Association Between PM2.5 Exposure and Ferroptosis in Seizures Patients

Association Between PM2.5 Exposure and Nrf2-depended Ferroptosis Changes in Seizures Patients

This cohort study aims to discover the possible effects of PM2.5 exposures on the Nrf2- dependent ferroptosis pathway in seizure patients. This observational cohort's main question is whether PM2.5 exposures will affect the Nrf2- dependent ferroptosis pathway in seizure patients.

Participants will be divided into two groups: the control group and the air pollution to detect the biomarkers of the Nrf2- dependent ferroptosis pathway in seizure patients

Background: Despite the exact mechanism of the effects of air pollution (PM2.5) on the central nervous system is still under debate. There is increasing evidence that air pollution (PM2.5) will affect neurodegenerative diseases including epilepsy. It is well known that air pollution (PM2.5) will increase the level of proinflammatory cytokine released by microglia, and advanced research indicated the level of interleukin-6 will affect epilepsy symptoms. In addition, our previous research indicated that Nrf2 plays a crucial role in seizure symptoms and cognitive function (NCT05269901). However, whether PM2.5-induced proinflammatory cytokine released by microglia will change Nrf2- dependent ferroptosis to facilitate seizure symptoms and cognitive function is still unexplored. Therefore, this pilot cohort study aims to discover the potential association between PM2.5 exposures and the changes in the interleukin-6 level and Nrf2- dependent ferroptosis pathway

Methods:

1. The investigators first enrolled 30 low PM2.5 exposure epileptic children and aged-matched high PM2.5 exposure children who were recently diagnosed with epilepsy (< 2 months)from two cities (Yangzhou and Xuzhou) as the control and air pollution group based on previous PM2.5 records;
2. Recorded the PM2.5 level in two cities every 5 days between 2022.12 and 2023.3 to compare the difference;
3. Interleukin-6 ELISA（IL6） KIT was used to compare the proinflammatory cytokine level in two groups
4. 4 Hydroxynonenal （4HNE） ELISA KIT was used to compare the lipid reactive oxygen species level in two groups
5. Real-Time Quantitative PCR（RT-qPCR） was used to compare messenger RNA (mRNA) expression of the Nrf2-dependent ferroptosis pathway (Nrf2, SLC7A11, GPX4) Detailed Methods protocol

1. PM2.5 level source: the PM2.5 levels were recorded via the data from the Ministry of Ecology and Environment of the People's Republic of China (air.cnemc.cn:18007/) 2. Sample collection: At the end of this study, the enrolled participant will come to the hospital to collect peripheral Blood (Affiliated Hospital of Jiangnan University and Xuzhou Children's Hospital, respectively), and the blood samples were collected into ethylene diamine tetraacetic acid（EDTA） vacutainer tubes and then divided into leukocytes and plasma for further experiments 3. Interleukin-6 and 4HNE: Plasma IL-6 and 4HNE concentrations were detected by the competitive ELISA method 4. RTqPCR sequence: h-GPX4 forward: 5'-GAGGCAAGACCGAAGTAAACTAC-3' h-GPX4 reverse: 5'-CCGAACTGGTTACACGGGAA-3' h-SLC7A11 forward: 5'-TCTCCAAAGGAGGTTACCTGC-3' h-SLC7A11 reverse: 5'-AGACTCCCCTCAGTAAAGTGAC-3' h-Nrf2 forward5'-ATAGCTGAGCCCAGTATC-3' h-Nrf2 reverse 5'- CATGCACGTGAGTGCTCT-3' h-GAPDH forward: 5'-GGAGCGAGATCCCTCCAAAAT-3' h-GAPDH reverse: 5'-GGCTGTTGTCATACTTCTCATGG-3'

Inclusion Criteria:

1. Recently diagnosed with epilepsy (< 2 months)
2. living in this city for more than two years
3. no exposure to any chemistry pollution before

Exclusion Criteria:

1. abnormal IL-6 level before starting this pilot cohort
2. other neurological disorders except for epilepsy;
3. endocrine diseases;
4. cancer
5. receive anti-proinflammatory cytokines therapy