All protocols in this study were approved by the Committee on the Ethics of Animal Experiments of Yangzhou University, in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no.85 − 23, revised 1996). The study was performed in accordance with ARRIVE guidelines too.
This study mainly used the following reagents: HCAECs purchased from Shanghai Zeye Biotechnology(Shanghai,China).Fetal bovine Serum (FB55011) were bought from ClarkBioscience(Virginia,USA).Anti-β-actin(#4970), anti-caspase1 (#24232), anti-NLRP3 (#15101), and anti-ASC (#67824) antibodies were from Cell Signaling Technology (USA), BeyoClick™, propidium iodide (PI; ST511), the Beyotime Reactive Oxygen Species Assay Kit(S0033),Crystal Violet Staining Solution(C0121) were bought from Beyotime Biotechnology. Mounting Medium With DAPI -Aqueous,Fluoroshield (ab104139)was purchased from Abcam(Boston,USA). Hoechst33342/PI apoptosis detection kit (R21807-100) purchased from Shanghai Shangbao Biological, H2O2 staining kit (S0038), cell cycle detection kit (KGA512)were bought from KeygenBiotech(Jiangsu,China). Cell-Light™ Edu Apollo In Vitro Kit was bought from RiboBio(Guangzhou,China).
2.1 Establishment of HCAECs pyroptosis model
The prepared HCAECs were revived and replenished with fresh medium and pyroptosis was initiated with 600 µmol /L H2O2. The relevant experiments were then performed on the cells at the corresponding time points. After exposing the HCAECs to 600 µmol /L H2O2 for 0h, 1h, 2h, 4h and 8h, the LDH levels in HCAECs culture medium were determined using a kit. In addition, the changes in the expression level of pyroptosis-related proteins NLRP3, Caspase1 and ASC, was assessed by Western blot analysis. HCAECs were treated with H2O2 for 8 hours and the levels of ROS in the cells were assessed using DFCH-DA probes.
2.2 Changes in the expression levels of Sestrin2 protein and miR-3160-5p in HCAECs culture medium after treatment with H2O2
HCAECs were incubated with 600 µmol/L H2O2 for different treatment durations (0, 1h, 2h, 4h, 8h) and the expression levels of Sestrin2 protein and miR-3160-5p were determined.
2.3 Establishment of high and low expression models of Sestrin2 in HCAECs
The expression levels of Sestrin2 protein and miR-3160-5p in HCAECs exhibited an inverse relationship under pyroptosis state. Further analysis using gene prediction webservers revealed that Sestrin2 protein can specifically bind to miR-3160, with the corresponding binding sites illustrated in Fig. 1. Consequently, we hypothesized that Sestrin2 protein modulated the level of pyroptosis in HCAECs through its interaction with miR-3160.
The target sequence of Sestrin2 siRNA was identified as GCAGAGACCCATTGAACAA using gene prediction software. We established high expression and low expression models of Sestrin2 protein in HCAECs through lentivirus transfection. Sestrin2, sh-Sestrin2, and Sestrin2-NC were used to represent the high expression group, low expression group, and plasmid control group, respectively. The cells were incubated with puromycin and stable total RNA was extracted from the cells in the Sestrin2, sh-Sestrin2, and Sestrin2-NC groups. The expression level of Sestrin2 protein was assessed by qRT-PCR.
2.4 Determination of LDH levels, expression profiles of pyroptosis-related proteins, and Hoechst33342/PI staining in HCAECs transfected with Sestrin2 protein after treatment with H2O2
The LDH cytotoxicity detection kit was used to assess the changes in LDH levels in the Sestrin2, sh-Esterin2, and Sestrin2-NC groups after treatment of HCAECs with 600 µmol/L H2O2 for 8 hours. Western blot analysis was performed to evaluate the expression levels of pyroptosis-related proteins in HCAECs in the Sestrin2, sh-Sestrin2, and Sestrin2-NC groups. Additionally, Hoechst33342/PI kit was used to determine the number of PI + cells in HCAECs in the Sestrin2, sh Estrin2, and Sestrin2-NC groups. Fluorescence microscopy was used to determine the number of PI + cells in each group. These experiments were conducted to explore the effect of Sestrin2 protein on the pyroptosis of HCAECs after treatment with H2O2.
2.5 qRT-PCR analysis
The changes in miR-3160-5p levels in HCAECs in the Sestrin2-NC, Sestrin2, and sh- Sestrin2 groups were evaluated using qRT-PC to explore whether the Sestrin2 protein modulated the expression of miR-3160-5.
2.6 Dual-luciferase assay
A dual-luciferase assay was performed to explore the potential targeting relationship between miR-3160-5p and the Sestrin2 protein. TargetScan database (http://www.targetscan.org) was used to predict the potential binding sites between Sestrin2 protein and miR-3160-5p (Fig. 1). Subsequently, a reporter gene plasmid containing the Sestrin2 protein sequence was constructed using the Gemma gene. This plasmid was co-transfected with miR-3160-5p mimics or miR-NC into 293T cells then dual-luciferase assays were conducted after 24 hours.
2.7 Construction of HCAEC models with high and low expression of miR-3160-5p and qRT-PCR analysis
miR-3160-5p mimics and miR-3160-5p inhibitor were transfected into HCAECs to construct HCAECs models exhibiting high expression and low expression miR-3160-5p to explore the effect of miR-3160-5p on the pyroptosis of HCAECs.
The forward and reverse primers used in this study are presented below:
forward: 5'-TCAGGGGATGTGAGAGTGTGG-3',
reverse: 5'-TCAGGGGATGTGAGAGTGTGG-3',
β-actin,
forward: 5'-CATGTACGTTGCTATCCAGGC-3',
reverse: 5'-CTCCTTAATGTCACGCACGAT-3'.
miR-NC, miR-3160-5p mimics, and miR-3160-5p inhibitor were used to represent the NC group, high expression group, and low expression group, respectively. qRT-PCR was performed after 24 hours of transfection.
2.8 Assessment of LDH levels in HCAECs transfected with miR-3160-5p under the action of H2O2
The pyrogenic model of HCAECs was constructed by stimulating HCAECs with 600 µM H2O2 for 8 h. LDH cytotoxicity detection kit was used to evaluate the levels of LDH in HCAECs transfected with miR-NC, miR-3160-5p mimics, and miR-3160-5p inhibitor to explore the effect of miR-3160-5p on LDH levels in H2O2-treated HCAECs.
2.9 Changes in Hoechst33342/PI staining of HCAECs transfected with miR-3160-5p after treatment with H2O2
The Hoechst33342/PI assay kit was used to evaluate the number of PI + cells in HCAECs transfected with miR-NC, miR-3160-5p inhibitor, and miR-3160-5p mimics to determine the effect of miR-3160-5p on the pyroptosis of HCAECs treated with H2O2.
2.10 Determination of the expression levels of pyroptosis-related proteins in HCAECs transfected with miR-3160-5p after treatment with H2O2
Western blot was performed to assess the expression profile of pyroptosis related proteins in HCAECs transfected with miR-NC, miR-3160-5p inhibitor, and miR-3160-5p mimics to explore the effect of miR-3160-5p on the expression profile of pyroptosis-related proteins.