2.1. Fundamental Experiment Study
H9C2 cardiomyocytes (rat): American Type Culture Collection, USA; Fetal bovine serum, G1: Gibco, USA; Trypsin, LPS: Solarbio Science & Technology Co., Ltd., China; DMEM culture medium: Invitrogen, USA; TNF-α, IL-6 ELISA kits: Hengyuan Biotechnology Co., Ltd., China; Annexin V/PI apoptosis detection kit: TaKaRa, Japan; PVDF membrane: Millipore, USA; BCA protein assay kit, ECL chemiluminescence kit: Biyun Tian Biotechnology Research Institute, Shanghai, China; NF-κB activator Phorbol 12-myristate 13-acetate (PMA), NF-κB P65 antibody, NF-κB p-P65 antibody, p-IκB-α antibody, IκB-α antibody, Bcl-2 antibody, Bax antibody, β-actin: CST, USA; Horseradish peroxidase-conjugated secondary antibody: Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China.
The H9C2 cardiomyocytes were cultured in DMEM supplemented with 10% fetal bovine serum. The cells were maintained in a humidified atmosphere of 5% CO₂ at 37˚C. Upon reaching 80% confluence, cells in good growth conditions were selected for subsequent experiments.
To prepare an LPS-induced myocardial inflammation model, H9C2 cardiomyocytes were treated with 10 µg/mL LPS for 24 hours, as described in reference
[10]
. The groups consisted of normal control, an LPS group, and three G1 pretreatment groups at concentrations of 1, 3, and 10 μmol/L. The LPS group was incubated with 10 μg/mL LPS for 24 hours. The G1 pretreatment groups were pre-incubated with respective G1 concentrations for 6 hours before adding 10 μg/mL LPS for another 24 hours. To explore G1’s regulatory effects on sepsis-induced myocardial inflammation and apoptosis via the NF-κB pathway, 1 μmol/L PMA was added to the LPS + 3 μmol/L G1 group for 24 hours before assessment.
Log-phase cardiomyocytes were adjusted to a concentration of 1 × 104 cells/mL. Next, 200 μL of the cell suspension was seeded into each well of a 96-well plate and cultured until adhesion. After treatment, as described in the ‘Cell Treatment’ section, 50 μL of CCK-8 solution was added to each well and incubated for 4 hours. The supernatants were removed, and 150 μL/well of DMSO was added, shaking for 10 minutes. The absorbance (OD values) were measured using a microplate reader to calculate the relative cell viability: Relative Cell Viability = (Experimental group OD value/Control group OD value) × 100%.
Cardiomyocytes from each group were collected, lysed on ice for 30 minutes, and centrifuged to obtain supernatants. Protein concentration was quantified using the BCA method. Samples were denatured by heating in the loading buffer. 40 μg of protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk for 2 hours, then incubated with primary antibodies overnight at 4˚C (dilutions: NF-κB p-P65 and P65 at 1:2000, p-IκB-α and IκB-α at 1:2000, Bax at 1:1000, Bcl-2 at 1:1000, β-actin at 1:20,000). Horseradish peroxidase-conjugated secondary antibody was applied at a 1:5000 dilution for 1 hour at room temperature. Detection was performed using ECL reagents, with images captured in a darkroom. Relative protein expression was analyzed using ImageJ software, and normalized to β-actin.
The cell density was adjusted to 1 × 104 cells/ml, and 200 μL of cell suspension was seeded into each well of a 96-well plate. After treatment as described in the ‘Cell Treatment’ section and collection of supernatants, TNF-α and IL-6 levels were measured using ELISA kits, following the manufacturer’s instructions. The absorbance was measured at 450 nm.
After treatment as described in the ‘Cell Treatment’ section, cells were digested with trypsin and washed twice with PBS. Approximately 5 × 105 cells were collected, resuspended in binding buffer, and stained with 5 μL Annexin V-FITC and 5 μL PI for 15 minutes in the dark at room temperature. Apoptosis was analyzed within 1 hour using flow cytometry.
Each experiment was repeated three times, and the data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software version 6.0 (GraphPad, San Diego, CA, USA). Differences between two groups were assessed using the Student’s t-test, while differences among more than two groups were assessed using one-way ANOVA followed by Tukey’s post hoc test. A p-value of <0.05 was considered significantly different.
2.2. Mendelian Randomization Analysis
The study data were obtained from publicly available GWAS summary datasets, which included eQTL (expression quantitative trait locus) data for the GPER gene variations and GWAS data for sepsis (28-day death)
[11]
. The original studies obtained ethical approvals and informed consent. A two-sample Mendelian randomization approach was used to investigate the causal association between GPER gene variations and sepsis (28-day death).
The eQTL data of GPER genes were derived from the IEU Open GWAS database (
https://gwas.mrcieu.ac.uk/
), and the GWAS ID of the dataset was eqtl-a-ENSG00000164850. The criteria for selecting instrumental variables were as follows: Instrumental variables achieving genome-wide significance (P < 5 × 10–8), and ensured minimal linkage disequilibrium (r² < 0.001) within a clump window exceeding 10,000 kb to ensure independence. The LD level was based on the European population-based 1000 Genomes Scheme estimation
[12]
. To ensure a strong association between instrumental variables and the exposure, the F-statistic of SNPs was used to assess the strength of association, with an F-statistic >10 indicating no bias from weak instrumental variables, calculated as F-statistic = (β/SE).
The data on sepsis (28-day death) was obtained from the UK Biobank (UKB) and IEU Open GWAS database (https://gwas.mrcieu.ac.uk/), with the GWAS ID for the dataset being ieu-b-5086. The dataset included genotype data for 1896 sepsis patients and 484588 controls of European descent.
MR analysis was conducted using R software (version 4.1.2,
http://www.R-project.org
) and the Two-Sample MR package (version 0.5.6)
[13]
. The causal relationship between the GPER gene variations and sepsis (28-day death) was assessed using the inverse variance weighted (IVW) method, The IVW method assumes all instrumental variables are valid; any SNP not meeting this assumption introduces bias
[14]
. Additional analyses were performed using the Weighted median and MR-Egger methods. The Weighted median approach requires at least 50% of the SNPs to be valid
[15]
, while MR-Egger regression provides an unbiased estimate even in the presence of pleiotropic instrumental variables
[16]
. The MR-Egger intercept test was used to assess the presence of pleiotropic associations among genetic variants and potential confounders. Significant causal relationship between GPER gene variations and sepsis (28-day death) was considered if: (1) the IVW method showed a significant difference (P < 0.05). (2) Estimates from IVW, Weighted median, and MR-Egger methods were directionally consistent. (3) The MR-Egger intercept test was not significant (P > 0.05)
[17]
. Cochran’s Q test and funnel plots were used to assess heterogeneity among SNPs
[18]
. The leave-one-out method, akin to classical statistical iteration, was applied to determine if the exclusion of any SNP significantly altered the results
[19]
.