NSUN2 alleviates doxorubicin-induced myocardial injury through Nrf2-mediated antioxidant stress

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Animal feeding and model building

All animals were kept on a 12-hour light/dark cycle in a temperature-controlled room with ad libitum access to food and water. The use of animals and all animal protocols have been approved by the Institutional Animal Care and Use Committee (IACUC) of Hubei Medical University. One hundred healthy male C57BL/6J mice aged 810 weeks (weighed 2325 g) were purchased from the Animal Center of Hubei Medical University. Mice were divided into saline group (NS), DOX group, shNC + saline group (shNC NS), shNC + DOX group (shNC DOX), shNSUN2 + saline group (shNSUN2 NS), shNSUN2 + DOX group (shNSUN2 DOX), Control Vector + Salt Group (NS Control), Control Vector + DOX Group (Control DOX), NSUN2 Vector + Salt Group (NSUN2 NS), and NSUN2 Vector + DOX Group (NSUN2 DOX). The shNC group, shNUSN2 group, control group and NSUN2 group were injected into the tail vein with 51011vg corresponding to AAV9 virus, respectively. Four weeks later, the four groups of mice were randomly divided into saline group (NS) and doxorubicin (DOX) group. The NS group received an intraperitoneal injection of 0.9% saline, while the DOX group received 20 mg/kg (diluted to 2 mg/ml with 0.9% saline) of DOX.

Ultrasonic collection of small animals

Three days after treatment with 0.9% saline or 20 mg/kg DOX, mice were anesthetized with 1.02.0% isoflurane. After the respiration of the mice was stable and the heart rate was approximately 400 beats per minute, the B-mode and M-mode ultrasound images of the left ventricular long-axis slice, left ventricular short-axis slice, and of the four-chambered heart were collected by the Vevo 2000 MS-400 probe. The modeler and the experimenter were independent of each other and the experimenter was unaware of the model clustering.

Establishment of the DOX-induced damage model in H9C2 cells

The H9C2 cells were donated by Dr. He [9], and cells in the logarithmic growth phase were plated in 6-well plates. After the cell density reached more than 95%, 0.5 mg/ml DOX or 0.9% saline was added to the medium, respectively, for 24 hours.

Transfection of NSUN2 siRNA

H9C2 cells in the logarithmic growth phase were plated in 6-well plates. When the cells reached 4050% fusion, the 50nM siNC or siNSUN2 mixture was transfected with the lipo 3000 kit according to the operating instructions. After 48 hours, cells were harvested and total RNA or protein was extracted. The siNSUN2 sequences are presented in Table 1.

Table 1 The sequence of NSUN2 siRNA and Nrf2 primers. Transfection of NSUN2-overexpressing lentiviruses

H9C2 cells in the log growth phase were seeded in 6-well plates. When the cell density reached 60%, 2l GFP or GFP-NSUN2 lentivirus solution (WZ Biosciences) was added to the cell medium, respectively. Twenty-four hours later, 2 g/ml puromycin was added to the screen for stably overexpressed NSUN2 cells.

TUNEL detection

Cell apoptosis was detected by TUNEL kit (Beyotime, C1090) and the operation process was performed according to the instructions. After dyeing, 1 liter of DAPI dye solution (Beyotime, C1005) was added and reacted for 510 min at room temperature, then dead cells were observed and photographed with a fluorescence microscope.

Immunohistochemistry

Heart tissues fixed with 4% paraformaldehyde were sectioned continuously at a thickness of 5 m and the slices were processed according to our previous method [10]. Rabbit polyclonal antibodies NSUN2 (Proteintech, 20854-1-AP, 1:200) and Nrf2 (Proteintech, 16396-1-AP, 1:200) were added and incubated at 37°C for 2 hours, then sections were incubated with goat anti-rabbit second antibody (ZSGO-BIO, PV-9001) at room temperature for 1 hour. Finally, the results were tested using the DAB chromogenic kit (ZSGO-BIO, ZLI-9018) according to the instructions for use.

ROS detection

Reactive oxygen species were detected using the ROS kit (Beyotime, S0033S) according to the operating instructions. The cells were observed under a microscope and photographed.

Quantitative real-time PCR (RT-PCR)

Total RNA was extracted from Trizol (Beyotime, R0016). The expression level of Nrf2 mRNA was detected by SYBR Green PCR kit (Beyotime, D7260). The primers have been presented in Table 1.

Cells treated with actinomycin D

The control group and NSUN2-overexpressed H9C2 cells in the logarithmic growth phase were seeded in 12-well plates. When the cell density reached 95%, 2 g/ml of actinomycin D was added for 0, 4, 8, 12, 16 and 24 hours, respectively. Then the expression level of Nrf2 was detected by RT-PCR and the half-life times were calculated.

Western scrub

Cells were lysed with RIPA (Beyotime, P0013B) containing 1% PMSF (Beyotime, ST505). Protein concentrations were determined using the BCA kit (Beyotime, P0010S) according to the instructions, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 20 mg/well. After electrophoresis, proteins were transferred to the membrane NC and rabbit NSUN2 (Proteintech, 20854-1-AP, 1:200), Nrf2 (Proteintech, 16396-1-AP, 1:200), Bax (Beyotime , AF0057, 1:500), Bcl-2 (Beyotime, AB112, 1:500), HO-1 (Beyotime, AF1333, 1:500), NQO1 (Beyotime, AF7614, 1:500), and polyclonal antibody -Tubulin Rabbit (Beyotime, AF5012, 1:500) were incubated overnight at 4°C. Then horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:2000) was added and incubated at room temperature for 1 hour. Finally, the protein concentration was measured using the Enhanced Chemiluminescence (ECL) Chemiluminescence Kit (Beyotime, P0018).

m5C MeRIP

Total RNA of H9C2 cells was extracted with Trizol and divided into IgG and m5C groups, and 7 l of IgG or m5C antibody was added to the two groups, respectively, followed by overnight incubation at 4°C. The next day, 50 L of magnetic beads were added and the mixtures were incubated in a shaking table at 4°C for 6 hours. After centrifugation at 12,000 rpm at 4°C for 2 minutes, the supernatant was discarded and 1 ml of RIPA containing 1% PMSF was added. Then, the RNA was extracted with Trizol and the m5C Nrf2 mRNA level was detected by RT-PCR.

Dot stain

Total RNA was diluted to 200, 400, 600 and 800 ng/l with DEPC water and denatured at 95°C for 10 minutes. One microliter of denatured RNA was transferred to NC films, then the NC films were crosslinked with ultraviolet light at 500 J for 2 minutes. The remaining steps were the same as for the Western blot.

statistic analysis

Graphpad Prism version 9.0.0 and SPSS 25.0 were used to analyze the data. All data were shown as mean standard deviation and the samples were in the normal distribution. Student’s t-test was used for comparisons between the two groups. The survival curve was tested by KaplanMeier. P<0.05 was considered as statistical significance.

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