Rutin (Figure 1 purity > 98%) was purchased from MCE Biotechnology Co., Ltd. (United States, #153184). 0.5% sodium carboxymethyl cellulose (NaCMC) was purchased from Sigma Biotechnology Co., Ltd. (United States, #419273). Rutin was dissolved in 0.5% NaCMC and was given to mice by gavage.

Male C57BL/6 mice (23 ± 2 g, 8–10 weeks old) were obtained from Beijing WTLH Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were adapted for 1 week (temperature: 23 ± 1°C, humidity: 45%–50%) and kept in a 12 h/12 h light/dark cycle. All animals were raised with standard water and food. The CLP surgery was performed as follows. Surgery was performed under anesthesia by isoflurane (3%) delivered in oxygen at a rate of 1 L/min in an anesthetic chamber via a face mask. Buprenorphine (0.05 mg/kg) was administered ip 5 min before surgery to obtain adequate analgesia. The cecum was exposed by a 1 to 2 cm midline incision in the anterior abdomen, subjected to ligation of the distal half of the cecum, and punctured once with a 22 G needle in the ligated segment. The cecum was then repositioned, 1 ml of sterile saline (pyrogen-free 0.9% NaCl) was subcutaneously administered, and the incision was closed with 9 mm steel wound clips.

Our experiments were carried out in strict accordance with international ethical guidelines and the National Institutes of Health Guide on the care and use of laboratory animals. Animals were checked daily for signs of distress and endpoints. Specific criteria used to determine when the animals should be euthanized were in accordance with Remick lab report (Nemzek et al., 2004). All protocols were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University. All animal studies followed the ARRIVE guidelines 2.0 (Percie du Sert et al., 2020).

Male C57BL/6 mice received rutin (50, 100, 200 mg/kg per day) dissolved in NaCMC or NaCMC only for 7 days before being subjected to CLP-induced lethal sepsis. Animals were checked daily and recorded for up to 7 days. The survival rate was analyzed by the log-rank test in GraphPad Prism software and presented as Kaplan-Meier curves.

Eighteen mice were randomly divided into a sham group (n = 6), CLP group (n = 6), and rutin+CLP group (n = 6). Sham group and CLP group received 0.5% NaCMC for seven consecutive days prior to surgery, while the rutin + CLP group received rutin (100 mg/kg) dissolved in 0.5% NaCMC for seven consecutive days prior to CLP (Figure 2). Cardiac function was determined by echocardiography 24 h after surgery. Immediately after this, the mice were sacrificed, and blood samples and heart tissues were retained for further investigation (three independent experiments were performed). The design of our study adheres to most of the points described in the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) Consensus Recommendations (Osuchowski et al., 2018).

The level of TNF-α, IL-6, IL-10, and cTnT in plasma was determined by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. The absorbance of each group was measured by a microplate reader at the wavelength of 450 nm. TNF-α, IL-6, and cTnT ELISA kits were purchased from Senbega Biotechnology Co., Ltd. (Nanjing, China), IL-10 ELISA kits were purchased from Beijing Dakewe Biotechnology Co., Ltd. (Beijing, China).

Heart tissues isolated from mice were completely fixed with 4% formaldehyde, then dehydrated with a gradient ethanol solution, soaked in wax, and embedded in paraffin in blocks. They were cut into 5 μm slices, and then deparaffinized and hydrated. The sections were stained with hematoxylin-eosin (H&E). The degree of cardiomyopathy was evaluated according to the degree of myocardial cell damage and the percentage of loss of myocardial fiber in this section. 0=no lesions, 1=myocardial damage <25%, 2=myocardial damage 25%–50%, 3=myocardial damage 50%–75%, 4=myocardial damage >75%.

The mice were anesthetized and placed on a heating pad to maintain their temperature. Measured echocardiographic indicators (VisualSonics, Canada) included the following: left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), EF was calculated using the following formula: EF (%) = (LVEDV-LVESV)/LVEDV × 100%. At the same time, left ventricular fractional shortening (FS) was calculated, and all echocardiograms were performed by the same trained investigator.

A commercial kit that uses fluorescein-dUTP to label DNA fragmentation sites (Servicebio, G1501) was used to detect cardiomyocyte apoptosis. Cut the myocardial tissue fixed with 4% paraformaldehyde into 4 μm slices. Heart sections were deparaffinized, rehydrated, and equilibrated in Tris buffered saline (TBS). Proteinase K (2 mg/ml) diluted with PBS (final concentration 20 μg/ml, pH 7.4–8.0) was then applied to the samples and incubated for 20 min at room temperature. The FITC-12-dUTP labeling mix was thawed and mixed with the buffer solution. The glass slide was immersed in the staining jar containing the DAPI solution in the dark and kept for 15 min at room temperature. Images were taken with a fluorescence microscope (Olympus, Japan), TUNEL-positive nuclei (fragmented DNA) were observed in green, while the nuclei locations (DAPI) were in blue.

Murine heart tissue was homogenized in homogenization buffer (20 mM HEPES, pH 7.9, 1 mM MgCl₂, 0.5 mM EDTA, 1% Nonidet P-40, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 μl/ml PIC) and centrifuged by 18,800 g for 40 min at 4°C. The supernatant was transferred to a fresh tube, and protein concentrations were determined using a BCA protein kit. Using 10% SDS-PAGE, separate the proteins (Solarbio, China), then transfer them to a polyvinylidene fluoride (PVDF) membrane (Millipore, United States). The membrane was blocked with TBST that contained 5% non-fat milk for 1 h at room temperature, then incubated with primary antibodies overnight at 4°C. Antibodies include Bcl-2 (Cell Signaling Technology, #3498; 1:1000), Bax (Cell Signaling Technology, #14796; 1:1000), Caspase-9 (Cell Signaling Technology, #9508; 1:1000), β-actin (Cell Signaling Technology, #3700; 1:1000). After three washing cycles, the membrane was incubated for 30 min with the HRP-conjugated secondary antibody. Protein bands were detected with an enhanced chemiluminescent (ECL) detection system. Finally, the membrane was exposed with a gel imaging system (Bio-Rad, United States). The immunoreactive bands were analyzed with ImageJ software.

After obtaining fresh myocardial tissue samples from mice, they were quickly cut into small pieces on ice and fixed in 2% glutaraldehyde precooled for 2 days, tissues were placed in PBS buffer for 6 h and fixed with 1% osmium acid fixative for 3 h, then gradient ethanol dehydration and embedding with epoxy resin at room temperature. The samples were cut with an ultrathin microtome, then stained with 3% uranyl acetate lead citrate, observed, and photographed under a transmission electron microscope (HT7700, Japan). Then randomly select 5–8 fields, observe the characteristics of the mitochondrial structure, numbers, and calculate the mean mitochondrial area using ImageJ software.

Statistical analyzes were performed using Prism 9.0 (GraphPad Software, Inc., San Diego, CA). Data were expressed as mean ± SEM. A one-way ANOVA followed by the Bonferroni test was used for multiple comparisons. Statistical significance was established at a p value less than 0.05. Survival is analyzed using the log-rank test in GraphPad Prism software and presented as Kaplan-Meier curves.

Male C57BL/6 mice were orally given rutin (50, 100, 200 mg/kg per day) dissolved in NaCMC or NaCMC only for 7 days before being subjected to lethal sepsis induced by CLP. There was no significant difference between the 50 mg/kg and the non-intervention group. Compared to the nonintervention group, doses of 100 mg/kg and 200 mg/kg significantly improved the survival rate of sepsis mice. There is no difference in survival between the 100 and 200 mg intervention groups (Figure 3). This result indicated that pretreatment with rutin (100 and 200 mg/kg per day) could protect mice against CLP-induced lethality. We chose the 100 mg/kg dose for further studies.

Male C57BL/6 mice were precautionarily treated with rutin (100 mg/kg) for 7 days, then subjected to CLP. Mice were sacrificed and blood samples were collected 24 h after CLP. From the measured data, we can see that serum levels of IL-6 (96.76 ± 7.69 vs. 137.90 ± 7.44 pg/ml, p < 0.05), TNF-α (329.80 ± 16.52 vs. 401.90 ± 13.77 ng/L, p < 0.05), and cTnT (101.30 ± 2.46 vs. 109.70 ± 2.39 ng/L, p < 0.05) decreased (Figures 4A–C) and IL-10 (20.52 ± 0.83 vs. 14.01 ± 0.83 pg/ml, p < 0.05) increased (Figure 4D) in the rutin pretreatment group compared with the non-intervention CLP group.

To determine whether pretreatment rutin attenuates cardiac inflammation, we used H&E staining to measure the histopathology of cardiac tissue. The results showed that the pathological changes of myocardial inflammation (Figures 5A–C1), such as inflammatory cell infiltration, interstitial edema, and myocardial fiber breakage, were significantly alleviated by pretreatment with rutin (2.250 ± 0.214 vs. 1.417 ± 0.154, p < 0.05) (Figure 5D).

To investigate the protective effect of rutin on the cardiac function of the sepsis model, we use Two-dimensional and M-mode images to record left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (FS). As shown in Figure 6A, pretreatment of rutin in CLP induced sepsis mouse markedly improved the LVEF (58.37 ± 3.21 vs. 49.82 ± 0.82, p < 0.05) and FS (30.28 ± 2.24 vs. 24.68 ± 0.46, p < 0.05), when compared with the non-intervention CLP group (Figures 6B,C).

After demonstrating that the pretreatment of rutin improved cardiac function, we next investigated the effect of rutin on cardiac apoptosis in CLP mouse model (Figure 7). Compared to the sham group, the CLP group resulted in a significant increase in Bax levels and caspase-9 activation (Figure 7C). Compared with the non-intervention CLP group, the pretreatment of the rutin group, however, significantly attenuated the increases in Bax levels (1.179 ± 0.004 vs. 1.770 ± 0.012, p < 0.05) and the associated activation of caspase-9 (1.422 ± 0.061 vs. 2.590 ± 0.055, p < 0.05) (Figures 7D,E). Furthermore, CLP induced a decrease in Bcl-2 levels in the heart, which was also significantly alleviated by rutin pretreatment (0.837 ± 0.008 vs. 0.751 ± 0.008, p < 0.05) (Figure 7F). The effect of rutin on Bcl-2/Bax showed a similar trend to that of Bcl-2 (Figure 7G). We also performed TUNEL staining to confirm apoptosis in heart tissue. The CLP group resulted in an increase in relative TUNEL fluorescence compared to the sham group, and the CLP group with rutin pretreatment resulted in a decrease in relative TUNEL fluorescence compared to the non-treatment CLP group (3.421 ± 0.228 vs. 4.974 ± 0.664, p < 0.05) (Figures 7A,B).

To clarify the mechanism of rutin protection in cardiomyocytes, we observe the morphology of mitochondria in cardiomyocytes with transmission electron microscopy (TEM). The results showed that the number of myocardial mitochondria in the CLP group was reduced and there was swelling, vacuole-like changes and broken mitochondrial cristae compared to the sham group (Figure 8A). Furthermore, pretreatment with rutin in CLP mice significantly alleviated mitochondrial morphology change of mitochondria (1.009 ± 0.027 vs. 1.307 ± 0.0326, p < 0.05) and increased mitochondrial number of mitochondria (11.500 ± 0.428 vs. 8.833 ± 0.792, p < 0.05) (Figures 8B,C).