Specific pathogen free (SPF) healthy male C57BL/6 mice (n = 40, weight 22 $\pm$ 2 g, 8–10 weeks) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China). The experiments were conducted in accordance with the principles approved by the Animal Care and Use Committee of the Affiliated Hospital of Jining Medical University (Permission number: 2021C104). All animal procedures conformed to the US of Health Guidelines for the Care and Use of Laboratory Animals. Mice were kept in a temperature- and humidity-controlled environment (22 $\pm$ 2 °C and 12 h dark/light schedule) with free access to chow and water. One week after acclimation, the mice were divided into four groups (N = 10 per group) using a random number table method. According to published articles and our preliminary experiments [30], mice in the DOX-treated group were intraperitoneally injected with DOX at 2 mg/kg every alternate day with a cumulative dosage of 28 mg/kg. Mice in the control group (CON) received an intraperitoneal injection of an equivalent volume of saline. The AS-IV treatment groups were intragastrically administered 40 mg/kg AS-IV (dissolved in saline) daily for 4 weeks after the first injection of DOX, according to previous experiments [31]. The AS-IV group was given only the same dose of AS-IV to eliminate the effect of AS-IV alone on the results. Mice were sacrificed by over-anesthesia with 100 mg/kg sodium pentobarbital (P3761, Sigma-Aldrich (Shanghai) Trading Co.Ltd., Shanghai, China) after 4 weeks. The bodyweight of the mice was recorded, and blood was obtained from the carotid artery of mice. DOX (D8740) was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China), and AS-IV (T2973) was purchased from TOPSCIENCE Biotech Co., Ltd. (Shanghai, China). The purity of DOX was 99%, and the purity of ASIV was 99.29%.

The cardiac function of mice was assessed after 4 weeks. Mice from the four groups were anesthetized with 1–2% isoflurane (1349003, Sigma-Aldrich (Shanghai) Trading Co.Ltd., Shanghai, China), and transthoracic echocardiography was performed using an animal-specific ultrasound imaging system with a 40 MHz transducer (M9Vet, Mindray, Shenzhen, China). Mice were placed supine and fixed on a board, the parasternal long axis was taken, and M-mode images were obtained at the inferior border of the mitral papillary muscle. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal dimension at end-diastole (LVIDd), and left ventricular internal dimension at end-systole (LVIDs) were measured and calculated to evaluate cardiac function.

Blood samples were collected from the carotid artery after over-anesthesia. Serum was obtained after centrifugation of whole blood. According to the instructions of each kit, ELISA was performed to detect the serum concentration of the heart failure marker brain natriuretic peptide (BNP, E-EL-M0204c, Elabscience Biotechnology Co., Ltd., Wuhan, China), the myocardial injury markers lactate dehydrogenase (LDH, 12239, MEIMIAN Industrial Co., Ltd., Yancheng, China), creatine kinase isoenzyme (CK-MB, E-EL-M0355c, Elabscience Biotechnology Co., Ltd., Wuhan, China), cardiac troponin I (cTnI, E-EL-M1203c, Elabscience Biotechnology Co., Ltd., Wuhan, China), the inflammatory markers IL-1$\beta$ (ab197742, Abcam, Cambridge, UK) and IL-18 (ab216165, Abcam, Cambridge, UK), and the oxidative stress markers superoxide dismutase (SOD, JL12237, j&l Biological, Shanghai, China), malondialdehyde (MDA, JL13329, j&l Biological, Shanghai, China), and glutathione (GSH, JL20360, j&l Biological, Shanghai, China). Absorbance was measured at 450 nm using a microplate reader (BioTek Cytation 5, American Berten Instrument Co., Ltd., Santa Clara, CA, US).

Mice were sacrificed after 4 weeks of DOX and AS-IV treatment. The heart of each mouse was removed and the weight was recorded. Cardiac tissue was rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for at least 48 h, and embedded in paraffin. The slices were cut into 5 $\mu$m sections and stained with HE, Masson’s trichrome, and wheat germ agglutinin (WGA) as previously described [31, 32]. Five random fields of cardiac tissue were imaged using a microscope (Nikon Eclipse E100, Nikon, Tokyo, Japan). ImageJ software version 1.8.0 (National Institutes of Health, Bethesda, MD, USA) was used to evaluate myocardial fibrosis and the cross-sectional areas of the cardiomyocytes ($\ge$2 random areas in $\ge$3 mice/group).

Fresh myocardial tissue was dissected into small pieces of approximately 1 mm${}^3$ and fixed in 2.5% glutaraldehyde (pH 7.4) for 2 h at room temperature (RT). After rinsing with PBS, the tissues were then fixed in 1% osmic acid at 4 °C for 2 h. The samples were then dehydrated in gradient ethanol and embedded in Epon-Araldite resin. Ultrathin sections were prepared and collected for microstructure analysis and then observed using a HITACHI transmission electron microscope.

To determine the expression of NLRP3, caspase-1, and GSDMD in the heart, immunohistochemical analysis was performed using anti-NLRP3 (GB11300, Servicebio, Wuha, China), anti-caspase-1 (sc-56036, Santa Cruz, Dallas, TX, USA), and anti-GSDMD (sc-81868, Santa Cruz, Dallas, TX, USA) antibodies as previously described [33]. Briefly, after dewaxing and antigen repair, the paraffin sections were placed in 3% hydrogen peroxide for 25 min at RT. The sections were subsequently incubated with primary antibodies overnight (16–18 h) at 4 °C and secondary antibodies at RT for 50 min. The sections were stained with diaminobenzidine (G1211, Servicebio, Wuha, China) and hematoxylin (G1004, Servicebio, Wuha, China). After sufficient drying, the slides were photographed and evaluated using the ImageJ software.

Heart tissues were fully dissolved in RIPA buffer (P0013B, Beyotime, Shanghai, China) containing protease inhibitor (P1045, Beyotime, Shanghai, China) and phosphatase inhibitor (P1045, Beyotime, Shanghai, China). After examination of protein concentrations using a BCA kit (P0010, Beyotime, Shanghai, China), 30 $\mu$g of protein samples were separated using 12.5% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with protein-free rapid blocking solution (1$\times$) at RT for 30 min, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies used were NLRP3 (ab263899, Abcam, Cambridge, UK), ASC (#67824, CST, Boston, MA, USA), caspase-1 (A0964, ABclonal, Wuhan, China), GSDMD (ab219800, Abcam, Cambridge, UK), GSDMD-N (#10137, CST, Boston, MA, USA), cleaved caspase-1 (#89332, CST, Boston, MA, USA), IL-1$\beta$ (ab283818, Abcam, Cambridge, UK), IL-18 (ab191860, Abcam, Cambridge, UK), Nrf-2 (A1244, ABclonal, Wuhan, China), pNrf-2 (#DF7519, Affinity Biosciences, Jiangsu, China), HO-1 (#82206, CST, Boston, MA, USA), and $\beta$-actin (#4970, CST, Boston, MA, USA). The bands were visualized using a sensitive ECL chemiluminescence kit (PK10002, Proteintech, Chicago, IL, USA) after incubation with horseradish peroxidase-conjugated secondary antibody (AS014, ABclonal, Wuhan, China) at RT for 2 h. Images were obtained using the ChemiDoc system (Tanon 5800, Shanghai Tanon Technology Co., Ltd., Shanghai, China), and quantitative analysis of the blots was conducted using ImageJ software with $\beta$-actin as the loading control.

All data are expressed as the mean $\pm$ standard error of the mean (SEM). One-way analysis of variance followed by Tukey post-hoc test (homogeneous variance) or Dunnett T3 post-hoc test (Unequal variances) was performed using SPSS 26 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 9.0 (GraphPad Software LLC, San Diego, CA, USA). Statistical significance was set at p 0.05.

To investigate the cardioprotective effects of AS-IV, DOX-treated mice were subsequently treated with AS-IV. After 4 weeks of intervention, transthoracic echocardiography was performed to evaluate cardiac structure and function in the four groups of mice (Fig. 1A). Our data indicated that intraperitoneal injection of DOX caused cardiac dysfunction, as evidenced by reduced LVEF and LVFS, compared with that in the control group. However, LVEF and LVFS significantly improved in mice treated with AS-IV (Fig. 1B). In terms of heart structure, LVIDd and LVIDs were increased in the DOX group compared to the control group, and LVIDd and LVIDs were lower in the AS-IV-treated mice than in the DOX-treated mice (Fig. 1C). To further evaluate the effects of DOX and AS-IV on cardiac function, we measured serum BNP, CK-MB, cTnI, and LDH levels, which are classical biomarkers of cardiac injury. As shown in Fig. 1D, the serum levels of BNP, CK-MB, cTnI, and LDH of the mice in the DOX group were significantly higher than those in the control group. Compared with the model group, the serum concentrations of these heart injury markers were reduced in the AS-IV-treated group. The results indicate that AS-IV alone does not alter cardiac function but can improve DOX-induced cardiac dysfunction.

Fig. 1.

AS-IV attenuates DOX-induced cardiac dysfunction. (A) Representative M-mode images of echocardiograms of experimental animals in each group. (B) Quantitative analysis of left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF). (C) Quantitative analysis of left ventricular internal dimension at end-diastole (LVIDd) and left ventricular internal dimension at end-systole (LVIDs). (D) Quantitative analysis of serum concentration of brain natriuretic peptide (BNP), creatine kinase isoenzyme (CK-MB), cardiac troponin I (cTnI) and lactate dehydrogenase (LDH). CON, mice treated with an equal volume of saline. AS-IV, mice treated with 40 mg/kg AS-IV. DOX, mice treated with 2 mg/kg BW doxorubicin every other day with a cumulative dosage of 28 mg/kg. DOX + AS-IV, mice treated with doxorubicin and AS-IV. Data are shown as mean $\pm$ SEM, N = 3–10, * p 0.05, ** p 0.01, *** p 0.001, **** p 0.0001. Using Shapiro-Wilk test to test the normality of the distribution, all the data are in accordance with the normal distribution. All the data were compared by one-way analysis of variance followed by Tukey post-hoc test for multiple comparisons.

Histological examinations were then performed to examine the microstructural abnormalities of mice in the four groups. HE staining showed that myocardial fibers in the control group were neatly and tightly arranged, with distinct nuclei, while myocardial tissue in the DOX group was disorganized. However, treatment with AS-IV effectively improved the histopathological features of the myocardial tissue (Fig. 2A). Masson-stained collagen was significantly increased in the DOX group compared to that in the other groups (Fig. 2B). In addition, WGA staining showed that the mean area of cardiomyocytes in the DOX-induced myocardial injury group was significantly lower than that in the control group, and AS-IV treatment increased the area of cardiomyocytes (Fig. 2C), which was consistent with the ratio of heart weight to body weight (HW/BW) (Fig. 2D). TEM was conducted to further detect the effects of DOX and AS-IV on the ultrastructure of the cardiomyocytes. In the absence of DOX, mitochondria were arranged normally in organized sarcomeres. However, in the DOX-treated group, mitochondrial damage was observed and was characterized by swelling and vacuolization as well as disarrangement of myofilaments, the disappearance of the H-band, reduced ridge lysis, and widened Z-rays. Fewer injuries were observed after AS-IV administration (Fig. 2A). These results demonstrate that AS-IV attenuates myocardial fibrosis and mitochondrial damage.

Fig. 2.

AS-IV ameliorated DOX-induced myocardial remodeling and myocardial injuries. (A) Representative images of HE (magnification, 400$\times$), Masson (magnification, 400$\times$), WGA (magnification, 400$\times$), and transmission electron microscopy (TEM) images of the four groups (magnification, 15,000$\times$). (B,C) Quantitative analysis of myocardial fibrosis area and the cross-sectional area of myocardial cells. (D) the ratio of heart weight to body weight (HW/BW). N = 10, data are shown as mean $\pm$ SEM; * p 0.05, ** p 0.01. Using Shapiro-Wilk test to test the normality of the distribution, all the data are in accordance with the normal distribution. Myocardial fibrosis area and HW/BW were compared by one-way analysis of variance followed by Dunnett T3 post-hoc test for multiple comparisons and cross-sectional area (CSA) followed by Tukey post-hoc test.

As the activation of inflammasomes and release of inflammatory factors are important in the process of pyroptosis and the fact that oxidative stress can cause pyroptosis [34], we measured the levels of serum IL-1$\beta$, IL-18, SOD, GSH and MDA. The results showed that administration of DOX increased the serum levels of IL-1$\beta$, IL-18, MDA and decreased the serum levels of SOD and GSH compared to that in the control group, while AS-IV treatment decreased serum IL-1$\beta$, IL-18, MDA and increased SOD and GSH levels (Fig. 3A–E). In addition, treatment with AS-IV alone did not alter the levels of serum inflammatory factors and oxidative stress indicators. The results showed that DOX increases the release of inflammatory factors and the level of oxidative stress, whereas AS-IV treatment reduces the levels of the inflammatory and oxidative stress markers.

Fig. 3.

AS-IV ameliorated the release of DOX-induced serum inflammatory factors and oxidative stress levels. (A–E) Quantitative analysis of serum concentration of IL-1$\beta$, IL-18, SOD, MDA and GSH. N = 5–7, data are shown as mean $\pm$ SEM; * p 0.05, ** p 0.01, *** p 0.001, **** p 0.0001. Using Shapiro-Wilk test to test the normality of the distribution, all the data are in accordance with the normal distribution. All the data were compared by one-way analysis of variance followed by Tukey post-hoc test for multiple comparisons.

To further investigate whether AS-IV alleviates myocardial injury by inhibiting DOX-induced myocardial pyroptosis, we first examined the expression of pyroptosis-related proteins in myocardial tissue using immunohistochemistry (Fig. 4A). Quantitative immunohistochemical analysis showed that the expression of pyroptosis-related proteins NLRP3, caspase-1, and GSDMD dramatically increased after DOX treatment, and subsequent treatment with AS-IV markedly attenuated the expression of these markers (Fig. 4B–D). In addition, the protective role of AS-IV was confirmed using immunoblotting (Fig. 5A). As shown in Fig. 5B,C, DOX significantly upregulated the expression of NLRP3 and ASC and activated the cleavage of caspase-1, GSDMD-FL, IL-1$\beta$, and IL-18. Treatment with only AS-IV did not affect myocardial pyroptosis; however, it rescued DOX-induced activation of pyroptosis. The remaining two western blots images can be found in the supplemental file.

Fig. 4.

AS-IV attenuates the expression of pyroptosis proteins in myocardial tissue. (A) Representative immunohistochemical images of the four groups. (B–D) Quantitative analysis of caspase-1, GSDMD and NLRP3. N = 4, data are shown as mean $\pm$ SEM, * p 0.05, ** p 0.01, **** p 0.0001. Using Shapiro-Wilk test to test the normality of the distribution, all the data are in accordance with the normal distribution. All the data were compared by one-way analysis of variance followed by Tukey post-hoc test for multiple comparisons.

Fig. 5.

AS-IV inhibits the expression of DOX-induced pyroptosis-associated proteins. (A) Representative images of western blot from cardiac tissue following DOX and AS-IV treatments. (B,C) Quantitative analysis of NLRP3, ASC, caspase-1, cleaved caspase-1, GSDMD-FL (full length gasdermin D), GSDMD-N (activated gsdmd), IL-1$\beta$, cleaved IL-1$\beta$, IL-18 and cleaved IL-18. N = 3, data are shown as mean $\pm$ SEM, * p 0.05, ** p 0.01, *** p 0.001, **** p 0.0001. Using Shapiro-Wilk test to test the normality of the distribution, all the data are in accordance with the normal distribution. All the data were compared by one-way analysis of variance followed by Tukey post-hoc test for multiple comparisons.

The Nrf-2 signaling pathway has been found to have a potential protective effect on neuronal, vascular endothelial, and cardiac myocyte injury caused by pyroptosis [35, 36, 37]. To further explore the possible regulatory pathways of pyroptosis, we examined the expression of Nrf-2, pNrf-2 and HO-1 (Fig. 6A). Western blotting results in Fig. 6 reveal a reduced expression of Nrf-2, pNrf-2, and HO-1 in the DOX-treated group. AS-IV activates Nrf-2 to phosphorylate it and increases the expression of HO-1. The remaining two western blots images can be found in the supplemental file.

Fig. 6.

AS-IV inhibits DOX-induced pyroptosis by activating the Nrf-2/HO-1 signaling pathway. (A) Representative images of western blot analysis of cardiac tissue following DOX and AS-IV treatments. (B–D) Quantitative analysis of Nrf-2, pNrf-2 and HO-1. N = 3, data are shown as mean $\pm$ SEM, * p 0.05, *** p 0.001, **** p 0.0001. Using Shapiro-Wilk test to test the normality of the distribution, all the data are in accordance with the normal distribution. All the data were compared by one-way analysis of variance followed by Tukey post-hoc test for multiple comparisons.