Apoptosis of THP-1 Macrophages Induced by Pseudohypericin-Mediated Sonodynamic Therapy Through the Mitochondria-Caspase Pathway

Atherosclerosis is the major cause of myocardial infarction and stroke, which together constitute the leading cause of mortality in cardiac patients worldwide [1, 2]. Coronary atherosclerosis plaque rupture is the main cause of myocardial infarction Macrophages are one of the most important inflammatory cells in unstable atherosclerosis plaques and play an indispensable role in stages of atherosclerosis [3, 4, 5]. Decreasing the infiltration of an atherosclerotic plaque with macrophages could stabilize the plaque and inhibit its progression. Targeting macrophages is one avenue for the growing interest of identifying and treating vulnerable plaques. The most frequently used treatment approaches include atherectomy, bypass surgery, endarterectomy, balloon angioplasty and stenting. However, the above techniques are correlated with restenosis resulting from intimal hyperplasia or constrictive remodeling [6]. Recently, photodynamic therapy (PDT) has been used in the regression of atherosclerotic plaques [7]. However, the application of PDT is limited to superficial lesions because of its definite penetration. Unlike PDT, sonodynamic therapy (SDT) can penetrate deep into tissues [8, 9], which is a promising noninvasive approach for the treatment of atherosclerotic plaque based on the synergistic effect of low intensity ultrasound and sonosensitization [10, 11, 12]. The sonosensitizer plays a crucial role in SDT. However, sonosensitizers have the obvious disadvantage of high dosage requirements [13, 14]. It is important to find a more readily available sonosensitizer that can overcome this. Most sonosensitizers come from photosensitizers [15, 16, 17], such as hematoporphyrin and aminolevulinic acid (ALA). Recently our group indicated that hydroxyl acetylated curcumin, and hypericin mediated SDT decreased macrophage viability and induced apoptosis of macrophages in vitro [18, 19].

Pseudohypericin and its congener hypericin have been used as photosensitizers [20]. Pseudohypericin possesses an interesting hydroxymethyl substituent [21], and the median elimination half-lives were determined to be 24.8 and 48.2 h for pseudohypericin and hypericin, respectively [20]. Our group previously showed that hypericin could be used as a sonosensitizer at a low dose[19]. Therefore, the effectiveness of P-HY as a potential sonosensitizer for SDT attracts interest for to be further investigation.

In this study, we presented a possible application for P-HY-SDT to induce THP-1 macrophage apoptosis, and subsequently determined whether the ROS generation was able to induce the collapse of the mitochondrial membrane potential through the translocation of BAX to the mitochondria leading to the release of Cytochrome C to the cytosol, which ultimately induced the activation of the mitochondria-caspase pathway.

Pseudohypericin (P-HY) was purchased from Sigma-Aldrich (St Louis, MO, USA) and was dissolved in RPMI 1640 medium (HyClone, Logan, UT, USA) and stored at -20°C in the dark.

The ultrasonic generator and power amplifier used in this study were assembled by the Condensed Matter Science and Technology Institute at the Harbin Institute of Technology (Harbin, China). The transducer (diameter 3.5 cm, resonance frequency 1.0 MHz, duty factor 10%, and repetition frequency 100 Hz) was fixed using upward aluminum stents and submerged in a special vitreous container containing degassed water at a 30 cm vertical distance from the cells. The average spatial ultrasonic intensity in the Petri dish was 0.5 W/cm2 as measured using a hydrophone (Onda Corporation, Sunnyvale, CA, USA). During the sonication procedure, the temperature of the solution inside the Petri dishes increased by less than 0.5°C and was measured with a thermometer.

Human THP-1 monocytic leukemia cells were obtained from the American Type Culture Collection [ATCC] (Manassas, VA, USA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and Penicillin-Streptomycin (56.1 µmol/L penicillin and 27.4 µmol/L streptomycin) at 37°C in a humidified atmosphere with 5% CO₂. For experiments, cells were seeded in the 35 mm Petri dishes or 96-well plates and were differentiated into THP-1 macrophages by adding 100 ng/mL phorbol-12-myristate-13-acetate (La Jolla, CA, USA) for 72 h.

THP-1 derived macrophages were divided randomly into four groups: (1) control, (2) ultrasound alone (ultrasound), (3) P-HY alone (P-HY), and (4) P-HY plus ultrasound (SDT). For the P-HY and SDT groups, the cells were incubated with 0.4 µg/mL P-HY for a 4 h drug-loading time in RPMI 1640 medium supplemented with 10% FBS. For the control and ultrasound groups, an equivalent volume of medium was used to replace P-HY. The cells in the ultrasound and SDT groups were exposed to an ultrasound at a frequency of 1.0 MHz and an intensity of 0.5 W/cm² for 15 min. After the treatment, the cells were cultured in fresh medium for a further 6 h and then prepared for different analyses.

For inhibitory studies, 10 mM sodium azide (NaN₃, Sigma, Aldrich, USA), mannitol (Beyotime Biotechnology, Inc., Beijing, China), 100 µg/mL superoxide dismutase (SOD, Beyotime Biotechnology, Inc., Beijing, China), 100 µg/mL catalase (CAT, Beyotime Biotechnology, Inc., Beijing, China), 1 mM N-acetyl cysteine (NAC, Sigma, Aldrich, USA), 20 µM z-VAD-FMK (z-VAD, BioVision Inc., USA), 0.5 µM cyclosporin A (CsA, Sigma, Aldrich, USA), 100 µM bongkrekic acid (BA, Sigma, Aldrich, USA) and 1 µM 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium (DIDS, Sigma, Aldrich, USA) were incubated together with P-HY for 4 h.

The survival rate of the cells was measured using a CCK-8 colorimetric assay (Beyotime Biotechnology, Inc., Beijing, China) according to the manufacturer's protocol. During the experiments, the cells treated with phorbol-12-myristate-13-acetate (100 ng/mL) were seeded into a 96-well plate at a density of 1 × 10⁵ cells per milliliter and incubated for 72 h to differentiate into THP-1 macrophages followed by different experimental treatments. At the indicated time after the treatments, the medium was removed and 100 µL of medium containing CCK-8 (the medium and CCK-8 volume ratio was 9:1) without FBS was added to each well. After incubation for 2 h at 37°C in a CO₂ incubator, the absorption at 450 nm of each well was measured using a microplate reader (Varian Australia Pty Ltd., Australia).

To study the intracellular metabolic kinetics of P-HY, THP-1 macrophages were incubated with P-HY in 35 mm Petri dishes for different lengths of time (0 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h and 6 h) at 37°C in the dark. After washing twice with PBS, the cells were examined under a florescence microscope (Olympus IX81, Olympus Corporation, Japan) using a filter with a 500-575 nm excitation wavelength and a 620-780 nm emission wavelength.

Cell apoptosis and necrosis were assessed by the Annexin VFITC apoptosis kit (Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Cells were divided randomly into four groups (control, ALA-SDT, HY-SDT and P-HY-SDT). In the SDT group, the concentration of P-HY, HY and ALA was 0.4 µg/mL. After 4 h incubation in the dark, the cells in the ultrasound alone and SDT groups were exposed to the ultrasound for 15 min. After 6 h, the cells were incubated with 5 mL Annexin V and 5 mL propidium iodide for 10 min at room temperature in the dark. Cells from each sample were then analyzed by FacsCalibur flow cytometer (BD Biosciences). The data were analyzed using CELLQuest software (BD). The results were interpreted in the following fashion: cells in the lower-left quadrant (Annexin-V^—/PI^—) represent living cells; those in the lower-right quadrant (Annexin-V⁺/PI^—) represent early apoptotic cells; those in the upper-right quadrant (Annexin-V⁺/PI⁺) represent late apoptotic cells; and those in the upper-left quadrant (Annexin-V^—/PI⁺) represent necrotic cells. Experiments were repeated at least three times independently.

The florescence intensity of 2′, 7′-dichloroflorescein (DCF; Molecular Probes, Eugene, OR, USA) was used to determine intracellular ROS generation. Within the cell, esterase cleaved the acetate groups on 2′, 7′-dichlorofluorescein diacetate (DCFH-DA; Applygen Technologies Co., Ltd, Beijing, China), intracellularly trapping the reduced probe dichlorodihydroflorescein (DCFH). ROS in the cells would then rapidly oxidize DCFH, yielding the highly florescent product DCF. Six h after SDT, the cells were washed twice with PBS and incubated with DCFH-DA (20 µM, diluted in serum-free medium) for 30 min at 37°C in the dark. Then, the cells were carefully washed twice with PBS. Immediately after washing, florescence was measured via CLSM (LSM 510 Meta; Zeiss, Gottingen, Germany) at 488 nm excitation and 525 nm emission wavelengths. The obtained images were subsequently processed using Image-Pro Plus software and Zeiss CLSM software.

The florescent, lipophilic, cationic probe jc-1 (Beyotime Biotechnology Inc. Beijing, China) was used to assess the mitochondrial membrane potential (∆Ψm). Six h after P-HY-SDT, macrophages were incubated with 10 mg/mL jc-1 for 20 min at 37°C in the dark. Then, the cells were washed twice with PBS. Immediately after washing, florescence was monitored with florescence microscopy. Red-orange florescence produced by jc-1 aggregation in the mitochondrial matrix suggested a high ∆Ψm. Green florescence generated by jc-1 monomers in the cytosol indicated ∆Ψm collapse. The florescence intensity was measured at 488 nm excitation and 530 nm and 590 nm emission wavelengths with a fluorospectrophotometer (Varian Australia Pty Ltd). To investigate the relationship between mitochondria, ROS, and singlet oxygen, the cells were pretreated with 0.5 µM CsA, 1.0 mM NAC, or 10 mM NaN₃. Then, cells were photographed with the fluorescence microscope (Olympus IX81, Olympus Corporation, Japan). The images were subsequently processed using Image-Pro Plus software.

The mitochondrial permeability transition pore (mPTP) opening in THP-1 macrophages was measured by detecting the fluorescence intensity of calcein-AM and cobalt chloride (GENMED Scientifics Inc., USA). The cells were loaded with 5 µM calcein-AM in the presence of 5 mM cobalt chloride in the dark for 20 min at 37°C as previously described. After various treatments, the cells were stained with 5.0 µM calcein-AM in the presence of 5.0 mM cobalt chloride in the dark for 15 min at 37°C. Then, the cells were washed twice with PBS before detection. Calcein-AM fluorescence was measured at 525 nm following excitation at 488 nm. For analysis of the effects of different agents on P-HY-SDT induced mitochondrial permeabilization, the cells were pretreated with inhibitors (DIDS, BA and CsA) and atractyloside, which acts to open mPTP.

The protein concentration was measured using a bicinchoninic acid kit (BCA, Beyotime Biotechnology, Inc., Beijing, China) according to the manufacturer's instructions. Denatured protein samples (50 µg) were resolved using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated at room temperature for 1 h in blocking buffer (5% low-fat milk powder in tris-buffered saline-Tween 20). The membranes were incubated with primary antibodies at 4°C overnight. After washing, the membranes were treated with an AP-IgG secondary antibody at room temperature for 2 h. After washing again, the immune complexes were detected using enhanced chemiluminescence reagents. The volumes of the protein bands were quantified using a Bio-Rad Chemi EQ densitometer and Bio-Rad Quantity One software (BioRad Laboratories, Hercules, USA).

Antibodies against BAX (1:1,000), Cytochrome C (1:1,000), caspase 9 (1:1,000), cleaved caspase 9 (1:1,000), caspase 3 (1:1,000), cleaved caspase 3 (1:1,000), PARP (1:1,000), cleaved PARP (1:1,000), and HSP60 (1:1,000) were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., USA). Antibodies against β-actin (1:1,000) were purchased from Proteintech Group (Wuhan, China). The AP-IgG secondary mouse and rabbit antibodies (1:1,000) were purchased from ZhongShan Company (Beijing, China).

All experiments were independently repeated at least three times. The difference between groups was analyzed using a one-way analysis of variance (ANOVA), and the results were presented as the mean ± standard deviation (SD). Values that reached a P<0.05 level of significance were considered statistically significant.

The structure of P-HY was shown in Fig. 1A. Intracellular florescence intensity of P-HY increased with time, peaked at 4 h, then faded over time (Fig. 1B).

To choose the optimized treatment conditions, the cell viability after treating with different P-HY concentrations with/without ultrasound exposure durations was determined by the CCK-8 assay (Fig. 2). When THP-1 macrophages were treated with increasing concentrations of P-HY, the cell viability decreased significantly until P-HY concentration increased to 1.5 µg/mL (Fig. 2A). The concentration of 0.4 µg/mL P-HY had no effect on cell viability at different incubation time (Fig. 2B). There was no cell viability difference observed when THP-1 macrophages were exposed to ultrasound for different durations (Fig. 2C). Cell viability significantly decreased with increasing P-HY concentrations accompanied by ultrasound exposure for 15 min (Fig. 2D). When the cells were treated with 0.4 µg/mL P-HY and subjected to different durations of ultrasound exposure, cell viability decreased with the increasing time (Fig. 2E). According to optimized conditions (0.4 µg/mL P-HY and 15 min of ultrasound exposure), the survival rate decreased significantly in the SDT group (Fig. 2F). The cell viability of macrophages post-SDT also decreased significantly with the increasing time (Fig. 2G). P-HY-SDT and HY-SDT induced cell viability decline at a concentration of 0.4 µg/mL, while ALA-SDT had no obvious effect on cell viability at the same concentration (Fig. 2H).

The flow cytometry analysis results showed that P-HY-SDT treatment induced higher apoptosis than necrosis in THP-1 macrophages, compared with ALA-SDT and HY-SDT (Fig. 3A). As shown in Fig. 3B, the activation of apoptosis-related proteins was also detected in the SDT group, indicating the burst of caspase-dependent apoptosis in the THP-1 macrophages. Fig. 3C showed that cell viability declines could be prevented by the broad-spectrum caspase inhibitor z-VAD. These findings suggested that activation of caspase-related proteins after SDT treatment is the key to induce apoptosis in THP-1 macrophages.

ROS-dependent cellular damage is considered to be the main mechanism involved in SDT, and the results demonstrated ROS generation (Fig. 4A). The relative ROS level significantly increased in the SDT group compared to the control group. ROS generation significantly decreased in the SDT+NAC group and SDT+NaN₃ group. To further determine which free radical scavenger was more efficient in modulating SDT-induced cell viability, the following were tested before the treatment: mannitol, SOD, CAT and NaN₃ (Fig. 4B). Cell viability in the SDT group significantly increased in the presence of NaN₃, but there was no obvious difference when cells were treated with the scavenger, indicating that singlet oxygen might be the main factor involved in cell damage induced by SDT. After pre-treatment with NAC, cell viability was rescued and induced by P-HY-SDT (Fig. 4B). The expression of proteins pretreated with NAC indicated that the expression levels of cleaved caspase 9 and cleaved PARP decreased in the SDT group pretreated with NAC, suggesting that ROS were a key player in the activation of caspase-related proteins (Fig. 4C).

We used jc-1 which selectively enters the mitochondria to further confirm the changes of ∆Ψm. The ∆Ψm began to decrease at the onset of the SDT, and this decrease was gradual within the first 2 h before becoming more precipitous (Fig. 5A). As showed in Fig. 5B, there was no effect on the intensity of red-orange fluorescence among the cells in the control, ultrasound alone or P-HY alone groups. The SDT group showed the highest green fluorescence intensity. The red-orange fluorescence intensity increased, and the green fluorescence decreased in the SDT group pretreated with CsA, NAC and NaN₃, when compared to the SDT group.

To explore whether mPTP opening was involved in apoptosis induced by P-HY-SDT, changes in the opening of the mPTP were assessed using calcein-AM. Control cells displayed high-intensity fluorescence, demonstrating the closed configuration of the mPTP. The SDT group demonstrated calcein fluorescence loss, which also showed that the mPTP was opening (Fig. 6A). After pre-treatment with BA and DIDS, the mPTP opening induced by P-HY-SDT was partially inhibited. When pre-treated with CsA, P-HY-SDT induction of mPTP opening was more effectively inhibited. From these results, we found that P-HY-SDT could induce mPTP opening through the cyclophilin D functional site.

To further confirm that the mechanism of P-HY-SDT induced apoptosis, changes in the expression of key apoptosis-related proteins, BAX and Cytochrome C, were shown in Fig. 6B. BAX was increased in the mitochondria and decreased in the cytosol over time. Cytochrome C, which was released into the cytosol as a result of BAX translocation, activated the caspase cascade. Cytochrome C levels significantly increased in the cytosol after SDT treatment. The translocation of BAX and the release of Cytochrome C in P-HY-SDT were both prevented by NAC (Fig. 6C).

Atherosclerosis (AS) is a lipid-driven chronic inflammatory disorder. The inflammatory response is considered a chief driving force in atherosclerotic plaque formation and growth and progression towards instability and rupture [22, 23, 24]. Remarkably, macrophages are the most abundant inflammatory cell type in plaques and form modified cell formations when there is low density lipoprotein uptake. Their subsequent death within lesions fuel the formation of the highly pro-inflammatory and thrombogenic lipid-rich necrotic core [25, 26]. Moreover, macrophages proliferation is critical for their accumulation in lesions, which accelerates the formation of vulnerable plaques in established atherosclerotic lesions [27]. Therefore, a key step in the reduction of plaque formation is the clearance of macrophage cells.

SDT has attracted more interest as a promising therapy for the directed treatment of AS due to its minimal undesirable damage to surrounding normal tissues [28] and its deep penetration depth in tissues [8, 9]. Our group found that low-intensity ultrasound induces little damage to tissue compared with high-intensity ultrasound [18, 19, 29]. In the previous reports, curcumin, hydroxyl acetylated curcumin, emodin and hypericin were confirmed as a sonosensitizer according to production of single oxygen and induction of THP-1 macrophage apoptosis mediated SDT in vitro. We attempted to investigate the possibility for a lower-dose P-HY as a sonosensitizer on macrophages and the underlying molecular mechanism of P-HY-SDT. In this article, we adopted a lower intensity of 0.5 W/cm², rather than the intensity of 2.0 W/cm² used in previous in vitrostudies [30, 31]. Our results revealed that the accumulation of P-HY in THP-1 macrophages was time-dependent (Fig. 1B), which was different from HY in the previous paper. There are two hypotheses for such phenomenon. First, pseudohypericin possesses an interesting hydroxymethyl substituent [32]. Second, the median elimination half-live of pseudohypericin was shorter than that of hypericin [20]. In the experiments of CCK-8 assay, cell viability was dependent on the ultrasound exposure time at the concentration of 0.4 µg/mL (Fig. 2E). The cell survival rate in the SDT group was much lower than that in the control, P-HY alone and ultrasound alone (Fig. 2F), and was decreased with the time after SDT (Fig. 2G), Therefore, a concentration of 0.4 µg/mL P-HY combining with 15 min of ultrasound exposure was considered optimal for inducing efficient macrophage death in vitro.

Compared with ALA, P-HY reduced THP-1 macrophage viability significantly with the same condition (Fig. 2H). However, the mode of cell death was not indicated. Clinically, apoptosis is preferred way to kill THP-1 macrophages compared to necrosis because it triggers less inflammation [33, 34]. We used two methods to investigate the mode of cell death. First, flow cytometry analysis indicated apoptosis dominated cell death induced by P-HY-SDT. Apoptosis was the main mode of cell death after SDT (Fig. 3A), which was higher than that in the ALA-SDT group and HY-SDT group. Second, the expression levels of the apoptosis-related proteins cleaved caspase 9, cleaved caspase 3, and cleaved PARP increased with the SDT (Fig. 3B). This process interacted with apoptotic protease via caspase recruitment domains, thus activating both caspase 9 and caspase 3, leading to PARP cleavage and initiation of the caspase cascade and ultimately resulting in apoptosis. Based on these results, we concluded that P-HY-SDT induced THP-1 macrophage apoptosis via the caspase-dependent pathway, which was consistent with previous studies [35, 36].

ROS have been recognized as crucial players in the process of apoptosis [35, 37]. Intracellular ROS generated from the mitochondrial electron transport chain directly interacts with mitochondrial proteins and lipids, causing their dysfunction [11, 38]. In the present study, P-HY-SDT increased the levels of ROS within THP-1 macrophages. Several scavengers were selected, including NAC (ROS scavenger), NaN₃ (singlet oxygen scavenger), mannitol (hydroxyl radicals scavenger), SOD (superoxide anion radicals scavenger) and CAT (hydrogen peroxide scavenger), to determine the type of ROS mainly involved in P-HY-SDT. The group with NaN₃ significantly attenuated ROS generation, indicating that P-HY-SDT produced singlet oxygen in macrophages.

The collapse of ∆Ψm and mPTP opening indicate that mitochondrial dysfunction was related to the activation of the apoptotic program through the mitochondrial-caspase pathway [39, 40]. To investigate whether P-HY-SDT induce dmitochondrial damage, we analyzed the mitochondrial depolarization and mPTP opening. The ∆Ψm of THP-1 macrophages in the SDT group was significantly decreased (Fig. 5A) but could be rescued when pretreated with NAC and NaN₃ which was triggered by SDT in a time-dependent manner (Fig. 5B), suggesting that ROS resulted from the collapse of ∆Ψm. Previous studies reported that BAX translocated to the mitochondria, which caused the collapse of the ∆Ψm through regulating the mPTP opening [6, 19, 41]. The loss of ∆Ψm allowed for the release of Cytochrome C and subsequently induced apoptotic cell death through a synergic effect [42]. The proportion the P-HY-SDT group with loss of calcein fluorescence increased significantly, suggesting that P-HY-SDT induced the mPTP opening (Fig. 6A). P-HY-SDT triggered mPTP opening as the pretreatment of cells with CsA significantly inhibited ∆Ψm loss and cell death. Therefore, mPTP opening was the key event in SDT-induced cell death. Following this line, we also observed both BAX translocation from the cytosol to the mitochondria and Cytochrome C release from the mitochondria to the cytosol after SDT (Fig. 6B). This process was prevented by NAC, which indicated that ROS may be an exogenous initiator of this biological process in the mitochondria (Fig. 6B). These results demonstrated that P-HY-SDT induced mitochondrial damage, including the loss of ∆Ψm and the opening of mPTP, which was activated by ROS generation following SDT.

This paper demonstrated P-HY-SDT induce apoptosis through the mitochondria-caspase pathway via ROS generation in vitro, which may provide a potential use in the treatment of AS. However, given that the role of macrophages in the disease process is complicated, further investigations of P-HY-SDT in animal models of atherosclerosis should be performed.

This study was supported by the National Natural Science Foundation of China (81000688, 81271734, 81571833), the Heilongjiang Provincial Science Foundation (H2015006), the Foundation of Science and Technology Innovation Talent of Harbin Science and Technology Bureau (2015RAQXJ100), the Wu Liande Youth Science Foundation of Harbin Medical University (WLD-QN1104), the Postdoctoral Science-research Developmental Foundation of Heilongjiang Province (LBHQ12049), the China Postdoctoral Science Foundation funded project (20090460911), Key Labolatory of Myocardial Ischemia, Harbin Medical University, Chinese Ministry of Education (KF 201212).

The authors report no conflicts of interest in this work.

X. Zheng, J. Wu and Q. Shao contributed equally to this work.