Animal study design
All animal experiments were conducted following the guidelines of animal welfare and approved by the Animal Care and Use Committee of Guangzhou Medical University (GY2023-108).
In the first acute experiment, mice aged 6–8 weeks were purchased from GemPharmatech Co., Ltd., China and raised at the Guangzhou Medical University Laboratory Animal Center. The mice were randomly divided into saline and DOX groups. The DOX group was injected intravenously with 10 mg/kg of DOX (S1280, Selleck, China). Different groups of mice were harvested at 8, 16, 24, 48, and 72 h after injection. The saline group was injected intravenously with the same dosage of saline.
In the second chronic STING inhibitor C-176 experiment, C57BL/6 male mice aged 6–8 weeks were randomly divided into Saline + Corn Oil, Saline + C-176, DOX + Corn Oil, and DOX + C-176 groups. Mice were injected intravenously with 5 mg/kg of saline or DOX once a week and injected intraperitoneally with 10 mg/kg of corn oil or C-176 (S6575, Selleck, China) dissolved in DMSO and diluted in corn oil at the same time. After 4 weeks, the treatment was stopped for 1 week.
In the third chronic Sting1 siRNA experiment, Sting1 siRNA (5′-GGAGCCGAAGACTGTACAT-3′) was purchased from Guangzhou RiboBio Co., Ltd. C57BL/6 male mice aged 6–8 weeks were randomly divided into NC + NS, NC + DOX, and Sting1 siRNA + DOX groups. Mice were injected intravenously with 5 mg/kg of NS or DOX once a week and injected intravenously with 10 nmol NC or Sting1 siRNA twice a week before DOX treatment.
Echocardiography
Before treating with DOX, mice were subjected to transthoracic echocardiography (Vevo 2100 Imaging System, VisualSonics, Canada) to obtain baseline data. Then, every week before injecting, mice were also subjected to echocardiography to obtain cardiac function data. The body temperature of mice was maintained at 36.9°C–37.3°C, while the heart rate was maintained at 550–650 bpm. Echocardiographic M-mode tracing records were used to measure cardiac structure, and at least five cardiac cycles were utilized to calculate the left ventricular EF (LVEF) and the left ventricular FS (LVFS). The measurement application was Vevo Lab.
Histological and immunofluorescence analyses
One week after the DOX treatment for 4 weeks, mice were sacrificed, and hearts were fixed with 4% paraformaldehyde for 24 h at 4°C, dehydrated with a gradient concentration of ethanol, embedded with paraffin, and sectioned. Tissue sections were stained with Masson’s trichrome immunohistochemical reagent and Picro-Sirius Red Solution to analyze cardiac fibrosis. To observe inflammatory cell infiltration, immunohistochemistry analysis was performed on the above paraffin sections with IL-1β antibody (11E5; Santa Cruz Biotechnology; Cat#sc-52012; 1:100).
Mice were harvested at the end point of the experiment, and hearts were fixed with 4% paraformaldehyde for 24 h at 4°C, dehydrated in 30% sucrose solution for 24 h at 4°C, embedded with OCT, and sectioned to acquire 5 µm frozen sections for immunofluorescence analysis. The above frozen sections were blocked and permeabilized for 45 min in a blocking buffer, comprising 1% bovine serum albumin and 0.3% Triton X-100 in PBS. Slides were incubated with primary antibodies diluted in the blocking buffer for 12 h at 4°C and with secondary antibodies diluted in the blocking buffer for 45 min at 37°C. A DAPI staining solution (Beyotime; Cat#C1006) was used for staining for 10 min at room temperature. The following antibodies were applied: anti-phosphor-Histone H2A.X (Ser139) rabbit antibody (20E3; Cell Signaling Technology; Cat#9718; 1:100), anti-cGAS rabbit antibody (D3O8O; Cell Signaling Technology; Cat#31659; 1:100), anti-phosphor-STAT1 (Ser727) rabbit antibody (SN67-04; HUABIO; Cat#ET1611-20; 1:100), anti-NF-κB rabbit antibody (D14E12; Cell Signaling Technology; Cat#8242T; 1:100), goat anti-rabbit IgG DyLight 488 (Abbkine; Cat#A23220; 1:200), goat anti-mouse IgG DyLight 488 (Abbkine; Cat#A23210; 1:200), goat anti-rabbit IgG DyLight 594 (Abbkine; Cat#A23420; 1:200), and goat anti-mouse IgG DyLight 594 (Abbkine; Cat#23410; 1:200).
Western blot assay
Heart tissue and cells were collected with RIPA lysis buffer (Thermo Fisher Scientific; Cat#89901) containing protease and phosphatase inhibitor (Thermo Fisher Scientific; Cat#78442), fully ground, and vortexed for complete total protein lysis. The protein was separated on 8–15% SDS-polyacrylamide gels by electrophoresis and transferred to 0.45 µm PVDF membranes. After blocking with 5% nonfat milk diluted in TBST for 45 min at room temperature, the membranes were incubated with primary antibodies diluted in a blocking buffer for 12 h at 4°C and then incubated with an HRP-conjugated secondary antibody diluted in a blocking buffer for 45 min at room temperature. SuperSignal West Pico PLUS (Thermo Fisher Scientific; Cat#34580) was used to detect by Amersham Imager 600. The following antibodies were used: anti-phosphor-Histone H2A.X (Ser139) rabbit antibody (20E3; Cell Signaling Technology; Cat#9718; 1:1000), anti-Histone H2A.X rabbit antibody (D17A3; Cell Signaling Technology; Cat#7631; 1:1000), anti-cGAS rabbit antibody (D3O8O; Cell Signaling Technology; Cat#31659; 1:1000), anti-cGAS rabbit antibody (HUABIO; Cat#HA500023; 1:1000), anti-phosphor-TMEM173/STING (Ser366) rabbit antibody (Affinity Biosciences; Cat#AF7416; 1:1000), anti-STING rabbit antibody (D2P2F; Cell Signaling Technology; Cat#13647; 1:1000), anti-phosphor-TBK1/NAK (Ser172) rabbit antibody (D52C2; Cell Signaling Technology; Cat#5483; 1:1000), anti-TBK1/NAK rabbit antibody (E813G; Cell Signaling Technology; Cat#38066; 1:1000), anti-phosphor-IRF3 (Ser396) rabbit antibody (D6O1M; Cell Signaling Technology; Cat#29047; 1:1000), anti-IRF3 rabbit antibody (D83B9; Cell Signaling Technology; Cat#4302; 1:1000), anti-phosphor-STAT1 (Ser727) rabbit antibody (SN67-04; HUABIO; Cat#ET1611-20; 1:1000), and anti-STAT1 rabbit antibody (HUABIO; Cat#ET1606-39; 1:1000).
Nucleocytoplasmic separation
Heart tissue was ground with a grinding rod in CER Ⅰ reagent in a nucleocytoplasmic separation kit (Thermo Fisher Scientific; Cat#78833), then CER Ⅱ was added in tubes. After vortex and centrifugation, the supernatant was aspirated, and cytoplasm protein was extracted and collected. The precipitate was washed in PBS, and NER reagent was added into tubes. After vortex and centrifugation, nucleus protein could be extracted. Afterward, the protein expression levels could be detected by Western blot.
Cell isolation, culture, and stimulation
Cardiac fibroblasts were isolated by digesting neonatal SD rat ventricular tissue with Trypsin without EDTA (Thermo Fisher Scientific; Cat#15050065) and type II collagenase (MP Biomed; Cat#100502), filtrated through a 70 µm cell strainer, and purified by differential anchoring velocity. Primary cardiac fibroblasts were resuspended in high glucose DMEM with 10% FBS for 24 h and passaged to the second generation.
DOX powder was dissolved in sterile water and diluted to 1 mM. The STING inhibitor C-176 was dissolved in DMSO and diluted to 1, 2, and 4 mM. When the cell density reached 70% of the dish, cells in the logarithmic growth phase were treated with 1 µM DOX for 2, 4, 8, 16, and 24 h. To inhibit the pathway, 2 µM DOX and 1, 2, and 4 µM STING inhibitor C-176 were used for different groups of cardiac fibroblasts; two control groups for DMSO and 4 µM C-176 were also set up to control the influence of dissolvent.
When the cell density of cardiac fibroblasts reached 50% of the dish, Sting1 siRNA (F: 5′-CAACAGUGUCUAUGAACUUTT-3′; R: 5′-AAGUUCAUAGACACUGUUGTT-3′) was transfected into cardiac fibroblasts by transfection reagent RNA iMAX (Thermo Fisher Scientific; Cat#13778030). After transfecting in Opti-MEM (Gibco; Cat#31985070) without FBS for 6 h, cells were continuously cultured in a high glucose DMEM culture medium with 10% FBS for 16 h and then treated with 1 µM DOX for 8 h.
The above cells were collected in RIPA, and the cell crawling slides were fixed with 4% PFA and then kept at − 20°C.
Statistical analysis
All data are expressed as mean ± standard error of the mean (mean ± SEM). Data were analyzed with GraphPad Prism 9.0 software (GraphPad, San Diego, CA). The student t test (2 tailed) was performed to compare 2 groups. If SD un-equation with Welch’s correction. One-way ANOVA followed by the Tukey post hoc test was used to compare multiple groups. A value of P < 0.05 was considered significant.