2.3.1 Preparation of CHX extracts

The dried CHX was pulverized into a coarse powder capable of passing through a 24-mesh sieve. Subsequently, we used 10 volumes of 80 % ethanol to extract 60 g of CHX coarse powder 2 times by reflux, and each extraction lasted for 1.5 h. The extraction solution was combined twice and concentrated under vacuum conditions with a rotary evaporator. Finally, the concentrated extract was freeze-dried to obtain CHX freeze-dried powder.

2.3.2 Preparation of CHX-containing serum

Eight male SD rats were orally administered 1200 mg/kg CHX. After continuous administration for 7 d, the rats were fasted for 12 h. After the last gavage, the rats were anesthetized by intraperitoneal injection of 3 % pentobarbital sodium (30 mg/kg), and blood was collected from the abdominal aorta. The whole blood samples were placed at room temperature and centrifuged to obtain the serum from the upper fraction. Three times the amount of methanol was added to the serum, which was mixed for 3 min and then centrifuged to remove the precipitated substance. A nitrogen blowing instrument was used to blow dry the liquid solvent from the serum samples. Finally, the serum samples were redissolved in 100 μL of methanol to obtain CHX-containing serum samples.

2.3.3 Identification of CHX extracts and blood entry constituents

CHX freeze-dried powder was diluted to 50 μg/mL with methanol. A 0.22 μm microporous membrane was used to filter the CHX sample. Next, 5 μL aliquots of the CHX sample and CHX-containing serum were analyzed with a Waters ACQUITY UPLC BEH C₁₈ column (1.7 μm, 2.1 × 50 mm) at 35 °C using an ACQUITY UPLC I-Class PLUS system (Waters Corporation, Massachusetts, USA). The mobile phase consisted of 0.1 % phosphate water (A) and acetonitrile (B). The gradient elution program was as follows: 0–5 min, 10 % B; 5–12 min, 10–15 % B; 12–17 min, 15–20 % B; 17–33 min, 20–40 % B; 33–48 min, 40–80 % B; 48–50 min, 80–10 % B; and 50–55 min, 10 % B. The flow rate was 0.3 mL/min.

Using a Waters SYNAPT XS Quadrupole-Time-of-Flight Mass Spectrometer equipped with an electrospray ionization source (Waters Corporation, Massachusetts, USA), we performed mass spectrometry on the CHX sample and CHX-containing serum. In the range of m/z 100–1000, mass spectrometry was performed under the following conditions: capillary voltage, 3.0 kV; sample cone voltage, 40 V; source temperature, 150 °C; desolvent gas temperature, 450 °C; desolvent gas flow rate, 800 L/h; molecular ion scanning collision energy, 6 V; and MS/MS collision energy, 2–4 V. The composition was analyzed by MassLynx V4.2 software.

2.4.1 Cell culture

DMEM containing 10 % FBS, 1 % penicillin and streptomycin was used to cultivate H9c2 cells. The environment consisted of humid air with 5 % CO₂ at 37 °C. The medium was changed every two days. Cells were passed when they reached the exponential growth stage.

2.4.2 Iso-induced H9c2 cells

We cultivated H9c2 cells in 96-well plates (1 × 10⁴ cells/well). When the cell growth reached 70–80 %, different concentrations (20, 40, 60, 80, and 100 μg/mL) of ISO were used to treat the cells for 24 h. The culture medium was discarded, and the cells were incubated with 100 μL of CCK-8 buffer (10 μL of CCK-8 concentrate + 90 μL of media) in each well under dark conditions for 30 min. Finally, the absorbance at 450 nm was measured using a microplate reader to determine the half maximal inhibitory concentration (IC₅₀) of ISO (Zhu et al., 2022).

Cell viability (%) = (measured value − blank value)/(normal value − blank value) × 100 %.

The measured value refers to the absorbance value of the treatment group. The blank value refers to the absorbance value of the CCK-8 blank solution without cells. The normal value refers to the absorbance value of the normal cell group.

2.4.3 The effect of blood entry constituents on H9c2 cells

In 96-well plates, the cells were treated with ferulic acid (4, 8, 16, 32, or 64 μg/mL), senkyunolide I (5, 10, 20, 40, or 80 μg/mL), senkyunolide H (2, 4, 8, 16, or 32 μg/mL), senkyunolide A (0.625, 1.25, 2.5, 5, 10 μg/mL), butylphthalide (5, 10, 20, 40, 80 μg/mL), ligustilide (2.5, 5, 10, 20, 40 μg/mL), or N-butylidenephthalide (5, 10, 20, 40, or 80 μg/mL) for 12 h and then treated with the IC₅₀ concentration of ISO for 24 h. Finally, the cell viability was measured by CCK-8 assay on a microplate reader.

2.5.1 Preparation of the CHX sample and mixed reference solution

CHX extracts were diluted to 40 mg/mL with methanol. The standard reference materials of ferulic acid, senkyunolide A, senkyunolide I, N-butylidenephthalide, butylphthalide, senkyunolide H, and ligustilide were weighed accurately and dissolved in a 10 mL volumetric flask with methanol. Then, CHX mixed reference solutions with concentrations of 0.122, 0.848, 0.120, 0.178, 0.190, 0.125, and 0.674 mg/mL were obtained.

2.5.2 Methodology validation

We used a Thermo UltiMate 300 system (Thermo Fisher Scientific Inc., Massachusetts, USA) to detect the contents of the Q-markers in the CHX extract. Then, 0.1 % phosphate water (A) and acetonitrile (B) were combined to form the mobile phase. The elution procedure was performed at a rate of 1 mL/min and a temperature of 35 °C. The optimized gradient elution program is shown in Table S1, Supporting Information. The high-performance liquid chromatography (HPLC) chromatograms of the CHX sample and mixed reference solution are shown in Fig. S1, Supporting Information.

We used a CHX sample solution to verify the precision of the instrument, the method's repeatability, and the sample's stability. We used different concentrations of mixed reference solution to investigate the linear relationship of the Q-markers. The CHX sample solution and the mixed reference solution were mixed 1:1 to investigate the recovery rate.

2.5.3 Analytical hierarchy process (AHP) calculation of component weights

The contents of multiple components in CHX were observed, so we used the overall score method to evaluate the extraction rate of CHX. Based on the contents of Q-markers in CHX and relevant literature reports, the weights of each component were determined by the AHP method (Table S2, Supporting Information shows the judgment matrix scoring table), and the overall score was calculated.

2.5.4 Orthogonal experiment

First, we used a single-factor method to detect the impact of extraction time (A), solid–liquid ratio (B), ethyl alcohol (EtOH) concentration (C) and extraction time (D) on the extraction rate of CHX. Fig. S2, Supporting Information shows the results of the single-factor experiment. When the extraction time was 2.5 h, the CHX extraction rate did not increase; however, the extraction rate decreased when the extraction time was 3 h; this may be because the volatile components contained in CHX were transformed into other substances or volatilized under prolonged heating. Therefore, 1.5, 2 and 2.5 h were selected as the three extraction times. When the solid–liquid ratio was 1:15, the CHX extraction rate reached its peak. Therefore, in the orthogonal experiment, 1:10, 1:15, and 1:20 were selected as the three solid–liquid ratios. When the EtOH concentration was 80 %, the extraction rate of CHX no longer increased. Therefore, in the orthogonal experiment, 40 %, 60 %, and 80 % EtOH were selected as the three EtOH concentrations. When the number of extractions was 3, the CHX extraction rate did not increase. Therefore, in the orthogonal experiment, 1, 2 and 3 times were selected as the extraction times.

Then, 9 aliquots of CHX coarse powder (20 g each) were weighed. CHX was extracted by heating reflux according to the L₉(3⁴) orthogonal experimental design table. The obtained CHX extract was diluted to 40 mg/mL, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. Finally, we carried out a range analysis to determine the best extraction process for CHX according to the orthogonal experimental design results. The optimized extraction process was used to extract CHX, and subsequent experiments were carried out.

2.6.1 Grouping, modeling, and administration of animals

We divided 42 male SD rats (200 g ± 20 g) into 6 groups: the normal group, ISO model group, positive drug Dil group, low-dose CHX group (600 mg/kg) (Li et al., 2014), medium-dose CHX group (900 mg/kg), and high-dose CHX group (1200 mg/kg). The formulation of the tested dosage of CHX in this study was based on its standard clinical dosage (6–10 g crude herb) (Committee, 2020), which was converted into a rat dosage according to body surface area. The CHX groups were given CHX freeze-dried powder extracted by an optimized process. The rats in the CHX groups were given the drug for 14 d (Xiao et al., 2019). On Days 1–12 of the experiment, the rats in the CHX groups were orally administered CHX extract according to their weight, while those in the normal group, model group, and Dil group were orally administered distilled water. The clinical dose of Dil was 3.2 mg/kg, which was translated to 20 mg/kg for rats. On Days 13–14 of the experiment, CHX and Dil were administered to the rats according to their weights. The rats in the normal group and model group were orally administered distilled water. On Days 13–14 of the experiment, rats in the model group, CHX groups and Dil group were intraperitoneally injected with ISO (40 mg/kg) to establish the MI model after 1 h of intragastric administration. Rats in the normal group were injected with normal saline.

2.6.2 Electrocardiogram (ECG) and echocardiography detection

On the 14th day of the experiment, the rats were anesthetized with pentobarbital sodium after ISO injection for 1 h. The rats were fixed in a supine position on a plane, and hypodermic needle electrodes were connected to the rat limb lead position II (Wu et al., 2019). A BL-420 N Biological Signal Collection and Analysis System (Taimeng, Chengdu, China) was used to record ECGs under stable baseline conditions.

A VEVO 3100 system (Visual Sound Wave, Toronto, Canada) was used to perform echocardiography to assess left ventricular function. In brief, the rats were placed on a temperature-controlled operating table in the supine position to maintain a stable heart rate. After hair removal and application of an ultrasound gel to the chest area of the rats, left ventricular function on the parasternal long axis was assessed using M−mode imaging, and echocardiographic measurements were performed (Zhou et al., 2022).

2.6.3 Cardiac index determination

After the ECGs and echocardiograms of the rats were assessed, the weights of the rats were recorded. After blood was collected from the aorta of each rat, the rats were euthanized by cervical dislocation. Complete rat heart tissue was isolated and cleaned. The weight of the heart tissue was recorded, and the cardiac indices of the rats were calculated.

Cardiac index = Heart weight (g)/Rat body weight (g).

2.6.4 Serum and myocardial tissue biochemical examinations

The whole blood sample was centrifuged, and the serum was extracted from the upper fraction. The LDH, CK-MB, cTnT, MDA, SOD and GSH-Px levels in the serum were detected according to the manufacturer’s instructions.

The left ventricular tissues were removed from each group of rats and cut after weighing. After adding cooled PBS (tissue:PBS = 1:9) to the tissue, tissue homogenization was performed with an ice bath for 30 min. The homogenate was centrifuged, and the supernatant was collected. The levels of MDA, SOD and GSH-Px in cardiac tissue were detected according to the manufacturer's instructions.

2.6.5 Histopathological analysis of the left ventricle

We fixed heart tissue for 24 h with 4 % paraformaldehyde. Then, the heart tissue was dehydrated with gradient concentrations of alcohol successively and dipped in wax. The wax-impregnated heart tissue was embedded and cut into 3 μm paraffin sections. After the paraffin sections were dewaxed in water, they were stained with H&E and Masson’s solution according to the manufacturer’s instructions.

For TUNEL-stained sections, after paraffin sections were dewaxed in water, antigenic repair was performed with Proteinase K working solution, membrane disruption was performed with 0.5 % Triton X-100, and endogenous peroxidase activity was blocked with 3 % H₂O₂. Then, TUNEL staining was performed on the sections.

2.6.6 Transmission electron microscopy (TEM)

The left ventricular tissue of the rats was prefixed with 3 % glutaraldehyde, fixed with 1 % osmium tetroxide, progressively dehydrated with acetone, and embedded in Epon812. Semithin sections of left ventricular tissue were stained with methylene blue for optical localization. The sections were cut into ultrathin sections with a diamond knife. Uranyl acetate and lead citrate were used to stain the ultrathin sections. Finally, the sections were observed and imaged using a JEM-1400FLASH transmission electron microscope (Japan Electron Optics Laboratory, Tokyo, Japan).

2.7.1 Effects of CHX on H9c2 cells

H9c2 cells were treated with CHX at concentrations of 6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL in 96-well plates. After incubation for 24 h, the cytotoxicity of CHX was detected by the CCK-8 method.

After detecting the cytotoxicity of CHX, we pretreated cells with CHX (6.25, 12.5, 25, 50 and 100 μg/mL) for 12 h and then induced them with 80 μg/mL ISO for 24 h to observe whether CHX had a protective effect on ISO-induced cell viability reduction.

2.7.2 Flow cytometry

H9c2 cells (1 × 10⁵ cells/well) were inoculated in 6-well plates. We divided the cells into a normal group, an ISO group and CHX groups. In the CHX groups, the cells were pretreated with 25, 50, or 100 μg/mL CHX and 80 μg/mL ISO for 12 h or 24 h. The ISO group did not undergo CHX preprocessing but was treated with ISO. EDTA-free trypsin was used to collect the cells. The cells were washed with PBS, diluted to 1 × 10⁶ cells/mL and resuspended in Annexin V binding buffer. Then, 100 μL of cells were cultured with Annexin V-FITC and PI dyes. Finally, H9c2 cells were detected with a FACSCanto II flow cytometer (BD Company, New York, NY, USA).

After treatment with CHX and ISO, the 6-well plates were treated with DCFH-DA and JC-1 fluorescent probes for 30 min to detect reactive oxygen species (ROS) and the mitochondrial membrane potential (MMOP). Then, the staining solution was removed, and the cells were washed 3 times with PBS. Finally, the ROS and MMOP levels in H9c2 cells were detected by a FACSCanto II flow cytometer.

2.7.3 Immunofluorescence

H9c2 cells were seeded and treated in laser confocal plates as described above. PBS and paraformaldehyde were used to clean and incubate the cells. Then, the cells were infiltrated with 0.3 % Triton X-100 and incubated with 10 % serum for 1 h. The cells were incubated with primary antibodies against Dvl-1 (1:100), Bcl-2 (1:100), and Bax (1:200) overnight at 4 °C. The cells were then incubated with secondary antibodies (1:150) at room temperature for 2 h. Finally, fluorescent sealant was added to the cells after the cells were washed. The cells were immediately observed by laser confocal microscopy (Leica, SP8 SR, Wetzlar, Germany).

We used a DCFH-DA fluorescent probe to detect ROS and a JC-1 fluorescent probe to examine the MMOP intracellular content. Briefly, H9c2 cells (1 × 10⁵ cells/well) were treated for 12 h with CHX (25, 50, or 100 μg/mL) or for 24 h with ISO. Then, the culture medium was removed, and the cells were incubated with DCFH-DA and JC-1 fluorescent probes. The excess fluorescent probe was removed by washing with PBS. Laser confocal microscopy was used to detect and analyze the fluorescence intensity.

2.7.4 Cell SOD, MDA and GSH-PX levels

As described above, H9c2 cells were incubated with CHX and ISO in 6-well plates. We used PBS to wash the cells (1 × 10⁵ cells/well) and collected total protein in RIPA buffer. Finally, following the manufacturer’s instructions, we used commercial assay kits to determine the MDA, SOD and GSH-Px levels in the cells.

2.7.5 Western blot analysis

H9c2 cells (7 × 10⁶) were cultured in petri dishes. After the cells were treated with CHX (100 μg/mL) and ISO, RIPA buffer was used to obtain total cell protein. For nuclear protein extraction, cells were collected using a cell scraper. Two hundred microliters of plasma protein extraction reagent containing PMSF was added to 2 × 10⁶ cells, and the cells were swirled into a single-cell suspension. After centrifugation, the supernatant was removed, and 100 µL of nuclear protein extraction reagent was added to the precipitate. After vortexing in an ice bath, the supernatant was collected as the nuclear protein. Next, we used a BCA protein assay kit to determine the protein concentration and SDS–PAGE to isolate the prepared proteins. Then, the proteins were transferred to activated PVDF membranes. The PVDF membranes were incubated overnight with antibodies against C-Caspase-3 (1:1000), Caspase-3 (1:1000), Bcl-2 (1:1000), Bax (1:6000), Dvl-1 (1:1000), Nrf2 (1:1000), Akt (1:3000), p-Akt (1:1000), Wnt-1 (1:2500), Wnt-3 (1:1100), β-Catenin (1:300), GSK-3β (1:1000), and p-GSK-3β (1:500) at 4 °C and HRP-conjugated secondary antibodies (1:2000) for 1 h at room temperature. Finally, we visualized the protein bands with an enhanced chemiluminescence (ECL) kit. ImageJ software was used to analyze the grayscale value of each blot.

In our study, GAPDH (1:50,000) was selected as the internal reference for total cell protein (Lee et al., 2016). However, GAPDH is not expressed in the nucleus. Lamin B1 is a nuclear membrane structural component that is highly conserved between species and is often used as a reference in samples with a nuclear envelope. Therefore, we selected Lamin B1 (1:1000) as the internal reference for nuclear protein.

2.7.6 Use of the Akt inhibitor MK-2206

To further verify the molecular mechanism of the anti-MI effect of CHX, cells were pretreated with the Akt inhibitor MK-2206 along with 25, 50, or 100 μg/mL CHX for 12 h and then treated with ISO for 24 h. Images of the cells in the normal group, ISO model group and MK-2206 + CHX groups were observed and recorded under an optical microscope. Finally, an Annexin Ⅴ-FITC/PI apoptosis detection kit was used to detect the percentage of apoptotic cells after the addition of MK-2206 by flow cytometry.

3.1.1 Blood entry constituents and their protective effects on H9c2 cells

The total positive/negative ion flow diagrams of CHX and CHX-containing serum are shown in Fig. 1A–1B. Table 1 shows the details of the CHX compounds. A total of 49 compounds were detected in the CHX extract. In negative ion mode, 24 compounds, such as caffeic acid, vanillic acid and ferulic acid, were obtained. A total of 25 compounds, such as butylphthalide, senkyunolide I and tetramethylpyrazine (TMP), were obtained in positive mode. Seven kinds of components were analyzed from CHX-containing serum: ferulic acid, ligustilide, senkyunolide I, senkyunolide H, butylphthalide, senkyunolide A, and N-butylidenephthalide (Table 2).

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Fig. 1

TIC chromatograms of CHX extracts and blood samples. (A) TIC chromatograms of CHX extracts in positive (upper) and negative (lower) modes. (B) TIC chromatograms of blood samples in positive (upper) and negative (lower) modes.

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ISO induction in H9c2 cells can cause cell damage and oxidative stress and increase the levels of inflammatory factors and the rate of apoptosis (Yang et al., 2022). As shown in Fig. 2A, when the ISO concentration was 80 μg/mL, the inhibition rate of H9c2 cells was 50 %. Therefore, in subsequent experiments, 80 μg/mL ISO was selected as the modeling concentration for the cell experiments.

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Fig. 2

Protective effects of the active compounds in CHX against cardiomyocytes. (A) The impact of ISO on cell viability. (B)–(H) Protective effects of active compounds in CHX against cardiomyocytes according to CCK-8 assays. B–H represent ferulic acid (B), senkyunolide I (C), senkyunolide H (D), senkyunolide A (E), butylphthalide (F), ligustilide (G), and N-butylidenephthalide (H), respectively. The data are expressed as the means ± SDs (n = 3). *p < 0.05, **p < 0.01, ^&p > 0.05 vs. the Model group.

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Fig. 2B–2H shows that all 7 blood entry constituents of CHX had protective effects on ISO-treated H9c2 cells. Therefore, we used these 7 components as quality markers (Q-markers) of CHX to optimize the extraction method of CHX.

3.1.2 Optimized CHX extraction process

The relative standard deviations (RSDs) for the methodological verification of instrument precision, method repeatability and sample stability were all less than 2 %. The Q-marker linear regression equations are shown in Table S3, Supporting Information. The recovery rates of ferulic acid, senkyunolide A, senkyunolide I, ligustilide, senkyunolide H, N-butylidenephthalide, and butylphthalide were 93.96 %, 94.70 %, 94.53 %, 92.75 %, 95.58 %, 99.05 % and 94.60 %, respectively.

According to the AHP method, the weight coefficients of ferulic acid, senkyunolide I, senkyunolide H, senkyunolide A, butylphthalide, ligustilide and N-butylidenephthalide were 0.3517, 0.1040, 0.0678, 0.1596, 0.0449, 0.2412 and 0.0308, respectively. The consistency scaling factor (CR) = 0.03 < 0.1 conformed to the consistency test, indicating that the weight coefficient was valid. Overall score = 0.3517* content of ferulic acid + 0.1040* content of senkyunolide I + 0.0678* content of senkyunolide H + 0.1596* content of senkyunolide A + 0.0449* content of butylphthalide + 0.2412* content of ligustilide + 0.0308* content of N- butylidenephthalide.

The results of the range analysis of the orthogonal experiments are shown in Table 3. According to our analysis, the influence sequence of each factor was A > B > C > D, and the optimal extraction process was A₁B₃C₃D₃. That is, the extraction time was 1.5 h, the solid–liquid ratio was 1:20, the EtOH concentration was 80 %, and the number of extractions was 3.

3.2.1 CHX protects heart integrity and function

ECG showed that rats in the MI group 1 h after intraperitoneal injection of ISO exhibited ST segment elevation (Fig. 3A), while no such phenomenon was observed in the Dil and CHX (600, 900, 1200 mg/mL) groups. These findings indicate the success of the ISO-induced MI model and the inhibitory effect of CHX on MI. This finding was further demonstrated by echocardiography, which revealed that CHX and Dil can increase ISO-induced fractional shortening (FS) and decrease the ejection fraction (EF) (Fig. 3B–3D).

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Fig. 3

Effects of CHX on ISO-induced myocardial injury in MI rats. (A) Rat electrocardiogram detection. (B) M−mode echocardiography. (C) Rat fractional shortening. (D) Rat ejection fraction. (E) Serum cTnT levels. (F) Serum LDH levels. (G) Serum CK-MB levels. (H) Rat heart index. The data are expressed as the means ± SDs (n = 6). *p < 0.05, **p < 0.01, ^&p > 0.05 vs. the Model group.

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Rats pretreated with CHX had lower serum cTnT, LDH and CK-MB levels than rats in the MI group (Fig. 3E–3G). Compared with those in the normal group, the cardiac indices of the rats in the MI group were significantly greater (p < 0.05). However, neither Dil nor CHX significantly affected the reduction in the cardiac indices (p > 0.05) (Fig. 3H).

3.2.2 H&E and Masson staining

The most common method for clinicopathological diagnosis is H&E staining (Varasteh et al., 2019). In our study, H&E staining revealed that compared with those of normal rats, the myocardial fibers of the left ventricular heart tissue of MI rats were denatured or broken. Intercellular collagen fibers were significantly increased. Myocardial cells were necrotic. The heart tissues were infiltrated by inflammatory cells. In the Dil group, myocardial fibrosis or rupture was observed in the rats. In the CHX 600 mg/mL group, cardiomyocyte necrosis, inflammatory cell infiltration and myocardial fiber shrinkage were observed. In the CHX 900 mg/mL group, myocardial fibroblast proliferation was observed. In the CHX 1200 mg/mL group, inflammatory cell infiltration was observed (Fig. 4A).

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Fig. 4

Effects of CHX on myocardial pathological injury in MI rats. (A) Representative images of H&E staining; black arrows show the location of myocardial injury. (B) Representative images of Masson staining; blue arrows show collagen deposition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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The heart, liver, spleen, kidney, and lung tissues of the normal group and the 1200 mg/kg CHX group were stained with H&E to determine whether CHX was toxic to the internal organs of the rats. The results (Fig. S3, Supporting Information) showed that CHX had no toxic effect on rat organs.

The principle of Masson staining is related to the size of anionic dye molecules and the degree of tissue penetration. The macromolecule aniline blue can only enter loose structures and high-permeability collagen fiber tissue to appear blue. Ponceau combines with muscle fibers to form red fibers. Therefore, Masson’s trichrome staining can be used to observe organ fibrosis (Hao and Jiao, 2022). Masson staining showed that ISO increased the amount of collagen fibers in the hearts of the rats. CHX and Dil reduced the increase in collagen fibers and prevented cardiac fibrosis (Fig. 4B).

3.2.3 Heart TEM and TUNEL oxidative stress assays

TEM revealed mitochondrial swelling, myoplasmic reticulum expansion, myofibrillar dissolution and lipid drop formation in the heart tissue of the MI group. However, 1200 mg/mL CHX prevented these changes (Fig. 5A). Immunohistochemical TUNEL results showed that myocardial cell apoptosis occurred in the MI group rats. CHX and Dil reduced ISO-induced cardiomyocyte apoptosis (Fig. 5B–5C). MDA, SOD and GSH-Px in the serum and heart tissue of the rats were detected (Fig. 5D–5I). An increase in the content of MDA and decreases in the activity of SOD and GSH-Px were detected in the MI group. However, after ISO induction, CHX and Dil decreased the MDA content and upregulated the activity of GSH-Px and SOD in serum and heart tissue.

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Fig. 5

Effects of CHX on myocardial ultrastructure, apoptosis, and oxidative stress in MI rats. (A) Images of the ultrastructure of the rat myocardium observed by transmission electron microscopy. The yellow arrows represent myofibrillar dissolution, and the red arrows represent mitochondrial swelling. (B)–(C) The results of TUNEL staining; the black arrows represent apoptotic cells. (D)–(E) MDA levels in the serum and heart tissues of MI rats. (F)–(G) SOD activity in the serum and heart tissues of MI rats. (H)–(I) GSH-Px activity in the serum and heart tissues of MI rats. The data are expressed as the means ± SDs (n = 6). *p < 0.05, **p < 0.01, ^&p > 0.05 vs. the Model group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.3.1 CHX prevents ISO-induced apoptosis

The CCK-8 assay results showed that concentrations of 6.25, 12.5, 25, 50, and 100 μg/mL CHX extract were not toxic to H9c2 cells (p > 0.05). However, CHX concentrations of 200 and 400 μg/mL decreased cell viability (Fig. 6A). CHX at concentrations of 25, 50, and 100 μg/mL protected ISO-induced H9c2 cells (p < 0.01) (Fig. 6B). Flow cytometry showed that ISO induced cell apoptosis, while CHX pretreatment reduced the apoptosis rate (Fig. 6C–6D).

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Fig. 6

Effects of ISO-induced CHX on H9c2 cells. (A) Effects of CHX at various concentrations on H9c2 cells. (B) Effects of CHX on ISO-stimulated H9c2 cells. (C)–(D) Effects of CHX on the apoptosis of ISO-stimulated H9c2 cells. (E)–(F) Effects of CHX on the Bax/Bcl-2 ratio of ISO-stimulated H9c2 cells. The data are expressed as the means ± SDs (n = 3). **p < 0.01, ^&p > 0.05 vs. the Model group.

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We further detected the expression of apoptosis-related proteins by immunofluorescence. As shown in Fig. 6E–6F, Bcl-2 is represented by red fluorescence, and Bax is represented by green fluorescence. By determining the fluorescence intensity of Bax and Bcl-2, the Bax/Bcl-2 ratio was calculated, and the relative expression levels of the two proteins were determined. The results showed that ISO increased the Bax/Bcl-2 ratio, while different concentrations of CHX decreased the Bax/Bcl-2 ratio.

3.3.2 CHX protects against ISO-induced MMOP reduction

JC-1 is an ideal MMOP fluorescent probe. A change in JC-1 from red to green fluorescence is an early indicator of apoptosis. In our study, the red fluorescence intensity of H9c2 cells decreased sharply after ISO treatment, as did the ratio of red to green fluorescence (p < 0.01). However, CHX increased the MMOP of the cells (Fig. 7A–7B). Flow cytometry analyses of MMOP are shown in Fig. 7C, where red cell clusters represent cells with normal MMOP and green cell clusters in the rectangular box represent cells with decreased MMOP. The statistical results showed that the effect of ISO on MMOP reduction can be prevented by CHX (Fig. 7D).

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Fig. 7

Effects of CHX on the MMOP of ISO-treated H9c2 cells via confocal microscopy (A)–(B) and flow cytometry analysis (C)–(D). The data are expressed as the means ± SDs (n = 3). **p < 0.01 vs. the Model group.

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3.3.3 CHX reduces ISO-induced cellular oxidative stress

GSH-Px, MDA and SOD in total cell protein were detected by a commercial kit. ISO increased the MDA content and decreased the activities of GSH-Px and SOD in cells. However, 50 and 100 μg/mL CHX increased the activity of GSH-Px and SOD. CHX (25, 50 and 100 μg/mL) decreased the MDA concentration (Fig. 8A–8C). Intracellular ROS levels were measured by flow cytometry and immunofluorescence. DCFH-DA is a cellular permeability indicator of ROS, and in a nonoxidizing environment, it has no fluorescence. In an oxidizing environment, DCFH-DA transforms into 2,7-dichlorodihydrofluorescein, which emits green fluorescence. According to the statistical analysis of fluorescence intensity, ISO increased the level of ROS in cells, while CHX reduced the level of ROS (Fig. 8D–8G).

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Fig. 8

Effects of CHX on oxidative stress levels in ISO-treated H9c2 cells. (A)–(C) Effects of CHX on GSH-Px, MDA, and SOD in ISO-treated H9c2 cells. (D)–(E) ROS levels determined via confocal microscopy. (F)–(G) Flow cytometry analysis. The data are expressed as the means ± SDs (n = 3). *p < 0.05, **p < 0.01, ^&p > 0.05 vs. the Model group.

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3.3.4 Effect of CHX on the Dvl-1/Akt/GSK-3β/Nrf2 signaling pathway

Immunofluorescence results (Fig. 9A–9B) demonstrated that CHX promoted Dvl-1 protein expression. With GAPDH as the extracellular reference and Lamin B1 as the intracellular reference, the western blot results showed that CHX can increase the phosphorylation of Akt and GSK-3β, promote Nrf2 entry into the nucleus, and inhibit the cleavage of Caspase-3. In addition, the Bax/Bcl-2 ratio decreased, and the expression levels of β-catenin, Wnt-3 and Dvl-1 increased in response to CHX. However, CHX had no effect on Wnt-1 (Fig. 9C-9 M).

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Fig. 9

Effects of CHX on Dvl-1/Akt/Nrf2 signaling in ISO-treated H9c2 cells. (A)–(B) Effects of CHX on Dvl-1 in ISO-treated H9c2 cells as determined by confocal microscopy. (C)–(M) Effects of CHX on Dvl-1/Akt/Nrf2 signaling in ISO-treated H9c2 cells as determined by western blotting. The data are expressed as the means ± SDs (n = 3). *p < 0.05, **p < 0.01, ^&p > 0.05 vs. the Model group.

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3.3.5 MK-2206 inhibits the anti-Mi effect of CHX

As shown in Fig. 10A, cell damage increased in the CHX groups after the addition of the Akt inhibitor MK-2206. Flow cytometry was used to measure apoptosis after adding MK-2206, and MK-2206 increased the percentage of apoptotic cells in the CHX groups (Fig. 10B–10C). These two studies showed that the protective effect of CHX on H9c2 cells was reversed by MK-2206.

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Fig. 10

The effect of Akt inhibitor MK-2206 on the cell protective effect of CHX. (A) MK-2206 reduces the cellular protective effect of CHX. (B)–(C) MK-2206 increases cell apoptosis rate. The data are expressed as the means ± SDs (n = 3). *p < 0.05, **p < 0.01, ^&p > 0.05 vs. the Model group.

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