board of Jinshan Hospital of Fudan University (Approval No. JIEC2021S18). Written informed consent was obtained from all participants prior to their inclusion in the study. The ethical considerations were meticulously adhered to, ensuring the rights, safety, and well-being of all participants were protected throughout the research process.

Study design and participants

This study employed a case-control design to investigate the association between the rs4354668 polymorphisms in the EAAT-2 gene, EAAT-2 mRNA expression, EAAT-2 protein concentration, and plasma glutamate levels in patients with CNP following HZ infection. The study was conducted between March 20, 2021, and March 2, 2022, and involved 102 consecutive patients who were clinically diagnosed with HZ and associated pain.

Participants were recruited from the dermatology department and were divided into two groups: the CNP group (n = 51), which included patients who had experienced CNP for at least three months, and the acute pain (ACP) group (n = 51), which included patients who had experienced acute pain for less than three months following the onset of HZ. The inclusion criteria for both groups were: (1) a clinical diagnosis of HZ according to the International Classification of Diseases, 10th Revision (ICD-10), and (2) the ability to undergo a numerical rating scale (NRS) pain assessment. Exclusion criteria included: (1) the presence of concurrent autoimmune diseases, (2) concurrent tumors or other acute or chronic pain conditions, (3) severe liver, kidney, or cardiovascular diseases, and (4) inability to complete pain assessments.

Pain assessment

Pain severity was assessed using the NRS, a validated tool for quantifying pain intensity. The NRS scores range from 0 to 10, with 0 representing no pain and 10 representing the worst pain imaginable. Pain characteristics were documented for all participants (including continuous and/or episodic burning, stabbing, or electric shock-like pain, touch-induced pain, and hypersensitivity). The duration and peak time of pain were also recorded, with the duration of pain defined as the time elapsed since the onset of pain, and peak time defined as the time taken to reach maximum pain intensity.

Blood sample collection and processing

Fasting venous blood samples (4 mL) were collected from each patient in the morning. The blood was drawn into anticoagulant tubes and immediately stored at – 80 °C until further analysis. Peripheral blood mononuclear cells (PBMCs) were isolated from the collected blood samples for subsequent DNA and mRNA analyses. Plasma was used for protein and glutamate analyses. All blood samples were collected within 7 days of the onset of HZ.

EAAT-2 genotyping analysis

DNA was extracted from PBMCs using the standard phenol-chloroform extraction method. Polymerase chain reaction (PCR) was performed to amplify the rs4354668 polymorphism region of the EAAT-2 gene. The following primers were used: forward primer 5’-CTGCCACCTGTGCTTTGCT-3’ and reverse primer 5’-GAGGGATCGCCTGCAAATCC-3’. The amplified DNA fragments were subjected to restriction fragment length polymorphism analysis to identify EAAT-2 genotypes. The PCR products were digested with the appropriate restriction enzyme, and the resulting fragments were separated by gel electrophoresis to determine the genotype.

EAAT-2 mRNA expression analysis

Total RNA was extracted from PBMCs using TRIzol reagent according to the manufacturer’s instructions. The quality and concentration of the extracted RNA were assessed using a spectrophotometer. Reverse transcription of the RNA into complementary DNA (cDNA) was performed using a commercially available reverse transcription kit. Quantitative real-time PCR (qPCR) was then conducted to measure EAAT-2 mRNA levels using specific primers designed for EAAT-2. GAPDH was used as the reference gene to normalize the expression levels. The relative expression of EAAT-2 mRNA was calculated using the 2^−ΔCt method. The threshold cycle (Ct) values of EAAT-2 and GAPDH were first recorded. Then the difference between EAAT-2 and GAPDH Ct values was calculated as ΔCt. Last, 2^−ΔCt was calculated to represent the mRNA levels of EAAT-2.

EAAT-2 protein expression analysis

To assess EAAT-2 protein levels, plasma was separated from blood samples by centrifugation at 3,000 rpm for 10 min. The plasma EAAT-2 protein levels were quantified using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (manufacturer: Abnova, catalog number: abx494583), following the manufacturer’s protocol. Briefly, plasma samples were added to wells coated with an anti-EAAT-2 antibody, followed by the addition of a secondary antibody conjugated to an enzyme. After the addition of a substrate solution, the enzyme reaction produced a colorimetric signal proportional to the amount of EAAT-2 protein present in the sample. The absorbance was measured at 450 nm using a microplate reader, and EAAT-2 protein concentrations were calculated based on a standard curve.

Plasma glutamate measurement

Plasma glutamate levels were measured using an ELISA kit (manufacturer: Abbexa, catalog number: KA1909) specifically designed for the quantification of glutamate. Plasma samples processing and ELISA procedure were performed in the similar manner as for the EAAT-2 protein analysis.

Statistical analysis

Statistical analyses were performed using R software version 4.3.0. Descriptive statistics, including mean ± standard deviation, and frequency distributions, were used to summarize the demographic and clinical characteristics of the study participants. After normal distribution test, independent-samples t-tests or Mann-Whitney U test were used to compare continuous variables between the CNP and ACP groups. Chi-square tests were employed to assess differences in categorical variables. Pearson’s correlation coefficient was used to evaluate the relationships between EAAT-2 mRNA expression and protein concentration and plasma glutamate level. Statistical significance was set at p < 0.05.

A power analysis was conducted to ensure that the sample size was adequate to detect significant differences between the groups with sufficient statistical power. The analysis indicated that a sample size of 25 per group was sufficient to detect a moderate effect size with a power of 0.80 at an alpha level of 0.05.