2.1 Chemicals and reagents

Balanophora dioica was collected from Yunnan Province of China in 2022 (Zhang et al., 2008), kept in desiccators and authenticated by Professor Jincai Lu of the Shenyang Pharmaceutical University. The voucher specimen (No. 20220126) was deposited in the Herbarium of Shenyang Pharmaceutical University (Shenyang, China). ISO was purchased from Sigma (America), CK, LDH, SOD, CAT, GSH-Px and MDA kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), Anti-caspase9 Rabbit pAb, TGF-β1 was purchased from Wanleibio (Shenyang, China). Anti-GAPDH Rabbit pAb was purchased from Servicebio (Wuhan, China). Other antibodies were all obtained from Beyotime Biotechnology (Shanghai, China).

2.2 Preparation and GC-MS, HPLC-FT-ICR-MS analysis of BDEE

2.3 Animal studies

Sixty C57/BL6 mice (male, 6 weeks old, 18–22 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd, license number: SCXK (Liao) 2020–0001. The study adhered strictly to the National Institutes of Health Guide for the care and use of laboratory animals and was approved by the Laboratory Animal Committee of Shenyang Pharmaceutical University. All mice were kept at a temperature of 23 ± 1℃ and a relative humidity of 50–60 %, with a 12 h light/dark cycle, and with free access to water and food. After 1 week of acclimatization, the animals were randomly divided into 6 groups (10 in each group). The normal control group was given 0.9 % saline (10 mg/kg/d) by intragastric injection and subcutaneous injection. The ISO model group was given 0.9 % saline by gavage and subcutaneous injection of ISO (10 mg/kg/d) to induce myocardial injury for 21 days (Han et al., 2020, Xue et al., 2021, Zhang et al., 2023). 100, 200 and 400 mg/kg/d of BDEE were gavaged in the BDEE low-dose group, BDEE medium-dose group and BDEE high-dose group, and ISO (10 mg/kg/d) was injected subcutaneously. The BDEE drug group was given 400 mg/kg/d of BDEE by gavage. The experiments in each group lasted for 21 days. On the 22nd day, the mice were executed at anesthesia. Eyeballs were taken for blood. The mouse was fixed with one hand, and the eye skin on the blood side was gently pressed to make the eyeball congested and protruding. The eyeball was picked up with elbow forceps and quickly removed. At the same time, the mouse's heart was lightly pressed to increase the rate at which the hearts pumped blood. When the blood ran out, the mice were killed by dislocating. The hearts and livers were taken for follow-up experiments. The myocardial tissue of the left ventricle of mice was taken, fixed with 4 % paraformaldehyde, paraffin-embedded, and other myocardial tissue were stored at −80℃ for subsequent experiments. The blood was removed from the heart and liver in normal saline, dried with filter paper, weighed, and the heart index HW/BW were calculated. Then the whole blood was centrifuged at 3500 r/min for 15 min, and the supernatant was carefully sucked out and stored at −80℃ for subsequent experiments.

2.4 Histopathological examination

Take the embedded paraffin blocks and cut into 4 μm sections using standard histological methods. Left ventricular sections were stained with H&E and Sirius red. Liver sections were stained with H&E and Masson.

2.5 Biochemical index analysis

Serum LDH and CK activities were measured with LDH and CK kits. 0.1 g of the heart tissue was mixed in normal saline at a ratio of 1:9, and the supernatant fluid was taken after centrifugation at 3500 r/min for 15 min for the detection of oxidative stress markers such as SOD, MDA, CAT and GSH-Px. These indicators were achieved using the corresponding kit according to the instructions.

2.6 Immunohistochemistry

The protein expression of COL1A1, α-SMA and TLR4 in the heart was determined by immunohistochemistry. The paraffin-embedded of the left ventricle of mice was taken, and sliced. After dewaxing paraffin sections to water, the slices were immersed in pH 6.0 citric acid resistant solution and placed in microwave oven for antigen repair. The following experimental steps were as follows: natural cooling, block endogenous peroxidase, serum seal, adding primary antibody, adding secondary antibody, DAB color rendering, overdyed nucleus, and the results are interpreted under a white light microscope (Nikon, E100).

2.7 TUNEL staining

The paraffin-embedded of the left ventricle of mice was taken, according to the TUNEL kit requirements for reaction. Rinsing, dehydration, sealing, fluorescence microscope observation. Normal cardiomyocytes showed blue, and apoptotic cells showed green. The apoptosis rate of myocardial cells in each group was obtained by Image analysis software.

2.8 Western blotting

Myocardial tissues of mice were taken and weighed, and they were lysed with lysis solution (RIPA: Phosphoproteinase inhibitor A: Phosphoproteinase inhibitor B: PMSF = 100:1:1:1), then were grinded using a tissue grinder. The homogenates were centrifuged at 12,000 g, −4℃ for 15 min to collect the supernatant. Protein content in each group were determined by BCA kit. We obtained protein samples through adding buffered solution, boiling for 10 min after mixing. Then the protein in each group was separated by SDS-polyacrylamide gel electrophoresis. Transferring of separated proteins onto nitrocellulose filter membrane (NC membrane). blocking with 5 % skimmed milk powder at room temperature for 1.5 h. NC membrane was incubated with primary antibody overnight at 4℃. Then wash the membrane with TBST at 100 r for 5 min a total of 5 times to remove unbound primary antibodies. Incubation with horseradish peroxidase-labeled goat anti-mouse (1: 2000) and anti-rabbit antibody (1:1000) for 1 h. Washing membrane with TBST 5 times, 5 min/time. Finally, the ECL luminescence kits were used, the gray value was quantified by image analysis software image-J.

2.9 Statistical analysis

All the data were presented as the mean ± SEM and were analyzed with one-way ANOVA using GraphPad Prism version 9 (GraphPad Software Inc., San Diego, CA, USA). The data were statistically significant when P < 0.05.

3.1 Dentification of main compounds in BDEE

GC–MS analysis results indicated that BDEE mainly contains 15 compounds (Fig.S1 and Table S1), while eight components identified from BDEE by HPLC-MS at positive ion mode (Fig.S3 and Table S2), and 17 components identified by HPLC-MS at negative ion mode (Fig.S4 and Table S3). The major components are luteolin-7-O-β-D-glucoside, 1-O-(E)-caffeoyl-3-O-galloyl-β-D-glucopyranose, palmitic acid, phloridzin, trilobatin, quercimeritrin, and ethyl brevifolin carboxylate. The information on all preliminarily identified compounds is in supporting information. Therefore, we speculate that phenols and flavonoids of BDEE maybe play a role in improving ISO-induced myocardial injury.

3.2 BDEE improves myocardial injury induced by ISO

In this study, the heart of the control group was smooth, bright red in color and normal in shape and size, while the heart of the ISO group was dark red in color and significantly increased in size. Compared with the ISO group, the heart of the BDEE administration group was close to normal in color and significantly reduced in size (Fig. 1A). The HW/BW in the ISO group was significantly higher than that in the control group, while the HW/BW in the BDEE group was significantly reduced in a dose-dependent manner (Compared with the Con group, the ISO model group increased HW/BW by 56.12 %, compared with the ISO model group, the ISO + 100, 200, 400 mg/kg BDEE group decreased by 18.06 %, 26.19 %, 34.55 %, respectively), suggesting that BDEE could alleviate ISO-induced myocardial hypertrophy (Fig. 1B). Histopathology indicated that the control group showed normal myocardial tissue structure by H&E staining, while the ISO group showed inflammatory cell infiltration vacuoles and interstitial edema, which were improved after BDEE administration. And the myocardial fibrosis was observed by Sirius red staining. The results showed that the degree of myocardial fibrosis in the ISO group was significantly higher than that in the control group, while the degree of myocardial fibrosis in the BDEE group was significantly improved (Fig. 1C). These results suggest that BDEE has a myocardial protective effect. Liver tissue H&E and Masson staining showed that BDEE had no drug toxicity compared with the control group (Fig. 1D).

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Fig. 1

BDEE improves myocardial injury induced by ISO. (A) Morphology of the hearts of mice in each group. (B) The HW/BW were calculated to determine the effects of BDEE. (C) H&E staining and Sirius Red staining of heart sections, magnification: 20 ×, scale bar = 50 μm. (D) Masson staining and H&E staining of liver section, magnification: 20 × and 40 ×, scale bar = 50 μm. Values are given as mean ± SEM, n = 6. *P < 0.05 compared with Con group; ^#P < 0.05 compared with ISO group.

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3.3 BDEE reduces ISO-induced oxidative stress

When the body produces too much reactive oxygen species (ROS) or clears them slowly, it can cause damage to cells and tissues. The heart is one of the organs more strongly affected by ROS. ISO can produce excessive free radicals through oxidative stress and lead to myocardial injury. Its morphological changes and energy metabolism are similar to that of human myocardial ischemia. In this process, SOD activity decreased, lipid peroxidation increased, and MDA content increased. In the process of oxidative stress, myocardial cells rupture or membrane permeability increases, so that CK, LDH and other enzymes exudate, resulting in increased enzyme activity in the serum of myocardial injury mice.

The results of this experiment showed that BDEE could significantly reduce the leakage of CK and LDH (Fig. 2A and B) (Compared with the ISO model group, the ISO + 100, 200, 400 mg/kg BDEE group decreased CK by 18.35 %, 30.39 %, 58.61 %; the ISO + 100, 200, 400 mg/kg BDEE group decreased LDH by 11.71 %, 27.05 %, 55.25 %, respectively), inhibit the accumulation of lipid peroxidation metabolite MDA in myocardium (Fig. 2C) (Compared with the ISO model group, the ISO + 100, 200, 400 mg/kg BDEE group decreased MDA by 17.51 %, 38.69 %, 66.16 %, respectively), and increase the levels of SOD, CAT and GSH-Px (Fig. 2D-F) (Compared with the ISO model group, the ISO + 100, 200, 400 mg/kg BDEE group increased SOD by 12.17 %, 37.01 %, 41.15 % and increased CAT by 12.71 %, 21.51 %, 55.25 % and increased GSH-Px by 7.22 %, 12.15 %, 40.69 %, respectively). It is suggested that it can reduce ISO-induced oxidative stress, stabilize the cell membrane and protect the myocardium of ISO-induced myocardial injury in mice.

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Fig. 2

BDEE reduces ISO-induced oxidative stress. Effects of BDEE on the levels of CK (A), LDH (B). Effects of BDEE on concentration of MDA and activities of SOD, CAT, GSH-Px. (C-F). Note: The “+” means to give the drug, and “-” means not to give the drug. The results are presented as means ± SEM, n = 6. ^***P < 0.001, ^**P < 0.01, *P < 0.05, compared with Con group; ^### P < 0.001, ^##P < 0.01, ^#P < 0.05, compared with ISO group.

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3.4 BDEE inhibits myocardial fibrosis induced by ISO

The immunohistochemical results of COL1A1 and α-SMA were shown in Fig. 3A-B. Compared with the control group, the expressions of COL1A1 and α-SMA were increased in the model group, but decreased in a dose-dependent manner after BDEE administration. Western blotting results were shown in Fig. 3C-G. Compared with the control group, the protein expressions of COL1A1, COL3A1, α-SMA, TGF-β1, P-Smad3/Smad3 in myocardial cells in model group were significantly increased. (Compared with the Con group, the ISO model group increased the protein expression levels of COL1A1, COL3A1, α-SMA, TGF-β1, P-Smad3/Smad3 by 2.05, 2.10, 1.01, 1.55, and 1.51 times respectively). Compared with the model group, the protein expression levels of COL1A1, COL3A1, α-SMA, TGF-β1, P-Smad3/Smad3 in the BDEE administration group were decreased in a dose-dependent manner. It was consistent with the literature and suggested that BDEE may improve ISO-induced myocardial fibrosis in mice through TGF-β1/Smad3 pathway (Sun et al., 2021b).

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Fig. 3

BDEE inhibits myocardial fibrosis induced by ISO. (A) Photomicrographs of histological sections of heart depicting expression of COL1A1 and α-SMA, magnification: 20 ×, scale bar = 50 μm. (B) Quantitative image analysis for immunohistochemical staining of COL1A1 and α-SMA, expressed as optical densities (OD). (C) Effects of BDEE on the expression of COL1A1, COL3A1, α-SMA in heart tissue were determined by western blotting. (D) Quantification of the COL1A1, COL3A1, α-SMA protein expression normalized to GAPDH. (E) Effects of BDEE on the expression of TGF-β1, P-Smad3 and Smad3 in each group. Quantification of the TGF-β1 (F) normalized to GAPDH, P-Smad3/Smad3 (G) protein expression. Results are represented as means ± SEM, n = 3. ^***P < 0.001, ^**P < 0.01, compared with Con group; ^### P < 0.001, ^##P < 0.01, ^#P < 0.05, compared with ISO group.

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3.5 BDEE can reduce the inflammation of ISO-induced myocardial injury mice

TLR4 is an important target protein in inflammatory pathways. Immunohistochemical results of TLR4 (Fig. 4A and B) showed that compared with the control group, TLR4 expression in ISO group was significantly increased, while after BDEE administration, TLR4 expression decreased in a dose-dependent manner. These results suggest that TLR4 plays a role in that BDEE improves the inflammatory response of ISO-induced myocardial injury mice. The role of TLR4 in reducing inflammation and other target proteins related to TLR4 were further verified by Western blotting. Western blotting results (Fig. 4C and D) showed that, compared with control group, TLR4, MyD88, IRAK-1, TRAF-6, NF-κB protein expression in ISO group increased. (Compared with the Con group, the ISO model group increased the protein expression levels of TLR4, MyD88, IRAK-1, TRAF-6, NF-κB by 1.64, 3.12, 0.43, 3.19 and 1.16 times, respectively) However, after BDEE administration, there were different degrees of decline, these results indicated that BDEE could reduce inflammatory response by regulating the expression of TLR4, MyD88, IRAK-1, TRAF-6 and NF-κB. It was further revealed that BDEE may reduce ISO-induced myocardial inflammation in mice and improve myocardial injury by regulating TLR4/MyD88/NF-κB pathway.

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Fig. 4

BDEE can reduce the inflammation of ISO-induced myocardial injury mice. (A) Photomicrographs of histological sections of heart depicting expression of TLR4, magnification: 20 ×, scale bar = 50 μm. (B) Quantitative image analysis for immunohistochemical staining of TLR4, expressed as optical densities (OD). (C) The effects of BDEE on the protein expression of TLR4, MyD88, IRAK-1, TRAF-6 and NF-κB in heart tissue were determined by western blotting. (D) Relative intensities of TLR4, MyD88, IRAK-1, TRAF-6 and NF-κB were calculated by normalization to the GAPDH in each group. Values are given as mean ± SEM, n = 3. ^***P < 0.001, ^**P < 0.01, *P < 0.05, compared with Con group; ^###P < 0.001, ^##P < 0.01, ^#P < 0.05, compared with ISO group.

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3.6 BDEE may achieve myocardial protection by inhibiting ISO-induced apoptosis

TUNEL staining results were shown in Fig. 5A and B. Compared with the control group, the apoptosis rate of myocardial cells in the model group was significantly increased. Compared with the model group, the apoptosis rate was decreased after BDEE administration. (Compared with the Con group, the ISO model group increased TUNEL-positive cells by 6.99 times, compared with the ISO model group, the ISO + 100, 200, 400 mg/kg BDEE group decreased by 2.28, 0.52, and 7.73 times, respectively). Western blotting results showed that (Fig. 5C–H), compared with the control group, Bcl-2 protein expression was decreased and Bax, Caspase-9 and Caspase-3 protein expression were significantly increased in the model group. Compared with the model group, the expression level of Bcl-2 increased in a dose-dependent manner, while the protein expression levels of Bax, Caspase-9 and Caspase-3 decreased in a dose-dependent manner. It was consistent with the literature and indicated that BDEE inhibited ISO-induced apoptosis by regulating Bcl-2, Bax, Caspase-9 and Caspase-3, thus improving myocardial injury (Bi et al., 2018, Nagoor Meeran et al., 2019).

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Fig. 5

BDEE may achieve myocardial protection by inhibiting ISO-induced apoptosis. (A) Representative microscopic photographs of TUNEL-stained heart sections, magnification: 50 ×, scale bar = 20 μm. (B) The histogram showed the relative proportion of TUNEL-positive cells in the heart of mice. (C) Effects of BDEE on the expression of Bcl-2, Bax in each group. Quantification of the Bax (D), Bcl-2 (E) protein expression normalized to GAPDH. (F) Quantification of Bcl-2/Bax ratio in protein expression level. (G) Effects of BDEE on the expression of Caspase-9 and Caspase-3 in each group. (H) Quantification of the Caspase-9, Caspase-3 protein expression normalized to GAPDH. Results are represented as means ± SEM, n = 3. ^***P < 0.001, ^**P < 0.01, compared with Con group; ^###P < 0.001, ^##P < 0.01, ^#P < 0.05, compared with ISO group.

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