Pachymic acid ameliorates myocardial ischemia and reperfusion injury by attenuating cardiomyocyte apoptosis through the regulation of the mTOR signal pathway

PA inhibits apoptosis in H/R-treated NMVCs

To explore the function of PA on cardiac function in myocadiac I/R, NMVCs were pretreated with PA (1, 3, 5, 10 μg/ml, Figure 1A) for 30 min. Then the NMVCs were then exposed to hypoxia 1h and reoxygenation for 12h. MTT assay was performed to detect cell viability. The results showed that 3, 5, 10 μg/ml PA could inhibit the decreased cell viability induced by H/R (Figure 1B). TUNEL assay performed that PA10 (μg/ml) prevented the apoptosis level in H/R NMVCs (Figure 1C). Calcein-AM/PI staining revealed that PA reduced the dead cells in NMVCs after H/R (Figure 1D). LDH was increased in NMVCs after H/R, while PA partially recovered the LDH level (Figure 1E). Then we assessed the apoptosis-associated protein level in NMVCs. PA blocked the increased level of Cytochrome-c (Cyt-c), and Bax induced by H/R and reversed the decreased level of Bcl2 induced by H/R (Figure 1F). Flow cytometry also confirmed that PA inhibited apoptosis levels in H/R NMVCs (Figure 1G). Taken together, PA could inhibit apoptosis in H/R induced NMVCs.

Figure 1. PA inhibits apoptosis in H/R NMVCs. (A) The chemical structure of PA. (B) Cell viability was detected by MTT. n=6, **P<0.01, ^#P<0.05, ^##P<0.01. (C) TUNEL assay was to detect apoptosis level in H/R induced NMVCs treated with 10 μg/ml PA. Scale bar 20 μm. n=4, **P<0.01, ^#P<0.05. (D) Calcein-AM/PI staining revealed the live cells and dead cells in NMVCs after H/R. n=4, *P<0.05, **P<0.01, ^#P<0.05. (E) LDH level was determined in NMVCs. n=4, **P<0.01, ^#P<0.05. (F) The apoptosis-associated protein level in NMVCs, Cytochrome-c (Cyt-c), Bax, and Bcl2. n=4, *P<0.05, ^#P<0.05. (G) Flow cytometry was used to assess apoptosis level in NMVCs. n=4, **P<0.01, ^#P<0.05.

PA alleviates mitochondrial function in H/R induced NMVCs

When the NMVCs was hypoxia, the content of ATP was decreased, while PA treated recovered the ATP content (Figure 2A). We also found that PA attenuated ROS production induced by H/R (Figure 2B). The of NMVCs was significantly decreased in the H/R group, while the mPTP opening was recovered in the PA group (Figure 2C). JC-1 staining showed the decreased mitochondrial membrane potential (MMP) in H/R induced NMVCs, while PA-pretreatment reversed the mitochondrial membrane potential (Figure 2D). Meanwhile, PA reversed the GSH and GR activity level in H/R NMVCs (Figure 2E, 2F). In summary, PA could improve the mitochondrial function in H/R induced NMVCs.

Figure 2. PA improves mitochondrial function in H/R NMVCs. (A) The ATP content was explored in NMVCs. n=4, *P<0.05, ^#P<0.05. (B) The ROS production in NMVCs was determined. Scale bar 20 μm. n=4, **P<0.01, ^#P<0.05. (C) The mPTP opening rate in NMVCs was explored. n=5, **P<0.01, ^#P<0.05. (D) Mitochondrial membrane potential detection in NMVCs. n=4, *P<0.05, ^#P<0.05. The level of GSH (E) and GR activity (F) in NMVCs. n=4, **P<0.01, ^#P<0.05.

PA attenuates excessive autophagy in H/R induced NMVCs

Mitochondrial dysfunction is a significant cause of poor prognosis in ischemic heart disease. Mitochondrial autophagy is a crucial process to regulate mitochondrial homeostasis and eliminate damaged mitochondria in time. However, excessive autophagy causes damage to cardiomyocytes. The over-activation of autophagy can promote cell apoptosis, which is the mechanism of injury. We found that excessive autophagy was induced by H/R in NMVCs, which was prevented by PA treatment (Figure 3A–3C). In summary, PA could prevent excessive autophagy in H/R induced NMVCs.

Figure 3. PA inhibited the excessive autophagy in H/R NMVCs. (A, B) Immunofluorescence assay for LC3II and p-mTOR in NMVCs. Scale bar 20 μm. (C) The protein level of LC3, Beclin-1, p62, ATG5, ATG7, mTOR in NMVCs. n=4, *P<0.05, ^#P<0.05.

PA improves cardiac function in I/R mice

To further explore the PA function on cardiac I/R injury, the I/R model was created in mice. The serum levels of LDH, CK-MB isoenzyme, ROS, MDA, SOD, and GSH-px were assessed by ELISA. PA inhibited the increased level of LDH, CK-MB isoenzyme, ROS, and MDA induced by I/R and recovered the level of SOD and GSH-px in I/R mice (Figure 4A–4F). EF and FS were detected by echocardiography. PA recovered the EF and FS in I/R mice (Figure 5A–5C). Then TTC staining was performed to calculate the infarct size. PA-treated mice revealed a lessened infarct size (Figure 5D). TUNEL assay showed that PA reduced the apoptosis level in I/R mice (Figure 5E). PA reduced the number of inflammatory cells and fibroblasts in the myocardium and reduced the degree of edema of the myocardium and interstitial hyperemia (Figure 5F). The results of the transmission electron microscope (TEM) showed that the mitochondria morphology was swollen and ruptured in the I/R group, while the morphology of mitochondria in the PA group was improved (Figure 5G). Similarly, the apoptosis-associated protein level was consistent with in vitro experiments (Figure 5H). In summary, PA improved apoptosis and autophagy level in I/R mice.

Figure 4. Myocardial injury and oxidative stress indices. Serum CK-MB isoenzyme levels (A), serum LDH levels (B), serum ROS levels (C), serum MDA levels (D), serum SOD levels (E), and serum GSH-px levels (F). n=10, *P<0.05, **P<0.01, ^#P<0.05.

Figure 5. PA ameliorates cardiac function in I/R mice. (A) Cardiac function was detected by echocardiography measurement: EF (B) and FS (C). (D) Infarct sizes as the percent of area at risk (AAR). n=4. *P<0.05, ^#P<0.05. (E) Representative images of TUNEL staining. (F) Respective HE-stained images of hearts. (G) Transmission electron microscopy was used to detect mitochondrial morphology. (H) The apoptosis-associated protein level in cardiac tissues, Cytochrome-c (Cyt-c), Bax, and Bcl2. n=4, *P<0.05, ^#P<0.05.

PA inhibited autophagy through the mTOR/p70S6K signal pathway in cardiac I/R injury

The level of autophagy was constantly changing in the stage of I/R. (Figure 6A). Previous research showed that the mTOR/p70S6K signal pathway played a central regulatory role in cell survival, growth and proliferation, and the abnormality of the signal transduction pathway mediated by it is related to a variety of diseases. Here, we assessed the protein level of mTOR and p70S6K in cardiac tissues and cardiomyocytes. Western blot assay performed that PA recovered the protein level of p-mTOR and p-p70S6K in cardiac tissues and cardiomyocytes (Figure 6A, 6B). We detected the autophagy-associated factors in H/R cells after treating them with Rapamycin. We found that Rap co-treated blocked the function of PA on H/R cells (Figure 6C, 6D). Taken together, PA could regulate autophagy via the mTOR/p70S6K signal pathway in cardiac I/R injury.

Figure 6. PA regulates autophagy in I/R via mTOR/p70SK6 signal pathway. (A, B) The protein level of mTOR and p70S6K in cardiac tissues and NMVCs. n=4, *P<0.05, ^#P<0.05. (C) The protein level was detected by Western blot. n=4, *P<0.05 vs. Control group, ^#P<0.05 vs. H/R group, ^&P<0.05 vs. H/R+PA group. (D) He apoptosis-associated protein level in cardiac tissues, Cytochrome-c (Cyt-c), Bax, and Bcl2. n=4, P<0.05 vs. Control group, ^#P<0.05 vs. H/R group, ^&P<0.05 vs. H/R+PA group.

Animal model

30 SPF grade of C57BL/6 mice, weighing (22 ±1) g. I/R model was created as follows [15]. The skin was prepared and disinfected in the operation area of the I/R model, the skin was cut between the third and fourth ribs on the left side of the sternum, the subcutaneous tissue and muscle were bluntly separated layer by layer, the chest cavity was opened, the heart was fully exposed with a chest opener, and the LAD was carefully identified under stereoscopic microscope. The suture needle with 6-0 thread was used to pass through the surface layer of the myocardium at the inferior edge of the auricle 1mm, and the LAD was ligated under pressure. After ligation of LAD, it was found that the anterior wall of the left ventricle was whitened, the local contractile movement was limited, the left atrial appendage was filled and inflated, and the ST segment was continuously arched and raised, indicating the success of myocardial ischemia. The chest cavity was closed by simple suture, and the chest cavity was opened again after ischemia for 30 min. The polyethylene tube was removed, and the ligation line was cut. The surface of the heart changed from pallor to red, and the elevated part of the ST segment of ECG fell back to judge the success of reperfusion. When the mice wake up, extubate the trachea and put it on a 37° C blanket for the night. After 12 hours, the heart function of mice was detected by echocardiography, and the heart was separated and stored at-80° C.

The PA-treated mice were given suspensions by gavage containing PA (5 mg/kg body weight) for 3 days before the model was induced, Then the mice were constructed for the I/R model.

All animal work has been reviewed and approved by the Animal Care and Use Committee our hospital and carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals.

NMVCs cultured

C57BL/6 mice (1-3 days old) were selected, soaked in alcohol for about 3 min, thoracotomy was performed to remove the apex of the heart under sterile conditions, and pre-cooled with PBS at 4° C for 3-4 times to remove residual blood. Then the heart was cut into a size of about 1 mm³, mixed digestive juices of 0.25% trypsin were added, and digested in a water bath at 37° C. After the first digestion, natural precipitation was required, the supernatant was discarded, and the precipitation was taken for digestion. After natural precipitation, the supernatant was taken for digestion. DMEM containing 10% FBS was added to terminate digestion. After repeated digestion for 5-6 times, all the collected supernatant was filtered with a 200-mesh stainless steel screen to remove the residual myocardial tissue mass, and the cell suspension was collected. After centrifugation for 5 min at 1000 r/min, the supernatant was discarded, and the cells were suspended once in DMEM medium containing 10%FBS. The supernatant was centrifuged at 1000 r/min, and the cells were resuspended and inoculated in a 100 mm culture plate. The cells were adhered to the wall at differential speed for 60min. The supernatant was absorbed and replanted to the plate, which was the primary cultured cardiomyocytes. In order to inhibit the growth of fibroblasts, Brdu with a final concentration of 0.1 mmol/L was added to the medium, and the solution was changed after 24 h culture, and then once every 2 days. Cells from the third day of culture were collected for subsequent experiments.

NMVCs were pretreated with PA (MEC, USA) for 1, 3, 5, 10 μg/ml for 30min. Then the NMVCs were then exposed to hypoxia 1h (0.1% O₂, 5% CO₂) and reoxygenation for 12h.

Transmission electron microscope (TEM)

The left ventricular tissues were fixed with glutaraldehyde and Osmic acid, dehydrated step by step with ethanol acetone, embedded with epoxy resin, ultra-thin section of LKB- V type ultrathin section mechanism, ultra-thin section, double staining of uranyl acetate and lead citrate, and autophagy bodies in cardiomyocytes were observed and counted under TEM.

Western blot

The cardiac tissues were extracted by RIPA 200 μL, and the protein level of the sample was detected by BCA method. After balancing the protein concentration of each group, the protein was separated by SDS-PAGE gel electrophoresis. The protein was transferred to NC membrane by wet method, 5% skimmed milk powder was incubated for 2h, primary antibody was added and incubated overnight in a closed bag at 4° C, and the fluorescent second antibody was incubated at room temperature for 1 h. The gray level of the target strip is analyzed, and the result is expressed by the ratio of the target to the loading control. The primary antibody was shown as follow. Bax Polyclonal Antibody (Proteintech, 50599-2-Ig, 21kDa); Bcl2 Polyclonal Antibody (Proteintech, 12789-1-AP, 25kDa); Cytochrome C Polyclonal Antibody (Proteintech, 10993-1-AP, 12kDa); LC3 Polyclonal Antibody (Proteintech, 14600-1-AP, 15-20kDa); Beclin 1 Polyclonal Antibody (Proteintech, 11306-1-AP, 52kDa); P62/SQSTM1 Polyclonal Antibody (Proteintech, 18420-1-AP, 62kDa); ATG5 Polyclonal Antibody (Proteintech, 18420-1-AP, 52kDa); ATG7 Polyclonal Antibody (Proteintech, 10088-2-AP, 78kDa); Phospho-mTOR (Ser2448) Monoclonal Antibody (Proteintech, 67778-1-Ig, 289kDa); mTOR Monoclonal Antibody (Proteintech, 66888-1-Ig, 289kDa); P70(S6K) Polyclonal Antibody (Proteintech, 14485-1-AP, 70kDa); Phospho-p70 S6 Kinase (Thr389/Thr412) Antibody (Affinit, AF3228, 70kDa); GAPDH Monoclonal Antibody (Proteintech, 60004-1-Ig, 36kDa).

TUNEL assay

Left ventricular tissues or NMVCs were fixed with 4% paraformaldehyde, ethanol gradient dehydration, paraffin-embedded and sectioned, operated according to TUNEL kit, and cardiomyocyte apoptosis was observed by fluorescence microscope. The specific experimental operation refers to the instruction manual of DeadEed^™Fluorometric TUNEL detection kit.

ROS kit assay

The cells were inoculated into 24-well plates, and after treatment, the cells were cleaned 3 times with PBS preheated at 37° C for 5 min each time. DCFH-DA was diluted with PBS buffer (1:1000), and working solution was prepared. 200 μL working solution was added to each well, and incubated in the incubator against light for 30 min. Then the incubator was cleaned with PBS at 37° C for 3 times, 5 min each time, and then observed and photographed under a fluorescence microscope. The image-Pro Plus software was used to analyze the fluorescence intensity, and the average fluorescence intensity represented the relative ROS level.

Apoptosis analysis

The cells were inoculated in 24-well plates with a density of 5×10⁴ cells per well. After treatment, the cells were fixed with poformaldehyde and permeated with 0.01%Triton X-100 (Sigma, USA). The cells were incubated by TUNEL enzyme reaction mixture for 60 min, and then the cells were washed with PBS for 3 times and observed under a fluorescence microscope.

Echocardiography

Mice were anesthetized by isoflurane, the left interventricular septum thickness (IVS) and the left interventricular septum thickness (IVS) were measured by high resolution small animal ultrasound imaging system. S), left ventricular posterior wall end-diastolic thickness (LVPW, d), LVPW (S), ejection fraction, EF), each index was measured and averaged over 2-3 cardiac cycles.

Statistical analysis

The data were analyzed by GraphPad 8.0. the experimental data were expressed in ±SEM, and the difference was statistically significant by T-test or one-way ANOVA. The difference was statistically significant (P < 0.05).