2.1. Clinical study
The clinical study was carried out as outlined in Fig 1 (Fig 1.).
2.1.1. Participant selection
Inclusion criteria
- Han nationality, male, 18~45 years old
- Group HACE: Diagnosed with HACE within two weeks of moving from the low to middle altitude area (<1000 m) to the Maduo area (4500 - 5000 m) of Qinghai Province, with no less than 20 cases.
- Group QH: Long-term residence in the Maduo area for more than 3 years, without leaving the place of residence for six months before enrollment, with no less than 80 cases.
- Group HH: Long-term residence in Sanya city (0-200 m) of Hainan Province for more than 3 years, without leaving the place of residence for six months before enrollment, with no less than 80 cases.
Exclusion criteria
- Clinically diagnosed with a malignant tumor, cor pulmonale, hepatitis, nephritis, immune disease, and serious infection.
2.1.2. Clinical information and sample collection
We recorded age, sex, and standard blood test results. After the clinical blood tests, residual EDTA anticoagulated blood was collected for plasma Zn, CA1, and HIF-1a level detection. For the RNA-Seq test, peripheral blood was drawn from 6 HACE patients, along with 6 QH and 6 HH healthy individuals matched by age with the HACE patients. Blood was collected using PAXgene Blood RNA Tubes (Becton, Dickinson and Company, USA, 762165), 2.5 mL/tube, 2 tubes per individual. Blood samples from the QH and HH groups were taken during their health check-ups, while for HACE patients, samples were collected within 4 hours post-diagnosis.
2.1.3. RNA-Seq and data analysis
The RNA-seq analysis (comprising 6 samples each from HACE, QH, and HH groups) was conducted by Hangzhou DiAn Biotechnology Co., Ltd. The procedural synopsis is as follows: Total RNA was isolated from these peripheral blood samples utilizing the PAXgene Blood miRNA Kit (Becton, Dickinson and Company, USA, 76216). Subsequently, libraries were constructed employing the SMART-RNAseq Library Prep Kit (Hangzhou Kaitai Biotech, China, AT4201). After validation and pooling, the sequenced libraries were run through a NovaSeq 6000 (Illumina, USA). The sequencing results were then interpreted via HISAT2, Stringtie, HTSeq, and edgeR software. Gene expression magnitudes were appraised using the Fragments per Kilobase per Million Mapped Fragments (FPKM) methodology. By juxtaposing the FPKM values of genes across the HACE, QH, and HH groups in a pairwise manner, differentially expressed genes (DEGs) were filtered employing parameters |log2FC| > 2 and padj < 0.05.
Furthermore, the principal component analysis (PCA) was conducted for the three groups. The Gene Ontology (GO) term enrichment analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the volcano map, and the heatmap of DEGs were executed using OmicShare, an online real-time interactive data analysis platform with significance set at p-value < 0.05 (http://www.omicshare.com).
2.1.4. Measurement of plasma zinc, CA1, and HIF-1α
Blood samples were centrifuged at 3000 rpm for 10 minutes at 4°C to isolate the plasma. The assays employed for analysis included the Blood Zinc Content Assay Kit (BC2815, CHN, Solarbio), Human Carbonic Anhydrase 1 (CA1) ELISA kit (CSB-EL004364HU, CHN, Cusabio), and Human Hypoxia-Inducible Factor 1α (HIF-1α) ELISA kit (CSB-E12112h, CHN, Cusabio). These assays were meticulously executed in adherence to the guidelines specified within their respective user manuals. Upon completion of the assays, quantification of analytes was performed using an enzyme-linked immunosorbent assay reader (Infinite M Nano, CHL, Tecan).
2.2. In vitro studies
2.2.1. Cell culture and model construction
Human brain microvascular endothelial cells (HBMECs) were procured from Fenghui Biotech (CL0116) and cultivated in endothelial cell-specific medium (ScienCell, USA, 1001) within fibronectin-coated (F8180, CHN, Solarbio) dishes or plates, under 5% CO2 and 37 ℃. Experimental procedures commenced once the cells progressed to the fourth passage and achieved approximately 80% confluence. Initially, a range of final concentrations (0, 2, 4, 6, 8, 10, 12, 16, 20, 40, 100 μM) of Zn2+ (zinc gluconate, C12H22O14Zn, XW44680241, Vocay, China) were administered to the culture medium.
Subsequently, the cells were segregated into four distinct groups, designated Con, Zn, Hy, and Hy+Zn. The Con group was cultivated under normoxic conditions (ambient 16-17% O2, 5% CO2) for 24 hours, while the Zn group received Zn supplementation at a specific concentration and was maintained under identical normoxic conditions as the Con group. Conversely, the Hy group was exposed to hypoxic conditions (ambient 8-8.5% O2, 5% CO2), and the Hy+Zn group was supplemented with Zn while being cultured under the identical hypoxic conditions as the Hy group.
2.2.2. Cell biology characterization
CCK8 assay: The CCK-8 kit (CA1210, CHN, Solarbio) was used following the manufacturer's instructions to measure the optical density (OD) values of each cell group. The proliferation rate was then calculated using the following formula: Proliferation Rate = (OD experimental - OD blank)/(OD control - OD blank) × 100%.
Acridine orange/ethidium bromide staining (AO/EB): The four groups of cells were stained using the AO/EB Staining Kit (GS0267, CHN, Biorab). AO permeates living cells, binding to the nucleus and emitting green fluorescence. EB only permeates damaged cell membranes, binding to the nucleus and emitting red fluorescence. After staining with AO/EB, the cells were observed under a fluorescence microscope.
Scratch assay: Using a marker, five equidistant parallel lines, spaced 5 mm apart, were delineated on the reverse side of the cell culture plate. Cells were cultivated until they reached confluence. The standard culture medium, containing 10% fetal bovine serum, was then substituted with a low-serum medium, containing 1% fetal bovine serum. Subsequently, two parallel lines, spaced 5 mm apart, were inscribed on the cell surface, oriented perpendicularly to the preexisting lines. As outlined in section 2.1.1., cells were divided into respective groups and cultured accordingly.
Flow cytometry: After 24 hours of cell culture, cells were collected and stained with the ANNEXIN V-FITC/PI apoptosis detection kit (CA1020, CHN, Solarbio), and subsequently analyzed using a flow cytometer (Navios, USA, Beckman).
2.2.3. Western blot
Total cellular proteins were extracted using the Membrane and Cytosol Protein Extraction Kit (P0033, CHN, Beyotime) and supplemented with a protease and phosphatase inhibitor cocktail (P1048, CHN, Beyotime). Protein concentrations were equalized using SDS-PAGE protein sample buffer (P0015, CHN, Beyotime). Gels were prepared using the SDS-PAGE Gel Quick Preparation Kit (P0012AC, CHN, Beyotime) and loaded with samples and a Prestained Protein Molecular Weight Marker (6.5-270 kD) (P0072, CHN, Beyotime). Electrophoresis and blotting were performed using an electrophoresis and blotting system (1658033, USA, Bio-Rad). Immunoblotting and visualization were carried out using specific primary and secondary antibodies (details in supplementary materials), Western blotting auxiliary reagents (P0023, containing blocking solution, washing solution, primary and secondary antibody diluents), and an ECL chemiluminescence kit (P0018), all purchased from Beyotime (China). The procedure was executed according to the kit's instructions. The results were captured using the SYSTEM GelDoc XR+ IMAGELAB (Bio-Rad, USA) and subjected to grayscale value analysis.
2.2.4. Multicolor fluorescent immunocytochemistry
The unlabeled primary antibodies, CA1 (1:200, PA5-19177, USA, Thermo Fisher) and SLC39A2/ZIP2 (1:200, PA5-99743, USA, Thermo Fisher), were diluted using antibody dilution solution (A1800, CHN, Solarbio) and stored at 4℃ for up to one week. Cells were cultured and treated on multiwell chamber slides (07-2104, CHN, BIOLOGIX). After the treatment, the medium was aspirated, and cells were washed with 4℃ PBS, followed by fixation in 4 ℃ acetone for 30 minutes. Then the cells were washed with PBS and blocked with 2.5% normal chicken serum (S9080, CHN, Solarbio) diluted in PBS at 25 ℃ for 20 minutes. Subsequently, the cells were immersed in working solutions of CA1 and ZIP2 and incubated at 37 ℃ for 1 hour. This was followed by thorough washing with PBS. FITC-labeled SLC30A1/ZNT1 (1:100, LMAI Bio, CHN, LMAI Bio) was diluted using fluorescent antibody dilution solution (A1840, CHN, Solarbio) and mixed with Alexa Fluor 594 labeled chicken anti-rat IgG (1:500, A-21471, Thermo Fisher) and Alexa Fluor 647 labeled Chicken anti-Goat IgG (1:500, A-21469, Thermo Fisher). The cells were completely submerged in this mixture and incubated at 37℃ for another hour, followed by washing with PBS. The plastic container was then removed, and a drop of anti-fade mounting solution containing DAPI (S2135, CHN, Solarbio) was added to the slide. The slide was stored in a dark, room-temperature environment and examined and imaged under a microscope within 4 hours.
2.3. Statistical analysis
Quantitative data are represented by the median (25th percentile, 75th percentile). The data were analyzed using SPSS 26.0 software (USA, IBM). Clinical sample variances were examined either by one-way ANOVA (for data conforming to a normal distribution) or by the Kruskal-Wallis test (for nonnormally distributed data). The Spearman correlation analysis was utilized to assess the correlation between various clinical sample indicators. Differences in cellular sample indicators were analyzed using the t-test. A p-value of less than 0.05 was considered statistically significant.